Autism is a developmental disorder characterized by decreased interest and engagement in social interactions and by enhanced self-focus. While previous theoretical approaches to understanding autism have emphasized social impairments and altered interpersonal interactions, there is a recent shift towards understanding the nature of the representation of the self in individuals with autism spectrum disorders (ASD). Still, the neural mechanisms subserving self-representations in ASD are relatively unexplored.We used event-related fMRI to investigate brain responsiveness to images of the subjects' own face and to faces of others. Children with ASD and typically developing (TD) children viewed randomly presented digital morphs between their own face and a gender-matched other face, and made “self/other” judgments. Both groups of children activated a right premotor/prefrontal system when identifying images containing a greater percentage of the self face. However, while TD children showed activation of this system during both self- and other-processing, children with ASD only recruited this system while viewing images containing mostly their own face.This functional dissociation between the representation of self versus others points to a potential neural substrate for the characteristic self-focus and decreased social understanding exhibited by these individuals, and suggests that individuals with ASD lack the shared neural representations for self and others that TD children and adults possess and may use to understand others. In describing another child he writes that he “…behaved as if people as such did not matter or even exist” and describes one young girl as giving “the impression of being self-absorbed” Autism spectrum disorder (ASD) is a complex developmental condition in which fundamental social development and communication are compromised interpersonal cognition in autism, a recent shift has been towards understanding altered intrapersonal cognition in ASD. One recent study highlights broad impairments in both self-referential cognition and empathy as measured by a battery of scales designed to measure these constructs Despite recent efforts to use a cognitive neuroscientific approach to understand key components of the ASD symptomatology, there is still little consensus as to its precise neurobiological underpinnings. Several imaging studies have shown both structural and functional brain abnormalities in individuals with ASD  children were recruited and compensated for their participation in this fMRI study. Due to excessive head movements (>3 mm mean displacement), the final analyses include 12 children with ASD  and 12 TD children . The two groups did not differ significantly with respect to age or IQ See . All parImages were acquired using a Siemens Allegra 3.0 Tesla MRI scanner. Each child completed 2 functional runs lasting 5 min 8 sec each , each with 36 transverse slices, 3mm thick, 1 mm gap, and a 64×64 matrix yielding an in-plane resolution of 3 mm×3 mm. Co-planar high-resolution EPI structural images were acquired to aid image registration .http://www.macbrain.org/faces/index.htm). We chose faces from this stimulus set for the Other face because subjects had previously viewed faces from this stimulus set in other experiments. Thus, novelty effects were minimized. MorphEditor  was used to create digital morphs between the subjects' face and the other face (a gender- and race-matched face chosen from the MacBrain Face Stimulus Set), resulting in 6 unique faces, each morphed to a varying extent . See http://www.neuro-bs.com/) was used to present stimuli and record responses. Stimuli were presented through magnet-compatible goggles (Resonance Technology Inc.) and responses were recorded using two buttons of an fMRI compatible response pad. During each functional run, each of the six morphed faces and the scrambled control were presented 10 times in a random sequence optimized and counterbalanced using the optseq algorithm (http://surfer.nmr.mgh.harvard.edu/optseq/), which provides temporal jitter to increase signal discriminability Stimuli were individually tailored to each child, and consisted of a series of static color images constructed from pictures acquired on a Kodak 3400C digital camera of the child's own face (self) and the face of another person (other). The Other face was chosen from the MacBrain Face Stimulus Set (www.fmrib.ox.ac.uk/fsl). After motion correction, images were temporally high-pass filtered with a cutoff period of 50 seconds and smoothed using an 8 mm Gaussian FWHM algorithm in 3 dimensions. The BOLD response was modeled using a separate explanatory variable (EV) for each of the seven stimulus types. For each stimulus type, the presentation design was convolved with a gamma function to produce an expected BOLD response. The temporal derivative of this timecourse was also included in the model for each EV. Data were then fitted to the model using FSL's implementation of the general linear model, with motion components included as confound EVs.Functional imaging analysis was carried out using FEAT , part of FSL  to register the functional data to the atlas space in two stages. Functional images were aligned with the high-resolution co-planar T2-weighted image using a 6 degrees-of-freedom rigid-body warping procedure. The co-planar volume was registered to the standard MNI atlas with a 12 degrees-of-freedom affine transformation.Statistical analyses were conducted with FSL using mixed-effects models to compute group differences. Higher-level analysis was carried out using FLAME  a priori region of interest  contrast for the ASD group revealed activations in the right lateral occipital cortex, right occipital fusiform gyrus, right temporal occipital fusiform cortex, right precentral gyrus, right inferior frontal gyrus, right insular cortex, left lateral occipital cortex, left occipital pole, and left temporal occipital fusiform cortex. The Self>Rest contrast for the TD group revealed a nearly identical pattern of activation . Direct The Other>Rest  contrast for the ASD group showed activations in right occipital fusiform gyrus, right lateral occipital cortex, left occipital fusiform gyrus, and left temporal occipital fusiform cortex. The Other>Rest contrast for the TD group showed activations in right occipital fusiform gyrus, right lateral occipital cortex, right inferior frontal gyrus, right precentral gyrus, right insular cortex, right frontal operculum cortex, left occipital fusiform gyrus, and left temporal occipital fusiform cortex. While direct statistical comparisons between groups  revealed no significant differences for the contrast of Other>Rest in whole-brain, cluster corrected analyses , the within-group results for the Other>Rest contrast revealed striking differences between TD and ASD children in the right prefrontal cortex. Specifically, while TD children activated the right inferior frontal gyrus (rIFG) while viewing images of others, there was no change in activity between Other versus Rest for the ASD children . For thiUsing anatomically defined ROIs provided by the Harvard-Oxford Structural Atlas, signal intensities were estimated within the following brain regions in the right hemisphere: BA 44, BA 45, middle frontal gyrus, precentral gyrus, angular gyrus, superior parietal cortex, lateral occipital cortex, and fusiform gyrus . For allinterpersonal  and intrapersonal  cognition The representation of self and other may be fundamentally altered in autism spectrum disorders Previous work on the neural basis of self-face recognition in adults suggests that while the right inferior frontal gyrus responds most strongly to self-faces, it also shows signal increases  above baseline to personally familiar faces Our paradigm required participants to explicitly evaluate facial identity to decide whether the image presented resembled themselves. Both groups of children behaviorally demonstrated a diminishing “self” response as the image presented contained a smaller percentage of the self face, indicating successful self-other discrimination. This result is consistent with behavioral work demonstrating successful visual self-recognition in most children with ASD Social cognition researchers have previously suggested that understanding of others' experiences may involve the activation of shared affective neural networks that enable us to “feel the emotions of others as if they were our own” One possible explanation for the lack of rIFG response to others observed in this group of children with ASD may be the fact that from a young age autistic individuals seem to lack the motivation for orienting to social cues that their typically developing counterparts demonstrate In conclusion, we find that children with ASD do not activate shared regions for self- and other-face processing, as do TD children. Reduction of activity in right prefrontal areas during other-face processing may be a neural signature of reduced social engagement and understanding in these individuals.


