Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency.Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first  The primary causative defects in approximately 85% of FH cases are mutations or deletions in the plasma membrane Low Density Lipoprotein Receptor (LDLR) encoding gene that is responsible for clearing LDL-cholesterol (LDL-C) from the blood stream by endocytosis and intracellular degradation . Over 10/Current/. The sec/Current/. Mutatio/Current/. The pat/Current/. The con/Current/. The cliOwing to mutations in both alleles of the LDLR locus, homozygous LDLR-associated FH patients present with markedly elevated total serum cholesterol  and LDL-cholesterol levels . The deposition of insoluble cholesterol causes xanthomata on the tendons of the hands and feet, cutaneous planar and corneal arcus in early life . AtheromIn most investigated populations, the heterozygote form occurs in at least 1:500 and the homozygous form in one in one million individuals , althougCholesterol levels alone are not sufficient to confirm a diagnosis of FH because blood cholesterol levels vary with age, gender and are population specific . In addiAlu family of middle repetitive DNAs [The human LDLR is a multi-component single-chain glycoprotein, which contains 839 amino acids in its mature form, encoded by a gene of 45 kb in length . The genive DNAs . LDL-Recive DNAs . Their five DNAs .The mature human LDLR of 160 kDa is composed of five domains, Figure Analyses of LDLR-associated FH variants estimated that there were 1066 LDLR gene mutations/rearrangements, 65% (n = 689) of which were DNA substitutions, 24% (n = 260) small DNA rearrangements (<100 bp), and 11% (n = 117) large DNA rearrangements (>100 bp) [To date there is no cure for FH. The primary goal of clinical management in heterozygotes is to control hypercholesterolaemia by lifestyle modification and/or drug treatment in order to decrease the risk of atherosclerosis and to prevent CAD. Lifestyle modification involves educating patients to adhere to a low-fat diet, exercise and to reduce overweight or maintain an optimal body weight. An effective low-fat diet could lower LDL-C (LDL cholesterol) by 20% to 30% -30. For Bile acid binding resins are non-absorbable anion exchange resins that bind bile acids in the intestinal lumen, preventing their absorption from the ilium and therefore increasing their fecal excretion. The liver responds by up-regulating cholesterol 7-alpha hydroxylase, which increases the conversion of cholesterol to bile acids, thereby reducing the cholesterol concentration in the hepatocyte . GastroiNiacin, or nicotinic acid, is the oldest lipid-lowering drug dating back to the 1950s . DependiA cholesterol absorption inhibitor, more commonly known as Ezetimibe, impairs dietary and biliary cholesterol absorption at the brush border of the intestine without affecting the absorption of triglycerides or fat-soluble vitamins . It has For patients who do not respond to a maximum dose of a statin and those who develop side effects with higher doses, a combination therapy of statin with one of the above agent, rather than an increase in the statin to high doses, may be more effective in achieving LDL-C goals and improving CAD outcomes while remaining at an acceptable safety profile . Adding Surgical interventions involving a portocaval shunt or an ileal bypass have yielded transient lowering of plasma LDL in these patients . The preThe most significant but also most aggressive metabolic correction is orthotopic liver transplantation in homozygous patients -56. HoweSeveral novel therapeutic approaches have also been developed recently to lower LDL-C, either as monotherapy or in combination with statins  includinIn August 2010, Genzyme Corp. and Isis Pharmaceuticals Inc. announced the completion of the four phase 3 clinical trials that are required in the initial United States and European of regulatory filings for mipomersen. Filings for therapeutic use in homozygous FH are expected in the first half of 2011 . These dAt the end of the study, these patients had an average LDL-C reduction of 36-37% with no serious adverse effects. The reductions observed in the study were in addition to those achieved with the patients' existing maximally tolerated statin regimens. The trial also met each of its three secondary endpoints with statistically significant reductions in apo-B, non-HDL-cholesterol and total cholesterol. All trials also demonstrated manageable safety and tolerability profile of mipomersen.Although each of these novel therapies effectively lowers LDL-C, challenges remain for clinical development in the assessment of long-term safety.Patients who have undergone liver transplantation and have experienced substantial reductions in LDL-C levels provide indirect evidence that gene therapy targeted towards the liver could be effective for this disease. While LDLR is expressed by the majority of body cells, hepatic reconstitution of LDLR expression alone may be sufficient for metabolic correction ,72. The The homozygous form of FH would be an excellent candidate for gene therapy since the plasma lipid profile, total cholesterol, LDL-C, HDL-C and LDL/HDL ratio, can be measured providing a convenient clinical endpoint to evaluate the response to therapy ,72. In aex vivo, involving isolation of autologous cells from the patient, their in vitro genetic modification and selection followed by reimplantation of the transduced cells, or it can be done in vivo, where the vector is delivered directly to the organ [ex vivo approach is that the transduction/transfection conditions can be carefully controlled and optimised and individual clones with the most desirable characteristics can be isolated to eliminate unmodified cells or cells with deleterious mutations before re-implantation. While this approach is laborious and time consuming, it may also offer significantly greater safety and control with respect to vector mediated mutagenesis and possible germline transmission of the transferred genes, which is a risk of in vivo gene delivery. The disadvantages of the ex vivo approach are failure of cell engraftment and difficulties in returning the cells to the patient due to disease manifestations such as portal vein hypertension [Gene transfer can be performed either he organ . The advrtension ,84.in vivo approach eliminates the need for engraftment after re-implantation and is therefore easier to perform, more cost effective and may be more applicable for use in countries with limited laboratory resources. The gene transfer vector is injected into the bloodstream (systemic delivery) aiming at somatic cell delivery only or by use of specific cell targeting, preferentially to the tissues of interest (targeted delivery). Organ specific delivery of the gene transfer vector includes intrahepatic injection or selective intravasular application routes. Disadvantages of in vivo gene transfer are vector dilution, ectopic transgene expression and non-targeted, random, potentially genotoxic insertion into the host genome.The In addition to the method chosen for delivery, successful treatment of FH would ideally require safe and efficient gene transfer vectors that provide appropriate and sustained levels of transgene expression and long-term survival of treated cells. The use of a liver specific promoter would be the most physiological approach to achieve this. However, because of present problems in transfection-efficiency, strong heterologous promoters are commonly used instead for proof of principles studies on the effectiveness of lipid-lowering. The development of more effective vectors to achieve this remains a formidable challenge to gene therapy. The properties required of such a vector system and those that should be avoided are listed in Table Gene transfer vectors are generally classified under two categories; they are either non-viral or virus mediated gene transfer systems.Gene therapy vectors based on modified viruses are unquestionably the most effective gene delivery systems in use today. Their efficacy at gene transfer is however tempered by their potential toxicity ,86. An iet al reported that a standard tail vein injection of naked DNA into mice resulted in almost no gene expression in major organs due to its rapid in vivo degradation by nucleases and clearance by the monocular phagocyte system [via the same route induced efficient gene transfer particularly in the liver [in vivo following hydrodynamic injection [Most of the non-viral vectors that have been described for gene therapy are based on complimentary DNA (cDNA) gene sequences driven by highly active promoters. The DNA in these vectors is typically formulated with cationic agents to form complexes, which protect the DNA and facilitate cell entry ,88. DNA e system . Howeverhe liver . This prnjection , this prin vitro studies finally lead to the successful administration of genomic LDLR vectors in vivo via hydrodynamic delivery [In contrast to using a cDNA expression cassette, the use of a complete genomic DNA locus to deliver an intact transgene with its native promoter, exons, all intervening introns, and regulatory regions with flanking non-coding genomic DNA sequences may allow regulated complementation of LDLR deficiency in the liver of hypercholesterolaemic animals. In 2003, a bacterial artificial chromosome containing the entire LDLR genomic locus and based on the Epstein Barr Virus (EBV)-retention system was delivered to LDLR deficient Chinese hamster ovary cell line (CHOldlA7) , and achdelivery ,96. WhenWhile the majority of studies were focused on treating FH by inhibition of hypercholesterolaemia through up-regulation of LDLR or other surrogate lipoprotein receptors (as will be discussed later), an alternative approach was to down-regulate apoB-100 LDLR-ligand or PCSK9  expressiin trans in order to support vector production. This can be achieved by use of a helper virus or a packaging (production) cell