Wound management can often be a challenging experience, especially in the presence of diabetes mellitus, vascular or immunological compromise. While no single technique can be considered by itself to be ideal, vacuum-assisted healing, which is a recent innovation, is fast becoming a necessary addition as adjuvant therapy to hasten wound healing.To determine the efficacy of vacuum-assisted healing.Plastic surgery centre. Ministry of Health Hospital, Kuwait.Patients from Kuwait in a wide variety of clinical situations were chosen for study: Patients (n=50) were classified by diagnosis: Group 1: pressure sore- sacral (n= 3), trochanteric (n=6), ischial (n= 2); Group 2: ulcers (n= 11); Group 3: traumatic soft tissue wounds (n =15); Group 4: extensive tissue loss from the abdominal wall perineum, thigh and axilla (n =5); Group 5: sternal dehiscence wounds (n =4) and Group 6: wounds from flap necrosis (n =4). All wounds were subjected to vacuum by wall unit or portable unit, using pressure of 100-125 mm - continuous or intermittent. Closure of wounds, significant reduction in size and refusal by patient for continuation of vacuum-assisted closure therapy were end points of vacuum application.Sixteen per cent of patients showed complete healing of the wound. Seventy per cent of patients showed 20-78% reduction in wound size. In 14% of patients treatment had to be discontinued. All patients showed improvement in granulation tissue and reduction in bacterial isolates and tissue oedema.The application of subatmospheric pressure or negative pressure promotes healing in a wide range of clinical settings and is an advanced wound healing therapy that can optimize patient care, promote rapid wound healing and help manage costs. It may be used in most instances in both hospital and community settings. All patients were explained the choice of available treatment options and consented to undergo VAC therapy. Patients were classified by diagnosis: Group 1: pressure sore - sacral (n= 3), trochanteric (n=6), ischial (n= 2); Group 2: ulcers (n =11); Group 3: traumatic soft tissue wounds (n =15); Group 4: extensive tissue loss from the abdominal wall perineum, thigh and axilla (n =5); Group 5: sternal dehiscence wounds (n =4) and Group 6: wounds from flap necrosis (n =4). The technique of VAC application used in all patients was similar to that described by Argental In the majority of patients (n=32), vacuum pressure (125 mm of Hg) was applied using a wall suction unit. Staphylococcus aureus, Streptococcus, Klebsiella, Proteus, Pseudomonas, Methicillin-resistant staphylococcus aureus and Acinobacter. These organisms were significantly less isolated on termination of therapy. In 40 patients micro-organisms were isolated prior to VAC therapy while in post VAC, they were isolated only in 22 (n=20). The time period of VAC application ranged from five days to four weeks while the healing period of the wounds ranged from two to six weeks.A total of 50 patients were subjected to VAC therapy. Of these 33 were males and the rest females. The age of the patients ranged from 5-72 years. In eight patients VAC therapy resulted in complete healing and closure of the wounds. Seven patients refused to continue with the treatment. Further, all wounds were less exudative following VAC therapy. Four patients refused treatment because the wall unit restricted their mobility significantly. Two patients had pain on VAC application while in one patient severe allergy resulted in the surrounding skin from the adherent occlusive dressing warranting termination of VAC therapy. In the rest of the 35 patients, the wounds showed reduction in sizes ranging from 20-78%. All patients showed some improvement in the quality of granulation tissue. Microrganisms isolated from the wound prior to VAC therapy (n=40) included A 55-year-old gentleman was admitted with a post-traumatic wound of the heel along with osteomyelitis of the calcaneum. A 43-year-old diabetic male, on insulin for the past 10 years, was referred with an abscess wound over the dorsolateral aspect of the right foot . The wouA 12-year-old boy was referred with history of blast injury to the right lower limb. The patient had a fracture of the tibia and fibula with vascular compromise. The fracture was fixed by external fixation following radical debridement and referred for soft tissue coverage. On examination, there was a defect of 10×12 cm with tissue loss and tibialis anterior tendon lay exposed along with the fracture site . Though A 42-year-old male was referred following a road traffic accident to the right lower limb. The patient sustained a fracture of the right ankle joint which was managed by external fixation and debridement of the medial ankle wound had been done. He had a 5×7 cm ankle wound with slough and exposed lower end of tibia and ankle joint . The wouWound healing is the culmination of a complex interaction between the reticuloendothelial and immune systems, with a number of internal and external factors influencing the outcome. In the past, a number of adjuvant therapies like hyperbaric oxygen, growth factors and skin substitutes have been used to enhance wound healing. Over the past few years, the VAC has become a major player with a number of surgical specialities including it in their armamentarium for wound healing. VAC referred to as vacuum-assisted fascial closure (VAFC) has been used in the management of open abdomen, especially where gross contamination, massive bowel oedema and continued bleeding at re-exploration may be present. The VAC system can be an effective and economically viable method of containing fistula effluent, protecting the skin of patients with enterocutaneous fistulae and promoting healing of the fistula.3 VAC is 6VAC has been effectively applied to heal or improve wounds in a variety of circumstances: exposed bone in burn patients, exposed tendons, extensive wounds following necrotizing fasciitis, salvage of exposed alloplastic materials in irradiated wounds, exposed instrumentation in spinal wounds, calciphylaxis-induced chronic wounds, improving bioengineered tissue grafts in diabetic foot wounds, in the management of infected vascular grafts in vascular surgery and in chronic pilonidal sinuses.–16 It ha17In vitro studies of VAC therapy have demonstrated fibroblast migration, increased proliferation through mitosis and the creation of a micro-environment that promotes granulation tissue formation.[ormation.–30In an experimental study in rabbits, it was found that VAC increased capillary calibre and blood volume, stimulating angiogenesis thereby improving the blood circulation in wounds. Further, it narrowed endothelial spaces and restored the integrity of capillary basement membranes causing a decrease in the permeability of blood vessels and wound oedema by removing excessive fluid from the wound bed. Signific5 colonies of bacteria per gram of tissue, studies have found that there is no consistent effect of bacterial clearance with the application of VAC in wound management. While some studies have found tissue bacterial counts to have significantly decreased after four days of application, other studies have shown that bacterial colonization increased significantly with wound VAC therapy and remained in a range of 104 −106.[Staphylococcus aureus showed a significant increase in VAC-treated wounds. Despite this finding, the beneficial effects of this treatment modality on wound healing were noted in most cases.[The bacterial colonization of a wound is a recognized detrimental factor in the multifactorial process of wound healing. While the harmful effects on wound healing are recognized to correspond to a level of > 10104 −106.26 Non-fest cases.In vitro studies have revealed that cells allowed to stretch tend to divide and proliferate in the presence of soluble mitogens, whereas retracted cells remain quiescent. In a computer model (finite element) of a wound, which simulated VAC application it was shown that most elements stretched by VAC application experienced deformations of 5-20% strain, similar to in vitro strain levels shown to promote cellular proliferation. The application of micromechanical forces through VAC may be a useful method with which to stimulate wound healing through promotion of cell division, angiogenesis, and local elaboration of growth factors.[Wound surface area reduction was significantly larger in VAC-treated wounds compared to conventionally managed wounds. While cl factors. The cycl factors. The appl factors. VAC can  factors. Studies  factors. SubatmosWhile both continuous suction and alternating pressure cycles have been used, the optimal subatmospheric pressure for wound, healing appears to be approximately 125 mm Hg utilizing an alternating pressure cycle of 5 min of suction followed by 2 min off suction. In most In patients with diabetes mellitus, presence of a high wound bacterial load and complications such as polyneuropathy and peripheral angiopathy lead to foot ulcers, gangrene, and osteoarthropathy which may necessitate minor or major amputation. Free tissue transfer or local flap surgery is often difficult or even impossible owing to an impaired arterial circulation. The VAC can be of great benefit in such cases by helping to heal the wounds satisfactorily. Because it is a closed system of treatment, it has the added benefit of minimizing exposure of staff and other patients to communicable diseases.Pain was not a predominant feature although some patients complained of discomfort during initial application of VAC; reduction in suction pressure helps to minimize it. In all our patients where VAC therapy was used, wound care was simplified and healing accelerated. The VAC device allowed optimal wound closure by preparing the wound for skin grafting or flap closure when required, in cases where the wounds did not heal completely with VAC therapy. Its use in the perineum presents a challenge, but with proper application, even the most complex perineal wounds can be healed. A cost-benefit analysis revealed that VAC therapy was genuinely cost-effective. VAC wounThe VAC should be used with caution over exposed vessels as it has been shown to cause erosion and haemorrhage of the left anterior tibial artery. ApplicatThe VAC device is an extremely versatile tool in the armamentarium of wound healing. Application of controlled subatmospheric pressure creates an environment that promotes wound healing. In order to heal a chronic wound, a number of factors need to be addressed like oxygen delivery to the tissue, wound nutritional requirements, a moist environment, debridement of slough and necrotic tissue and correction of associated medical illnesses. While in some patients these factors may be easily dealt with, in others adjunctive therapies like hyperbaric oxygen, growth factors and skin substitutes can help to heal the wounds. The VAC is an additional tool that can be of great benefit in healing wounds in selected patients. If utilized selectively and judiciously, VAC is a valuable tool; but its prolonged application over several weeks at the expense of early surgical reconstruction needs to be deplored. Ideologically, the VAC was never meant to replace surgical interventions, only to complement them. VAC is generally well tolerated and with few contraindications or complications, is fast becoming a mainstay of current wound care.


