A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test. Infectious diseases including malaria, pulmonary tuberculosis, and viral infections ) remain major public health problems particularly in the developing world. As individuals unaware of their infection status represent a high risk of transmission, rapid and accurate diagnostics are crucial to decrease incidence and allow for immediate therapeutic intervention. Rapid test devices (RTDs) are available for many infectious diseases allowing appropriate initial screenings. However, these RTDs typically require confirmation by a second test. Currently, confirmatory diagnostics are conducted in well-equipped clinics staffed with trained personnel. Moreover, confirmatory tests generally require a second visit to the clinic, and patients often do not return to collect confirmatory test; this reduces effectiveness in respect to prompt treatment of the infectious disease , 2.Many of the available RTDs used in point-of-care (POC) settings use lateral flow in combination with a visual interpretation of the test result, and these devices do not always exhibit the expected sensitivity and specificity . Often, Microfluidic lab-on-a-chip (LOC) devices performing high complexity assays may bring confirmatory testing from the specialized laboratories to the POC setting . SeveralTo demonstrate active/acute infection, on-chip NA amplification methods have been developed based on their versatility, speed, and high sensitivity and specificity –11. MicrLOC devices with highly functional modules are particularly useful when they include the capability for multiplexing, allowing for rapid screening and, if necessary, a confirmatory test. Here we describe the development of a device for simultaneous detection of antibody and nucleic acid using a test platform from Rheonix with their previously described CARD technology , 19. Thelink collector (previously available from OraSure Technologies) and the newly developed collector with solid Porex matrix were tested for potential application in the dual-path CARD. The dual-path CARD was eventually designed for use with the solid Porex matrix collector. Whole mouth saliva samples (WMSSs) clarified using either the collectors or centrifugation (or a combination) were spiked with plasma-based control samples as provided with the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test (OraSure Technologies): a borderline positive and a negative control sample. Samples were furthermore spiked with Armored RNA particles (Asuragen Inc.) as noninfectious replacement for HIV.Saliva was collected from a healthy volunteer according to protocols described earlier . The UPl2O as required. Proprietary wash buffers from Rheonix with reduced EtOH concentrations were also successfully used to replace the wash buffers provided with the High Pure Viral RNA kit. RNA isolated using the Rheonix wash buffers (either by the benchtop or by on-chip protocols) improved downstream RT-PCR amplification yields. The Roche and Zymo kits utilize spin columns in the RNA isolation; the microfluidic chips were provided with a Rheonix proprietary silica membrane suitable for filtration by vacuum.RNA was isolated using either the High Pure Viral RNA kit (Roche) or the ZR Viral RNA kit (Zymo Research). In the procedures provided with both kits, the lysis/binding buffer could be replaced with Pluronic Lysis Buffer which contained 15% (w/v) of the nonionic detergent heteropolymer Pluronic F-68 (Sigma-Aldrich) in 50 mM Tris-Cl pH 6.6 and 4.5 M Guanidine Hydrochloride. Pluronic lysis buffer can be lyophilized to dryness, stored as a dry reagent, and readily dissolved in Hgag fragment from RNA isolated from noninfectious Armored RNA particles (Asuragen Inc.). Amplification was performed using primers developed for the COBAS Amplicor HIV-1 Monitor Test v1.5 [RT-PCR kits were obtained from Roche, Qiagen, GE Healthcare, and Zymo. In most experiments the QIAGEN OneStep RT-PCR Kit (QIAGEN) was used as its hot-start capability allowed convenient on-chip addition of the assay reagents to the microfluidic chip before initiation of the assay. Also, the kit materials could be lyophylized for storage in 0.2 mL microfuge tubes by the proprietary technology of Tetracore Inc., which is a format compatible with the dual CARD NA amplification compartment. The Transcriptor One-Step RT-PCR Kit (Roche) allowed the largest reduction in RT-PCR assay time. The illustra Ready-To-Go RT-PCR Beads  were the only commercially readily available dry reagents successfully applied in on-chip RT-PCR