line transfected with the plasmids expressing the genes coding for the required structural virus components and replication proteins. Helper virus must be purified away from the final vector batches intended for safe gene delivery.Recombinant viral vectors are usually more effective than non-viral vectors in mediating cell entry and nuclear transfer of therapeutic genes in the target cells. In addition natural tropism of viral envelopes and serotypes can be employed to achieve targeting selectivity for particular host cells. Most of these vectors have mechanisms to avoid intracellular degradation and overcome cellular and immunological barriers to the delivery of the genetic cargo. Generation of a virus vector requires the transformation of a potentially harmful virus from a pathogen into a gene transfer agent whilst retaining the viral infectivity. Hence, the first step is to make the vector replication defective . Replication deficient viral vectors are developed by deletion of crucial genes in the virus genome, which are then generally replaced by the therapeutic gene. The elements removed in this way have to be provided Recombinant viral vectors presently used are generally classified under two categories; integrating or non-integrating viral vectors . This diNon-integrative vectors such as adenovirus and herpes simplex viral vectors allow transient episomal expression of a foreign gene in the target cells. Because of their episomal maintenance, the transferred genes are usually lost over time by dilution at cell division in actively dividing cells or by degradation in non-dividing cells . A non-igag coding region. The genome is surrounded by a spherical or cylindrical shaped core and an enveloped glycoprotein,  are a large family of enveloped RNA viruses, which are generally classified into three subfamilies; oncoretroviruses, lentiviruses and spumaviruses (foamy viruses). The RV particle is composed of two copies of an RNA genome held together primarily by a sequence called the dimer linkage, which is termed the leader region and found in some cases in , Figure . Upon ingag), the polymerase encoding gene (pol), and the envelope glycoprotein encoding gene (env), which are flanked by elements called long terminal repeats (LTRs), Figure gag gene encodes the viral structural core proteins, which form the matrix, capsid and nucleocapsid, generated by protease cleavage of the Gag precursor protein. The pol gene expresses a complex of enzymes that are involved in particle maturation (protease), DNA metabolism (reverse transcriptase) and proviral integration (integrase). These enzymes are usually derived from the Gag/Pol precursor. The env gene encodes the surface glycoprotein and the transmembrane protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. The genes in the viral DNA are bracketed by the LTRs, which define the beginning and the end of the viral genome. The LTRs are identical sequences that can be divided into three elements. U3 is derived from a sequence unique to the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The genesis of the LTR elements lies in the process of reverse transcription. U3 contains most of the transcriptional control elements of the provirus , which include the promoter and multiple enhancer sequences responsive to cellular and in some cases viral transcriptional activator proteins. The site of transcription initiation is at the boundary between U3 and R of the 5' LTR and the site of poly(A) addition is at the boundary between R and U5 at the 3' LTR, as shown in Figure U3 and U5 are determined by the primer binding site (PBS) and the polypurine tract (PTT), which are important for reverse transcription. Just downstream of the 3' end of the 5' LTR, is a short packaging sequence , which extends into gag and is responsible for encapsidation of the two viral RNA genomes into the capsid. The att sequences at the ends of the 5' and 3' LTRs are necessary for proviral integration [The genome size of simple RVs is approximately 8-12 kb and comprises three main genes; the group specific antigen encoding gene . The PIC translocates to the nucleus where the viral enzyme integrase, which is part of PIC, mediates integration of the provirus DNA sequence into the chromosomal DNA of the host cell. The inserted sequence (provirus) is flanked by complete copies of cis elements of the vectors and is generated by replacing viral protein encoding sequences later to be provided in trans with the transgene of interest. Both the Ψ signal that is essential for packaging of the vector genome into the capsid and the viral LTRs, which are necessary for proviral integration, remain in the vector genome construct. Expression of the transgenic protein is driven by the promoter in the U3 region of the 5' LTR. The packaging construct provides all of the viral proteins in trans to the vector genome construct . The packaging signal is deleted from the packaging construct to prevent its incorporation into viral particles. When both the vector genome and packaging constructs are present in a producer cell, retroviral vector particles, which are capable of delivering the vector genome with its inserted gene into new target cells, are released [The first generation replication defective retroviral vectors were developed using Molony Murine Leukaemia Virus (MoMLV) as a prototype. Replication defective vector particles were produced by a deconstruction strategy, which aims to dissect/segregate the viral genome into two transcriptional units (plasmid constructs). They are the vector genome and the packaging constructs, Figure released . The progag gene are retained in the vector construct to enhance the efficiency of packaging, although Gag protein production is prevented by mutation. In addition, overlapping sequences also exist because the LTRs are retained in the packaging construct to provide both promoter and poly-adenylation sequences. Secondly, the early MoMLV based vectors were established in murine NIH 3T3 packaging cell lines, therefore, the possibility for RCR generation through recombination between vector constructs and defective endogenous MoMLV-like sequences present in the target cells cannot be excluded [in vivo gene delivery [The basic arrangement described above is functional but unsatisfactory for several reasons. Firstly, the sequence overlap that remains between the vector and packaging constructs could result in recombination to form infectious replication competent retrovirus (RCR). The overlap exists principally because extensive sequences of the excluded . Thirdlydelivery ,111.gag/pol and env genes present on the packaging construct, onto discrete expression units, Figure gag sequence present on the first generation MoMLV vector is removed from the vector genome without significant loss of packaging efficiency  [In order to minimise the risk of RCR production, an improved vector system was designed by segregating the ckaging) , Figure LTRs that give low-level gene expression in producer cell lines not of murine origin. In the vector genome construct itself, heterologous promoters have been used to replace the 5' U3 promoter. In addition, the 3' U3 sequences can be significantly deleted as long as the sequences necessary for recognition by the integrase protein are retained. This is the basis of self inactivating (SIN) vectors where deletion of the viral promoter and enhancer regions in the 3' U3 are duplicated during reverse transcription in the 5' LTR to prevent LTR-driven transcription in infected host cells which could result in the expression of downstream inserted proto-oncogenes [The problem of overlapping sequences between the vector and the packaging construct has been solved by using heterologous promoters and polyadenylation signals to drive structural gene expression from the packaging constructs. Strong heterologous promoters like cytomegalovirus (CMV) can provide high virus titre production circumventing the limited titre offered by he MoMLV ncogenes . Transgencogenes .In the latest generation of RV based-vectors, improvements have also been made in the vector titre (number of colony-forming units per ml) by the development of transient plasmid co-transfection systems, which are capable of producing very high vector titres for a short period of time in the highly transfectable HEK 293  cell line . Also soin vitro of close to 100%. They can be produced at a high titre (109 infectious units (lU)/ml) and have the capacity to infect a wide variety of dividing cells including hepatocytes. The RV vector genome can also provide transfer of RNA of approximately 7.5 kb in length.Recombinant MoMLV-based vectors produced by the strategy described above are efficient gene transfer vehicles, reaching transfer levels in vivo transduction of the liver, cells have to be either in a naturally dividing state or to be induced to divide. Alternatively, the vectors can be used for ex vivo treatment.The critical limitation to the use of RVs is their inability to infect non-dividing cells and as the liver is an only slowly proliferating tissue these vectors are not ideal for LDLR gene delivery to hepatocytes. Therefore, for direct ex vivo gene delivery in numerous experimental studies. The original procedure used for liver-directed gene therapy of FH was based on the ex vivo approach, which involved re-infusion of autologous hepatocytes that had been removed from a WHHL rabbit and subjected to in vitro genetic correction with RV vectors based on MoMLV. Animals transplanted with LDLR transduced celIs demonstrated a 30-50% reduction in total serum cholesterol levels persistent for the duration of the experiment (122 days). Recombinant derived LDLR mRNA was detected in liver cells for 6 months. There was no apparent immunological response to the recombinant derived rabbit LDLR [ex vivo approach to ameliorate hyperlipidaemia associated with FH using a RV-based vector.Hypercholesterolaemia has been