Based on neurochemical and genetic evidence, we suggest that both prevention and treatment of multiple addictions, such as dependence to alcohol, nicotine and glucose, should involve a biphasic approach. Thus, acute treatment should consist of preferential blocking of postsynaptic Nucleus Accumbens (NAc) dopamine receptors (D1-D5), whereas long term activation of the mesolimbic dopaminergic system should involve activation and/or release of Dopamine (DA) at the NAc site. Failure to do so will result in abnormal mood, behavior and potential suicide ideation. Individuals possessing a paucity of serotonergic and/or dopaminergic receptors, and an increased rate of synaptic DA catabolism due to high catabolic genotype of the COMT gene, are predisposed to self-medicating any substance or behavior that will activate DA release, including alcohol, opiates, psychostimulants, nicotine, gambling, sex, and even excessive internet gaming. Acute utilization of these substances and/or stimulatory behaviors induces a feeling of well being. Unfortunately, sustained and prolonged abuse leads to a toxic" pseudo feeling" of well being resulting in tolerance and disease or discomfort. Thus, a reduced number of DA receptors, due to carrying the DRD2 A1 allelic genotype, results in excessive craving behavior; whereas a normal or sufficient amount of DA receptors results in low craving behavior. In terms of preventing substance abuse, one goal would be to induce a proliferation of DA D2 receptors in genetically prone individuals. While in vivo experiments using a typical D2 receptor agonist induce down regulation, experiments in vitro have shown that constant stimulation of the DA receptor system via a known D2 agonist results in significant proliferation of D2 receptors in spite of genetic antecedents. In essence, D2 receptor stimulation signals negative feedback mechanisms in the mesolimbic system to induce mRNA expression causing proliferation of D2 receptors.The authors propose that D2 receptor stimulation can be accomplished via the use of Synapatmine™, a natural but therapeutic nutraceutical formulation that potentially induces DA release, causing the same induction of D2-directed mRNA and thus proliferation of D2 receptors in the human. This proliferation of D2 receptors in turn will induce the attenuation of craving behavior. In fact as mentioned earlier, this model has been proven in research showing DNA-directed compensatory overexpression (a form of gene therapy) of the DRD2 receptors, resulting in a significant reduction in alcohol craving behavior in alcohol preferring rodents. Utilizing natural dopaminergic repletion therapy to promote long term dopaminergic activation will ultimately lead to a common, safe and effective modality to treat Reward Deficiency Syndrome (RDS) behaviors including Substance Use Disorders (SUD), Attention Deficit Hyperactivity Disorder (ADHD), Obesity and other reward deficient aberrant behaviors. This concept is further supported by the more comprehensive understanding of the role of dopamine in the NAc as a "wanting" messenger in the meso-limbic DA system. The abusive behaviors also lead to neurotransmitter dysfunction via depletion. Therefore both substance seeking and pathological behaviors as ways of providing a feel good response (FGR) "fix" result in ever escalating and uncontrollable craving behavior. It has been well established that individuals possessing certain genetic polymorphisms (variations) are particularly prone to amplified polymorphic expressions with environmental or lifestyle insult and will be at increased risk for impulsive, compulsive and addictive behaviors  etc.) ,60. The With this said we must consider these results with caution, especially in terms of in vivo studies by Chen et al  showing It is noteworthy that diminished DA receptors are not inevitably associated with depression or addictive behaviors. In fact, while the lower incidence of Parkinson's disease (PD) among smokers may be explained by a protective effect of cigarette smoke, or by a tendency to avoid addictive behaviors among future PD cases, this does not hold true for alcoholism. Hernan et al  conducteThere is an abundance of studies showing that acute blockade of DA receptors will result in an attenuation of substance seeking as in the case observed for the Cannabinoid CB1 receptor antagonist, Rimonabant, which neuronal blocks DA-release . This anIn brief, the site of the brain where one experiences feelings of well being is the mesolimbic system. This part of the brain has been termed the "reward center". The chemical messages include serotonin, enkephalins, GABA and dopamine, all working in concert to provide a net release of DA at the NAc (a region in the mesolimbic system). It is well known that genes control the synthesis, vesicular storage, metabolism, receptor formation and catabolism of neurotransmitters. The polymorphic versions of these genes have certain variations, which could lead to an impairment of the neurochemical events involved in the neuronal release of DA. The cascade of these neuronal events has been termed "Brain Reward Cascade". A breakdown of this cascade will ultimately lead to a dysregulation and dysfunction of DA. Since DA has been established as the "pleasure molecule" and the "anti-stress molecule," any reduction in function could lead to reward deficiency and resultant aberrant substance seeking behavior. Our physiology is motivationally programmed to drink, eat, have sex and desire pleasurable experiences. Impairment of the mechanisms involved in these natural processes leads to multiple impulsive, compulsive and addictive behaviors governed by genetic polymorphic antecedents. While there are a plethora of genetic variations at the level of mesolimbic activity, polymorphisms of the serotonergic-2A receptor (5-HTT2a), dopamine D2 receptor (DRD2) and catechol-o-methyl-transferase (COMT) genes predispose individuals to excessive cravings and resultant aberrant behaviors. An umbrella term to describe common genetic antecedents of multiple impulsive, compulsive and addictive behaviors is RDS. Individuals possessing a paucity of serotonergic and/or dopaminergic receptors and a increased rate of synaptic DA catabolism due to a high catabolic genotype of the COMT gene are predisposed to self-medicating with any substance or behavior that will activate DA release, including alcohol, opiates, psychostimulants, nicotine, gambling, sex, and even excessive internet gaming, among others. Acute utilization of these substances induces a feeling of well being. But unfortunately, sustained and prolonged abuse leads to a toxic pseudo feeling of well being resulting in tolerance and disease or discomfort. Thus, low DA receptor levels consequent on carrying the DRD2 A1 allelic genotype result in excessive cravings and consequential behavior, whereas normal or high DA receptors levels result in low craving-induced behavior. In terms of preventing substance abuse, one goal would be to induce a proliferation of DA D2 receptors in genetically prone individuals. Experiments in vitro have shown that constant stimulation of the DA receptor system via a known D2 agonist results in significant proliferation of D2 receptors in spite of genetic antecedents. In essence, D2 receptor stimulation signals negative feedback mechanisms in the mesolimbic system to induce mRNA expression, causing proliferation of D2 receptors. This molecular finding serves as the basis for inducing DA release naturally, also causing the same induction of D2-directed mRNA and thus proliferation of D2 receptors in the human. This proliferation of D2 receptors will in turn induce the attenuation of craving behavior. In fact, as mentioned earlier, this has been proven with work showing DNA-directed overexpression (a form of gene therapy) of the DRD2 receptors and significant reduction in alcohol craving-induced behavior in animals . FinallyA new but emerging concept provides a more comprehensive understanding of reward behaviors and the role of DA. In fact, interfering with accumbens DA appears partially to dissociate the process of primary reinforcement from processes regulating instrumental response initiation, maintenance and selection . The facWe therefore suggest further that the biochemical and molecular changes that take place in dopaminergic and enkephalinergic systems following continuous neutraceutical treatment with dopamine agonists may underlie the mechanisms by which certain dopamine-mediated behaviors may be influenced. It is our intention to perform micro-dialysis studies showing that precursor amino acid therapy and enkephalinase inhibition induce DA release at the nucleus accumbens of both animals and humans, as well to perform additional clinical trials using nutrigenomic principles ,77. FinaKenneth Blum, Lonna Williams, B William Downs, and Roger Waite are officers of LifeGen Inc. and current stock holders. LifeGen, Inc. is the worldwide distributor of Synaptamine.™KB – Investigator and major contributor to writing of manuscript; ALHCC – Co-investigator and contributor to hypothesis; TJHC – Editorial contributions; ERB – Co-writer and editorial support; SHB – Literature search; KC – Editorial review; BWD – Major editor and co-writer; RLW – Contributor to clinical aspects of the commentary; LW – Editorial contributions; TJP – Statistical contributions and co-contributor to overall concept; MK – Editorial assistant and scientific review; TP – Editorial review; DEC – Contributor to scientific validity and editorial; HT – Editorial review; JR – Editorial review; PR – Editorial review; MOB – Contributor to writing manuscript and editorial and literature review.