Solid tumours account for 90% of all cancers. Gene therapy represents a potential new modality for their treatment. Up to now, several approaches have been developed, but the most efficient ones are the viral vector based gene therapy systems. However, viral vectors suffer from several deficiencies: firstly most vectors currently in use require intratumoural injection to elicit an effect. This is far from ideal as many tumours are inaccessible and many may have already spread to other parts of the body, making them difficult to locate and inject gene therapy vectors into. Second, because of cell heterogeneity within a given cancer, the vectors do not efficiently enter and kill every cancer cell. Third, hypoxia, a prevalent characteristic feature of most solid tumours, reduces the ability of the viral vectors to function and decreases viral gene expression and production. Consequently, a proportion of the tumour is left unaffected, from which tumour regrowth occurs. Thus, cancer gene therapy has yet to realise its full potential.The facultative or obligate anaerobic bacteria have been shown to selectively colonise and regerminate in solid tumours when delivered systemically. Among them, the clostridial spores were easy to produce, stable to store and safe to use as well as having extensive oncolytic ability. However, research in animals and humans has shown that oncolysis was almost always interrupted sharply at the outer rim of the viable tumour tissue where the blood supply was sufficient. These clostridial spores, though, could serve as "Trojan horse" for cancer gene therapy. Indeed, various spores harbouring genes for cancerstatic factors, prodrug enzymes, or proteins or cytokines had endowed with additional tumour-killing capability. Furthermore, combination of these "Trojan horses" with conventional chemotherapy or radiation therapies often significantly perform better, resulting in the "cure" of solid tumours in a high percentage of animals.It is, thus, not too difficult to predict the potential outcomes for the use of clostridial spores as "Trojan horse" vectors for oncolytic therapy when compared with viral vector-mediated cancer therapy for it be replication-deficient or competent. However, to move the "Trojan horse" to a clinic, though, additional requirements need to be satisfied (i) target tumours only and not anywhere else, and (ii) be able to completely kill primary tumours as well as metastases. Current technologies are in place to achieve these goals. Gene therapy represents a potential new modality for the treatment of cancer and it is developing with a very fast pace . By the The key to a successful gene therapy is the vector system. Various vectors have been developed with unique features, including viral and non-viral based therapy systems. Although each has its own advantages and disadvantages, the replication-competent oncolytic viral vectors are the most promising amongst existing ones ,5. HowevClostridia have several advantages over others as clostridial spores specifically colonise and germinate into vegetative cells in the hypoxic regions of solid tumours, causing tumour lysis and destruction. Early trials in the 70's of non pathogenic strains in human had shown plausible safety. However, existing knowledge indicated that oncolysis was almost always interrupted sharply at the outer rim of viable tumour tissue, thus, combinational approaches have to be implemented, such as with radiofrequency therapy [C. novyi in combination with microtubule-interacting chemotherapeutic agents, including vinorelbine and docetaxel and demonstrated very promising results. A phase 1 trial of the combined approach in patients is in progress [Of all cancer diagnosed, 90% of these are solid tumours. Recent understanding of the unique pathology of solid tumours has shed light on the disappointing nature of these new therapies and now demands rational and innovative design of vectors. All solid tumours, when they grow more than 2 mm diameter in size, undergo angiogenesis that results in biological changes and adaptive metabolisms, i.e.: formation of defective vessels, appearance of hypoxic areas, and emergence of heterogeneous tumour cell population . This mi therapy ,9. A newprogress .The intrinsic property of tumour-targeted colonisation of clostridia enables them to serve as "Trojan horses" for the delivery of genes for cancer therapy. Indeed, clostridial spores that were genetically manipulated to harbour genes for cancerstatic factors, prodrug converting enzymes, or cytokines to improve their innate oncolytic activity have been developed, including our work and others ,12. FurtThis review briefly describes the viral vectors, including the replication defective vectors, the targeted vectors and replication-competent oncolytic vectors, and their use in cancer gene therapy as well as their advantages and disadvantages. Subsequently, we will focus on the development of clostridial spores as "Trojan horse" vectors for cancer therapy, the mechanisms involved, and the foreseeable promises and problems when compared with existing viral vector systems.in vivo use of the vector system. However, studies in animal models have shown that poor vector penetration is always a problem in vivo. MoMLV vector rarely travelled away from the injection sites [i.c.) injection of replication-competent MoMLV [Murine Moloney leukaemia virus (MoMLV)-based retroviral vector is one of the earliest systems that were developed for gene therapy. The vector has unique ability to transduce dividing cells . Tumour on sites . Due to nt MoMLV . Viral eOverall, more than 23% of all gene therapy trials in patients for various diseases have used a replication-defective or competent MoMLV vector system. Existing studies in animal experiments have shown that the vector system was relatively safe and non-toxic. The case against retroviral vector systems is potential problems related to activation of cellular oncogenes and inactivation of tumour suppressor genes by insertional mutagenesis. This was true in the case of using MoMLV to transducer bone marrow stem cells for the therapy of severe combined immune deficiency syndrome (SCIDs), 4 out of 11 treated children have now developed leukaemia . In addiin vitro and in vivo [8 plaque forming unit (PFU)/ml of virus in the plasma), and being able to efficiently integrate into chromosomes of non-dividing and terminally differentiated cells. Most of the lentiviral vectors were pseudotyped with a glycoprotein from the vesicular stomatitis virus (VSV), VSV-G, as it provided not only a broad tropism, but also physical strength that enabled concentration by centrifugation.Lentiviral vector is a new type, more complicated retroviral vectors, which are primarily based on Human or Bovine Immunodeficiency Virus. They have all the unique features of MoMLV and have been shown to transduce post mitotic cells  in vivo . Studies in vivo . Thus, s in vivo ,21. JDV Adenovirus vector is the most commonly studied and most widely used system in cancer gene therapy. They are of particular utility for cancer gene therapy applications, where temporary gene expression is acceptable or even beneficial. The currently employed AVs in clinical trials are all based on serotype 5. AV can be produced at high titre and has demonstrated efficient gene transfer to various types of cancer cells . Two AVsSeveral other adenoviruses, based on canine, porcine, bovine, ovine and avian have all been developed. The ovine AV is based on serotype 7 and developed in Australia. Preclinical testing on prostate cancer in animal models has shown therapeutic efficacy .in vivo recombination. With regard to AV-mediated cancer treatment, high-level tumour transduction remains a key developmental hurdle. To this end, AV vectors possessing infectivity enhancement and targeting capabilities should be evaluated in the most stringent model systems possible. Advanced AV-based vectors with imaging, targeting and therapeutic capabilities have yet to be fully realized; however, the feasibilities leading to this accomplishment are within close reach [AVs contain many viral genes encoding major proteins that elicit a strong host immune response. Of particular concern is the development of cytotoxic T lymphocytes that lyse cells expressing the recombinant genes. Newer generations of AV vector were designed to overcome some of these problems and the initial results were encouraging. New techniques involved in removing the recombinant viral genes and transfecting the non-recombinant plasmid with a helper virus and then separating the helper virus with sedimentation techniques were developed. Improvements in helper virus have also been trialled that reduces "floxed" helper virus production 1000-fold, but this method still has a 1% wide type (WT) contamination thus still allowing the possibility of se reach .AAV-based vectors have been shown to be non-toxic and undergo widespread cellular uptake in preclinical evaluation . A recenVectors based on herpesviruses are well-developed and have progressed to clinical trials. As with other viral vector, replication defective vectors did not show much of use. The first replication competent vector was based on a mutant strain, where the vectors are deleted of the main neurovirulence gene r34.5, which restricted its ability to replicate in adult central nervous system and to form latency. However, later study showed that the mutant strain that had the deletion of the r34.5 gene also reduced the capacity of replication inside tumour cells . The newPre-existing immunity may pose a problem that limits the clinical efficacy of herpesvirus-based vectors. The immunity prevented the transduction of peripheral organs and also caused liver toxicity. However, a recent mutant strain-secreting cytokine granule macrophage colony stimulatory factor (GM-CSF) or IL-12 was shown to be effective in liver cancer therapy in a murine model which likely involves both direct viral oncolysis and actions of specific immune effector cells .Semliki Forest virus (SFV) subgenomic replicons (i.e. non toxic replication) have been developed that allow stable expression of a required gene e.g. beta-galactosidase  in mammalian cell lines. Expression remained high (approximately 150 pg per cell) throughout cell passages .mariner fish element [Since construction of the Sleeping Beauty transposon from defective copies of a Tc1/ element , new verWhile efforts have been focused on the continuing refinement of various vector systems, several obstacles remain, primarily the low efficiency of gene delivery into target tumour cells. The vascular endothelial wall is a significant physical barrier prohibiting access of systemically administered vectors to the tumour cell. To overcome this obstacle, strategies are currently being developed to take advantage of transcytosis pathways through the endothelium. An AV vector targeted to the transcytosing transferrin receptor pathway, using the bifunctional adapter molecule had been constructed . The tra18F-penciclovir, providing an imaging system for viral replication that can directly be translated for clinical applications. Interestingly, HSV TK is an enzyme that has utility in so-called suicide gene therapy, in which the expressed enzyme converts a substrate such as ganciclovir to its phosphorylated metabolite, which can then be further phosphorylated by cellular kinases to a toxic metabolite, causing cell death [Recently, a concept of multifunctional particles (MFPs) based viral vectors has emerged. The idea incorporated viral vectors' tumour targeting, imaging and amplifying tumour killing capacities. AAV has been developed as MFP, by virtue of genetic capsid modifications, to incorporate additional functionalities, such as modified fibres, combined with imaging motifs on the pIX protein, to simultaneously target tumour cells while monitoring viral replication and spread. HSV TK has been incorporated at pIX site of the AAV capsid. This enzyme is compatible with available PET imaging ligands such as ll death . Also, tNanotechnology has also been introduced recently in the context of MFP. This is defined as the development of devices of 100 nm or smaller, having unique properties due to their scale. The devices that are being developed generally incorporate inorganic or biological material. In this regard, the coupling of inorganic nano-scale materials to targeted AV vectors has much potential. For example, magnetic nano-particles have recently received much attention due to their potential application in clinical cancer treatment; targeted drug delivery and magnetic resonance imaging (MRI) contrast agents . Howeverwt-p53 in cancer