ameliorated by RV-based vectors using bit LDLR . This stex vivo gene therapy for FH was further documented in non-human primates [ex vivo approach for delivery of gene transduced hepatocytes via the mesenteric circulation was further documented in a canine model [In preparation for human trials with RV-based vectors, the efficacy, safety and feasibility of primates ,118. Thrprimates . The safex vivo approach, which was then carried out on a human patient in the first clinical trial for FH published in 1994. In this trial a 29 year-old woman with a homozygous receptor defective FH was subjected to ex vivo gene therapy using an amphotropic RV-based vector expressing human LDLRcDNA under control of the CMV enhanced chicken β-actin promoter. The patient tolerated the procedure and in situ hybridisation of liver tissue four months after therapy revealed evidence for engraftment of transgene expressing cells. The patient's LDL/HDL ratio declined from 10-13 before vector delivery to 5-8 after vector delivery, an improvement that remained stable for the duration of the reported observation (18 months). However, kinetic studies of LDL metabolism including LDL binding, uptake and degradation were not presented [The above studies demonstrated the feasibility and safety of the resented . This trresented .in situ hybridisation in all four subjects. One of four patients had a significant and prolonged reduction of about 20% in his LDL-C levels. Kinetic studies of the LDL metabolism demonstrated that LDL catabolism was increased in the same patient, which was consistent with increased LDLR expression [ex vivo liver-directed gene therapy with RV based vectors, pending improvement of vector efficiency. The following sections review the preclinical work towards this goal with alternative vector system.Grossman et al then reported four additional homozygous FH patients subjected to a surgical resection of the left lateral segment of the liver and re-infusion of the genetically modified hepatocytes . The patpression . The reapression . The varAdenoviruses (Ads) are icosahedral particles consisting of linear, double stranded DNA with a non-enveloped virion, Figure . There ain vitro [in vivo [Because of the ability of adenoviral vectors to infect a broad range of mammalian cell types regardless of their replication status, they have been widely used for a variety of gene transfer applications in vitro , in vivo[in vivo  and in c[in vivo . Most ad[in vivo .Vectors derived from serotypes 2 and 5 enter the cells after attachment to the cellular receptor CAR (coxsackievirus and adenovirus receptor), through the knob of the fiber . Virus e11-1012 Ad particles per ml, can be generated and allow high efficiency Ad mediated gene transfer with strong tropism for the liver. Cells that were transduced with these vectors express adenoviral genes at low levels, in addition to the transgenic protein [The first generation of replication deficient Ad vectors was constructed by replacing one or two viral early (E1 and E2) genes, which are essential for viral replication, with the transcriptional cassette of interest containing an enhancer-promoter element and the desired gene. Vectors in such a configuration have a packaging capacity of 6.5-8.3 kb. The recombinant vectors are replicated in cells that express the products of the E1 and/or E2 genes. Purified high titre stocks of 10 protein .The utility of replication defective first-generation recombinant Ad to mediate hLDLR gene transfer in hepatocytes derived from FH patients was first examined and documented in 1993 , using tin vivo approach resulted in reduction of the elevated intermediate density lipoprotein (IDL)/LDL ratio to normal levels, four days after vector delivery [Reversal of hypercholesterolaemia was then demonstrated in LDLR-/- mice fed with a high cholesterol diet after intravenous injection of a replication-defective Ad encoding the hLDLR driven by CMV promoter. This delivery . Similardelivery . Similardelivery  or humandelivery . These sdelivery ,133. Thidelivery .The transient expression was not solely due to the episomal nature of Ad infection but also a result of host immune responses against adenoviral proteins ,135,136.13 vector particles (vp)/kg) and low (5 × 1012 vp/kg) doses of HD-Ad. Throughout the 24-week experiment, plasma cholesterol of LDLR-treated mice was lower than that of VLDLR-treated mice. Anti-LDLR antibodies developed in 2 of 10 mice treated with high-dose HD-Ad-LDLR but in none (0/14) of the other treatment groups. The antibody titre in the high-dose experiments was significantly above background, but was three orders of magnitude lower than that seen following first generation Ad-LDLR treatment, indicating that the marked pro-inflammatory adenoviral protein expression following FG-Ad-LDLR gene transfer could have acted as an adjuvant that stimulated antibody production in these mice. Long-term efficacy of low-dose HD-Ad-LDLR injected into 12-week old LDLR-/- mice was tested and after 60 weeks, atherosclerosis lesions covered approximately 50% of the surface of aortas of control mice whereas aortas of treated mice were essentially lesion-free. The lipid lowering effect of HD-Ad-LDLR lasted at least 108 weeks (>2 years) when all control mice had died [In direct response to these immunological reactions and vector cytotoxicity, helper dependent adenovirus (HD-Ad) vectors were developed, in which additional viral coding sequences were deleted . This alhad died .13 lU/mI) administration of a 2nd generation adenoviral vector, which led to the unfortunate death of a patient in a non-FH clinical trial [in vivo adenoviral vector delivery trials, pending improvement in vector design. In an attempt to address this issue, Jacobs and colleagues investigated the use of a relatively low dose (5 × 1010 particles) of second generation E1E3E4-deleted adenoviral vectors for transfer of the LDLR or VLDLR, under control of the hepatocyte-specific human α1-antitrypsin promoter and 4 copies of the human apo E enhancer, into C57BL/6 LDLR-/- mice [Despite the reported improvements achieved by HD-Ad, the cytotoxic effect resulting from immune response to high titre , a small (20-25 nm) non-enveloped DNA virus Figure  that is There are several current obstacles to AAV gene therapy that need to be addressed. Although AAV is not known to cause human disease, 85% of the adult population is sero-positive for AAV capsid proteins  and wildCurrently a high multiplicity of infection is needed to achieve therapeutic AAV mediated gene transfer. Efficient transduction of target cells is blocked at several levels during AAV cell infection and movement of the vector into the nucleus. The host gender appears to be an important consideration, since in mice exogenous androgens can increase stable hepatocyte gene transfer in females to levels observed in male mice . StrategThe dsDNA genome of the AAV vector can persist as an episomal element in transduced cells for long periods of time in a variety of molecular forms, including circular monomers, linear monomers and linear concatemers by head to tail recombination of the ITRs. Integration of single and concatemeric genomes into the chromosomal DNA of the host cells occurs at low frequency . BecauseSlow conversion of the virus single stranded (ss) to the double stranded (ds) DNA genome is another issue. After the AAV virus enters the nucleus, the virus single stranded DNA genome (ssDNA) is converted to a transcriptionally active double stranded DNA (dsDNA) . Unless 12 AAV-2 vector particles encoding the murine VLDLR driven by the CMV enhanced chicken β-actin promoter [One of the earliest studies on AAV vectors for FH gene therapy found promising results. Reversal of hypercholesterolaemia was demonstrated in LDLR-/- mice fed with a high cholesterol diet after intraportal vascular injection of 1 × 10promoter . Western12 genome copies (gc) per mouse was injected into the portal veins of LDLR-/- mice that were fed a high-fat diet. Transduction efficiency was increased to 50 gc/cell and 10 gc/cell after treatment with an AAV-8 or AAV-7 vector respectively, compared with 2 gc/cell after administration of an AAV-2 vector. Animals receiving the AAV-LDLR serotype 7 and 8 achieved nearly complete normalization of serum lipids and failed to develop the severe atherosclerosis that characterized the untreated animals, with no apparent toxicity observed. Animals treated with the AAV-2 vector achieved partial lipid correction and only a modest improvement in atherosclerosis. Serotype 8 virus achieved stable transduction and expression of the transgene in up to 85% of the hepatocytes. These results are encouraging especially since no expression-terminating immune responses were detected [Lebherz and colleagues  compareddetected . There wdetected .Another approach using AAV vectors has been to try to counteract the development of atherosclerosis by gene transfer of interleukin-10 (IL10), an anti-inflammatory cytokine. Injection of AAV IL10 vector into the tail vein  of LDLR-More recently, a single intravenous injection of an AAV8 vector containing the mouse LDLR gene to a humanized mouse model of FH, the LDLR-/-Apobec-/- mouse, was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at low doses. Treated mice realized an 87% regression of atherosclerotic lesions with substantial remodeling, after 3 months compared to baseline mice .In summary, modifying the AAV vector system by altering the capsid (reviewed in ), includgag, pol, and env organised in the 5' to 3' orientation. Lentiviruses have additional unique small ORFs located between pol and env at the 3' terminus, which contain genes for regulatory proteins [Lentiviruses (LVs) are a complex sub-group of RVs responsible for a variety of immunological and neurological diseases. Their biological and molecular and properties have been used to classify them as lenti-(sIow) retroviruses. They can be subdivided into primate and