The discovery of BRAF mutations in melanoma has not yet translated into clinical success, suggesting that BRAF/MEK inhibitors will need to be combined with other agents. In the current review, we discuss other pathways likely to be important for melanoma progression and suggest possible drug combinations for future clinical testing. BRAF in over 60% of melanomas has raised expectations that melanoma may be amenable to targeted therapy. The most prevalent mutation in BRAF is at the V600E position, resulting in the substitution of valine to glutamate and destabilisation of the inactive kinase conformation switching the equilibrium towards the active form (BRAF V600E is thought to result through the activation of the mitogen-activated protein kinase (MAPK) pathway . A number of studies have now shown that sorafenib induces melanoma cell apoptosis BRAF V600E mutation compared with wild-type BRAF, such as SB590885   is a kinase that occupies a pivotal position in growth factor receptor and nutrient availability signalling, and has important downstream effects on cell growth and survival. It is also a part of the AKT signalling pathway and regulates the activity of this kinase in complex ways. Mammalian target of rapamycin can either positively or negatively regulate AKT signalling, and this is dependent on the composition of the mTOR signalling complex. Thus, mTOR forms at least two protein signalling complexes called mTORC1 and mTORC2. mTORC1 consists of mTOR, as well as the regulatory proteins raptor and mLST8. Activation of the mTORC1 signalling complex leads to the phosphorylation and activation of p70 S6kinase (S6K) and the eukaryotic initiation factor 4E-binding protein (4EBP1) . In contAlthough most attention preclinically is focused on the combined inhibition of signal transduction pathways, most clinical interest is focused on the combination of targeted therapy agents with chemotherapy. Already, there are early suggestions that sorafenib may enhance the activity of carboplatin/paclitaxel and dacarbazine  localise to the asters and kinetochores during mitosis, as well as to the actin-microtubule cytoskeleton. Earlier studies have shown that the orally available MEK inhibitor CI-1040 enhances the efficacy of paclitaxel in lung cancer xenograft models , indicating that a subset of BRAF mutant melanomas may be sensitive to this agent. The trial was designed to detect an improvement in progression-free survival compared with single-agent temozolomide. As this activity was not seen, AZD6244 was deemed insufficient to warrant further single-agent clinical trials in melanoma.The MEK inhibitors—PD0325901 and AZD6244—have also progressed through phase I, and in the case of AZD6244 phase II clinical trials. PD0325901 has been evaluated in a phase I trial, in which most of the patients had melanoma. Of these patients, some experienced an objective response (2 out of 27) and 5 additional patients showed some disease stabilisation. Dose-limiting diarrhoea and rash precluded further dose escalation and possibly preclude the delivery of a dose required to adequately suppress the MAPK pathway. Phase II trials of this drug were suspended because of the occurrence of retinal vein thrombosis in several patients. AZD6244 was evaluated in a phase I trial among patients with advanced solid tumors . Similarly, in melanoma, there are early reports of success when selected groups of patients harbouring activating mutations in the receptor tyrosine kinase


Antigenic determinants of p30, the most abundant internal virion protein of C type RNA viruses, were detected on the surface of spleen cells from mice bearing Moloney leukaemia and on an in vitro line of Moloney sarcoma, MSC. On both cell types, these determinants on the p30 molecules served as cytotoxic targets in a xenogenic complement dependent antibody mediated 51Cr release assay. Two antisera were used: a rat anti MLV -M induced lymphoma serum, and an antiserum raised in goats to either disrupted FeLV. The cytotoxic target antigens of these antisera were analysed by inhibition of cytotoxicity with viral and cellular proteins.


Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents. Candida sp. is the fourth most common pathogen isolated from blood cultures Cryptococcus sp., Aspergillus sp., Fusarium sp., and zygomycetes, have an unacceptably high morbidity and mortality Members of the fungal kingdom are found in almost all habitats and exist as saprobes, commensals, and pathogens. Approximately 11,000 plant diseases have been attributed to fungi in over 120 genera The diverse nature of fungi can be attributed to such factors as increased capacity to utilize a wide range of carbon and nitrogen sources, the capability for rapid growth, and the ability to adapt to otherwise harsh environments. Fungi are constantly bombarded by toxic compounds from external sources. These compounds can be synthesized by other microorganisms to impede the fungus for a competitive advantage to limited resources, synthesized by hosts as a defense mechanism, or may be present in the environment. Regardless of the source, these toxic compounds force the fungus to evolve mechanisms in order to survive. One common method to overcome these antifungal compounds is active efflux, which prevents any intracellular build-up of the compound and, therefore, renders the fungus resistant or tolerant to the otherwise toxic compound.As research in mycology progresses it is becoming evident that transporters are important factors in pathogenicity. Several transporters have been described as being involved in fungal pathogenicity, including a range of cellular processes such as calcium entry, vescicle transport, stress tolerance, dimorphic switching, capsule synthesis, iron acquition, and virulence factor enzyme activity Aspergillus nidulans and the closely related human pathogen Aspergillus fumigatus both have 45 ABC transporters encoded in their genomes, and A. nidulans has more MFS transporters than A. fumigatus , of which three families are involved in efflux of toxic compounds. Members of these three families, ABCB, ABCC, and ABCG, also referred to as the multi-drug resistance (MDR), multi-drug resistance–associated protein (MRP), and the pleiotropic drug resistance (PDR) families, respectively, have been extensively studied in 6 design of half ABC transporters, which dimerize to form a fully functional protein 6 half transporters, creating one functional protein (The conserved architecture of ABC transporters is composed of a nucleotide-binding domain (NBD) followed or preceded by six transmembrane-spanning helices creating a transmembrane domain (TMD). These two domains together represent the NBD-TMD protein  [21],[22+ antiporter (14 spanner) (DHA14) and the drug:H+ antiporter (12-spanner) (DHA12), are involved in efflux of toxic compounds The MFS transporters are smaller in size compared to the ABC transporters since they do not require an NBD; however, they still contain 12 or 14 transmembrane-spanning helices . TransloVirulence-associated efflux pumps in plant pathogenic fungi can be classified into two categories. The first class of transporters is responsible for secretion of virulence factors or compounds produced by the fungus. Several fungi synthesize and secrete low molecular weight compounds that are not required for growth. These compounds, termed secondary metabolites, are bioactive and are sometimes involved in virulence Cochliobolus and Alternaria species Cochliobolus carbonum produces a cyclic tetrapeptide HST, termed HC-toxin, which functions as an inhibitor of histone deacetylases in several organisms and is a virulence factor on maize TOXA and TOXB, postulated to be responsible for efflux of HC-toxin into the host plant cell Cercospora species produces a host-nonselective toxin, cercosporin, which is also synthesized by a secondary metabolite gene cluster. Cercosporin is exported out of the fungal cell by the cercosporin facilitator protein (CFP) in C. kikuchii and by CTB4 in C. nicotinaeMagnaporthe grisea produces a secondary metabolite via the PKS-NRPS fusion protein termed ACE1 and the surrounding accessory proteins. This cluster contains a gene encoding an MFS transporter (MFS1), although it is not involved in efflux of the ACE1 metabolite as it has a deletion of a single base pair resulting in an early stop codon Phytotoxins produced by fungi that are involved in virulence on a specific host plant, and therefore expand host range, are termed host-specific toxins (HSTs), and while not all plant pathogens produce HSTs, they are important virulence factors for members of the atrD in Aspergillus nidulans, although not in a cluster, produce less penicillin than the wild-type, suggesting this transporter is involved in efflux of the compound Efflux pumps in secondary metabolite gene clusters that are responsible for synthesis of mycotoxins are less well understood. The expression of MFS transporters in mycotoxin secondary metabolite clusters appears not to be controlled by the same transcription factors as the rest of the secondary metabolite cluster but, instead, is induced by the accumulation of the synthesized metabolite Nectria haematococca MPVI and the pea phytoalexin pisatin suggested that a fungal efflux mechanism is involved in the tolerance to this plant-derived defense compound and, therefore, may be a pathogenicity factor M. grisea, the causative agent of rice blast disease The second class of transporters involved in plant virulence is responsible for efflux of molecules produced by the host plant. As a defense mechanism, plants produce antimicrobial compounds. These low molecular weight compounds, termed phytoalexins or phytoanticipins depending on when they are synthesized, have diverse chemical structures. Over 25 years ago, studies conducted on Botrytis cinerea and GpABC1 in Gibberella pulicaris provide tolerance to the host plant's phytoalexins, resveratrol and rishitin, respectively BcatrB mutant is reduced in pathogenicity on grape leaves GpABC1 mutant is essentially nonpathogenic on potato N. haematococca is able to confer an increase in pathogenicity on pea when placed in a nonpathogenic pea isolate PEP5 for pea pathogenicity, is induced upon exposure to the pea phytoalexin pisatin Since that time a number of ABC transporters have been identified as virulence factors on host plants. Most