cells through gene transfer techniques restores p53 function and triggers apoptosis leading to tumour cell destruction [wt-p53 following gene transfer.The p53 tumour suppressor gene has received a great deal of attention as a cancer therapeutic strategy due to the important role it plays in maintaining the integrity of the genome. It is involved in cell cycle regulation, DNA repair, and apoptosis, essentially controlling the integrity of the genome. Following exposure to DNA damaging agents, p53 activation results in cell cycle arrest, allowing for DNA repair or triggers cellular apoptosis if the damage is irreparable. Thus, p53 mutations in cancer allows for unregulated cellular proliferation in the face of genetic mutations and accumulation of genetic errors contributing to the malignant phenotype and genomic instability of cancer cells. Preclinical studies have demonstrated that replacement of truction ,37. The p53 gene repair clinical trials have been initiated. These trials have used different vector systems for gene transfer (retrovirus and adenovirus), different routes of vector delivery , and have focused on different subtypes of lung cancer . The first phase I clinical trial of such an approach was conducted by Roth et al. at the M.D. Anderson Cancer Centre [p53 via CT-guided or endobronchial injections. Effective gene transfer was demonstrated in biopsied tumours following injection and some degree of tumour regression of the injected lesion was seen in three subjects providing proof-of-concept for this gene therapy approach. All subsequent trials have utilized adenoviral vectors for gene transfer since such vectors are relatively easy to manufacture at large scale, can be produced at higher viral titres, and have the ability to transduce both dividing and non-dividing cells. Three adenoviral p53 (Ad-p53) single agent trials have been performed as well as three Ad-p53 combination trials. Two of the single agent trials were performed in advanced NSCLC using either single or multiple vector injections [p53 gene transfer as the sole treatment modality whereas more recent trials have combined p53 gene transfer with other cancer therapies, notably chemotherapy or radiation, as part of a combined modality treatment approach.Based on these preclinical studies a number of r Centre . In thisjections . These tjections . Neverthjections , or radip53 gene transfer approaches described above, preclinical data with adenoviral HSV TK gene transfer (AdHSV TK) followed by ganciclovir exposure results in a bystander effect in which neighbouring, non-transduced cells are also being killed, presumably due to transfer of toxic metabolites from the transduced cells as well as induction of a generalized immune response [This is also one of the well studied strategies and is based on the delivery of a "suicide-toxin producing enzyme" gene not normally found in mammalian cells to tumour cells that allows for selective sensitivity to a systemically administered pro-drug. One such suicide gene is the HSV TK gene that codes for an enzyme that converts the normally nontoxic nucleoside analogue, ganciclovir, in subsequent steps into a toxic compound that leads to tumour cell death. Like the adenoviral response . Preclinresponse . Gene trAt present, the hurdle to clinical use of this strategy is the low efficiency of gene transfer. To overcome the problem, we also developed a novel way of using a unique peptide to shuttle the HSV TK gene to neighbouring cells . Howeverex vivo and in vivo approaches. Early studies of adoptive transfer of ex vivo expanded tumour infiltrating lymphocytes (TIL) demonstrated responses in melanoma and renal cell cancer but activity in other solid tumours was limited [Several genetic strategies have been employed to enhance the immunogenicity of tumours with a goal of inducing immune-mediated tumour destruction. Unlike the gene repair and suicide gene transfer studies described above, immunogene therapies have the theoretical advantage of inducing a systemic anti-tumour response associated with immunologic memory. Such a response potentially allows for treatment of disseminated disease and a prolonged anti-tumour effect that persists beyond the immediate treatment period. Immunogene therapy strategies involve both  limited . Systemiex vivo approach using the lentiviral vector-mediated transfer of the tumour antigen gene into dendritic cells (DCs) cells. Therapeutic effects were demonstrated in up to 85% of the subjects [We have developed an subjects .Raf gene to angiogenic blood vessels using αvβ3-targeted liposomes. The integrin, αvβ3, is preferentially expressed in the angiogenic endothelium and contributes to viral internalization making it a good targeting molecule for anti-angiogenic gene therapy strategies. Raf is a cellular signalling molecule that plays an important role in neovascularization. Mice lacking Raf die early in development with vascular defects and a mutant form of Raf was shown to block angiogenesis in response to pro-angiogeneic factors in vitro. Systemic injection of targeted liposomes conjugated to a mutant Raf gene into mice with pre-established lung and liver metastases from colon carcinoma demonstrated predominant tumour endothelial cell uptake, tumour endothelial cell apoptosis, and pronounced tumour regressions.One of the features of the malignant tumours was the increased vasculature. Therefore, targeting tumour vasculature rather than the tumour cell itself as a treatment for cancer has gained increasing interest in recent years. A number of inhibitors of angiogenesis . VEGFR2 has relatively restricted expression on endothelial cells and is upregulated in proliferating tumour neovasculature. An orally available DNA vaccine encoding murine FLK-1 was shown to suppress angiogenesis in tumour vasculature, protect mice from lethal challenges with melanoma, colon, and lung carcinoma cells, and reduce the growth of established metastases .in the growth inhibition of NSCLC cells. A further study is underway to demonstrate the in vivo efficacy of EGFR silencing in animal models [One of the newer technologies in cancer gene therapy involves the silencing of genes in cancer cells that regulate tumour cell growth and proliferation. We have developed a double stranded RNA mediated silencing of the epidermal growth factor receptor (EGFR). In vitro studies have demonstrated effective silencing of the EGFR and resulted l models "C. histolyticum into mouse sarcoma caused oncolysis (liquefaction) and tumour regression [i.v. with spores of C. tetani, the causative agent of tetanus. Injected non-tumour bearing animals remained healthy. However, tumour bearing mice died within 48 h because of C. tetani colonisation and tetanus production. This provided evidence that the C. tetani were able to germinate/replicate selectively in the anaerobic environment found within tumours, and released their toxins systemically [C. butyricum M-55 has been isolated [C. oncolyticum and taxonomic studies have now clearly established that it is a C. sporogenes strain (ATCC13732). This is a proteolytic species causing liquefaction of colonised tumours. This was later verified by testing more isolates.It is obvious that a major step towards the development of an effective cancer therapy will be to construct a vector that targets the tumour alone, and is capable of spreading to and throughout the tumour found in tissues. Clostridial spores fit into this equation very well. Clostridia are strictly anaerobic. They are gram-positive, rod-shaped, and form spores under unfavourable conditions. There are about 80 species and several of these have been tested in solid tumours. All known species require anaerobic conditions to grow but do vary in their oxygen tolerance and their biochemical profile. Clostridial spores have been administered intravenously and showed a distinct advantage for use in cancer therapy as they are easy to produce and store. Germination of spores will only occur when they encounter the requisite anaerobic conditions. Spontaneous colonization of tumours in cancer patients and the apparent selectivity of Clostridia for tumours were noticed more than 50 years ago. The first experiment in 1947 showed that direct injection of spores of gression . Later eemically . Obviousisolated . M-55 waC. beijerinckii NCIMB8052 spores administered intravenously to EMT6 tumour-bearing mice germinated in the necrotic tumour regions while the oxygenated tumour areas remained free of spores [C. beijerinckii ATCC17778, C. acetobutylicum DSM792 (= ATCC824) or C. acetobutylicum NI-4082 (reclassified as C. saccharoperbutylacetonicum) showed tumour colonisation without complete tumour lysis [Saccharolytic clostridia, such as f spores . Equallyur lysis .C. sporogenes was the first Clostridium to be gene modified and this was performed with the E. coli Colicin E3 gene. Colicin E3 encodes a bacteriocin shown to have canceriostatic properties [E. coli cytosine deaminase (CD) into C. sporogenes NCIMB10696 by electroporation [C. sporogenes has a great capacity to colonise the tumour. At least 10e8 CFU/g of tumour was obtained following the intravenous injection of the spores  or C. acetobutylicum NI4082 (reclassified as C. saccharoperbutylacetonicum) and C. butyricum are non-pathogenic and their development has been industry funded. Therapeutic genes, encoding the cytokine tumour necrosis factor alpha (TNF-α), CD or nitroreductase (NTR) have been introduced into these strains [C. acetobutylicum using strain-specific electroporation protocols, CD expression was monitored in lysates and supernatants of early logarithmic growth phase cultures of recombinant C. acetobutylicum (pKNT19closcodA) [C. acetobutylicum strains NI4082 and DSM792 engineered to produce cytosine deaminase were able to express and secrete this enzyme at the tumour site [C. acetobutylicum, but not in control animals. Animals, concomitantly treated with antivascular chemical agent, CombreAp, showed higher incidence of CD-positive tumours (100 versus 58%). Moreover, the level of active CD in these tumour specimens was considerably higher (mean conversion efficiency of 5-FC to 5-FU ~11%) as compared to tumours not treated with the vascular targeting drug (mean conversion efficiency of 5-FC to 5-FU ~11%) when compared to untreated tumours (mean conversion efficiency of 5-FC to 5-FU ~3%) [in vivo, there was a consistent lack of significant tumour regression observed. Factors that may have contributed to this lack of efficacy include a low level of bacterial colonisation of the tumour or insufficient recombinant gene expression and secretion at the tumour site [Clostridial strains C. sporogenes or C. novyi-NT. Recombinant C. sporogenes and C. novyi-NT overexpressing NTR showed significant in vivo anti-tumour effects [Saccharolytic Clostridia strains including  strains ,59. FollloscodA) . A consiour site ,59. Funcour site . Recent  effects  when useAt present, there are various gene therapy vector systems under development against cancer. However, due to the complexity of the solid tumours involving angiogenesis, hypoxia, stromal cell, tumour cell heterogeneity and the emergence of de-differentiated stem cells, none of the existing vectors are holding any real promises. The clostridial spore-based vector system is not infectious, and has gained renewed interest, because of the following true advantages.C. novyi-NT has demonstrated that the strain was unable to colonise artificially created infarcted heart where the level of hypoxia was inadequate to support the replication of the Clostridia. Early trials of non-pathogenic Clostridia strains in patients have demonstrated safety. In the unlikely event of an adverse effect, clostridia can be eliminated from the blood stream with the use of readily available antibiotics such as metronidazole which showed total spore clearance from the blood stream after 9 days of treatment [Safety is always a concern when live vector systems are used for human gene therapy. Some of the hurdles of using viral vectors include: (1) whether the vector is sufficiently targeted to tumour alone; (2) whether the vector expresses low levels of viral genes that may lead to increased toxicity and immunogenicity ; (3) posreatment .The biological properties of virus-based vectors, in particular the ability to enter and replicate  within a tumour cell and then spread from cell to cell are highly relevant for effective cancer therapy. However, recent understanding of tumour pathology has revealed that several features of the tumour environment may not be conducive for viral replication ,66. HypoAll of the viral vector systems need sophisticated cell culture