non-primate viruses. The primate viruses are the human and simian immunodeficiency viruses (HIV and SIV), and the non-primate viruses include the feline and bovine immunodeficiency viruses, the caprine arthritis/encephalitis virus, the visna/maedi/ovine progressive pneumonia virus, and the equine infectious anaemia virus (EIAV) . As for proteins .Interest in LVs as putative gene transfer systems is derived from the fact that they have the potential to integrate efficiently into the genome of dividing and non-dividing cells providing the possibility for lifetime correction with a single administration of vector ,162. UnlAlthough integration of linear DNA episome, provirus precursor, is generally regarded as the end point of gene transfer, two circular episomal types with intact viral coding regions are also generated by cellular proteins from retro- or lenti-viruses and their derived vectors . The firUsing a LV backbone, two types of vector system can be produced and used for gene transfer, the first are integrated lentivirus based vectors (ILV) and the second are integration deficient lentiviral vectors (IDLV). Initial research on the development of lentivirus-based vectors has focused mainly on HIV-1 derived integrated LV vectors as prototype. This is facilitated by the abundance of knowledge that has been accumulated on this virus since its recognition in 1984 as the causative agent of acquired immuno-deficiency syndrome. Like other virally derived vectors, the initial problem to overcome is to maintain viral infectivity but to render the virus replication deficient ,162. TheDue to the increased concern of insertional mutagenesis (IM) caused by integrating retro- and lentivirus based vectors (as will be discussed later), IDLVs has been thought of as a logical alternative to alleviate the risk of IM. IDLV particles can be generated by the use of integrase mutations that specifically prevent proviral integration resulting in the generation of increased levels of circular vector episomes in transduced cells, but not to compromise its other functions, Because these lentiviral circular episomes lack replication signals, they are gradually lost by dilution in the transduced actively dividing cells, but are stable for several months in transduced quiescent cells -176. Comatt mutations in addition to those in the integrase to minimize integration in the event of vector mobilization . Lo9 IU) third-generation HIV-1 based lentiviral vectors encoding rabbit LDLR. LSP-driven transgene expression was detected after in vitro gene transfer into human hepatoma (HepG2) cells, but not after transfer into HeLa cells, HEK 293 cells, or WHHL rabbit skin fibroblasts [In vivo injection of 1 × 109 infectious virus particles into the portal vein of WHHL rabbits resulted in liver-specific expression of the LDLR and clinical chemistry and histological analyses showed normal liver function and morphology during the 2-year follow-up without safety issues. This vector dose resulted in low transduction efficiency (<0.01%) but demonstrated on average a147 ± 7% decrease in serum cholesterol levels during the first 4 weeks, a 44 ± 8% decrease at 1 year and a 34 ± 10% decrease at the 2-year time point, compared to the control rabbits infected with HIV-green fluorescent protein. During this period, 70% of the rabbits treated with the liver specific lentiviral LDLR vector demonstrated a positive treatment effect with lowered plasma cholesterol levels (25 ± 8%). However, the detailed pattern of bio-distribution after HIV-vector mediated gene transfer, to evaluate potential risks for possible IM and germ-line transmission, has not been investigated.Many labs including ours  experienroblasts ,184. In in vitro studies using model systems based on mutagenesis of the hprt locus or genes that control promotion of growth factor independence at frequencies between 10-5-10-7 per provirus insertion [The integration of RV and LV into the genome during gene therapy has caused concern because of the potential for vector-related deleterious side effects on the host. This is, in part, due to the fact that vector insertion occurs in a semi-random manner into actively transcribed genes. For RV vectors insertion preference is for gene promoter regions -188 whernsertion ,192. Hennsertion ,195-199.ex vivo trial carried out in France that used patients' own haematopoietic stem cells for transplantation after retroviral transduction to correct X-linked severe combined immuno-deficiency (X-SCID), clonally dominant clones have developed into leukaemias in 4 of these patients [LMO2 gene [BMI1 and CCND2 proto-oncogenes [NOTCH1, deletion of tumour suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement [In an patients ,86,196. patients . In 4 ofMO2 gene . In addincogenes . Althougangement .In a more recent trial for chronic granulomatous disease (CGD) clonal dominance has also been attributed to retrovirus mediated IM 5 month after vector delivery in 2 patients . Vector ex-vivo and in vitro models have been developed in order to examine RV and LV genotoxicity using haematopoietic cells. Ex vivo gene therapy using stem cells is considered a more controllable way of introducing genetic modification to the host than by direct systemic vector administration in vivo [In response to these findings,  in vivo -205. The in vivo ,201-206. in vivo ,201-206. in vivo .in vivo. In this model vector application in utero is performed via the fetal mouse circulation that results in gene transfer to most organs, although the liver is mainly transduced [et al. unpublished data). Most importantly in these tumours, we find insertions within genes assigned as candidate genes involved in cancer development . More than 50% of these genes are registered in the Mouse Retroviral Tagged Cancer Gene Database (RTCGD) [in utero gene delivery where genes are in a highly active transcription state, we are able to sensitively detect adverse effects caused by vector integration.In our laboratory we have developed a model more suited to gene therapy for FH where vectors may be delivered directly ansduced . We founansduced . This mo (RTCGD) . FurtherThe current models for vector associated genotoxicity all rely on the use of rodent cells as a measure of IM. As these cells are more predisposed to tumour development than human cells, each must be viewed with caution as reliable predictors for mutagenesis occurring in the clinic. The finding of vector genotoxicity in the clinic has, however, revived the use of models of genotoxicity to obtain useful information regarding safe vector design. They may also help to elucidate possible mechanisms relating to IM. In summary, the importance of genotoxicity assays to understand the cause and measure the risk of adverse effects by gene therapy of FH and indeed the treatment of any disease with these vectors cannot be overstated. With the current genotoxicity assays in place we are becoming more confident that gene therapy to FH homozygotes will be possible with minimal side effects.in vivo and/or ex vivo gene transfer. Vectors derived from AAV and LVs are very promising. However, the oncogenesis risk from semi-random integration into actively transcribing genes of the host by LVs [Several novel therapies have been developed recently to lower LDL-C in homozygous and heterozygous FH patients -65,211. t by LVs ,212,213,t by LVs  and somet by LVs ,215 are in vivo and/or ex vivo gene therapy of FH and many other human diseases.In addition to the LDLR gene augmentation approach, the successful use of the VLDLR as an effective surrogate lipoprotein receptor gene ,216,217 ex vivo transduction to reduce vector spread can also improve the safety outcome, particularly, if autologous or induced pluripotent stem cells are the target. The application of viral or non-viral, integrating or non-integrating vectors for long term persistence in stem cells with self renewal and differentiation capacities will also be important perspectives for gene-based stem cell therapy.Future work will generally focus on making gene transfer vectors safer by improving their immunogenic, integration, expression and targeting profile. Reducing the inherent oncogenic danger of integrating vectors by engineering conditional suicide genes into the vector backbone to provide a self-destructive mechanism in case of oncogenesis or by targeting their integration into specific pre-defined benign genomic sites, i.e. by zinc-finger nuclease technology, may help achieving this goal . In combThe authors declare that they have no competing interests.FAA is responsible for conceiving this work and writing the manuscript. CC, SW, ALD, RH, and MT participated in the drafting of this manuscript. All authors read and approved the final manuscript.F. A. A. is an Assistant Professor of Genetics and Molecular Medicine, Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, Al-Abedia Campus, Makkah 21955, Saudi Arabia. C. C. is an Emeritus Professor and Former Leader, Gene Therapy Research Group, Department of Molecular and Cell Medicine, Sir Alexander Fleming Building, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK. S. W. is a Lecturer and Group Leader, Prenatal Gene Therapy Research Group, Department of Haematology, Haemophilia Centre and Haemostasis Unit, Royal Free and University College Medical School, London NW3 2PF, UK. R. H. is a Research Fellow and Gene Therapy Group Leader in the Section of Molecular Medicine in the Sir Alexander Fleming Building, NHLI, Imperial College London, London SW7 2AZ, UK. A. L. D. is a Senior Lecturer and Honorary Consultant in Obstetrics and Maternal/Fetal Medicine, and leads the Prenatal Cell and Gene Therapy Group, Institute for Women's Health, University College London and UCLH, 86-96 Chenies Mews, London, WC1E 6HX, UK. M. T. is a Lecturer and Group Leader, Gene Therapy and Genotoxicity Research Group, Brunel University, Heinz Wolff Building, Uxbridge, Middlesex, West London UB8 3PH, UK.