amplification. All kits were used to amplify a 155 bp HIV est v1.5 ; the forμg mouse anti-digoxigenin antibodies or 25 μg protein-A as described [Upconverting phosphor (UCP) particles are a highly sensitive reporter suitable for LF format analysis , were obescribed , 23, 24.escribed , 23–25 wMicrofluidic chips were provided by Rheonix, Inc. Before designing the entire and comprehensive dual path CARD, various types of simpler intermediate devices were developed to allow the analysis of specific modules and assay steps. Required design changes were indicated by the users and implemented by Rheonix according to methods protocols described in Zhou et al.  and SpizμL of saliva added to the sample reservoir, the automated protocol was initiated. Individual on-chip compartments referred to in different steps are indicated in Saliva from an uninfected individual was used to evaluate the dual path chip. Clarified saliva was spiked with 5% (v/v) of the OraQuick antibody control and 10% (v/v) dilution of Armored RNA (Asuragen Inc.). After buffer and reagents were loaded and 100 μL PBS (PBS—Compartment B) is used to release and dilute 100 μL of sample from the oral collector (Sample—Compartment A) by pumping. Note that the use of oral collector with the Porex matrix is shown in  200 μL aliquot of the PBS diluted saliva sample to Compartment L. Part of the aliquot is further mixed/diluted with HSLF assay buffer from Compartment I while flowing to the antibody detection LF strip (μL) containing 100 ng UCP-protA conjugate is applied to the strip from Compartment J.  Initiated by transferring a 100 LF strip . HSLF (5μL) of the PBS-diluted saliva sample is mixed with 400 μL of 15% (w/v) Pluronic Lysis Buffer in the Lysis Buffer Compartment (C). The lysate is then drawn through the NA binding silica membrane in Compartment (F). The membrane is washed with 200 μL of Rheonix WB-I (containing EtOH) and twice with 200 μL EtOH free Rheonix WB-II and then air-dried for 5 min. Elution water is pumped (Compartment G) onto the top of the silica membrane and incubated for 1 min. RNA is subsequently eluted and directed through the silica back into Compartment G.  The remainder (200 μL RT-PCR reagent is added to RT-PCR reagent Compartment H and pumped to the microtube together with 1 μL of the eluted RNA from Compartment G. A drop of low melting paraffin (melt point <50°C) on the inner wall of the tube  from Compartment K. Immediately after completion of the RT-PCR, 100 A dedicated reader is used to read the UCP signal. Results are presented as the ratio of the Test line signal divided by the flow control signal. The design of the dual path CARD allowed convenient iterations of the various modules. In our evaluation we distinguish three units: (i) the sample application unit, (ii) the antibody detection unit, and (iii) the nucleic acid unit. Each unit has a modular design with variable complexity. link collector (OraSure Technologies) designed to deliver a metered fluid sample directly into a cassette with a LF strip was identified as the most suitable collector. The UPlink collector was successfully applied in this study, but as this collector is not easily available anymore, an alternative collector was designed. The new collector consists of a circular, solid, porous, and removable Porex disk  in a lever and a plastic handlebar. The disk can be conveniently forced out of the lever into a specially designed reagent vessel on the CARD  of PBS by pumping fluid reversibly through the disk. The porosity of the disk contributes to the efficient mixing of the saliva sample with PBS, which renders the sample less viscous for subsequent operations. Viscosity issues when analyzing untreated saliva in different types of antibody-only devices are part of a separate study (manuscript in preparation). The diluted sample was split between the antibody and NA analysis pathways.In a previous study  several the CARD . The curThe antibody detection path is directly modeled on the sequential flow assay format (referred to as consecutive flow (CF)) described previously for the detection of antibodies against infectious disease pathogens . The appThe NA analysis pathway is the most complex part of the dual path chip. It consists of several modules related to the various procedural steps in the benchtop protocols. The first step is the RNA isolation, by itself consisting of several subprocedures. The conventional protocol to isolate HIV RNA employed the High Pure Viral RNA Isolation Kit (Roche) including spin columns with a silica matrix. Centrifugation steps were