of these transporters are members of the PDR family of ABC transporters . A numbeB. cinerea and Mycospharella graminicola, as well as the model filamentous fungus A. nidulans, have been reviewed recently B. cinerea is also able to provide resistance to the fungicides fenpiclonil and fludioxonil in addition to resveratrol, as referenced above Fungicide resistance is also due to ABC and MFS transporters. Several transporters in the agriculturally relevant fungi C. albicans, has a total of 21 ABC transporters and 85 MFS transporters encoded within its genome , and SNQ2 in C. glabrataS. cerevisiae. The PDR family is one of the largest families of ABC transporters in fungi and is overrepresented when their numbers are compared to other organisms, i.e., there are five and nine ABCG/PDR transporters in humans and Caenorhabditis elegans, respectively A number of transporters that confer resistance to members of the antifungal class of triazoles have been described. These include the ABC transporters CDR1 and CDR2 in CDR2 retain a wild-type level of resistance to fluconazole; however, in a double mutant (ΔCDR1/ΔCDR2) the strain is more susceptible to fluconazole than either single mutant CDR2 genes, and further phylogenetic analysis suggested that as many as 33 codon changes between the two alleles may be selectively advantageous Of the transporters involved in fluconazole resistance in medically important fungi, CDR1 and CDR2 are the most studied. Despite their high degree of amino acid similarity, there are functional differences between the two. Mutants of r) and FLU1 in C. albicansMDR1 have been implicated in fluconazole resistance in C. dubliniensisC. tropicalis+ translocation -Cys(6) finger transcription factor TAC1 CDR1 and CDR2, the transcript levels did not increase trans-acting factors of the CDR genes C. glabrata (CgPDR1 and CgPDR3) have been demonstrated to bind the substrate, promoting the expression of the efflux pumps under its control MDR1FLU1 in clinically resistant isolates is not increased, and therefore FLU1 is not believed to contribute significantly to resistance to fluconazole Pathogenic isolates are able to develop resistance to prescribed antifungal treatment rapidly CDR1/CDR2 overexpressing C. albicans isolates require the TAC1 fluconazole-resistant transcription factor to be homozygous, as heterogygous isolates retain wild-type levels of fluconazole susceptibility MRR1 increases fluconazole resistance CDR1, CDR2, and MRR1 overexpression have been identified, but their role in clinical resistance is less understood. These transcription factors include CaNdt80 in CDR1 overexpression MDR1 overexpression CDR1 transcripts in fluconazole-resistant C. albicans isolates that results in an increase in the number of CDR1 transporters A major difference in the mutations of the major transcription factors that confer fluconazole resistance is the heritability of the resistance phenotype. The S. cerevisiae and C. albicans have relatively few ABC and MFS transporters compared to filamentous fungi, and the scope of research already conducted on these important yeast superfamilies, it appears there is much more to be discovered in other fungi.The role of efflux pumps, in particular ABC and MFS transporters, in fungal virulence cannot be understated. These transporters contribute to pathogenesis by 1) transporting HSTs and mycotoxins outside of the cell, 2) removing host-derived compounds that would otherwise inhibit the fungus, and 3) providing resistance to clinical antifungals. This concept is further supported by the vast amount of substrate redundancy already evidenced with the few transporters that have been extensively studied. Considering The redundant nature of the transporters creates difficulty in characterization of the efflux mechanisms, as single deletion mutants sometimes retain a wild-type phenotype, and it is only through multiple mutations of transporters or expression in heterologous systems that the function of the transporters becomes apparent. Although a daunting endeavor, understanding all of the contributing efflux factors of a given antimicrobial will provide a means to circumvent efflux transporters.


The midbrain dopaminergic (mDA) neurons of the substantia nigra and the ventral tegmental area play a fundamental role in the control of voluntary movement and the regulation of emotion, and are severely affected in Parkinson's disease. Recent advances in mouse genetics and vertebrate development have provided us with insight into the genetic cascades involved in the development of mDA neurons, including the induction of mDA neuron progenitors in the ventral mesencephalon, the specification of the mDA neuronal fate and the maintenance of postmitotic mDA neurons. In parallel, rapid progress has been made in the generation of DA neurons from pluripotent stem cells and the development of stem cell-based therapies for Parkinson's disease. Here, we summarize the new findings via the developmental progression of mDA neurons and outline how this knowledge has been exploited to develop novel paradigms for the in vitro generation of these neurons from embryonic stem cells. Dopamine containing neurons are present in different positions in the vertebrate central nervous system with the largest assembly in the midbrain. Midbrain dopaminergic (mDA) neurons are separated into functionally distinct subgroups called the substantia nigra compacta  and the ventral tegmental area  based on their position within the midbrain and the target structures which they innervate [Because of their involvement in Parkinson's disease and other mental disorders, mDA neurons have been a focus of clinical interest and a subject of intensive studies for a long time. For Parkinson's disease, a potential therapy is to replace the lost mDA neurons with healthy DA neurons that have been generated in vitro through the differentiation of stem cells. To achieve this, a comprehensive understanding of the genetic cues and extrinsic signalling cascade controlling the fate choice of pluripotent embryonic stem (ES) cells into neuroepithelial stem cells and subsequently into functional midbrain specific DA neurons is required. In this regard, recent studies have identified a number of regulatory factors that influence the emergence of mDA neurons during vertebrate embryogenesis. These studies not only have increased our understanding of mDA neuron development in vivo, they have also guided the development of new paradigms for the in vitro generation of mDA neurons from ES cells. In return, ES cell differentiation in vitro provides a powerful research tool for the genetic dissection of mDA neuron development and cell biology.Several excellent reviews have extensively analyzed the extrinsic signalling pathways and genetic programme governing mDA neuronal differentiation and functional maturation -7. In thIn the embryo, the pluripotent cells of the epiblast give rise to the multitude of different cell types of the adult animal. A complex series of extracellular signals and cell autonomous differentiation events transform these cells through germ layer specification, regionalisation and finally cell specific determination. In the generation of neurons of the SNc, research has focused on three major transitions: regionalisation of the neural plate, midbrain cell fate determination and finally terminal differentiation into mDA neurons Figure . DiffereRegionalisation begins early in neural plate development. At its inception the neural plate is axially defined: anterior-posterior and dorsal-ventral. The lateral edges of the neural plate roll up during neuralation becoming dorsal in the newly created tube. The neural plate midline becomes the ventral-most part of the developing nervous system. This positions the midline cells to receive extrinsic signals from the underlying mesoderm as well as signalling factors from within the neural plate  progenitors, more lateral is the Nkx2.2 expressing region and the dorsal cells express Pax3. Coordinated with the induction of a broader Shh domain is a dorsal shift of the Nkx6.1 and Nkx2.2 expressing regions and a reduction of the Pax3 region. This change in the dorsoventral pattern leads to an increase in mDA neurons and a loss of RN neurons. A second mutant, regulating Otx2 expression using the En1 promoter, produces a loss of both ventral and lateral Otx2 expression [Shh, a dorsal shift in Nkx6.1 expression and a dorsal reduction of the Pax expressing region but in this case the Nkx2.2 expanded ventrally into the normal Shh expressing territory. In contrast to the previous result, the combined loss of ventral and lateral Otx2 midbrain expression causes a severe reduction in mDA neurons. These experiments indicate that endogenous lateral Otx2 expression maintains Shh in its ventral domain preventing the Shh inhibition of Nkx6.1. Additionally, Otx2 represses ventral expression of Nkx2.2, which can inhibit mDA neuronal differentiationRecent work with conditional mutants precisely targeted to dissect the spatial regulatory activities of Otx2 has greatly broadened our understanding of the gene interactions necessary to create the midbrain dorsoventral pattern of neuronal fates ,31,32. OFgf8, Shh and Otx2, genes involved in the early regional specification, are capable of promoting dopaminergic cell specification in greater abundance in their normal location as well as ectopically in naïve hindbrain or forebrain tissue ,25,28,32The last few years have been rich in data contributing to understanding of the interactive molecular cascade involved in the multi-step differentiation of the mDA neuron progenitor population as well as identifying key players: Lmx1a, Ngn2, Fox2a, and Shh ,30,33,34Shh is downregulated in the ventral midbrain [Lmx1a in the developing midbrain with siRNA results in a dramatic reduction in Nurr1+ cells whilst its ectopic expression results in an expansion of the Nurr1 expressing midbrain region [Foxa2 expression which in turn positively regulates Ngn2 expression  is at its peak. This is based on the understanding, supported by developmental studies, that Shh induces a DA fate via acting on ES cell derived Nestin+ Sox1+ neural progenitors. However, we found that Shh and FGF8 were unable to stimulate more DA neurons in FACS (fluorescence activated cell sorting) purified Sox1+ neural progenitors than observed