Understanding the biological mechanisms used by microorganisms for plant biomass degradation is of considerable biotechnological interest. Despite of the growing number of sequenced (meta)genomes of plant biomass-degrading microbes, there is currently no technique for the systematic determination of the genomic components of this process from these data.We describe a computational method for the discovery of the protein domains and CAZy families involved in microbial plant biomass degradation. Our method furthermore accurately predicts the capability to degrade plant biomass for microbial species from their genome sequences. Application to a large, manually curated data set of microbial degraders and non-degraders identified gene families of enzymes known by physiological and biochemical tests to be implicated in cellulose degradation, such as GH5 and GH6. Additionally, genes of enzymes that degrade other plant polysaccharides, such as hemicellulose, pectins and oligosaccharides, were found, as well as gene families which have not previously been related to the process. For draft genomes reconstructed from a cow rumen metagenome our method predicted Bacteroidetes-affiliated species and a relative to a known plant biomass degrader to be plant biomass degraders. This was supported by the presence of genes encoding enzymatically active glycoside hydrolases in these genomes.Our results show the potential of the method for generating novel insights into microbial plant biomass degradation from (meta-)genome data, where there is an increasing production of genome assemblages for uncultured microbes. Lignocellulosic biomass is the primary component of all plants and one of the most abundant organic compounds on earth. It is a renewable, geographically distributed and a source of sugars, which can subsequently be converted into biofuels with low greenhouse gas emissions, such as ethanol. Chemically, it primarily consists of cellulose, hemicellulose and lignin. Saccharification - the process of degrading lignocellulose into the individual component sugars - is of considerable biotechnological interest. Several mechanical and chemical procedures for saccharification have been established; however, all are relatively expensive, slow and inefficient . An alteCytophaga hutchinsonii[Fibrobacter succinogenes[Dictyoglomus turgidum[For some lignocellulose-degrading species, carbohydrate-active enzymes (CAZymes) and protein domains implicated in lignocellulose degradation are well known. Many of these have been recognized by physiological and biochemical tests as being relevant for the biochemical process of cellulose degradation itself, such as the enzymes of the glycoside hydrolase (GH) families GH6 and GH9 and the endoglucanase-containing family GH5. Two well-studied paradigms are currently known for microbial cellulose degradation: The ‘free-enzyme system’ is realized in most aerobic microbes and entails secretion of a set of cellulases to the outside of the cell. In anaerobic microorganisms large multi-enzyme complexes, known as cellulosomes, are assembled on the cell surface and catalyze degradation. In both cases, the complete hydrolysis of cellulose requires endoglucanases (GH5 and GH9), which are believed to target non-crystalline regions, and exo-acting cellobiohydrolases, which attack crystalline structures from either the reducing GH7 and GH48) or non-reducing (GH6) end of the beta-glucan chain. However, in the genomes of some plant biomass-degrading species, homologs of such enzymes have not been found. Recent genome analyses of the lignocellulose-degrading microorganisms, such as the aerobe  and GH48 turgidum have revNasutitermes), the Australian Tammar wallaby (Macropus eugenii) [Metagenomics offers the possibility of studying the genetic material of difficult-to-culture (i.e. uncultured) species within microbial communities with the capability to degrade plant biomass. Recent metagenome studies of the gut microbiomes of the wood-degrading higher termites (eugenii) ,12 and teugenii) ,14 have eugenii) ,15. Notade novo discovery of protein domains and CAZy families associated with microbial plant biomass degradation from genome and metagenome sequences. It uses protein domain and gene family annotations as input and identifies those domains or gene families, which in concert are most distinctive for the lignocellulose degraders. Among the gene and protein domains identified with our method were known key genes of plant biomass degradation. Additionally, it identified several novel protein domains and gene families as being relevant for the process. These might represent novel leads towards elucidating the mechanisms of plant biomass degradation for the currently less well understood microbial species. Our method furthermore can be used to identify plant biomass-degrading species from the genomes of cultured or uncultured microbes. Application to draft genomes assembled from the metagenome of a switchgrass-adherent microbial community in cow rumen predicted genomes from several Bacteroidales lineages which encode active glycoside hydrolases and a relative to a known plant biomass degrader to represent lignocellulose degraders.We describe a method for the without requiring sequence homology to known proteins involved in lignocellulose degradation. Classification with SVMs has been previously used successfully for phenotype prediction from genetic variations in genomic data. In Beerenwinkel et al.[et al.[et al.[a priori assumptions about the problem domain. SVMs can be applied to datasets with millions of input features and have good generalization abilities, in that models inferred from small amounts of training data show good predictive accuracy on novel data. The use of models that include an L1-regularization term favors solutions in which few features are required for accurate prediction. There are several reasons why sparseness is desirable: the high dimensionality of many real datasets results in great challenges for processing. Many features in these datasets are usually non-informative or noisy, and a sparse classifier can lead to a faster prediction. In some applications, like ours, a small set of relevant features is desirable because it allows direct interpretation of the results.In technical terms, our method selects the most informative features from an ensemble of L1-regularized L2-loss linear Support Vector Machine (SVM) classifiers, trained to distinguish genomes of cellulose-degrading species from non-degrading species based on protein family content. Protein domain annotations are available in public databases and new protein sequences can be rapidly annotated with Hidden Markov Models (HMMs) or - somewhat slower - with BLAST searches of one protein versus the NCBI-nr database . Co-occuel et al., supportl.[et al. for predl.[et al., SVMs wel.[et al.,21 is a Postia placenta into our analysis. After training, we identified the most distinctive protein domains and CAZy families of plant biomass degraders from the resulting models. We compared these protein domains and gene families with known plant biomass degradation genes. We furthermore applied our method to identify plant biomass degraders among 15 draft genomes from the metagenome of a microbial community adherent to switch grass in cow rumen.We trained an ensemble of SVM classifiers to distinguish between plant biomass-degrading and non-degrading microorganisms based on either Pfam domain or CAZY gene family annotations . We used a manually curated data set of 104 microbial (meta-)genome sequence samples for this purpose, which included 19 genomes and 3 metagenomes of lignocellulose degraders and 82 genomes of non-degraders , Actinobacteria (5), Proteobacteria (3), Bacteroidetes (1), Fibrobacteres (1), Dictyoglomi (1) and Basidiomycota (1). Eighty-two microbial genomes annotated to not possess the capability to degrade lignocellulose were used as examples of non-lignocellulose-degrading microbial species genome sequence was represented by a feature vector with the features indicating the presence or absence of Pfam domains (see Methods). The nested cross-validation macro-accuracy of eSVMbPFAM in distinguishing plant biomass-degrading from non-degrading microorganisms was 0.91. This corresponds to 94% (97 of 103) of the (meta-)genome sequences being classified correctly. Only three of the 21 cellulose-degrading samples and three of the non-degraders were misclassified . Again, we determined the most relevant protein domains for identifying a plant biomass-degrading sequence sample from the models by feature selection. Among the most important protein families were, as before, GH5, GH10 and GH88 (PF07221: N-acylglucosamine 2-epimerase)  and binding domains PF10509  and PF03793 (PASTA domain) , carbohydrate-binding modules (CBM), glycosyltransferases (GT), polysaccharide lyases (PL) and carbohydrate esterases (CE). The annotations from the CAZy database comprised 64 genomes of non-lignocellulose-degrading species and 16 genomes of lignocellulose-degraders. There were no CAZy annotations available for the remaining genomes. In addition, we included the metagenomes of the gut microbiomes of the Tammar wallaby (TW), the wood-degrading higher termite and of the cow rumen microbiome  and eSVMCAZY_a (counts), based on genome annotations with all CAZy families.1) By training of the classifiers eSVMCAZY_B (presence/absence) and eSVMCAZY_b (counts), based on the annotations of the genomes and the TW sample with all CAZy families, except for the GT family members, which were not annotated for the TW sample.2) By training of the classifiers eSVMCAZY_C (presence/absence) and eSVMCAZY_c (counts) with the entire data set based on GH family and CBM annotations, as these were the only ones available for the three metagenomes.3) By training of the classifiers eSVMCAZY classifiers, except for the Firmicute Caldicellulosiruptor saccharolyticus. The best classification results were obtained with the presence-absence information for all CAZy families except for the GT families of the microbial genomes and the TW sample. In this setting (eSVMCAZY_B) only two species (Thermomonospora curvata and Actinosynnema mirum) were misclassified genomes . These CAZy families, as well as GH115 and CBM56, are not included in Figure Using feature selection, we determined the CAZy families from the six eSVMs Figure . This anbPFAM and eSVMCAZY_B models, which had the highest macro-accuracy based on Pfam domain or CAZy family annotations, respectively. The eSVMbPFAM classifier assigned seven of the draft genomes to plant biomass degraders  are of the order of Bacteroidales, and include all but one draft genomes affiliated to the Bacteroidales. The 6th and 7th predicted degrader, represented by genome AIa and AWa, belong to the Clostridiales, like genome APb. The eSVMCAZY_B classifier also assigned five of these genomes to the plant biomass degraders. Additionally it classified genome AH as plant biomass-degrading, while being ambiguous in the assignment of AFa -degrading GH5 endoglucanase as well as GH9 enzyme capable of degrading insoluble cellulosic substrates such as Avicel®. AIa demonstrated capabilities towards xylan and soluble cellulosic substrates with affiliations to four GH10 xylanases. Both AGa and AJ demonstrated broader substrate versatility and were linked to enzymes with capabilities towards cellulosic substrates CMC and Avicel® , hemicellulosic substrates lichenan  and xylan , as well as the natural feedstocks miscanthus and switchgrass (GH5 and GH9). Importantly, no carbohydrate-active enzymes were affiliated to draft genomes that were predicted to not possess plant biomass-degrading capabilities . Genomees Table . AC2a waFibrobacter succinogenes genome [http://csbl.bmb.uga.edu/dbCAN/ on April 19th 2011) on the same machine required approximately 1 hour and 55 minutes, a difference of two orders of magnitude. Because of their better scalability and also because they are well-established for identifying protein domains or gene families [Our method uses annotations with Pfam domains or CAZy families as input. Generating these by similarity-searches with profile HMMs rather than with BLAST provides a better scalability for next-generation sequencing data sets. HMM databases such as dbCAN contain a representation of entire protein families rather than of individual gene family members, which largely decreases the number of entries one has to compare against. For example, searching the ORFs of the s genome  for simifamilies -29, we rFibrobacter) where there is an absence of genes encoding exo-acting cellobiohydrolases (GH6 and GH48) and cellulosome structures [et al.