systems, expensive culture media, rounds of filtrations and purifications and dedicated centrifugation and storages. On the contrary, clostridial spores can be easily and inexpensively produced from anaerobic bacterial culture. There are only a few steps involved and the spores, once produced can be stored at room temperature for at least 3–6 months.While most viral vectors have to be intratumourally injected, intravenous injection of resuspended clostridial spores are possible and sufficient as they will be leaked out of the incomplete vessels in the solid tumour, thus specifically targeting to and colonising the hypoxic regions of the tumours.Solid tumours comprise not only malignant cells, but also extracellular matrix and many other non-malignant cell types, including stromal cells such as fibroblasts, endothelial cells and inflammatory cells. The mechanisms of clostridial vector-mediated tumour killing consist of several aspects: one is from its transgene that encodes prodrug converting enzymes for suicide-gene therapy or cytokines for immuno-gene therapy. These are essentially the same as the viral vectors. However, there is another side of the tumour killing effect that is resulting from the consequences of an innate antitumour effect of the clostridial strain due to production of hydrolytic enzymes including proteases, lipases, and nuclease. Furthermore, there is also a nutrients competition between the clostridia and cells surrounding them , where the clostridia multiplied much faster than the mammalian cells. The cumulative multiplications and the combined events of energy and substance metabolism effectively depleted the limited nutrient source and deprive the tumour cells, causing starvation and death. More recently, there were observations that indicate the germination of the clostridial spores, the transformation from spores to vegetative rods, and the continue multiplications of the vegetative rods inside the tumour activated the immune system, assisting the antitumour effects . These tWhile viral vectors need access to viable target cells and their cellular machinery to achieve transgene delivery and expression, this goal is often difficult to fulfil as some tumour cells are not viable at the time of gene delivery. Furthermore, none of the existing vector systems efficiently transfer genes to every tumour cell which subsequently allows for tumour regrowth. On the other hand clostridial spore replication is not tumour cell dependent and occurs via rod multiplication extra-cellularlly. Furthermore, the tumour killing mechanism of clostridial spores may operate independently of the requirement for gene transfer. Without the requirement for gene integration into the host cell genome removes the possibility of insertional mutagenesis when using Clostridia. Therefore, Clostridia may show tumour killing irrespective of the tumour cell heterogeneity found within the tumour environment.One of the primary limitations of most viral vectors has been the small size of the virion, which only permits the packaging of very limited sizes  of exogenous DNA that includes the promoter, the polyadenylation signal and any other enhancer elements that might be desired. However, for clostridia size limitations are far less restricted, not only because the plasmids used can harbour much larger DNA fragments, but in case the foreign gene is integrated in the host chromosome there is in fact unlimited capacity for insertion of therapeutic genes, forecasting the promising future for the development of ever powerful vectors.The unique pathophysiology of solid tumours presents a huge problem for the conventional therapies. Thus, the outcomes of current therapies are so far disappointing. Several new approaches aiming at developing effective treatments are on the horizon. These include a variety of virus-based therapy systems -71. AmonC. novyi-NT) with an antimicrotubuli agent has been initiated [The strictly anaerobic clostridia, on the other hand, have been shown to selectively colonise in solid tumours when delivered systemically and has resulted in high percentage of "cures" of experimental tumours. A phase I clinical trial combining spores of a non toxic strain ; Dendritic cells (DCs); Colony forming unit (CFU); Plaque forming unit (PFU); Epidermal growth factor receptor (EGFR); Herpes simplex thymidine kinase (HSV TK); Interleukin-2 (IL-2); Granule macrophage colony stimulatory factor (GM-CSF); Human Immunodeficiency Virus (HIV); Jembrana Disease virus (JDV); Magnetic resonance imaging (MRI); Murine Moloney leukaemia virus (MoMLV); β-galactosidase ; Multifunctional particles (MFPs); Nitroreductaes (NTR); Non small cell lung cancer (NSCLC); Semliki Forest virus (SFV); Severe combined immunodeficiency syndrome (SCIDs); tumour infiltrating lymphocytes (TIL); Tumour necrosis factor (TNF); Vascular endothelial growth factor (VEGF); Vesicular stomatitis virus (VSV); Wide type (WT).Adenoviral vector (AV); Adeno-Associated Virus (AAV); The author(s) declare that they have no competing interests.All authors participated in the production of the manuscript together and have read and approved the final manuscript.


Peripheral blood mononuclear cells (PBMC) serve a sentinel role allowing the host to efficiently sense and adapt to the presence of danger signals. Herein we have directly compared the genome-level expression patterns (microarray) of a human PBMC model (THP-1 cells) subjected to one of two canonical danger signals, heat shock or lipopolysaccharide (LPS).Based on sequential expression and statistical filters, and in comparison to control cells, we found that 3,988 genes were differentially regulated in THP-1 cells subjected to LPS stress, and 2,921 genes were differentially regulated in THP-1 cells subjected to heat shock stress. Venn analyses demonstrated that the majority of differentially regulated genes (≥ 70%) were uniquely expressed in response to one of the two danger signals. Functional analyses demonstrated that the two danger signals induced expression or repression of genes corresponding to unique pathways, molecular functions, biological processes, and gene networks. In contrast, there were 184 genes that were commonly upregulated by both stress signals, and 430 genes that were commonly downregulated by both stress signals. Interestingly, the 184 commonly upregulated genes corresponded to a gene network broadly related to inflammation, and more specifically to chemokine signaling.These data demonstrate that the mononuclear cell responses to the canonical stress signals, heat shock and LPS, are highly divergent. However, there is a heretofore unrecognized common pattern of gene network expression corresponding to chemokine-related biology. The data also serve as a reference database for investigators in the field of stress signaling. Peripheral blood mononuclear cells serve a sentinel role by allowing rapid host adaptations to danger signals associated with various biological stresses such as infection, trauma, or drastic environmental changes . Two of These two traditional paradigms involving the transcriptional responses to LPS or heat stress may represent oversimplifications. For example, transcriptional profiling experiments have suggested the existence of previously unrecognized genes that are expressed in response to LPS stimulation -12. Our In the current work, we sought to expand our understanding of the peripheral blood mononuclear cell transcriptional response to canonical stress/danger signals. We have exposed peripheral blood mononuclear cells to either LPS or heat shock, and have directly compared their respective genome-level responses using microarray technology. The focus of the analytical approach is coordinated expression/repression of genes corresponding to functional annotations and gene networks.2 incubator at 37°C using RPMI  supplemented with 10% fetal bovine serum. One group of cells was exposed to LPS  for 4 hours at 37°C. Heat shock was carried out by placing one group of cells in a dedicated room air/5% CO2 incubator set at 43°C for 1 hour. After heat shock, cells were returned to a 37°C, room air/5% CO2 incubator and allowed to recover for 4 hours. Control cells were maintained in basal growth media at 37°C. After exposure to the experimental conditions, cells were harvested for total RNA extraction as described below.Human THP-1 mononuclear cells  were used as a model of peripheral blood mononuclear cells as previously described . Cells wThe data and protocols described in this manuscript have been deposited in the NCBI Gene Expression Omnibus  and areTotal RNA was isolated from THP-1 cells exposed to the above experimental conditions using the Trizol reagent (Invitrogen) according to the manufacturer's specifications. Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at Cincinnati Children's Research Foundation as previously described using the Human Genome U133 Plus 2.0 GeneChip  ,22.Analyses were performed using one experimental condition per chip, with each experimental condition carried out in triplicate . Image files were captured using an Affymetrix GeneChip Scanner 3000. CEL files were subsequently preprocessed using Robust Multiple-Array Average (RMA) normalization and GeneSpring GX 7.3 software . All signal intensity-based data were used after RMA normalization, which specifically suppresses all but significant variation among lower intensity probe sets . All chiDifferences in mRNA abundance between LPS-stimulated cells and control cells, and between heat shock-treated cells and control cells were determined using GeneSpring GX 7.3 (Agilent Technologies). All statistical analyses used corrections for multiple comparisons. A list of genes differentially regulated between LPS-treated cells and control cells was generated using sequential expression and statistical filters as follows. Starting with all gene probes within the microarray  we selected the genes having ≥ 2-fold expression difference (increased or decreased expression) between the respective median values of LPS-treated cells and control cells. The genes passing this expression filter were then subjected to a statistical test  using LPS-treated cells and control cells as the comparison groups. A list of genes differentially regulated between heat shock-treated cells and control cells was generated using identical sequential expression and statistical filters, but using heat shock-treated cells and control cells as the comparison groups.Protein Analysis Through Evolutionary Relationships) Classification System [Gene lists of differentially expressed genes were primarily analyzed for functional annotation enrichment using the PANTHER  . The IPAThe functional annotations associated with the gene networks presented in Results section were generated using a relational database independent of the IPA application and the PANTHER database. The gene lists corresponding to the gene networks were analyzed using D.A.V.I.D. , which also allows public access to relational databases of functional gene annotations . In the Based on the filtering strategy described in the Methods section, there were 3,968 gene differentially expressed in THP-1 cells exposed to LPS. Of these 3,968 genes, 1,345 were upregulated and 2,623 were downregulated relative to control cells . The majority of the top ten genes expressed in response to LPS stress Table  are wellWe have characterized the genome-level responses of THP-1 cells  following stimulation with one of two canonical stress/danger signals at a single time point. While previous high throughput-centered studies have focused on either LPS -12 or heThe global view (Venn diagrams) of the genes differentially regulated in response to LPS or heat shock stress indicates that each stress signal regulates (increased or decreased expression) a relatively unique group of genes. Among the upregulated genes, > 85% of the genes were uniquely expressed in response to either LPS or heat shock stimulation. Among the downregulated genes, > 69% of the genes were uniquely repressed in response to either LPS or heat shock stimulation. Thus, the mononuclear cell response to LPS stress is generally divergent compared to that of heat shock stress.The divergence between the LPS response and the heat shock response is evident by the functional annotations derived for the respective individual lists of differentially regulated genes. The functional annotations derived for the upregulated LPS-responsive genes were predominantly in the areas of inflammation and immunity. This observation is consistent with the well known role of LPS as the major pathogen associated molecular pattern of gram negative bacteria, and the well