replaced with chip-based vacuum filtration . Chips wHigh viscosity buffers represent a problem for fluid transport through narrow channels on a microfluidic chip, and it is especially true for the lysis step. The first action in the on-chip RNA isolation protocol is the transport of diluted sample from the sample reservoir into the prefilled lysis buffer reservoir, which is then reciprocally pumped between compartments until the two fluids are homogenous. The porous matrix used to collect the oral clinical sample  version 1.5 [per PCR cycle, was achieved with the Transcriptor One-Step RT-PCR Kit (Roche). For most of the on-chip experiments, the Qiagen OneStep RT-PCR Kit was used since the HotStarTaq DNA Polymerase permitted retaining mixtures of RT-PCR reagents and primers at ambient temperature allowing preloading of the RT-PCR reagents. The use of hot start conditions and polymerases is necessary to limit the formation of primer-dimers and other PCR artifacts when primers and RT-PCR reagents are mixed and preloaded in advance. In the final version of the CARD, dry target-specific amplification reagents will be provided to the amplification compartment, a replaceable 0.2 mL microtube affixed to the bottom of the microfluidic chip. One currently available dry reagent kit that was tested was the Ready-To-Go RT-PCR Beads , This kit performed well on chip using the original protocol and control target (a 425 bp D. melanogaster fragment), but it did not accommodate drastic shortening of the cycle time. A complete, ready-to-go reagent cocktail made from reagents supplied with the Qiagen kit and gag-specific primers (supplied with a Dig and Bio hapten) was successfully transformed to a dry format by Tetracore Inc., using their proprietary technology [The second step in the NA pathway is the amplification procedure. Initially PCR-only dedicated chips were used to investigate on-chip RT-PCR conditions for the amplification of a 155 bp sion 1.5 . As the chnology ; other tchnology .αDIG reporter particles that are bound to the DIG tag. When LF is completed, both the antibody and NA strips are removed from the CARD and scanned for the presence of UCP label. In future versions the controller box may contain a built-in IR scanner.A typical experiment with the dual path CARD involves the simultaneous detection of antibody and RNA utilizing saliva containing HIV Armored RNA samples and antibody standards from the OraQuick ADVANCE Rapid HIV1/2 Antibody Test. The open structure allowed convenient manual addition of wet reagents and permitted visual observation of the on-chip fluid transport. In the on-chip protocol, the antibody detection path and the NA detection path proceed sequentially. Buffers and other reagents are loaded into their reservoirs prior to initiating computer control. Once initiated, the fully automated chip-based protocol dilutes the saliva sample with PBS and then transfers an aliquot to the antibody detection path for analysis. This aliquot is further diluted with HSLF assay buffer and transferred to the antibody LF strip containing a test zone to bind HIV-specific antibodies. The sample flow is followed by a wash and a flow of the IgG generic UCP-protA reporter conjugated to detect bound antibody. The strip is dried while the NA analysis path proceeds. NA is extracted from the remainder of the PBS diluted saliva sample by first mixing with Pluronic lysis buffer and then running the lysed sample over the NA-binding silica membrane. The membrane is flushed with the proprietary Rheonix wash buffers and air-dried to help remove residual EtOH prior to elution with nuclease-free water. RT-PCR reagents and eluted RNA are then added to the microtube through a plastic lumen , and theμL of the spiked saliva and run without operator interference. The dual path chip was tested with saliva samples spiked with the OraQuick positive control and Armored RNA or with the OraQuick negative control. Chips were loaded with 100 Schistosoma species, pathogen-derived NA targets and specific glycosylated-proteinaceous antigens can be explored [This study describes a portable processing system  for disposable microfluidic chips suitable for POC applications in the diagnosis, detection, and confirmation of infectious disease pathogens. Prototype devices were developed and assessed for their potential to analyze saliva samples. Each step of the process was evaluated independently, from collection to detection of targets specific to pathogen infection. We note here that besides oral-based fluids, the system was successfully tested with