[Clostridium thermocellum without the expression of GH48, albeit at significantly lower rates. Furthermore, genome analysis of cellulose-degrading microbes Cellvibrio japonicus[Saccharophagus degradans[We investigated the value of information about the presence-or-absence of CAZy families and Pfam protein domains, as well as information about their relative abundances, for the identification of lignocellulose degraders. Classifiers trained with CAZy family or Pfam domain annotations allowed an accurate identification of plant biomass degraders and determined similar domains and CAZy families as being most distinctive. Many of these are recognized by physiological and biochemical tests as being relevant for the biochemical process of cellulose degradation itself, such as GH6, members of the GH5 family and to a lesser extent GH44 and GH74. In contrast to widely accepted paradigms for microbial cellulose degradation, recent genome analysis of cellulolytic bacteria has identified examples , pectins , oligosaccharides  and the side-chains attached to noncellulosic polysaccharides . This was expected, since many cellulose-degrading microbes produce a repertoire of different glycoside hydrolases, lyases and esterases  thatSeveral Pfam domains of unknown function (DUFs) or protein domains which have not previously been associated with cellulose degradation are predicted as being relevant. These include transferases (PF01704) and several putative lipoproteins (DUF4352), some of which have predicted binding properties . The functions of these domains in relation to cellulose degradation are not known, but possibilities include binding to cellulose, binding to other components of the cellulolytic machinery or interaction with the cell surface.Butyrivibrio fibrisolvens. Cross-referencing of all draft genomes against a catalogue of enzymatically active glycoside hydrolases provided a degree of method validation and was in majority agreement with our predictions. Four genomes  predicted positive were linked to cellulolytic and/or hemicellulolytic enzymes, and importantly no genomes that were predicted negative were linked to carbohydrate-active enzymes from that catalogue of enzymatically active enzymes. Also, no connections to carbohydrate-active enzymes from that catalogue were observed for the three genomes  where ambiguous predictions were made. As both draft genomes as well as the catalogue of carbohydrate active enzymes in cow rumen are incomplete, in addition to our training data not covering all plant-biomass-degrading taxa, such ambiguous assignments might be better resolvable with more information in the future.Another result of our study are the classifiers for identifying microbial lignocellulose-degraders from genomes of cultured and uncultured microbial species reconstructed from metagenomes. Classification of draft genomes reconstructed from switchgrass-adherent microbes from cow rumen with the most accurate classifiers predicted six or seven of these to represent plant biomass-degrading microbes, including a close relative to the fibrolytic species Methanosarcina barkeri fusaro incorrectly labeled as a plant biomass degrader, according to information provided by IMG. In cross-validation experiments, our method correctly assigned M. barkeri to be a non-plant biomass-degrading species. We labeled Thermonospora curvata as a plant biomass degrader and Actinosynnema mirum as non-degrader according to information from the literature cellulose binding CBM domains CBM6 and CBM_4_9, which were identified as being relevant for assignment to lignocellulose degraders with the eSVMbPFAM classifier, are absent. All of the latter ones, GH26, CBM6 and especially CBM4 and CBM9, occur very rarely in eukaryotic genome annotations, according to the CAZy database.We trained a previous version of our classifier with the genome of e et al.  it was s beliefs . The aut species  and migh species ,37. IndeWe have developed a computational technique for the identification of Pfam protein domains and CAZy families that are distinctive for microbial plant biomass degradation from (meta-)genome sequences and for predicting whether a (draft) genome of cultured or uncultured microorganisms encodes a plant biomass-degrading organism. Our method is based on feature selection from an ensemble of linear L1-regularized SVMs. It is sufficiently accurate to detect errors in phenotype assignments of microbial genomes. However, some microbial species remained misclassified in our analysis, which indicates that further distinctive genes and pathways for plant biomass degradation are currently poorly represented in the data and could therefore not be identified.To identify a lignocellulose degrader from the currently available data, the presence of a few domains, many of which are already known, is sufficient. The identification of several protein domains which have so far not been associated with microbial plant biomass degradation in the Pfam-based SVM analyses as being relevant may warrant further scrutiny. A difficulty in our study was to generate a sufficiently large and correctly annotated dataset to reach reliable conclusions. This means that the results could probably be further improved in the future, as more sequences and information on plant biomass degraders become available. The method will probably also be suitable for identifying relevant gene and protein families of other phenotypes.The prediction and subsequent validation of three Bacteroidales genomes to represent cellulose-degrading species demonstrates the value of our technique for the identification of plant biomass degraders from draft genomes from complex microbial communities, where there is an increasing production of genome assemblages for uncultured microbes. These to our knowledge represent the first cellulolytic Bacteroidetes-affiliated lineages described from herbivore gut environments. This finding has the potential to influence future cellulolytic activity investigations within rumen microbiomes, which has for the greater part been attributed to the metabolic capabilities of species affiliated to the bacterial phyla Firmicutes and Fibrobacteres.http://csbl.bmb.uga.edu/dbcan) [We annotated all protein coding sequences of microbial genomes and metagenomes with Pfam protein do-mains (Pfam-A 26.0) and Carbohydrate-Active Enzymes (CAZymes) ,38. The u/dbcan) . To be cWe defined genomes and metagenomes as originating from either lignocellulose-degrading or non-lignocellulose-degrading microbial species based on information provided by IMG/M and in the literature. For every microbial genome and metagenome, we downloaded the genome publication and further available articles  are sought, in a second cross-validation experiment with the remaining data points. For determination of the best setting for the penalty parameter C, values for C = 10x, x = -3.0, -2.5, -2.25, …, 0 were tried. Values of the parameter C larger than 1 were not tested extensively, as we found that they resulted in models with similar accuracies. This is in agreement with the Liblinear tutorial in the appendix of [The assignment accuracy of a classifier was determined with a standard nested cross-validation (nCV) setup . In nCV,endix of  which stThe average of the recall and the true negative rate, the macro-accuracy, was used as the assignment accuracy to assess the overall performance:C with the best macro-accuracy by leave-one-out cross-validation. The parameter settings resulting in the most accurate models were used to each train a separate model on the entire data set. Prediction of the five best models were combined to form a voting committee and used for the classification of novel sequence samples such as the partial genome reconstructions from the cow rumen metagenome of switch-grass adherent microbes  genomes and metagenomes. The macro-accuracy for each model for a discrete set of values for the parameter Click here for file


Previous studies have demonstrated task-related changes in brain activation and inter-regional connectivity but the temporal dynamics of functional properties of the brain during task execution is still unclear. In the present study, we investigated task-related changes in functional properties of the human brain network by applying graph-theoretical analysis to magnetoencephalography (MEG). Subjects performed a cue-target attention task in which a visual cue informed them of the direction of focus for incoming auditory or tactile target stimuli, but not the sensory modality. We analyzed the MEG signal in the cue-target interval to examine network properties during attentional control. Cluster-based non-parametric permutation tests with the Monte-Carlo method showed that in the cue-target interval, beta activity was desynchronized in the sensori-motor region including premotor and posterior parietal regions in the hemisphere contralateral to the attended side. Graph-theoretical analysis revealed that, in beta frequency, global hubs were found around the sensori-motor and prefrontal regions, and functional segregation over the entire network was decreased during attentional control compared to the baseline. Thus, network measures revealed task-related temporal changes in functional properties of the human brain network, leading to the understanding of how the brain dynamically responds to task execution as a network. A large body of studies in neuroscience have investigated task-related changes in activation of different brain regions to infer functional specialization. Recent studies have extensively examined task-related changes in connectivity among the different brain regions Here we used MEG to examine task-related temporal changes in functional properties of the human brain network. To this end, we used graph-theoretical analysis to compute network measures from the MEG signal recorded in a multisensory cue-target attention task. Our primary interest was to examine whether graph-theoretical analysis of MEG can detect task-related changes in network properties. Previous studies using fMRI have found the involvement of prefrontal and posterior parietal areas in the attentional control system Recordings were obtained from nine healthy right-handed subjects (one woman and eight men), aged 24 to 52 years old. All subjects gave written informed consent prior to the study, which was first approved by the Ethics Committee of the National Institute for Physiological Sciences.A visual cue stimulus was followed 1.0–1.5 s later by an auditory or tactile stimulus. The interval between successive cue stimuli varied randomly between 4 and 5 s. Both auditory and tactile stimuli were used to examine the multisensory nature of task-related changes in the network properties. The cue stimulus was a right- or left arrow, which was presented in random order and with an equal probability on a screen through a digital light processing projector in front of the subjects at a distance of 2 m. The auditory or tactile stimulus had equal probability of presentation on the cue side or the opposite side, irrespective of the direction of the cue stimulus. The tactile stimulus was a single-pulse or double-pulse electrotactile stimulus presented in random order to the second digit of the left or right hand through ring electrodes . The stimulus lasted 0.2 ms and its intensity was 2.5 times the sensory threshold. In the case of the double-pulse stimulus, two electrotactile pulses separated by an interval of 150 ms were applied. The auditory stimulus was a single or double click presented in random order to the left or right side from card-type speakers  through cylinders with a diameter of about 10 cm, made of reinforced cardboard . One endThe subjects were instructed to placed the second digit of each hand near the auditory stimuli  before the measurement started. They were instructed to attend