known role of peripheral blood mononuclear cells as sentinels of the innate immune system . In contThe assertion of divergence is further supported by the functional annotations derived for the individual lists of differentially downregulated genes. The functional annotations derived for the downregulated LPS-responsive genes were predominantly in the areas of protein biosynthesis and sterol metabolism. These observations are indirectly supported by reports documenting LPS-mediated alterations of protein and lipid metabolism -42. In cIn the context of our previous studies, several of the genes upregulated in response to heat shock or LPS warrant further discussion. Dual specificity phosphatase 1  was substantially increased in THP-1 cells subjected to heat shock. MKP-1 is a dual specific phosphatase serving an important counter-regulatory function in the MAP kinase signaling pathway ,43,44. WSupplementary Data). All three of these genes appear to play important negative regulatory roles in the NF-κB signaling pathway. NFKBIZ is a member of the ankryin-repeat family of the IκB group of proteins that bind and retain NF-κB in the cytoplasm in an inactive state [Several laboratories, including our own, have previously reported that heat shock inhibits activation of the NF-κB signaling pathway ,13,16-18ve state . NKIRAS2ve state . Finallyve state  and has ve state .in vitro that LPS suppresses the expression of genes that either depend on zinc homeostasis or directly participate in zinc homeostasis.LPS is a key pathogen associated molecular pattern that is well known to play an important role in the pathobiology of human septic shock ,35. In sThe most novel information derived from the current data involves the list of genes commonly upregulated in response to LPS and heat shock stress. There were 184 genes common to both the upregulated LPS gene list and the upregulated heat shock-responsive gene list. This relatively limited number of genes corresponded to gene networks having functional annotations related to inflammation in general, and more specifically to chemokine-related biology. Thus, the response to heat shock stress is not completely divergent from that of the LPS response in that it shares a group of coordinately regulated genes related to chemokine signaling, a heretofore unreported observation.We have directly compared the genomic response of THP-1 cells subjected to LPS stress or heat shock stress. The global gene expression patterns demonstrate that the two responses are predominantly divergent. The response to heat shock, however, is not necessarily characterized by global downregulation of non-heat shock protein genes. In fact, a large number of "non-heat shock protein" genes are expressed in response to heat shock stress and a number of these genes have overlap with inflammation-associated gene expression patterns classically attributed to LPS stress. Thus, the heat shock response and the LPS response are not necessarily mutually exclusive. Finally, the data generated from these experiments can serve as a valuable reference database for investigators in the field of stress/danger signaling.HRW conceived and directed the overall study, conducted analyses, and drafted the initial manuscript. KO conducted all microarray experiments. BS assisted with data analysis and bioinformatics. All authors proof read the manuscript and agreed with its submission for review.3968 genes regulate in lps treated thp1 cells.xls, list of genes regulated in response to LPS alone.Click here for file2921 genes regulate in heat shocked thp1 cells.xls, list of genes regulated in response to heat shock alone.Click here for filemerged uregulated lps network.xls, list of genes corresponding to two merged, upregulated networks in response to LPS alone and shown in Figure Click here for filenetwork legend.pdf, legend for networks depicted in Figures Click here for filemerged upregulated heat shock network.xls, list of genes corresponding to two merged, upregulated networks in response to heat shock alone and shown in Figure Click here for filemerged downregulated LPS network.xls, list of genes corresponding to two merged, downregulated networks in response to LPS alone and shown in Figure Click here for filemerged downregulated heat shock network.xls, list of genes corresponding to two merged, downregulated networks in response to heat shock alone and shown in Figure Click here for filegenes common to both lps and heat shock.xls, list of genes commonly regulated in THP1 cells treated with either LPS or heat shock.Click here for file


By plotting response times of young and older adults across a variety of tasks, Brinley spurred investigation and debate into the theory of general cognitive slowing. Though controversial, Brinley plots can assess between-task differences, the impact of increasing task demand, and the relationship between responses in two groups of subjects. Since a relationship exists between response times and the blood-oxygen level dependent (BOLD) signal of functional MRI (fMRI), Brinley's plotting method could be applied as a meta-analysis tool in fMRI studies of aging. Here, fledgling “Peiffer plots” are discussed for their potential impact on understanding general cognitive brain activity in aging. Preliminary results suggest that general cognitive slowing may be localized at the sensorimotor transformation in the precentral gyrus. Although this meta-analysis method is naturally used with imaging studies of aging, theoretically it may be applied to other study pairs  or imaging datasets . In the mid 1960s, Brinley presented a novel plotting method to consider therelationship between response times in young and older adults across a varietyof tasks with varying levels of cognitive difficulty [The history of Brinley plots is rife with debate and discussion on what the plot is measuringand what it means. Feelings in opposing camps can even be so strong as toelicit T-shirts emblazoned with the No symbol over the word Brinley. Laterresearchers inferred that aspects of Brinley plots could provide information ongeneral cognitive functioning in older adults since Brinley's data containedboth task switching and nonswitching data subsets –7. For eIn addition to describing the behavioral appearance of cognitive slowing, researchhas pursued localizing the phenomenon within the aging brain. Generalizedcognitive slowing has been thought to result from pervasive slowing of allcognitive brain functions in older adults. With this in mind, some researchershave proposed that the locus of general cognitive slowing occurs where sensoryimpulses transfer to a common site of motor generation for the response andthat this sensorimotor dysregulation intensifies with more difficult tasks –13. YordAfter reviewing the body of research literature on general cognitive slowing inaging, we adopted the early Brinley method for plotting reaction times andapplied it to the blood-oxygen level dependent (BOLD) signal from multiple fMRIstudies. In doing so, an attempt is made to localize brain areas responsiblefor the deviant slope in the response time Brinley plot. Unlike reaction times,the BOLD signal has a legitimate negative value that occurs when contrasting activity during two different events. For example, certain brain areas are more active duringbaseline than during any particular task. These areas prominently includeposterior cingulate cortex and inferior parietal lobe areas and are known asthe default network . In apphttp://www.fmridc.org) contributed a complete dataset from Buckner and colleagues (Accession no. 2-2000-1118W) for 2 points inthe analysis  [BOLD data and behavioral response times were obtained from 4 simple detection tasks . In order to plot a BOLD signal value for young and older adults in each study, original fMRI data was needed. The fMRI Data Center (s = 14) . These Bs = 14) . These B0 in question was whether ornot there was equivalent change between young and older adults across tasks,the slope value for H0 was 1. To evaluate H0 : Using the 4 average young maps as observed As graciously pointed out by an anonymous reviewer, slopes of −1 should not be considered aspart of the null hypothesis, since the direction of activity across tasks forthe age groups would actually be opposite . Therefore, it is also important toinvestigate areas where the slope is significantly negative, since the above“residual” analysis would not just eliminate slopes of +1 but also slopes whichwere not significantly different from −1. An example of this can be seen in theplotted data of To evaluate a significant voxel's b-intercept, the 95% confidence interval was assessed. If this intervalcontained zero, the voxel's b-intercept was considered not to deviate fromzero. Again, b-intercept clusters were considered significant if they containedat lest 3 contiguous voxels with significantly nonzero b-intercepts.The traditional Brinley plot using average response times across the 4 tasks included in the fMRI meta-analysis showed the established differences between young and older adults see . The slFor the fMRI meta-analysis, five distinct clusters survived the stringent correction appliedfor multiple comparisons. The location of these clusters is summarized visuallyin When the Peiffer plot was explored for 3 contiguousvoxels with significantly negative slopes less than zero, 19 total clusters were identified. Nine of these clusters (47%) were located within the rightmiddle and superior frontal gyri and included a total of 68 voxels see . Across∼51 years of age). When the Peiffer plot wasconstructed for these new groups, no significant clusters were identified wherethe null hypothesis  suggesting a high predictabilityfor older adults' BOLD signal in several brain regions; however, within theclusters identified as significantly deviant from one, R-squared values werelower and ranged between 0.1–0.46.Here we report the preliminary use of a novel meta-analysis technique in studies onaging, which localizes one factor of general cognitive slowing to thesensorimotor transfer. These findings lend support to existing data fromevent-related potential work indicating that slowing occurs predominatelyduring the time for generation of the response in older adults and not when evaluatingincoming sensory material , 13. DeAn important caveat exists, however, since datasets within this analysis were obtained fromrelatively noncognitively demanding tasks. In other words, more brain areasmay be involved as loci of general cognitive slowing and would emerge as morecognitively demanding datasets become available for assessment. Comparing ourcurrent datasets to the existing literature on Brinley plots of reaction times,simple discrimination tasks show the least amount of response time slowing;therefore, these BOLD signal findings presumably will only become stronger withthe addition of datasets containing more cognitively demanding conditions. In addition, with more tasks requiring greater attentional demand, the negative slope found within right frontal cortex maysteepen and be found within the residual analysis which can control for themultiple comparisons inherent within imaging data. Power analyses of thesepreliminary results suggest that roughly 9 datasets are needed to perform a meta-analysis with a paired t-test to achieve a power-level of 80%.Interestingly, allsignificant clusters identified as deviant within the meta-analysis wereseparate from peak activity differences reported in any of the individual tasksused within the datasets. If traditional meta-analysis techniques were used –20, noneAn existing disadvantage of this meta-analysis technique is that it requires access to rawfMRI data to obtain subjects' normalized contrast weighted BOLD activity maps(task - baseline) from multiple tasks. With thecontinued increase of complete data sets maintained in accessible repositorieslike the fMRI Data Center, this shouldhopefully become less burdensome in the near future.Overall as a meta-analysis method in the fMRI field, this plotting addresses severallimitations of existing analysis methods. Specifically, it allows theassessment of between-task differences regardless of a task's paradigm domainor baseline condition. Further, it can identify areas of subthreshold effectsin addition to the suprathreshold within-task differences that are identifiedby performing a meta-analysis on voxel quadrants identified in individualstudies as the local maxima. Lastly, this method provides imaging researchersthe ability to localize between-task differences in BOLD signal and apply thatknowledge to existing behavioral evidence not only in aging but in other complex conditions as well.