blood-derived samples (mainly plasma and serum) spiked with cultures of HIV  and can be adapted for use of other body fluids as well. For the oral-based sample analysis the use of a dedicated saliva collector was implemented. The device includes oral fluid-based sample preparation steps required for NA purification and allows the integration of essentially any NA amplification method. The device is suitable for simultaneous detection of multiple types of biomolecules. Here, the detection of antibody and NA is described. Diagnosis of HIV infection was used as a model to screen for the infection through detection of anti-HIV antibodies and confirmation of the actual presence of the virus by detecting an HIV-specific RNA target. This combination of a diagnostic screening with a confirmatory test has major advantages: it allows immediate initiation of therapy and counseling and removes the need for the patient to return to the POC facility for either subsequent testing or a final test result. In addition for populations at high risk for HIV infection, it also provides a method to decrease the window between infection and seroconversion, which is a relatively short period when viral loads are highest and transmission (infection of others) is most likely. Other applications could apply to the detection of anti-HPV (subtype 16 and 18) antibodies , 30 and explored –36. The microfluidic chips are produced by a patented technology (CARD), which is adapted readily for both large- and small-scale production and provides a convenient strategy for rapid modification and testing of small batches of chips for research and development purposes. Different modules were tested and ultimately combined into a single microfluidic chip design for simultaneous detection of nucleic acid (HIV-RNA) and protein (anti-HIV antibody). Alternative methods and modules for several of the on-CARD assay steps described here have been explored (data not shown) and can be integrated depending on the constraints defined for the performance of particular assays. Nucleic acids are detected after amplification; in the example discussed here, RT-PCR was used to amplify an RNA target from HIV. RT-PCR amplification requires a relatively complex heat cycling system; thus we submit that a system capable of performing RT-PCR can be easily adapted for other simpler amplification technologies. To demonstrate this, the system was also successfully tested with a recently commercialized isothermal amplification technology LAMP (results not shown). The only constraint when considering a device with dry reagents is the requirement that the enzymes and other reagents required for amplification need to be provided with a sufficient stability. The dry reagents tested in this study were obtained from a commercial source  or were dried using a custom proprietary technology (Tetracore Inc.). The assay-specific reagents for the amplification of the RNA target including the target-specific primers can be supplied to the chip in a microtube attached to the bottom of the CARD. In this open architecture research prototype the tubes are easily installed by the operator. The final design will be a disposable and sealed chip that will be discarded after use as biohazard waste. However, the possibility to change the PCR tube with specific amplification reagents remains and adds flexibility with minimal complexity to the system. For nucleic acid detection the processing of the chip will differ only in the technical details required for the specific method of amplification. Amplicons labeled with digoxigenin (DIG) and biotin (BIO) haptens during amplification can be detected by the rapid LF-based method using phosphorescent upconverting phosphor reporters. Required capture zones and dry UCP reporter can be fully integrated in the LF strips which also can be added to the CARD at a later time point. Multiplexing at the NA level  can be iSupplementary Movie Legend: The operation of an open-structure prototype of the dual-path microfluidic device is shown. The chip was designed for simultaneous detection of antibodies and nucleic acid. The reagent chambers are pre-filled with colored solutions as surrogates for the various reagents and buffers in order to observe the fluid movement and mixing process. Automated processing is initiated after the manual addition of the sample to be analyzed. The antibody detection and the nucleic acid analysis paths proceed sequentially. Note: The timeline for the movie is compressed for demonstration purposes only.Click here for additional data file.