covertly to the side of the visual cue, and to press a button when a double-pulse or double click was presented there. The to-be-attended stimulus and action sides were compatible in space, i.e., the responding hand was the hand on the attended side, to exclude the effect of stimulus-response incompatibility. Each subject received 960 trials , divided into 12 blocks. Each block lasted about 4 minutes, and an experiment lasted about 50–60 minutes.The MEG signal was recorded with a helmet-shaped detector array , which in each of 102 locations has 3 sensors , two orthogonal planar gradiometers and one magnetometer coupled to a multi-SQUID (superconducting quantum interference device). Signals were filtered through a bandpass filter of 0.03–200 Hz and digitized at a sampling rate of 1,004 Hz. The subjects' head location relative to the MEG sensors was measured before the measurement using head position indicator coils. Eye movements and blinks were monitored with a near infrared camera .http://fieldtrip.fcdonders.nl/) and Brain Connectivity Toolbox (BCT) (https://sites.google.com/a/brain-connectivity-toolbox.net/bct/). We analyzed the MEG signals recorded from 102 pairs of 2 orthogonal planar-type gradiometers. Line noise removal was performed . The data were segmented from −2.5 sec before to 1.5 sec after the target stimulus. Trials with extremely high variance containing jumps and muscle artifacts were removed. Spectral analyses were performed using multitaper spectral estimates based on slepian sequences. We computed spectral estimates across logarithmically scaled frequencies f from 4 to 180 Hz and across 61 points in time t from −2.0 to 1.0 sec (0.05 sec step). The interval of logarithmically scaled frequencies was adjusted for equal spacing in time and frequency domains so that the number of frequency bins was 61. We adjusted the spectral smoothing to a 0.6 octave step, which means that spectral smoothing increases as the frequency of interest increases. The data for two orthogonal planar sensors were summed, resulting in a time-frequency representation at each of the 102 locations. Statistical analysis of the data segment was performed before target stimuli by means of cluster-based non-parametric permutation tests with the Monte-Carlo method .Data analyses were performed in Matlab (MathWorks) using custom scripts and open source toolboxes: Fieldtrip  is the number of shortest paths between h and j that through i. The averaged clustering coefficient C of the network is defined by the following equation,iC is the clustering coefficient of node i (iC = 0 for ik<2), ik is the degree of node i, and it is the number of triangles around node i,ija is the connection status between i and j: ija = 1 when a link  exists (when i and j are neighbors); ija = 0 otherwise .  is a link between nodes i and j. These measures were calculated in two segments of 2000 ms before target stimuli in each of the attend-left and right conditions to compare pre-cue (baseline) and cue-target  intervals. For the averaged clustering coefficient, two-way analysis of variance was performed with attention  and attended hemifield (right and left hemifield) as factors. Mauchly's sphericity test was also performed. We did not compute the characteristic path length, because this measure is not easy to interpret when computed from functional data In time-frequency analysis, beta desynchronization was observed during a cue-target interval. We thus focused on the beta frequency  to compute the phase locking value (PLV) Time-frequency analysis showed that beta desynchronization continued from 1000 msec before and up to the target stimulus . ClusterP<0.05). There was no interaction between the two factors.Global hubs reflected by betweenness centrality were found in the bilateral sensori-motor and prefrontal regions and remained stable over time . The aveWe found task-related temporal changes in network measures in a cue-target attention task using MEG. Functional segregation was decreased during attentional control versus the baseline interval, whereas global hubs found in the prefrontal and sensori-motor regions remained relatively stable over time. Previous fMRI studies have reported functional properties of the resting state brain network Three possible systems for the organization of spatial attention have been proposed: 1) a completely supramodal attentional system which acts on any sense, 2) a completely unimodal system where attention acts individually on each sense, and 3) a separable but linked system where modality-specific attentional systems interplay across different senses Modulation of beta oscillation was also found in the cue-target interval, which requires the control of attention to both stimulus and action. In the present study, the to-be-attended stimulus and action sides were compatible, excluding the effect of stimulus-response incompatibility. Thus, the beta desynchronization observed here may include the effects of attention to both stimulus and action. Studies of attention have found slow EEG deflections The present study revealed task-related changes in functional properties of the human brain with reduced functional segregation, while global hubs were found in prefrontal and sensorimotor areas. The present study is thus the most fundamental first step to examine task-related rapid changes in functional network structures associated with a variety of brain functions.


The two key elements of realistic simulation of LFP are plausible model of neural activity, and the physical properties of the setup, tissue, and electrodes. To reduce the computational burden yet obtain a trustful rendering of LFP in specific experimental context different strategies have been tried. For instance, using total synaptic activity from a population of spiking neurons as a proxy for LFP, or hybrid strategies with network simulations of spiking neurons followed by computation of contributions to LFP from single cells driven by pre-calculated network dynamics. Tests of those strategies are impossible without ground-truth data including all the necessary components.we investigated how the use of increasingly detailed physical models of slice tissue affects the resulting model LFPs. Traub's model of thalamo-cortical loop [hom) were computed post-hoc from tracked trans-membrane currents. To verify the need for inclusion of experimental setup context we modeled the field in the tissue and saline using finite-element approach (FEM) with FENICS package with mesh generated with gmsh. The slice  was placed in a saline solution  'no saline' case: slice and saline with the same conductivity of 0.3 mS/cmii) 'hom+iso+saline': homogeneous and isotropic slice of conductivity 0.3 mS/cm, saline: 3.0 mS/cmiii) 'inhom+anis+saline': inhomogeneous and anisotropic slice with conductivity profiles for the layers taken as mean values from [ues from .We see that the inclusion of slice setup noticeably modifies the observed activity as both the amplitude and shape of the potential profile is changed Figure . However


Another five male AL mice  served as control group. Descending aortas were scanned with nano-CT at (1.5 μm)3 isotropic voxel size. Number and area of adventitial VV as well as plaque cross sectional area were measured. Results were complemented by histology. Thalidomide inhibited proliferation and migration of HCAEC dose-dependently. VV neovascularization decreased in number per cross section  and in cross sectional area . Cross sectional area of plaque decreased significantly when treated with thalidomide . Nano-CT imaging revealed a reduced plaque growth and VV neovascularization after long-term application of thalidomide. Therefore, nano-CT can be considered as a new method to detect therapeutic effects in experimental models of atherosclerosis.Plaque progression in atherosclerosis is closely connected to angiogenesis due to vasa vasorum (VV) growth. Objective of this study was to determine the unknown long-term effect of thalidomide on adventitial VV neovascularization and plaque progression using nano-focussed computed tomography (nano-CT). Proliferation and migration assays in human coronary artery endothelial cells (HCAEC) measured number of viable cells after incubation with thalidomide. Male ApoE At the age of 35 weeks, AL mice demonstrate adventitial VV neovascularization and atherosclerotic plaques in the descending aorta  proliferation and migration in HCAEC in vitro, (2) adventitial VV growth and (3) growth of atherosclerotic plaque in the descending aorta of AL mice. It supports thalidomide’s effectiveness in an experimental model of atherosclerosis, which shows morphologically comparable findings to human atherosclerosis. The study underlines the value of nano-CT for quantitative imaging of atherosclerosis.Thalidomide was introduced to the European market in 1957 as a sedative and withdrawn in 1961 after press reports about intake of thalidomide during pregnancy and a series of birth defects such as phocomelia or aplasia of extremities. Recent investigations identified cereblon as a protein, responsible for limb outgrowth, which is inhibited by binding to thalidomide . It was Migration and proliferation of ECs are basic requirements for angiogenesis  as seen We hypothesized, that a long time application of thalidomide might inhibit lesion progression in concert with VV neovascularization in the descending aorta of AL mice and can be monitored using nano-CT. Similar therapeutic effects were observed for short time application of 3-deazaadenosine to AL mice in early atherosclerosis  indicatiFor studying this purpose, nano-CT is a versatile and helpful technique that offers additional features for quantitative assessment of atherosclerosis. Just as micro-CT, nano-CT combines non-disruptive ex vivo tissue characterization with 3D visualization and quantification of major vessels (aorta) and its pathognomonic VV micro vascularization. It exceeds micro-CTs spatial resolution to a level of sub micrometer voxel size and provides the opportunity to assess vessels of capillary size. Thereby nano-CT depicts the 3D architecture of the complete vascular tree. In the past, micro-CT measured “density” i.e. changes of gray scale values in atherosclerotic plaque often remained speculative between cellular plaque composition and contrast agent within intraplaque microvessels . Due to The study was performed in a limited number of animals (n = 5 in each group). Blinded image analysis revealed findings with clear statistical significant differences proving (1) the effect of thalidomide and moreover (2) the applicability of nano-CT for research in atheroscloris. On the other hand, feeding a WD means a metabolic stress leading to obesity and cutaneous xanthoma. Therefore we abstained from a further enlargement of groups. This study proves a reduction of HCAECs viability but was not designed to clarify the specific molecular targets and effects on cellular metabolism in HCAECs. Application of thalidomide began prior to the obvious appearance of VV and atherosclerotic plaque. It needs to be clarified if thalidomide even acts on growth of pre-existing plaque and VV and if it takes influence on the course of the apparent disease simulating a pharmacological intervention after the onset of atherosclerosis. Further research should comparatively investigate molecular similarities and differences of plaque development in AL mice and humans and thalidomide’s mechanistic influence on both. Against this background, 3D imaging with high spatial resolution as provided by nano-CT has the potential to prove the impact of molecular findings on morphological alterations in atherosclerosis.Vasa vasorum neovascularization is associated to plaque development. A continued application of thalidomide even over a longer time course reduces both in WD fed AL mice. The data demonstrate nano-CT’s value as a tool to investigate the influence of preclinical drug interventions on atherosclerosis at a microscopic level with 3D data sets in isotropic voxel size. Future research in the field of atherosclerosis inclusive research on novel therapeutics and other topics of microvascular research such as tumor, fracture and inflammation should consider nano-CT as a powerful imaging modality.