The morphology of human pollical distal phalanges (PDP) closely reflects the adaptation of human hands for refined precision grip with pad-to-pad contact. The presence of these precision grip-related traits in the PDP of fossil hominins has been related to human-like hand proportions (i.e. short hands with a long thumb) enabling the thumb and finger pads to contact. Although this has been traditionally linked to the appearance of stone tool-making, the alternative hypothesis of an earlier origin—related to the freeing of the hands thanks to the advent of terrestrial bipedalism—is also possible given the human-like intrinsic hand proportion found in australopiths.Orrorin, Australopithecus, Paranthropous and fossil Homo, in order to investigate their morphological affinities. Our results indicate that the thumb morphology of the early biped Orrorin is more human-like than that of australopiths, in spite of its ancient chronology (ca. 6 Ma). Moreover, Orrorin already displays typical human-like features related to precision grasping.We perform morphofunctional and morphometric (bivariate and multivariate) analyses of most available hominin pollical distal phalanges, including Homo, more encephalized than the previous australopiths. This hypothesis remains may be further tested by the finding of more complete hands of unequivocally biped early hominins.These results reinforce previous hypotheses relating the origin of refined manipulation of natural objects–not stone tool-making–with the relaxation of locomotor selection pressures on the forelimbs. This suggests that human hand length proportions are largely plesiomorphic, in the sense that they more closely resemble the relatively short-handed Miocene apes than the elongated hand pattern of extant hominoids. With the advent of terrestrial bipedalism, these hand proportions may have been co-opted by early hominins for enhanced manipulative capabilities that, in turn, would have been later co-opted for stone tool-making in the genus  PositivPan and Pongo, which can be roughly distinguished from each other thanks to the highest loads on PC 2 of orangs, which display very small shafts and apical tufts relative to the base  in the scatter plot indicates that they show overall proportions on PDPs more similar to those of humans than to great apes, although being relatively shorter and displaying a larger base (The position of baboons (ger base .Orrorin (BAR 1901′01) most closely resembles modern humans in both components (A. africanus) also fall close to modern humans in both components, although both La Ferrassie I and Stw 294 display slightly highest values on the PC 2. However, both Paranthropus robustus (SKX 5016) and OH 7 depart in PC 1 by having an extremely robust  shaft and apical tuberosity in the PDP.Concerning the fossil hominins, mponents . NeanderParanthropus robustus and Neandertals  falling within the 95% CI displayed by monkeys  and third (DP3) manual rays of extant taxa, together with OH 7, ndertals . In orankeys see .Pongo and Pan, in which locomotor selection pressures have probably favored the lengthening and increased strength of digits II-V Extant great apes do not significantly use the thumb during locomotion: it does not participate in below-branch suspensory behaviors, and it does not support body weight during knuckle-walking in African apes Gorilla hand therefore displays more balanced proportions between the thumb and index finger (presumably related to somewhat advanced manipulatory capabilities) than chimps and especially orangs Thus, although extant great apes display diminished thumbs, especially concerning extrinsic muscle insertions, they do have well-developed intrinsic muscles that enable efficient power and precision grips, the latter being used during food manipulation Pan and Pongo, would be related to the lack of a developed flexor apparatus . The overall reduced PDP of orangs, especially referring the shaft and apical tuft, obviously stems from their rudimentary thumbs, which are diminished like in Colobus and ProcolobusPan and PongoOur results show that extant great apes exhibit rod-like PDPs with barely discernible muscular impressions on the palmar side, further lacking developed tufts and associated palmar pads . The lacPapio spp.) show the same capabilities, although to a lesser degree flexor digitorium profundusTerrestrial monkeys, in its turn, exhibit proportionally short fingers in relation to their thumbs, thereby enabling an efficient precision grip Our results agree with previous findings showing that baboons display PDPs more similar in overall proportions to those of extant humans rather than those of extant great apes, although being shorter and displaying a larger base . This moA. africanus) and, particularly, in BAR 1901′1 (Orrorin), the latter showing, among early hominins, the greatest resemblance to extant humans (P. robustus) and OH 7, although more closely resembling extant humans than any other taxa examined, display extremely broad shafts and apical tuberosities  are characterized by a high degree of apical and midshaft robusticity, as it is found here , their manipulative capabilities being limited by their relative short thumbs Pierolapithecus, which displays relatively short manual phalanges—like other early and middle Miocene apes—as well as a relatively long PDP Be that as it may, the highest resemblance between the late Miocene Orrorin was an early biped hominin with a PDP adapted to refined manipulation, it follows that the whole thumb would be long relative to the hand, thus enabling an efficient pad-to-pad contact. Thus, this leads us to hypothesize that the manual proportions of Orrorin might have more closely resembled those of early and middle Miocene apes than those of extant apes, which would have diverged towards a different direction from the same ancestral morphotype. This idea is similar to that presented by Tuttle A. afarensisA. africanusFrom the evidence presented above, it can be suggested that the hand length proportions of humans are plesiomorphic to a large extent. If Homo ca. 2.5 Ma Homo would have involved an increase of overall robusticity at the several manual joints, an increase in robusticity of the whole thumb, and especially the development of distinct palmar pads on the distal phalanges.Most likely, these hand capabilities would have not been co-opted until much later for a regular use in stone tool-making, coinciding with the encephalization increase that took place in hominins with the advent of the genus Orrorin PDP. For the moment being, the most complete fossil hand around this time corresponds to the 4.4 million-years-old putative hominin Ardipithecus ramidusOrrorin, in spite of the older chronology of the latter. Thus, although A. ramidus has been claimed to be an early hominin close to the last common ancestor with PanMore complete fossil hands from the African Mio-Pliocene transition would be necessary to clarify this issue, especially regarding of the selection pressures underpinning the remarkable human-like features of the Orrorin (BAR 1901′01) unequivocally shows precision grasping capabilities in spite of its ancient chronology, most closely resembling modern humans than some later Plio-Pleistocene hominins—which show a derived robusticity pattern. This indicates that refined manipulation is an ancient acquisition already present by the late Miocene. This is consistent with the hypothesis that habitual terrestrial bipedalism and the possession of skillful hands do constitute a single adaptive complex. Both types of behaviors might have been simultaneously selected, by synergistically favoring each other.The pollical distal phalanx of the early bipedal hominin Homo.From the evidence reported by BAR 1901′01, it is reasonable to assume that human hand length proportions (i.e. short hands and relatively long thumbs) are plesiomorphic to some degree, thus more closely resembling the short hands with relatively long thumbs of Miocene apes, rather than the elongated hands of extant apes, which seem to be secondarily derived. These ancient proportions, suitable for refined manipulation, would not have been co-opted for stone tool-making until much later, coinciding with a significative increase in encephalization in the genus Pan; N = 29), gorillas , orangutans , baboons  and modern humans . The fossil sample includes the PDPs of Orrorin tugenensis from the Lukeino Formation (BAR 1901′01), Australopithecus africanus from Sterkfontein (Stw 294), the hand from Olduvai (OH 7 A), Paranthropus robustus from Swartkrans (SKX 5016), and H. neanderthalensis from La Ferrassie I and Kebara 2. Apart of the above-mentioned extant taxa, we further employed the following fossil third distal phalanges for computing ratios: OH 7 B, SKX 27504 (attributed to P. robustus), La Ferrassie I and Kebara 2. Measurements for fossil specimens were taken from originals, good quality casts or from the literature The comparative extant sample includes the following taxa: chimpanzees and bonobos  based on the covariance matrix was employed to perform morphometric comparisons between the pollical distal phalanges (PDP) of selected fossil specimens and those of other hominins and extant primates, including modern humans. This analysis, which does not assume group membership on a priori grounds, was based on six shape variables of the PDP, in its turn computed on the basis of the following six metrial variables: length (L); mediolateral width at the apical tuft (MLT), midshaft (MLS) and the base (MLB); and dorsopalmar height at midshaft (DPS) and the base (DPB). These measurements were transformed into shape variables by dividing each of them by the geometric mean (GM) of all the six phalangeal measurements  and then applying a logarithmic transformation , following Distal phalangeal robusticity was analyzed by means of a ratio between apical tuft width and maximum length, separately for both the distal pollical and middle finger phalanges. We further calculated the difference between both ratios, in order to quantify the relative robusticity of the pollical distal phalanx in relation to that of the middle finger. Summary statistics for extant and fossil taxa are reported in Video S1Pan troglodytes pollical distal phalanx.360° video render of (3.31 MB AVI)Click here for additional data file.Video S2Gorilla gorilla pollical distal phalanx.360° video render of (2.96 MB AVI)Click here for additional data file.Video S3Orrorin tugenensis pollical distal phalanx.360° video render of (3.72 MB AVI)Click here for additional data file.Video S4Homo sapiens pollical distal phalanx.360° video render of (3.46 MB AVI)Click here for additional data file.Video S5360° video render of Olduvai Hominid 7 pollical distal phalanx.(4.32 MB AVI)Click here for additional data file.