Glutathione is abundant in the lining fluid that bathes the gas exchange surface of the lung. On the one hand glutathione in this extracellular pool functions in antioxidant defense to protect cells and proteins in the alveolar space from oxidant injury; on the other hand, it functions as a source of cysteine to maintain cellular glutathione and protein synthesis. These seemingly opposing functions are regulated through metabolism by gamma-glutamyl transferase . Even under normal physiologic conditions, lung lining fluid (LLF) contains a concentrated pool of GGT activity exceeding that of whole lung by about 7-fold and indicating increased turnover of glutathione at the epithelial surface of the lung. With oxidant stress LLF GGT activity is amplified even further as glutathione turnover is accelerated to meet the increased demands of cells for cysteine. Mouse models of GGT deficiency confirmed this biological role of LLF GGT activity and revealed the robust expansiveness and antioxidant capacity of the LLF glutathione pool in the absence of metabolism. Acivicin, an irreversible inhibitor of GGT, can be utilized to augment LLF fluid glutathione content in normal mice and novel GGT inhibitors have now been defined that provide advantages over acivicin. Inhibiting LLF GGT activity is a novel strategy to selectively augment the extracellular LLF glutathione pool. The enhanced antioxidant capacity can maintain lung epithelial cell integrity and barrier function under oxidant stress. II and CuI , L-DON (6-diazo-5-oxo-L-norleucine) and azaserine (O-diazoacetyl-L-serine) are all chemically reactive and inhibit GGT irreversibly [in vitro experiments [in vivo studies to see the effect of chemical knockdown of GGT on tumor cells [ in vivo. Acivicin and the related glutamine antagonists are highly cytotoxic and inhibit a number of glutamine-dependent biosynthetic enzymes [de novo purine and pyrimidine biosynthesis. Glucosamine 6-phosphate synthase, asparagines synthetase, NAD synthetase and anthranilate synthase are also inactivated by these compounds [per se as inhibitors of GGT. Therefore, the inactivation of GGT by acivicin, L-DON and azaserine is a fortuitous event that derives from the fact that GGT has a nucleophilic and catalytically essential Thr residue at the binding site near the γ-carboxy group of glutamine derivatives such as glutathione. In contrast, the naturally occurring glutamine antagonists such as acivicin [L- was synthesized. This compound served as a potent inactivator of E. coli GGT as a transition-state analog for successful affinity labeling the N-terminal Thr-391 in the small subunit as the catalytic nucleophile [1), however, is chemically too reactive to be used as a general inhibitor of GGT. To attenuate the reactivity, a series of γ-(monophenyl)phosphono glutamate analogs were synthesized [E. coli and human GGTs with a reasonable rate and the inactivation rates toward the human enzyme surpassed that of acivicin when an electron-withdrawing group was introduced . In fact, compound 2  was used successfully to identify the catalytic nucleophile of human GGT  [3) were synthesized as second-generation inhibitors [2). To our surprise, the umbelliferone derivative (4) exhibited an extraordinarily high activity toward the human enzyme with the inactivation rate reaching 6000 times that of acivicin. This finding has led to design the third-generation inhibitors (5 and 6) which mimic the structure of glutathione and its C-terminal carboxy group to interact with the active site of the human enzyme. In particular, compound 6 with a simplified Cys-Gly moiety (a phenyl ring) and a carboxymethyl group at the meta position is highly promising in that it compromises chemical stability and high activity toward the human enzyme. In fact, compound 6 is reasonably stable  and inhibited human GGT with an inactivation rate more than 120 times higher than that of acivicin. No regain of enzyme activity was observed. Interestingly, the para-substituted analog (7) was 155-times less active than compound 6, indicating that the human enzyme strictly recognizes the meta-carboxy group that is equivalent to the C-terminal carboxy group of glutathione[6 does not inhibit glutamine-dependent asparagines synthetase, has no toxicity toward human fibroblasts up to 10 mM and has passed the GLP safety guidelines (unpublished results). Therefore, compound 6 is so far the most promising candidate for pharmaceuticals to chemically inactivate GGT activity in vivo. This compound does inhibit lung GGT activity, but like acivicin, our preliminary data shows that it must be delivered through the airway . Hence the advantages of compound 6 include: specificity, potency, and lack of toxicity. Compound 6 is now commercially available under the name of GGsTop from Wako Pure Chemical Industries, Ltd., Japan.In our effort to identify the catalytic residue of GGT, a γ-monofluorophosphonate derivative of glutamate  . In linehibitors . Due to utathione. FurtherKi = 53 µM, competitive with respect to γ-glutamyl 4-nitroanilide) toward rat kidney GGT, highlighting the importance of the binding of the Cys-Gly moiety of glutathione