in un-sorted control cultures [Induction with Shh and FGF8 during ES cell differentiation can result in a culture in which ~30% of the neuronal population expresses Th. In these studies Shh and FGF8 are applied at the time when neural progenitor production  produced by bone marrow derived PA6 stromal cells . This pron stage . Howeveron stage , authorsTo date, the most efficient production of dopaminergic neurons from ES cells is achieved via genetic manipulation of transcription factors that are normally active in midbrain DA neurons ,47,64-67The pathological hallmark of Parkinson's disease is the preferential loss of the substantia nigra subgroup of mDA neurons. Previous transplantation studies demonstrated that only midbrain dopaminergic cells are therapeutically useful in cell replacement therapy in animal models of Parkinson's disease . Further+, Aldh1+) either by RT-PCR or immunocytochemistry [+ neurons co-express almost completely with known midbrain DA markers including Pitx3, engrailed, Lmx1a, Raldh1 (Ahd2), Nurr1 and pan-DA neuron markers such as AADC, VMAT2 and Th [+DAT+ neurons were Pitx3-GFP+, demonstrating that ES cell-derived DA neurons are heterogeneous and do not all exhibit midbrain identity [+ neurons derived using a monolayer differentiation protocol [+ neurons expressing Pitx3-GFP [With increasing recognition of the necessity of generating DA neurons of 'true' midbrain property, many recent studies have investigated the induction, in ES cell derived neuronal cultures, of midbrain neural progenitor markers  and the generation of midbrain specific post mitotic DA neurons  or other types of lineage-specific stem cells ,83, for NSC can be routinely established by propagating foetal or adult neural tissues and ES cell derived neural progenitors in chemically defined culture media in the presence of FGF and EGF. However, whether propagated adherently or as cell aggregates (neurospheres), FGF and EGF expanded NSC primarily give rise to GABAergic neurons with no apparent regional identity -87. ThisElucidation of the transcription programme associated with R-NSC status may lead to the discovery of master regulators governing NSC potency. A list of candidate marker genes has been identified by transcriptome profiling for the primitive NSC and R-NSC ,89. ReguA similar approach may be taken to program dopaminergic competent NSC by exploiting transcription factors known to act in this specific neuronal lineage. For example, combined gene manipulation of Nurr1 (controlling dopaminergic neurotransmitter phenotype) with Lmx1a or components of the Wnt/Lmx1b-Pitx3 pathway (midbrain identity, Figure One should note that for clinical consideration, it is desirable to produce the therapeutically important somatic cell types using extrinsic growth factors or small molecules during the course of ES cell in vitro differentiation. While we are progressing toward discovering new soluble reagents, genetic based programming provides an important tool to decipher the pathways and mechanisms controlling stem cell fate choice which is fundamental for the establishment of culture parameters capable of sustaining the full complement of NSC potential. Furthermore, the timely investment in iPS related studies should prompt rapid technological developments such as protein transduction or small molecule mediated gene regulation.The past few years have seen a rapid progress in our understanding in the genetic control of mDA neuron fate specification during foetal development. Combinatorial expression of transcription regulators provides markers for ventral mesencephalic neuroepithelial cells of different regional zones and/or at different developmental stages. These mDA neuron lineage markers have greatly facilitated the assessment of 'authentic' mDA neural phenotype that can be derived from ES cells. In addition, the availability of defined mDA neuron progenitor populations with distinct differentiation capacity isolated by lineage marker expression from existing reporter mice and ES cells has initiated the search for the most suitable cell population for cell-based transplantation therapy ,90. MostThe authors declare that they have no competing interestsAll authors participated in developing the ideas, the writing, discussion and integration of information. All authors read and approved the final manuscript.


Wnts are expressed in the embryonic forebrain; however, it is poorly understood which specific Wnt performs which function and how they interact. Wnts are able to activate different intracellular pathways, but which of these pathways become activated in different brain subdivisions also remains enigmatic.Wnt signalling regulates multiple aspects of brain development in vertebrate embryos. A large number of in ovo electroporation approach, we show that Wnt4 expression in the thalamus is negatively regulated by Sonic hedgehog (Shh) signalling from the zona limitans intrathalamica (ZLI), a known organising centre of forebrain development.We have compiled the first comprehensive spatiotemporal atlas of Wnt pathway gene expression at critical stages of forebrain regionalisation in the chick embryo and found that most of these genes are expressed in strikingly dynamic and complex patterns. Several expression domains do not respect proposed compartment boundaries in the developing forebrain, suggesting that areal identities are more dynamic than previously thought. Using an Wnt4 expression in the thalamus is repressed by Shh from the ZLI we reveal an additional level of interaction between these two pathways and provide an example for the cross-regulation between patterning centres during forebrain regionalisation.The forebrain is exposed to a multitude of Wnts and Wnt inhibitors that are expressed in a highly dynamic and complex fashion, precluding simple correlative conclusions about their respective functions or signalling mechanisms. In various biological systems, Wnts are antagonised by Shh signalling. By demonstrating that  The vertebrate forebrain is the most functionally complex of biological structures and the centre of all higher brain functions, including learning, memory, cognition and, in humans, self-awareness, reasoning and personality. During embryogenesis, the forebrain is induced at gastrula stages when gradients of signalling molecules establish anteroposterior (AP) and dorsoventral (DV) polarity in the neural plate -5. One sFollowing neural induction, the prospective forebrain becomes progressively regionalised. The first major division is the establishment of the telencephalon anteriorly and of the diencephalon posteriorly. The dorsal telencephalon gives rise to the pallium  while the ventral part  gives rise to the striatum and globus pallidus . The hypothalamus evolves from neuroepithelium that is located ventral to the subpallium. The diencephalon consists of three major AP subdivisions: prethalamus, thalamus and pretectum (from anterior to posterior). Based on their topographical relationship within the postnatal brain, prethalamus and thalamus were previously referred to as ventral thalamus and dorsal thalamus, respectively. However, these terms obscure their embryonic origins as AP subdivisions of the diencephalic primordium. The area dorsal to the prethalamus constitutes the eminentia thalami; dorsal to the thalamus lies the epithalamus (habenula) and the dorsal part of the pretectum forms the posterior commissure. The epiphysis , a small endocrine gland that secretes melatonin and modulates wake/sleep patterns, is located at the dorsal midline of the epithalamus. For a detailed topographical description of forebrain subdivisions see .We are only beginning to understand the mechanisms that generate the complex regional diversity of the forebrain. As in other parts of the vertebrate neural tube, groups of forebrain cells are set aside to function as local signalling centres ('organisers') that regulate patterning and proliferation in adjacent areas ,20. TeleShh is expressed throughout the basal plate and at later stages also in the the zona limitans intrathalamica (ZLI), a stripe of cells that interfaces the prethalamus and the thalamus and acts as an organiser of thalamic development [Lunatic fringe (Lfng)-negative area in the early diencephalon [Lfng-negative wedge that gives rise to the ZLI seemingly narrows along its AP axis and elongates along its DV axis. Shh expression is subsequently seen within this territory as a characteristic sharp peak between the prethalamus and thalamus [In the diencephalon, elopment -29. Cellcephalon . The diethalamus . NeitherSfrp2, encoding the putative Wnt inhibitor secreted Frizzled-related protein (Sfrp) 2, is expressed along the PSB, raising the possibility that a gradient of Wnt signalling is established across the cortex/pallium between the cortical hem and the PSB  [Sfrp1 is expressed in a similar fashion at late developmental stages [Wnt signalling has been implicated in multiple aspects of central nervous system development, ranging from early pattern formation to the establishment of axonal connectivity . After ithe PSB) . In the l stages . Wnt sigl stages ,39 and hl stages -43. Howel stages .in situ hybridisation to relate their expression to known developmental domains within the diencephalon and telencephalon. Interestingly, we found that Wnt4 is strongly expressed in the emerging thalamus, but becomes downregulated with the appearance of Shh expression in the ZLI. To further probe the regulatory relationship suggested by this expression, we performed in ovo eletroporation experiments to demonstrate that Shh signalling is both necessary and sufficient for the downregulation of Wnt4, indicating that the organising function of the ZLI may be mediated not only directly, through Shh signalling, but also indirectly, by regulating other diffusible signals. Our study highlights the potential complexity of Wnt signalling during forebrain development, lays the foundation for a systematic functional analysis of Wnt signalling in this area and reveals a novel regulatory interaction between the Wnt and Shh signalling pathways.Although Wnt signalling has been shown to influence multiple aspects of brain development, few studies have succeeded in linking a specific developmental process to a single Wnt ligand, possibly due to extensive functional redundancy within the Wnt family. In this