Drosophila brain amplify their proliferation through generation of transit amplifying intermediate progenitor cells. Together, these DM neuroblast lineages comprise over 5,000 adult-specific neural cells and thus represent a substantial part of the brain. However, no information is currently available about the structure or function of any of the neural cells in these DM lineages. In this report we use MARCM-based clonal analysis together with immunocytochemical labeling techniques to investigate the type and fate of neural cells generated in the DM lineages.Specific dorsomedial (DM) neuroblast lineages of the Genetic cell lineage-tracing and immunocytochemical marker analysis reveal that DM neuroblasts are multipotent progenitors that produce a set of postembryonic brain glia as well as a large number of adult-specific protocerebral neurons. During larval development the adult-specific neurons of each DM lineage form several spatially separated axonal fascicles, some of which project along larval brain commissural structures that are primordia of midline neuropile. By taking advantage of a specific Gal4 reporter line, the DM-derived neuronal cells can be identified and followed into early pupal stages. During pupal development the neurons of the DM lineages arborize in many parts of the brain and contribute to neuropile substructures of the developing central complex, such as the fan-shaped body, noduli and protocerebral bridge.Drosophila brain.Our findings provide cellular and molecular evidence for the fact that DM neuroblasts are multipotent progenitors; thus, they represent the first identified progenitor cells in the fly brain that have neuroglioblast functions during postembryonic development. Moreover, our results demonstrate that the adult-specific neurons of the DM lineages arborize widely in the brain and also make a major contribution to the developing central complex. These findings suggest that the amplification of proliferation that characterizes DM lineages may be an important requirement for generating the large number of neurons required in highly complex neuropile structures such as the central complex in the  Drosophila brain is a highly complex structure composed of tens of thousands of neurons that are interconnected in numerous exquisitely organized neuropile structures, such as the mushroom bodies, antennal lobes and central complex. The neurons of the central brain, defined as the supraesophageal ganglion without the optic lobes, derive from approximately 100 bilaterally symmetrical pairs of neural stem cell-like neuroblasts, each of which is thought to generate a characteristic lineage of neural progeny [The  progeny . Several progeny -5).Neuroblasts begin to proliferate during embryonic development and during this initial phase of proliferation they generate the primary neurons of the larval brain. After a period of mitotic quiescence during the early larval period, most brain neuroblasts reactivate proliferation and produce secondary neurons that make up the bulk of the adult brain; these are referred to as adult-specific neurons ,7. IndeeMost neuroblasts in the central brain generate lineages comprising, on average, 100 to 120 adult-specific cells ,5,13-15.Drosophila brain [The six bilaterally symmetrical pairs of DM (type II) neuroblast lineages together generate over 5,000 adult-specific cells due to the amplification of neuroblast proliferation . Given cla brain , this ceDuring larval development, six DM neuroblasts generate large lineages, each of which consists of an average of 450 cells that are located at the DM midline of each hemisphere ,17. ThesAll postembryonic DM lineages contained a set of intermediate neuronal progenitors located near the neuroblast; these cells have been described previously ,16,17 anThe DM lineage glial cells had the general structural features of neuropile glia. In the late third larval instar, their cell bodies were generally clustered in the vicinity of the emergent secondary axon tracts of the DM lineages, and their processes were associated with these tracts Figure . FurtherTaken together, these findings uncover an unexpected feature of these neuroblast lineages; they are not only by far the largest in terms of overall cell number, they also comprise glial cells and thus represent the only known neuroglioblast lineages in the postembryonic central brain.To study the axonal projections of the adult-specific neurons of DM lineages in the larval brain, we recovered MARCM-labeled neuroblast clones corresponding to each of the six DM lineages and co-immunostained these with an anti-Neurotactin antibody that labeled secondary neurons and their axon tracts (clones were induced at early first instar stage and recovered at the late third instar). In all DM lineages, axons from the adult-specific neuronal cells fasciculated to form initial secondary axon tracts within the cortical layer. As soon as these initial secondary axon tracts reached the brain neuropile, they split into several subsidiary tracts. This contrasted with the behavior of secondary tracts in most other brain lineages, which generally formed a single, discrete fascicle of axons within the brain cortex and neuropile during larval stages ,11. In aThe DM lineages formed a set of secondary axon tracts with a characteristic and relatively invariant trajectory in the larval brain neuropile. Based on the rostral-to-caudal arrangement of the cell body clusters of the six DM lineages in the larval brain hemispheres, we numbered each of these as follows: DM1 , DM2, DM3, DM4, DM5, DM6. The characteristic neuroanatomical features of the secondary axon tracts formed by all six DM lineages in the late third instar larval brain are described below. For a given DM lineage, these anatomical features were the same irrespective of whether MARCM clones were induced in the stage 13 embryo or in the early larva.The DM1 lineage formed a secondary axon tract that projected towards the interhemispheric region, where it separated into two fascicles: the first fascicle entered a larval interhemispheric commissure and immediately split into multiple commissural fiber bundles; the second fascicle formed a descending projection, from which an additional small commissural bundle also branched off Figure . The comThe DM2 lineage formed a secondary axon tract that projected toward the commissural system and also split into multiple commissural fiber bundles and one descending fascicle Figure . The setThe DM3 lineage initial secondary axon tract split into three fiber bundles upon entering the neuropile Figure . Two of The DM4 lineage had the most complex projection pattern of all the larval DM lineages. Its initial secondary axonal tract first split into three main axon bundles, and each of these formed several subsidiary axonal bundles upon entering the neuropile Figure . The resThe DM5 lineage secondary axon tract split into several commissural fascicles and one major ascending fiber bundle upon entering the neuropile Figure . The sinThe secondary axon tract of the DM6 also split into several commissural fibers and one major ascending fiber bundle (and one minor descending bundle) upon entering the neuropile. The anatomical features of all of these fiber bundles were very similar to those of the DM5 lineage Figure . Thus, cTaken together, these findings indicate that all of the DM lineages generate highly complex secondary axon tract projections in the larval brain. Our data reveal five types of axonal projection patterns, all of which have pronounced  longitudinal as well as commissural components. Four different types of axonal projection patterns can be ascribed to the DM1, DM2, DM3, and DM4 lineages and a common fifth DM5/6 type can be attributed to both DM5 and DM6 lineages.distal-less gene [UAS-CD8::GFP (membrane labeling) or UAS-H2B::RFP (nuclear labeling)  Figure . This GaTo confirm that the Dll-Gal4 labeled cell groups at the DM brain midline were indeed the DM lineages, a more detailed cellular and molecular analysis of the labeled cells was carried out. A characteristic hallmark of DM lineages is the fact that they contain multiple mitotically active cells located at variable distance from the parental neuroblast ,16,17. BTaken together, these reporter line-based studies provide cellular and molecular data that confirm the identification of DM lineages based on anatomical criteria. Moreover, they provide a labeling method for DM lineages in early pupal stages.During pupal development, the relatively simple secondary axon tracts generated by adult-specific neurons in the larval brain arborize and undergo final morphogenesis -11. GiveTo investigate this, Dll-Gal4-based MARCM labeling of DM neuroblast clones was induced at the early first instar larval stage. Labeled DM clones were recovered at 24 hours after puparium formation.  In contrast to the larval DM lineages, each of which was easily identifiable in the larval brain, the recovered MARCM-labeled pupal DM lineages could not be unambiguously individually identified since lineage-related neurons sometimes changed position and no longer formed compact groups during pupal development. In consequence, we could not assign individual arborization patterns with confidence to any one of the six pupal DM lineages. Nevertheless, two features of the adult-specific DM neurons characterized all of the MARCM-labeled DM lineages in early pupal development. First, all DM lineages formed multiple axonal projections and widespread arborizations in the brain. Second, all DM lineages projected a subset of their axon fascicles into the central complex neuropile. Two examples of the type of adult-specific arborizations formed by DM lineages in the early pupal brain are shown in Figures Figure Figure For a more comprehensive characterization of the neuroanatomy of individual DM1-6 lineages in pupal development, lineage-specific labeling techniques will be required. For this, more selective Gal4 lines that subdivide the six DM neuroblasts into individuals will be needed. However, based on the ensemble of MARCM-labeled pupal DM lineages that were recovered and analyzed in this study, one common feature of all pupal DM lineages appears to be that a subset of the neurons in each of these lineages contributes to the developing central complex neuropile.In this report we have studied the postembryonic development of the neural cells generated by the DM lineages in the larval and early pupal brain. We report three main findings. First, DM lineages comprise both adult-specific neurons and glia; DM neuroblasts thus have features of neuroglioblasts. Second, during larval development, adult-specific neurons form complex secondary axon tracts composed of separate commissural and longitudinal fascicles. Third, during pupal development the commissural fascicles arborize in and contribute to the central complex neuropile. In the following, we discuss the major implications of these three findings.Drosophila, as in other insects, glial cells fall into three classes, surface glia, cortex glia and neuropile glia, each of which is represented in the larval brain [In al brain . The glial brain . Howeveret al. [Drosophila brain, only the perineurial surface glia and the neuropile glia are extensively generated during postembryonic development, whereas most of the subperineurial surface glia and cortex glial cells are thought to have their origin in embryogenesis. Moreover, this analysis has provided evidence that perineurial glial precursors are distributed around the brain surface, whereas neuropile (ensheathing and astrocyte-like) glial cells are derived from specific proliferation centers within the brain. However, the progenitors of these postembryonically generated glial cells as well as the mode of postembryonic glial proliferation still remained elusive. Here we identify the first postembryonic neuroglioblasts in the Drosophila brain.  