with mammalian GGT.Other synthetic GGT inhibitors reported to date include a series of L-homocysteine analogs  , 88. A set al. reported a high-throughput screening approach to find a drug-like, non-glutamate analog OU749 and its derivatives as inhibitors of human GGT [Ki of 17.6 µM. The inhibition is species-specific and is reported to inhibit human kidney GGT with 7- to 10-times potency than those from rat or mouse kidney. Despite the fact that these compounds target the acceptor-site of GGT, toxicity is still reported. Optimal activity also requires a high level of GGT enzyme activity which is not the case in the lung [Recently, Hanigan uman GGT . From kithe lung .LLF is composed of a highly concentrated pool of glutathione that serves a biological role in antioxidant defense and cysteine supply over the entire lung surface. This extracellular glutathione pool is dynamic and reduced glutathione predominates. That LLF glutathione content is related to the level of oxidant burden at the lung epithelial surface has been recapitulated in studies over time. Ongoing efforts to assess and manipulate this pool to protect against extracellular oxidant stress are warranted and novel mechanisms to accomplish this goal are available. Studies suggest that early identification and correction of LLF glutathione deficiency can prevent lung injury and disease. Glutathione metabolism plays a role in determining the size of the LLF glutathione pool. Induction of GGT activity with the onset of acute lung injury and inflammation contributes to a relative deficiency of LLF glutathione, even in children with cystic fibrosis . Inhibit


Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) have been shown to be involved in a spectrum of cellular processes. In a previous study, we constructed a novel multigenic vector that contained two separate transcription units, each consisting of a strong promoter and an efficient polyadenylation signal. The two promoters were chosen for their ability to work simultaneously. Dual gene transfer of bFGF and PDGF in a single plasmid resulted in a significant increase in collateral blood vessel formation in a rabbit model of hind limb ischemia. The aim of the present study was to investigate the effect of this dual gene transfer strategy in a rat model of acute myocardial infarction (AMI). AMI was induced in rats by ligation of the left anterior descending coronary artery. The animals were randomly divided into four groups and treated with the following therapeutic strategies: Empty plasmid (control), plasmid encoding bFGF (PL-bFGF), plasmid encoding PDGF (PL-PDGF) or plasmid encoding bFGF and PDGF (PL-F-P). Echocardiography and histological examinations were performed 28 days subsequent to gene transfer. Dual gene therapy with bFGF and PDGF resulted in a significant angiogenic effect accompanied by vessel maturation, along with a significant reduction in infarct size and improvement in cardiac function. In a rat model of AMI, single plasmid-mediated dual gene therapy with bFGF and PDGF decreased infarct size and improved cardiac function due to the formation of functionally and morphologically mature vasculature. These results are relevant to the ongoing clinical trials involving the use of single plasmid-mediated angiogenic factors for the treatment of myocardial ischemic disease. Mechanical revascularization during percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) is largely effective in treating patients with myocardial infarction; however, there are certain categories of patients in which mechanical revascularization techniques are not able to be applied, and this is affecting an increasing number of patients. This is particularly true for patients with extensive ischemic tissue and patients who have undergone multiple bypass and stenting procedures ,2. The nA number of growth factors have been demonstrated to induce angiogenesis. Basic fibroblast growth factor (bFGF) is a potent mitogen for cells of mesenchymal, neural and epithelial origin, including all the cell types found in the vascular wall  . PreclinIn our previous study, a novel multigenic vector with two transcription units, allowing the combined expression of two genes of interest from a single vector, was constructed . In the Male Sprague-Dawley rats  were obtained from the Laboratory Animal Center of Sichuan University . All animals were handled in strict accordance with good animal practice, as defined by the relevant national and/or local animal welfare bodies, and in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . The study was approved by the Ethics Review Board for Animal Experiments of Sichuan University.A CMV5 promoter/enhancer and β-globin poly(A) region from a pAdenovator-CMV5 vector were inserted upstream of the Simian virus 40 (SV40) enhancer and early promoter sequence in the pSI expression vector using multi-step polymerase chain reactions (PCRs). The bFGF and PDGF genes were amplified from pBLAST45-hbFGF and pBLAST49-hPDGF, respectively, and then cloned into the newly constructed double-promoter plasmid : i) Rats treated with control plasmid (PL-Null); ii) rats treated with plasmid