In most solid cancers, cells harboring oncogenic mutations represent only a sub-fraction of the entire population. Within this sub-fraction the expression level of mutated proteins can vary significantly due to cellular variability limiting the efficiency of targeted therapy. To address the causes of the heterogeneity, we performed a systematic analysis of one of the most frequently mutated pathways in cancer cells, the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Among others PI3K signaling is activated by the hepatocyte growth factor (HGF) that regulates proliferation of hepatocytes during liver regeneration but also fosters tumor cell proliferation. HGF-mediated responses of PI3K signaling were monitored both at the single cell and cell population level in primary mouse hepatocytes and in the hepatoma cell line Hepa1_6. Interestingly, we observed that the HGF-mediated AKT responses at the level of individual cells is rather heterogeneous. However, the overall average behavior of the single cells strongly resembled the dynamics of AKT activation determined at the cell population level. To gain insights into the molecular cause for the observed heterogeneous behavior of individual cells, we employed dynamic mathematical modeling in a stochastic framework. Our analysis demonstrated that intrinsic noise was not sufficient to explain the observed kinetic behavior, but rather the importance of extrinsic noise has to be considered. Thus, distinct from gene expression in the examined signaling pathway fluctuations of the reaction rates has only a minor impact whereas variability in the concentration of the various signaling components even in a clonal cell population is a key determinant for the kinetic behavior. Cancer heterogeneity is considered a result of clonal instability, followed by clonal evolution  and intermediate (Hepa1_6-D8) mCherry-AKT expression levels based on flowcytometric analysis Figure . In addiTo investigate if the overexpression of the mCherry-AKT construct is affecting the upstream signaling pathway, the time course of cMet phosphorylation and degradation dynamics in the two clones was compared Figure . The quakAkt and kAktc. We implemented the same procedure as employed for the primary hepatocytes to transform the deterministic model to the corresponding stochastic model based on Gillespie's algorithm using chemical master equation formalism to simulate single cell traces for the clones. The intrinsic fluctuation calculated in the form of CV (green line) could not recapitulate the experimentally obtained CV (blue line) for both clones  retains functionality comparable to the endogenous protein as demonstrated by its kinase activity and ligand-induced membrane translocation  induced mRNA expression and new protein synthesis, (2) a consequence of the experimental procedure with accumulated fluorescent bleaching due to the laser light, and hence (3) increased stress response and induced apoptosis as seen in some of the cells when imaged for more than 40 min. In the future, further fine-tuning of the modeling approach could be achieved by considering in addition mRNA concentrations, production and degradation rates, and correlation with the determined protein concentrations and their change over time depending on the treatment.Single cell responses can be very diverse but still give a robust population response to physiological ligand concentrations for many different signaling pathways  supplemented with aprotinin and AEBSF (Sigma-Aldrich) at different time points. For imaging purposes cells were seeded in 2-well Labtech chambers after collagen coating for 2 h at a density of 120.000 for primary hepatocytes and 80.000 for Hepa1_6 cells per ml per well. Cells were transfected in a total volume of 800 μl OptiMem using 6 μl Lipofectamine™ LTX and 4 μl Plus™ regency (Invitrogen), and 1 μg of Plasmid DNA. Transfection media was removed after 12 h and cells incubated in pre-starvation medium for at least 6 h. All biological assays and imaging where performed 24–48 h post transfection in starvation media.The procedure for hepatocyte isolation and HGF stimulation has been previously established in our lab   was established and the phosphorylation signal was detected using an anti-phosphotyrosine antibody 4G10 . For all other components the total amount and the activation by phosphorylation was detected and quantified in immunoblots or protein array analysis using the following antibodies: pAKT(S473) #4058L, pAKT(T308) #4056S, and total AKT #9272S , total cMet (B-2) #sc-8057 (Santa Cruz), for pPTEN(Ser380/Thr382/383) #9554 and total PTEN #9552 , and total p85 #50-172-006 polyclonal serum (Upstate). Blots were developed using ECL advanced  with acquisition on an Image Quant LAS 4000 system and quantification with the Image Quant TL software . Repeated measurements have been merged on log scale assuming signal scaling between different gels. The merged signals are represented as parameters in a generalized least squares problem. Parameter estimates and 1 sigma confidence bounds are depicted as dots and error bands in Figures For absolute quantifications using dilution series of known concentrations of recombinant calibrator-proteins, SBP, or GST-tagged versions of the proteins PTEN, cMet, and p85 were cloned by PCR amplification from cDNA with introduction of appropriate restriction enzyme sites for ligation into the expression vectors. The cDNA for human PTEN was a kind gift from Alex Toker , p85 from Michael D. Waterfield , and cMet from George Vande Woude . Calibrator-proteins were expressed in BL21 bacteria and purified using Avidin- or Gluthation-beads, respectively. The AKT calibrator was purchased as 6His-AKT (Millipore). SDS-Page with appropriate calibrator concentrations and biological replicates of the cellular lysates with subsequent quantitative immunoblotting was performed. Calibration curves were employed to determine the molecule number in the respective sample. Information on the used protein amount, number of the lysed cells, and the cell volume were used to estimate the molecules per cell and concentrations of the signaling components.To study the dynamic activation of the pathway components the hepatocytes were stimulated with HGF and time resolved data were generated by quantitative protein array analysis similar as previously published , Wheat-germ-agglutinine-Alexa488 (WGA-Alexa488) (Invitrogen) as membrane stain (green) and the transfected mCherry-AKT (red). Time series imaging every 20–30 s or for 3D z-Stacks every minute where acquired in unstimulated 6 h starved cells and post HGF stimulation for at least 30 min or up to 2 h if applicable.Live cell imaging was performed on a Zeiss LSM710 with an incubation chamber at 37°C and 5% COImage analysis and quantification of mCherry-AKT membrane recruitment was done using the LSM-Zen2009 software, ImageJ, and a newly developed MatLab script for tracing the membrane stain in one channel over time with adjustments to cell movements and shape changes of the membrane (WGA-Alexa488) and quantifying the first 5 pixels inside the cell as membrane fraction in the second fluorescent channel (mCherry-AKT) with an further inside cytoplasmic region as reference. All values were normalized for bleaching during acquisition by the overall cell fluorescence.Using the confocal Zeiss LSM710 with z-stack mode by consecutive imaging with adjacent not overlapping sections, the cell volume was determined by quantification of the cell area multiplied with the high of the confocal section summed up over all images.2, and humidity control. The total intensity changes over time where quantified using ImageJ software representing the kinetic of mCherry-AKT at the membrane of the cells at the glass bottom due to the TIRF settings.TIRF microscopy was performed at the Nikon Imaging Center of the University of Heidelberg with a Nikon Ti inverted microscope with perfect focus system for TIRF automated dual channel time-lapse imaging with laser lines of 488 and 561 nm. For detection an Andor iXon DU-897 Electron Multiplier CCD digital camera was used. Cells were imaged in 8-well labtech chamber slides in an environmental chamber from okolab, allowing for full temperature, COThe fluorescent tagging of AKT with mCherry was achieved by PCR amplification of mCherry-cDNA removing the stop-codon and replacing it with a short linker (Asp-Glu-Leu-Tyr-Lys-Gly-Thr-Gly-Ser-Ile) and the mouse AKT1 cDNA (Addgene #10841) sequence via an introduced BamHI restriction side in a similar fashion as described previously , Pen/Strep, and Glutamax with additional 1.2 μg/ml puromycin supplementation for further cultivation. Finally, two single cell clones with different but stable mCherry-AKT expression levels were selected, namely clone Hepa1_6-D8 and E2.Generation of retrovirally transduced stable clones was performed following standard protocols. Briefly, 10 μg of the final vector pMOWS-puro-mCherry-AKT was transfected into 293T-Phoenix-eco cells using CaCl13C6-phenylalanine were used as previously described  using FACS-tubes . The mCherry-AKT was detected with the PI-channel settings with 0.4% compensation. The logarithmic transformed FACS values of mCherry-AKT intensity were used to calculate the CV of the protein expression of the population.We constructed a mass action kinetics-based deterministic model for the simplified pathway scheme proposed in Figure To account for the cell-to-cell variability in the AKT phosphorylation kinetics, we first investigated the effect of intrinsic noise theoretically. The deterministic models, formulated in terms of mass action kinetics, were converted into stochastic model using the chemical master equation formalism and simulated by using Gillespie's algorithm (Kar et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.