All siDrosophila, are generated by amplifying intermediate progenitors. However, an exact clonal analysis of the relationship between glial cells and intermediate progenitors in DM lineages will be required to validate this notion. There is some evidence that DM derived glial are generated early in the lineage. If this is indeed the case, it is conceivable that these glial cells might be important for the differentiation of the subsequently generated neuronal cells in these lineages. For example, the extended processes of DM-derived glia located near the emergent secondary axon tracts or associated with the larval brain commissure might be important in guiding axons of DM-derived neurons  across the midline.DM neuroblasts proliferate through asymmetric division that involves intermediate progenitors with transit-amplifying cell features ,16,17. TDuring postembryonic development, secondary, adult-specific neurons generated by reactivated neuroblasts produce secondary lineages and axons of a given secondary lineage fasciculate with each other to form a discrete and generally unbranched secondary axon tract within the brain cortex and neuropile ,11. In cThe multiplicity of axonal bundles that emerge from a given DM lineage has features that are more reminiscent of a secondary axon tract system comprising the axon tracts of several lineages than a single secondary axon tract. This may be an indirect result of the fact that DM lineages contain three to five times more secondary neurons than do conventional neuroblast lineages, which, hence, would generate three to five times more axons than conventional lineages, resulting in an excessively large secondary axon tract in the neuropile if branching did not occur. While the underlying mechanisms are currently not known, it is also possible that this anatomical complexity is related to the particular mode of neurogenesis in the DM lineages, which involves amplifying intermediate progenitors. Thus, a given intermediate progenitor might produce neural progeny that develop a common type of axonal projection pattern, whereas progeny subsets derived from different intermediate progeny might develop different axonal projection types.Like other adult-specific neurons, DM secondary neurons differentiate during the pupal period, when they evolve into the long tracts that characterize the adult brain and send out proximal as well as terminal arborizations that form synapse-rich neuropile circuitry. During the early pupal differentiation period, some of the neuropile structures that specifically characterize the adult brain become visible. Among these are the principle components of the central complex. The central complex, one of the most prominent neuropile structures in the adult brain, is located centrally between the two hemispheres and consists of four substructures. These are the protocerebral bridge, the fan-shaped body, the ellipsoid body, and the noduli, all of which are interconnected by sets of columnar interneurons that form regular patterns of projection -27. A reIn the late larval brain, distinguishing the four substructures of the adult central complex is difficult. However, a putative midline neuropile primordium of the central complex has been described in the late larval brain in the form of a large commissural neuropile domain, consisting of the DPC1 and trCM and probably part of the DPC2 commissures . All DM From the first day of pupal development onward, the major components of the fan-shaped body, ellipsoid body, noduli and protocerebral bridge can be clearly recognized. In the early pupal brain, DM derived commissural neurons have already contributed to the arbors in the fan-shaped body, nodulus and protocerebral bridge. Although we were not able to identify them individually, it is very likely that the central complex-innervating DM neurons in the pupal brain are the same neurons that projected commissural axon bundles into the DPC1 commissure of the larval brain. This observation supports the notion that the DPC1 is indeed part of the central complex primordium.A noteworthy feature of the DM-derived projections in the fan-shaped body is the fact that two major fascicles are formed that project in parallel into the fan-shaped body and form two separate arborization domains. This may reflect a contribution of a given DM lineage to a pair of the eight modular subdomains ('staves') that make up the fan-shaped body ,25. A siMost neurons of the central complex belong to one of two categories: large-field elements or small-field elements . A largeDrosophila brain.We have used MARCM-based clonal analysis together with immunocytochemical labeling techniques to investigate the type and fate of cells generated in the DM lineages. With a combination of neuronal and glial cell markers we show that most of the cells in the DM lineages are neuronal but that glial cells are also generated in these lineages. The DM neuroblasts thus represent the first identified precursor cells in the fly brain that have neuroglioblast functions during postembryonic development. We also show that the adult-specific neurons of each DM lineage form several spatially widely separated axonal fascicles, some of which project along larval brain commissural structures that are primordia of midline neuropile. By taking advantage of a specific Gal4 reporter line, we identify and follow DM-derived neuronal cells into early pupal stages and demonstrate that neurons of the DM lineages make a major contribution to the developing central complex, in that the numerous columnar elements are likely to be DM lineage-derived. These findings suggest that the amplification of proliferation that characterizes DM lineages may be an important requirement for generating the large number of neurons required in highly complex neuropile structures such as the central complex in the Drosophila stocks were reared and maintained on standard yeast-cornmeal-agar medium and all experiments were performed at 25°C. Unless otherwise stated, fly stocks carrying transgenes and recombinant chromosomes were obtained from the Bloomington Stock Center and assembled using standard genetics. To generate positively marked MARCM clones: y, w, hsFLP; FRT40A, tubP-GAL80LL10/CyO, ActGFPJMR1; tubP-GAL4LL7, UAS-mCD8::GFPLL6/TM6, Tb, Hu were mated to w; FRT40A, UAS-mCD8::GFPLL5 (standard cell lineage labeling with membrane-tethered GFP); and; FRTG13, tub-Gal80, hs-Flp/ were mated to; FRTG13, Dll-Gal4, UAS-mCD8::GFP/. The distal-less Gal4 line was; Dll-Gal4/CyO; UAS-mCD8-GFP, UAS-H2B-mRFP1.All Generation of MARCM clones and larval staging was performed as previously described .Nervous systems were dissected from late third instar larvae and 24 h old pupae, and fixed and immunostained as previously described . PrimaryFluorescently stained nervous systems were imaged using a Leica TCS SP scanning confocal microscope using 40× and 63× oil-immersion objectives. Z stacks were collected with optical sections at 1-μm intervals. Scanned pictures were visualized in ImageJ  and analDHSB: Developmental Study Hybridoma Bank; DM: dorsomedial; GFP: green fluorescent protein; MARCM: mosaic analysis with a repressible cell marker; PAN: posterior asense-negative; PH3: phospho-histone 3.The authors declare that they have no competing interests.NI, JB and BB carried out all the experiments. HR conceptualized the project. NI and HR wrote the manuscript.


Pregabalin is a newly developed synthetic gamma-aminobutyric acid (GABA) that is approved for the treatment of fibromyalgia and several neuropathy. It has been proven to show analgesic, anxiolytic, anticonvulsant and sleep enhancement effects, which could be applicable in the treatment of a variety of psychiatric disorders. There have been consistent reports that unexplained somatic symptoms  may be a part of psychiatric disorders such as major depressive disorder (MDD) and anxiety disorders. Previous researches have also suggested the possible therapeutic potential of anticonvulsants as augmentation therapy or monotherapy in the treatment of mood disorders and anxiety disorders. Hence this short perspective tries to prompt and facilitate a shifting of researchers' attention to potential neuropsychotropic drug role of pregabalin to treat a wide range of neuropsychiatric disorders. A, GABAB, or benzodiazepine receptors. In addition it does not augment GABAA responses in cultured neurons, alter rat brain GABA concentration or have acute effects on GABA uptake or degradation. However, it was found that prolonged application of pregabalin increases the density of GABA transporter protein as well as the rate of functional GABA transport in cultured neurons. Pregabaline has no involvement with serotonin and dopamine receptors and does not inhibit dopamine, serotonin, or noradrenaline reuptake. It has no effects on sodium channels, opiate receptors, and cyclooxygenase enzyme activity.Pregabalin is a new synthetic molecule and a structural derivative of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Pregabalin does not bind directly to GABAPregabalin is characterized by its potent high-affinity binding to the α2-δ subunit on voltage-gated calcium channels,6The drug may have a strong potential as one of promising neuropsychotropic agents in the monotherapy or augmentation treatment of various neuropsychiatric disorders based on its currently available preclinical and clinical data. Hence this short perspective will review the potential neuropsychotropic effects of pregabalin.The recent study of brief treatment with alprazolam initiated immediately after exposure to a traumatizing experience revealed that treatment effectively curbed short-term anxiety-like behaviors, but had no prevention value and caused a statistically significant increase in individual vulnerability to a trauma-reminder and even more so to re-exposure to the index stressor.7Pregabalin was also excellent in sleep efficiency as showing an enhancement of slow-wave sleep, reduction of the number of midnight awakenings, rapid eye movement sleep and sleep-onset latency, which are commonly seen in depression, anxiety and pain disorders.9Pregabalin is a GABA analogue without abuse potential, although the action mechanism is not same with benzodiazepine agents. This property is important in patients who need to withdraw benzodiazepine or may have abuse propensity. Although the proper principal mechanism of pregabalin's effect on psychiatric disorders may not be fully explained, the modulation of calcium channels by which reduction of excitatory neurotransmitters release should account for its usefulness for various psychiatric disorders.Several line of preclinical data have also suggested the neuroprotective effect of pregabalin by anti-apoptotic and anti-inflammatory actions, which is useful in protection of cognitive function in patients with depression and geriatric psychiatric population.As stated, pregabalin has been U.S. FDA-approved medication for fibromyalgia, which is interesting since fibromyalgia and major depressive disorder (MDD) share some commonalities. Numerous studies have found that fibromyalgia patients are at increased risk for a lifetime history of MDD as compared to normal community samples.14Recently an emerging evidence of potential role of pregabalin in the treatment of psychiatric disorders such as generalized anxiety disorder (GAD) and social anxiety disorder (SAD) has been available. The potential anti-depressive and anti-anxiety effects of pregabalin have been established in a number of randomized, placebo-controlled clinical trials (RCTs).21According a post-hoc analysis of the existing clinical trial database, pregabalin at doses of 150 mg/d (32.1%), 300-450 mg/d (40.1%) and 600 mg/d (36.7%) was associated with statistically significant improvement in endpoint Hamilton Depression Rating Scale-17 item (HAMD-17) scores compared to placebo (22.8%).Pregabalin was also effective in the treatment of benzodiazepine withdrawal symptoms in even chronic abusers.In the author's clinical experience, pregabalin was very useful as augmentation therapy in the treatment of posttraumatic stress disorder (PTSD) patients who were partially responsive to current antidepressants as well.26We may expect a substantial role of pregabalin for patients with depression, anxiety disorders, substance abuse and dependence, aggression and violence, interictal psychosis, sleep disturbance, unexplained somatic complaints (predominantly widespread pain), PTSD, neurodegenerative diseases and bipolar affective disorders .Future researches should include whether or not pregabalin has early improvement effect in depression or anxiety disorders or any treatment effects as augmentation therapy for such patients who did not respond to conventional treatment options. Predictor analysis on response to pregabalin will be intriguing well. Given methodological pitfalls in currently available data, additional works are also needed to determine whether these favorable findings for pregabalin hold true over the long-term period, and whether patients having several comorbid psychiatric disorders are also responsive to pregabalin. Direct comparison studies will be also necessary to confirm differential outcomes between pregabalin and contemporary treatment regimes.