encoding bFGF (PL-bFGF); iii) rats treated with plasmid encoding PDGF (PL-PDGF); iv) rats treated with plasmid encoding bFGF and PDGF (PL-F-P). The PL-Null group was referred to as the control. Ten minutes subsequent to AMI, the border zone of the infarct area was directly injected with plasmid (100 μg in 100 μl) between the infarcted and normal tissue using a 1-ml syringe and a 25-gauge needle. All the rats were closely monitored and were treated with antibiotics.Echocardiography was performed in all the experimental subjects 28 days subsequent to gene transfer, under controlled anesthesia and using a 3–8 MHz phased-array transducer  and Philips Sonos 7500 ultrasound system . M-mode tracing and 2D echocardiography images were recorded from parasternal long- and short-axis views. The short-axis view was at the level of the papillary muscles. Left ventricular (LV) end-systolic and end-diastolic dimensions, as well as systolic and diastolic wall thickness were measured from the M-mode tracings. For each M-mode measurement, ≥3 consecutive cardiac cycles were sampled. The average LV end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs), LV ejection fraction (LVEF) and LV fractional shortening (FS) were measured. All measurements were performed by two experienced echocardiographers who were blinded to the treatment group.The rats were sacrificed 28 days subsequent to gene transfer. The hearts from each group were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned transversely and stained with Masson’s trichrome. Sections from each heart were evaluated in their entirety and quantified using a computer-assisted automated image analyzer . The extent of fibrosis was assessed by measuring the collagen area as a proportion of the total LV area. Morphometric studies were performed by two examiners blinded to the treatment group.2.The rats were sacrificed 28 days subsequent to gene transfer. Heart muscle samples were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned transversely. Endothelial cells were identified by the expression of von Willebrand factor (vWF), while smooth muscle cells were identified by the expression of smooth muscle actin (SMA). Immunohistochemistry was performed with specific primary antibodies against vWF  or SMA . Vessels from the infarction border zone were analyzed at ×200 and ×400 magnification for SMA and vWF immunostaining, respectively. All vessel measurements were performed in a blinded manner in six randomly selected fields on the infarction border zone from each section. Blood vessel density was expressed as vessel number/mmData are expressed as the mean ± standard error of the mean (SEM) and were analyzed using SPSS version 11.0 statistical software . Differences between groups were analyzed using analysis of variance (ANOVA) and the least significant difference (LSD) method. P<0.05 was considered to indicate a statistically significant difference.Twenty-eight days subsequent to infarction, the PL-F-P group had a significantly lower LVDd, and LVDs than the PL-bFGF and PL-PDGF groups (P<0.01). Each of these three groups had a significantly lower LVDd, and LVDs than the control group (P<0.01). Conversely, the PL-F-P group showed a significantly higher level of LVEF and FS than that of the control group P<0.01; . These rHistological examination revealed that the average infarct size in all the therapy groups was smaller than that in the PL-Null group at 28 days subsequent to infarction . CompareImmunostaining of the infarcted myocardium for the expression of vWF and SMA demonstrated the augmentation of neovascularization in all the therapy groups . Among tet al -α and PDGF R-β expression. In this manner, PDGF-BB (PDGF homodimer), an active ligand for the PDGF-α and PDGF-β receptors, may subsequently display increased and potent angiogenic activity .Compared with viral methods, the delivery of plasmid DNA is limited by lower gene transfer efficiency rates and a shorter half-life. However, skeletal muscle has been indicated to take up and express naked DNA more efficiently than other types of tissues . The preIn the present study, the results of the echocardiography and examination of infarct size were consistent with those obtained from blood vessel measurements. Echocardiography showed a significant improvement in cardiac function in the dual gene-treated group. The examination of infarct size revealed that the average infarct size was significantly decreased in the dual gene-treated group. These results suggest that neovascularization was important for recovery following myocardial ischemia. The number of mature vessels determines the number of surviving myocardial cells; these cells are important for the improvement of cardiac function.et al  and increases VEGF release . The reset al , in whicet al . In addil  thSingle plasmid-mediated dual gene therapy with bFGF and PDGF was effective in stimulating the functional and morphological maturation of the vasculature in cardiac muscle following infarction. The results of the present study may be relevant to future clinical trials involving angiogenic factors for the treatment of myocardial ischemic disease.