study, we have analysed the expression of a large number of Wnt ligands, Wnt receptors and intracellular targets of the Wnt signalling pathway during critical stages of forebrain regionalisation. We find that no fewer than twelve Wnts, seven Wnt receptors, three extracellular and three intracellular Wnt inhibitors as well as five nuclear factors that regulate the transcriptional output of the Wnt pathway are expressed in characteristic, regionalised and dynamic patterns in the developing chick forebrain. For selected genes, we performed double Fertilised hens' eggs  were incubated in a humidified chamber at 38°C until they reached the desired stages. Embryos were staged according to the tables by Hamburger and Hamilton (HH)  and fixein situ hybridisation was performed as described elsewhere [Wnt in situ constructs can be found in [NotI and transcribed with T3 RNA polymerase to generate probes for in situ hybridisation: Axin1, ChEST175L2; Axin2, ChEST755b16; Axud1, ChEST782g10; Ctbp1, ChEST765f10; Ctbp2, ChEST411c2; Drapc1, ChEST357h24; Idax, ChEST644a15.Gene expression analysis by lsewhere . The reffound in . The folin ovo as described [Δloop2 [in situ hybridisation. After in situ hybridisation, GFP expression was detected using a polyclonal antibody against GFP and a fluorescently labelled (Alexa) secondary anti-rabbit IgG antibody  using standard methods [HH13 to HH15 embryos were electroporated escribed  with expescribed ) or PtcΔ [Δloop2 . The Shh methods .Wnt expression during early neural plate stages has been previously performed [In order to investigate the role of Wnt signalling during vertebrate forebrain regionalisation, we initially set out to generate a spatiotemporal atlas of Wnt pathway gene expression in the embryonic chick forebrain. A comprehensive analysis of erformed . Thus, werformed . These serformed ,54 and ferformed ,56. AddiWnt expression data in three groups: those Wnts whose expression is restricted to the dorsalmost forebrain (Wnt1 and Wnt6), those that are expressed more widely in the dorsal half of the forebrain, but are not detected ventrally , and those whose expression also extends into the ventral forebrain . We did not observe any forebrain expression of Wnt2 [Wnt8 [Wnt11 [Wnt11B [We have loosely organised our /14; see  for nome of Wnt2 , Wnt8 [5t2 [Wnt8 , Wnt11 [8 [Wnt11  or Wnt11 [Wnt11B  at the sWnt1 has previously been described at HH5 in a crescent-shaped domain in the anterior neural plate, suggesting a role in the induction or initial regionalisation of the forebrain [Wnt1 transcripts; however, we have observed two previously undescribed regions of Wnt1 expression at HH20 - a distinctive patch in the epithalamic midline anterior to the epiphysis (arrow) and a region of weaker expression along the dorsal midline of the pallium  and around the MHB  are devoid of Wnt5A transcripts whereas Wnt5A overlaps with Shh expression in the ventral midbrain and that Wnt5A expression abuts that of Shh along the diencephalic alar/basal plate border and along the ZLI. Hence, areas of Shh expression in the forebrain are free from, but are flanked by, Wnt5A expression domains  family . In a prk embryo . At HH15y Figure . Weaker 9 Figure . In the Fz2 is strongly expressed in the ventral midbrain and diencephalon  and in the ventroanterior hypothalamus.By HH13, s Figure . Weak exFz9 has been described before in neural precursors of the chick embryo [Fz9 expression have appeared in the ventral midbrain and in the ventral pretectum as well as in a triangular region encompassing the epithalamus and the dorsal part of the thalamus  at this stage . Ventrally, Sfrp1 is expressed at the posterior end and along the ventral midline of the hypothalamus, but it is absent from the anterolateral hypothalamus and from the area around the base of the optic stalk  have a high sequence homology to the extracellular ligand-binding domain of the Frizzleds, the cystein-rich domain. They are thought to inhibit Wnt signalling by sequestering Wnt ligands in the extracellular space -76. Prevneuraxis -79. At Hk Figure . The comSfrp2 is widely expressed in the developing brain. At HH13, transcripts are found throughout most of the forebrain and midbrain with the exception of the MHB and a dorsal region between the telencephalon and the diencephalon  and the eminentia thalami are free from Sfrp2 expression at this later stage. Strong expression of Sfrp2 is detected throughout the hypothalamus at all stages examined  has been described in the anterior neural plate of the gastrulation chick embryo [Sfrp3 is weakly expressed throughout the anterior neural tube (lower transcript levels in the presumptive pretectum), but a distinctive domain of strong expression is apparent in the ventralmost diencephalon  are known mediators of Wnt signals in the nucleus . In the Tcf1 is expressed ubiquitously in the developing brain between HH13 and HH19 (not shown), Lef1 transcripts are absent from the anterior forebrain at HH13  are transcriptional co-repressors that interact with Tcfs and mediate their repressive function, possibly by competing with β-catenin ,84, althlsewhere . In orden Figure . At HH20m Figure . Taken tDown-regulated by adenomatosis polyposis coli 1 (Drapc1) is the orthologue of human Adenomatosis polyposis coli down-regulated 1 (APCDD1) and has been shown to be a target of Wnt signalling and a tumorigenic factor in colorectal carcinogenesis [Drapc genes lack significant homology to other known gene families or conserved domains, but hydrophobicity algorithms suggest that they form double-pass transmembrane proteins [Drapc1 is expressed in the prospective forebrain and midbrain areas from around the five-somite stage onwards. Between embryonic day 9.5 and 10.5, Drapc1 transcripts are found in the midbrain and diencephalon with highest levels of expression in the dorsal diencephalon and towards the ZLI as well as the MHB [Drapc expression is thought to reflect Wnt pathway activation, we were interested in investigating its expression pattern in the chick forebrain. At HH20, Drapc1 is strongly expressed in the ZLI, at the MHB and along the dorsal midline of the diencephalon and pallium /Cystein-serin-rich nuclear protein 1 (Csrnp1) is a putative tumor suppressor gene that is upregulated by exogenous Axin1 expression in a colon cancer cell line [Axud1 in the developing forebrain, as it may represent another readout of Wnt pathway activity. At HH20, chick Axud1 is expressed in the midbrain and in the diencephalon up to the ZLI as well as around the base of the optic stalk, bearing resemblance to Axin2 expression  in ovo [Wnt4. In line with our hypothesis, 13 of 15 embryos showed a reduction of thalamic Wnt4 expression on the electroporated side , which renders cells unresponsive to Shh signalling, into the diencephalon of HH14 to HH16 embryos [Wnt4. Electroporated areas in the presumptive thalamus failed to downregulate Wnt4 expression compared to the contralateral side  in ovo . Embryose Figure . To test embryos , incubatin ovo electroporation approach, we tested whether Wnt signalling regulates Shh expression in the ZLI, but we found that the forced expression of various Wnt pathway activators or inhibitors had no influence on Shh expression in the diencephalon (data not shown).Signals that are secreted from patterning centres in the developing brain often cross-regulate each other, mutually restricting their effect by forming negative feedback loops ,93. UsinShh expression in the forebrain, in particular the ZLI and the diencephalic basal plate, are flanked by stripes of Wnt5A expression at later stages of brain development . Hence, we assume that it reflects a general Shh-induced ventralisation of this brain area, rather than the direct induction of Wnt5A by Shh signalling.We found that domains of t Figure . Wnt5A ifollicle  and in bfollicle ; thus, wloop2 resulted in an even more complex outcome , those that are more widely expressed in the dorsal half of the forebrain  and those whose expression extends into the ventral forebrain . Four Wnts that we have examined are not expressed in the forebrain at the stages examined . Areas that are particularly rich in Wnt expression are the dorsal midbrain and dorsoposterior forebrain, the thalamus and the dorsal border of the pallium . Noncaniewed in .Wnt5/Wnt11 or interference with their function in fish or frog embryos results in disturbed convergence and extension movements during gastrulation, establishing these two Wnts as bona fide activators of the PCP pathway [Although certain Wnts have been found to preferentially activate either the canonical or the noncanonical Wnt pathway, a comprehensive picture as to which Wnt induces which pathway in a specific cellular context remains elusive. While Wnt1, Wnt3A and Wnt8 are known activators of the canonical pathway in animal models and in cell culture experiments, Wnt5 and Wnt11 fail to elicit comparable effects in those tests -114. How pathway -117. Yet pathway -121. A r pathway . Further pathway ).bona fide activators of the noncanonical pathway, only Wnt5A and Wnt5B are expressed in the chick forebrain between HH13 and HH24, whereas neither Wnt11 nor Wnt11B are detectable at these stages. Interestingly, both Wnt5A and Wnt5B transcripts are enriched along regional interfaces and boundaries within the developing brain such as the MHB (Wnt5B), the ZLI (Wnt5A + Wnt5B), the dorsal border of the pallium (Wnt5B) and the border between the alar plate and the basal plate (Wnt5A + Wnt5B). It is tempting to speculate that noncanonical Wnt signalling may be involved in the morphogenesis of these boundaries that often form physical ridges or constrictions in the neuroepithelium. However, Wnt5A seems to function like Wnt1 in inducing dopaminergic neurons in the midbrain via the canonical Wnt pathway [Of the  pathway ,125. In  pathway , so Wnt5Wnt and Fz expression patterns during forebrain development highlights an urgent requirement to identify which pathway is activated in which brain area at a given developmental stage. Future studies could address this problem by analysing the activation pattern of intracellular factors that are exclusive to either the canonical or the noncanonical Wnt pathway.The complexity of Sfrp1, Sfrp2 and Sfrp3 are expressed in multiple domains in the developing brain, many of which overlap with the expression domains of various Wnt ligands. Thus, dynamic domains of Wnt inhibition may be superimposed on the already complicated pattern of Wnt activator expression. Usually, Sfrps are regarded as inhibitors of canonical Wnt signalling; however, we cannot rule out that they also interfere with the noncanonical Wnt pathway. Furthermore, anecdotal evidence has indicated that Sfrp1 functions in a biphasic manner, activating or inhibiting the Wnt pathway depending on dose [ on dose . Sfrps m on dose  and they on dose ,129, sug on dose ). In add on dose -134.Sfrp1 expression at the ventral DMB - an area that is traversed by axons of the medial longitudinal fascicle [Frizzleds, including Fz2, are also expressed at high levels in this area . We speculate that Sfrp1 may be involved in guiding axons of this major longitudinal tract in the embryonic brain.Recently, Sfrp1 has been shown to act as an axon guidance molecule by interacting with Fz2 independently of Wnt ligands . Interesfascicle . NotablyFrizzleds and Sfrps in the developing forebrain is likely to add an additional level of controlling Wnt ligand distribution.It is important to keep in mind that signalling receptors themselves can also limit the spread of a secreted signal and that they may be involved in removing a signal from the extracellular space . Thus, tAxin1 expression has been reported to be ubiquitous [Axin2 is known to be a target of Wnt signalling [Axin2 is thought to be indicative of Wnt pathway activation. We found that the expression of Axin1 and Axin2 in the midbrain and posterior forebrain is very similar to that of Wnt1, Wnt3 and Wnt3A - bona fide activators of the canonical Wnt pathway - suggesting that canonical Wnt signalling is controlled by a negative feedback loop in this area of the brain. It is somewhat surprising that regions of the developing brain, such as the anterior forebrain and the hindbrain, which we show to express activators of the canonical Wnt pathway, do not express Axin2. On the one hand, it is well established that anterior neural development requires the repression of canonical Wnt signalling at early stages of development [masterblind phenotype that is characterised by an absence of the telencephalon and eyes is caused by a mutation in axin1 itself [Axin-negative region of the anterior neural tube - at least at later stages . More sensitive reporters may be useful to determine whether Wnt activation is really absent from Axin-negative areas of the developing brain.In the mouse embryo, iquitous  while Axgnalling . Hence, elopment ,139,140.1 itself ,13. On tLef1 and Tcf1 are widely expressed throughout the developing forebrain, suggesting widespread competence to respond to Wnt pathway activation. The tcf3 gene is inactivated in the zebrafish mutant headless and it was shown that ectopic activation of Wnt targets, which would normally be repressed by Tcf3 at the anterior pole of the embryo, causes the microcephalic phenotype of this mutant [Tcf3 expression in the prospective telencephalon and anterior hypothalamus of the chick embryo is consistent with a requirement for Wnt repression in this area [Tcf3 appears to be absent from the dorsal midline of the developing brain - a known Wnt signalling centre - and from the area encompassing the emerging ZLI, suggesting that this may also be a region of high Wnt activity. However, the Dishevelled antagonist Idax is expressed around the ZLI and may cap Wnt signalling intensity in this part of the diencephalon.Both s mutant . The dishis area . Tcf3 apTcf4 displays a surprisingly regionalised expression pattern in the developing forebrain. The expression domain in the ventral telencephalon and in the ventroanterior hypothalamus is consistent with a recent study in mouse where Tcf4 has been suggested to mediate the growth-promoting function of canonical Wnt signalling [Nkx2.2, thereby integrating dorsal and ventral patterning signals [Ctbp2, encoding a Tcf-binding corepressor, posterior to the ZLI suggests a complex transcriptional response to Wnt signalling in the presumptive thalamus. The observation that various bona fide target genes of the canonical Wnt pathway  are expressed in different patterns during brain development supports the idea that combinations of transcription factors and corepressors lead to divergent outputs of Wnt pathway activation.gnalling . Anothergnalling ,36. In t signals . It willVarious models have been proposed for forebrain regionalisation in vertebrates. Based on the analysis of the expression of a large number of regional marker genes, it was suggested that the forebrain, similar to the hindbrain, develops in a segmented fashion. However, many of the proposed segmental interfaces do not fulfil the criteria for true compartment boundaries (reviewed in ). A studLfng expression [in situ hybridisation analysis using Lfng, Shh, Tcf4, Wnt4 and Wnt8B suggests that these regional markers are expressed dynamically in the diencephalon between HH13 and HH19 and that they do not in all cases respect regional boundaries. Most notably, Wnt4 expression reaches from the thalamus into the Lfng-negative triangle without respecting the posterior boundary of the prospective ZLI. Moreover, Tcf4 is expressed in a patch within the Lfng-free wedge; however, Tcf4 expression does not follow the apparent narrowing of this wedge and is still expressed in a broad stripe at HH19. Similarly, Wnt7A expression is found in a domain that does not respect the DMB. These observations suggest that diencephalic development is more dynamic than previously thought and highlight the interpretative pitfalls of equating gene expression domains with regional identities. However, it is remarkable that the expression of a large number of genes examined in this study at later stages is most sharply defined around the ZLI - a key boundary in the developing forebrain that has been suggested to mark the interface between prechordal and epichordal neuroepithelium [The most widely accepted model for forebrain development proposes that the diencephalon consists of three neural segments called prosomeres 1 to 3  with p1 corresponding to the pretectum, p2 to the thalamus and p3 to the prethalamus . Previoupression . Our douithelium ,35.Wnt1 is expressed along most of the dorsal midline of the neural tube. However, while Wnt1 is excluded from the telencephalon and the anterior diencephalon in the mouse, we detected expression in the dorsal midline of these anteriormost brain regions at HH19. Interestingly, ectopic expression of Wnt1, similar to the expression described here in the anterior chick brain, is found in Emx2 mutant mice, indicating that the homeodomain transcription factor Emx2 normally serves to repress Wnt1 in the anterior brain of the mouse embryo. Such mice develop mild abnormalities in cortical layering that arise from defects in preplate development during early stages of cortical radial migration [Emx2 is expressed in the anterior neural plate from HH8 onwards [Wnt1 in the anterior neural tube cannot be explained by the absence of this repressor. Thus, temporal differences in Emx2 expression between chick and mouse or a lack of Emx2 binding sites in the chick Wnt1 enhancer regions are likely explanations for the differences in Wnt1 expression.In both chick and mouse, igration . Since c onwards , the expWnt3A is strongly expressed in the cortical hem of the mouse embryo [Wnt3A or of its close homologue Wnt3 in the chick embryo . It is ical hem .Wnt7A expression has been reported in the mouse telencephalon, where it is required for the vascularisation of this brain region [Wnt7A expression only in the chick midbrain and posterior diencephalon; hence, angiogenesis must be driven by another Wnt in the telencephalon of the chick embryo. As mentioned above, we also found a striking difference in the expression of an intracellular Wnt pathway antagonist: whereas mouse Axin1 is ubiquitously expressed during embryogenesis [n region . We founogenesis , we deteShh is expressed in the ventral midline along the length of the entire neural tube, consistent with its well characterised role in specifying different ventral neural identities in the spinal cord and brain [Wnts are expressed along the dorsal midline of the neural tube, suggesting that they may exert a complementary role in specifying dorsal neural character [Shh expression in the ventral midbrain, thereby enabling neurogenesis in the otherwise non-neurogenic floor plate [nd brain ,146. Mulharacter ,147-149.haracter ,100,142 haracter ,99,150. haracter . Recentlor plate .in ovo electroporation have demonstrated that canonical Wnt signalling influences neural fates in the spinal cord by promoting transcription of the Shh antagonist Gli3 [Gli3 [Xenopus embryos [Two recent studies using chick ist Gli3 ,154. Coni3 [Gli3 . An intei3 [Gli3 ,156, duri3 [Gli3  and in g embryos , suggest embryos -161 and  embryos . Thus, tShh expression in the ZLI forms a distinctive peak protruding into the dorsal diencephalon and, for a long period until much later in development, this is the only non-ventral expression domain of Shh in the neural tube [Wnts are in close proximity to, or even overlap with, Shh expression in the ZLI. For example, both Wnt3 and Wnt3A are expressed in the thalamus and they are believed to be essential for thalamic specification [Lrp5-/- mice suggest that activation of the canonical Wnt pathway is required in this tissue [Shh expression in the ZLI is also required for thalamic development [Gbx2 in the thalamus depends on the presence of Gli3, indicating a positive role of Gli3 in mediating Shh signalling in this area of the brain that is in contrast to its prevalent role as a Shh antagonist [From HH18 onwards, ral tube ,163,164.fication . The thas tissue . Shh expelopment -27, raistagonist . Since Gin ovo electroporation, we found that Shh signalling from the ZLI is necessary and sufficient to downregulate Wnt4 expression in the presumptive thalamus, revealing an additional level of interaction between Shh and Wnts. We could not find any evidence for a converse regulation of Shh expression by canonical Wnt signalling (data not shown). Interestingly, Wnt4 has been identified as a target gene of Shh signalling in basal cell carcinomas whose mRNA levels are downregulated compared to normal skin [Wnt4 by Shh may represent a conserved regulatory mechanism of clinical relevance.Using mal skin . Thus, twnt4 transcript levels using morpholino oligonucleotides enhanced the characteristic neurulation defects of wnt5/wnt11 mutant zebrafish embryos [Wnt4 exerts a similar role in the forebrain [Wnt4 has been implicated as an activator of the noncanonical Wnt pathway because its overexpression in zebrafish embryos results in gastrulation defects very similar to those produced by Wnt5, whereas none of the effects characteristic for activators of the canonical Wnt pathway are observed . A reduc embryos . Wnt4 in embryos . In comborebrain .Wnt expression, raising the question as to what extent and by what mechanism Wnts differ functionally. Furthermore, no areas were identified that exclusively express activators of either the canonical or the noncanonical pathway. Our study shows that the structural complexity of the vertebrate forebrain corresponds to the complexity of signalling events during its generation. In the future, it will be crucial to visualise the activation of canonical and noncanonical Wnt pathways in vivo, possibly by detecting the activation pattern of intracellular mediators of each pathway or by using particularly sensitive reporter assays.Here, we have shown that a large number of genes associated with Wnt signalling are expressed in complex and highly dynamic domains during the development of the chick forebrain. Whereas Wnts are particularly abundant in certain areas - such as the dorsal midline of the neural tube and the thalamus - there are no areas in the forebrain that are completely devoid of Lfng, Shh, Tcf4, Wnt4, Wnt8B) by double in situ hybridisation and found that their expression boundaries often change their relative positions and do not necessarily respect proposed compartment boundaries and regional interfaces. This observation supports a dynamic model of diencephalic development in which regional identities cannot always be equated to gene expression domains and, more generally, it emphasises the necessity of complementing the molecular characterisation of neuroepithelial subdivisions with cell lineage analysis.We have analysed a number of genes with distinctive expression patterns in the diencephalon , Shh , Bmps and Wnts  all cross-regulate each other during telencephalic arealisation ,93,170. Axud: Axin-upregulated; Bmp: bone morphogenetic protein; Ctbp: C-terminal binding protein; DMB: diencephalon-midbrain boundary; Drapc: Down-regulated by adenomatosis polyposis coli; DV: dorsoventral; Fgf: fibroblast growth factor; Fz: Frizzled; GFP: green fluorescent protein; HH: Hamburger and Hamilton; Lfng: Lunatic fringe; MHB: midbrain-hindbrain boundary; PCP: planar cell polarity; PSB: pallium-subpallium boundary; Sfrp: secreted Frizzled-related protein; Shh: Sonic hedgehog; ZLI: zona limitans intrathalamica.AP: anteroposterior; The authors declare that they have no competing interests.in situ hybridisations. CK performed electroporation experiments and supervised the study. IM and AL provided funding for this research. All authors have approved the manuscript.RQ and CK designed the study, performed the gene expression analysis and drafted the manuscript. MG helped with


Several oncogenes and tumour-suppressor genes have been identified that may have an important role in the development of human breast carcinoma. Furthermore, some of these gene alterations may be linked to the development of invasion and subsequent metastasis. Alterations in the expression of ras p21, p53 and c-erbB-2 have all been linked to tumours with rapid cellular proliferation, but the evidence that they are of prognostic importance in patients with breast cancer is conflicting. This study explores the relationship between expression of these oncoproteins and clinical outcome in 92 patients with either locally advanced or metastatic breast cancer treated with primary endocrine therapy. Specimens of the primary carcinoma were available for analysis of hormone receptor, Ki67 labelling index, epidermal growth factor receptor (EGFR), c-erbB-2, p53 and ras p21. Clinical response was measured according to UICC criteria after 6 months of treatment and all patients were followed for time to progression and overall survival. As shown previously, oestrogen receptor (ER) negativity, high Ki67 labelling index and EGFR overexpression were associated with a shorter time to progression and overall survival. However, no statistically significant relationship existed between expression of ras p21, p53 or c-erbB-2 and response to treatment, time to progression or overall survival. We conclude that staining for these three oncoproteins has no role in therapeutic decision-making in patients with advanced breast cancer. The negative finding implies that while abnormal expression of these genes may have an important role in the development of breast cancer, the variations in growth characteristics of advanced breast cancer may be influenced by other factors.


Activation of the epidermal growth factor receptor (EGFR) has been linked to tumour proliferation, invasion, metastasis and angiogenesis in epithelial tumours. Inhibitors of the EGFR have emerged as promising anticancer agents and two main approaches have been developed, humanised monoclonal antibodies and tyrosine kinase inhibitors. This review discusses the current status of EGFR tyrosine kinase inhibitors (EGFR-TKIs) that have entered clinical development. EGFR-TKIs are generally well tolerated and can sometimes produce impressive tumour regression in patients with advanced non-small-cell lung cancer. However, highly predictive or surrogate markers of activity have not been identified and there remains a need for translational research in their future development. EGFR signalling has been reported to be important for tumour cell proliferation, inhibition of apoptosis, angiogenesis, metastasis and sensitivity to chemotherapy and radiotherapy  is a member of a family of four closely related receptors: EGFR (or erbB1), HER2/neu (erbB2), HER3 (erbB3) and HER4 (erbB4). The EGFR mediates the actions of multiple ligands including epidermal growth factor, transforming growth factor-otherapy . EGFR siThe quinazolines, gefitinib and erlotinib, which are the most advanced in clinical development, are competitive inhibitors at the tyrosine kinase ATP binding site. Irreversible inhibitors that bind to specific cysteines in the ATP-binding pocket of EGFR family receptors have been developed. Examples include CI-1033 and EKB-569. The structural homology between EGFR receptor members has also been exploited for the development of inhibitors that block multiple members of the EGFR family  and phosphatidylinositol 3′–kinase/Akt (PI3/Akt) pathways. In A431 cells, the antitumour effects of gefitinib are mediated via inhibition of both MAP kinase and PI3/Akt pathways resulting in apoptotic cell death. In NSCLC cell lines, however, more limited antiproliferative effects were accompanied by evidence of continued activity in one or both of these pathways  and erlotinib  are in advanced phase II/III development and other agents are in phase I/II trials −1vs 500 mg day−1 in second or third line treatment of relapsed, advanced stage NSCLC , the median survival of 210 randomised patients from the start of ZD1839 was 7.8 months with a median survival in responding patients of approximately 11 months . Response rates of approximately 5–10% have been reported with up to a third of patients showing stable disease . A phase−1), and pharmacodynamic data showed that EGFR signalling was consistently inhibited in skin biopsies at all doses above 150 mg day−1  for most tumour cells in vitro  did not improve the response rates, time to progression or survival compared to combination chemotherapy alone  enhances the activity of single agent cytotoxic drugs reviewed . Such obsingle agent chemotherapy. Phase II/III studies of gefitinib or erlotinib are underway in a wide range of solid tumours including head and neck cancer, prostate, breast, colorectal, ovarian, cervical, endometrial, pancreatic, glioblastoma and renal tumours and preliminary results have been reported in recent reviews  or with cisplatin/gemcitabine (TALENT TRIAL) have completed patient recruitment, and in October 2003 OSI Pharmaceuticals announced that these trials were also negative and that the addition of erlotinib to these doublet chemotherapy regimens failed to produce a survival advantage over chemotherapy alone. An important unanswered question is whether either of these agents improves the efficacy of  reviews .The toxicity profile of gefitinib and erlotinib are remarkably similar; skin rash and diarrhoea being the most frequently encountered adverse effects. At current phase II/III dose levels, toxicity is usually grade 1 or 2 and rarely dose limiting. Diarrhoea usually responds well to antidiarrhoeal treatment; and it has been observed that skin toxicity can sometimes improve in the face of continued dosing , a 6-thiazolylquinazoline, is a reversible, dual ErbB2 and EGFR TKI under development by GlaxoSmithKline. Phase I data have recently been reported with notable tumour responses seen in patients with trastuzumab refractory breast cancer and in NSCLC  tyrosine kinase inhibitor of the quinazoline class. This compound has additional activity against EGFR and fms-like tyrosine kinase 4 (VEGFR3)  and the EGF/HER2 receptor pathways is associated with endocrine therapy resistance . Studies to date have relied upon tumour or skin biopsies, but while these have sometimes been used to guide subsequent trial design, they have not resulted in the identification of a validated, predictive marker for antitumour efficacy. Molecular imaging of pharmacodynamic effects and visualisation of target inhibition is a promising area of research that holds longer-term promise.We urgently need a more comprehensive understanding of the role of EGFR in human cancer. We must acknowledge that EGFR receptor expression in a tumour does not prove that its function is important for tumour growth, nor that inhibition will automatically result in cell death or therapeutic effect. While the process of clinically validating drug targets is notoriously difficult, EGFR TKIs have relatively specific mechanisms of action, and advances in pharmacodynamics, pharmacogenomics, and genomics/proteomics must be applied in clinical settings to help us realise the full potential of these agents.