A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases. Selective inhibition of specific panels of multiple protein targets provides an unprecedented potential for improving therapeutic efficacy of anticancer agents. We introduce a computational systems pharmacology strategy, which uses the concept of target inhibition networks to predict effective multi-target combinations for treating specific cancer types. The strategy is based on integration of two complementary information sources, drug treatment efficacies and drug-target binding affinities, which are readily available in drug screening labs. Compared to the cancer sequencing efforts, which often result in a huge number of non-targetable genetic alterations, the target combinations from our strategy are druggable, by definition, hence enabling more straightforward translation toward clinically actionable treatment strategies. The model predictions were experimentally validated using siRNA-mediated target silencing screens in three case studies involving MDA-MB-231 and MCF-7 breast cancer and BxPC-3 pancreatic cancer cells. In more general terms, the cancer cell-specific target inhibition networks provided additional insights into the drugs' mechanisms of action, for instance, how the cancer cell survival pathways can be targeted by synergistic and synthetic lethal interactions through multi–target perturbations. These results demonstrate that the principles introduced here offer the possibilities to move toward more systematic prediction and evaluation of the most effective drug target combinations. The two single most important reasons are , which makes the use of partly overlapping target subsets and supersets of promiscuous drug-target binding profiles in the estimation of anticancer efficacies for novel drug target combinations. The model construction and target combination predictions are based on functional data on drugs and their targets that are available from comprehensive target binding assays and from high-throughput drug sensitivity screens. We implemented a modified sequential forward floating search algorithm for model selection, which enables scaling-up to proteome-wide evaluation of the targets in terms of their relevance to cancer survival. Both simulation studies and an application to a canine osteosarcoma cell line data showed that TIMMA achieved improved prediction accuracy, when compared to a published algorithm 50). A drug with a smaller inhibitory concentration is usually considered as more potent. Drug treatment efficacy and potency can be also quantified based on the area under the dose-response curve, such as the activity area (AA) Consider a set of i can be binarized from drug-target binding affinities as a binary vector To relate a drug's treatment efficacy with its underlying mechanism of action, the cellular targets of the drug need to be mapped into a drug-target inhibition profile. Let the potential target set be Given the single drug efficacy and target inhibition profiles, our aim is to predict the treatment efficacy for novel drug combinations. We consider the target inhibition profile of a drug combination as a union of the target inhibition profiles of each component drug in the cocktail . HoweverLet Let In an earlier work by Pal and Berlow If a drug If a drug The rationale for the two rules is straightforward. First, it is assumed that a drug inhibits cancer growth by switching off certain survival signaling pathways via modulation of its targets. Second, the topology of cancer survival pathways is perceived to be conserved irrespective of drug perturbations. These assumptions generally hold for multi-target inhibitors that are tested for treatment efficacy in a specific cancer subtype  model, which make the full use of the information content in the screening data to predict i.e.For the subsets of i.e.For the supersets of a priori unknown and need to be inferred from the potential target set Construction of a TIMMA model for predicting drug efficacy requires a selection of cancer-specific target set Evaluate the objective function For the current target set After inclusion of In the temporary set a priori determined upper bound of The sequential search strategy allows a dynamic change of the target set dimensionality and thus can overcome the limitations of many monotonic search algorithms, such as the greedy search in the PKIM algorithm http://timma.googlecode.com/.We have further improved the scalability of the TIMMA algorithm to large and complex data in MATLAB by exploiting its matrix computation architecture. Briefly, the TIMMA model was represented as a 3- dimensional array, where each drug's contribution to the estimate of A, B) based on the multiplicative null model:For the optimal target set tion in  . Here, w1, d2d) whose targets are included in Using the model predictions, we can calculate the synergy score also for those drug pairs , we defined a synthetic lethality score as the ratio of the fitness function of these two models, given by the total sum of squares (TSS) of the predictions:When the target set size is fixed at two, the TIMMA model construction algorithm evaluates the pairwise target inhibitions without considering any higher-order interactions. This enables the TIMMA modeling strategy to systematically predict target pairs with synthetic lethality effect. By definition, synthetic lethality among a target pair states that: (i) inhibition of either of the single targets will result in incomplete cancer killing; and (ii) inhibition of both of the targets simultaneously will block the complete cancer survival sub-network. Therefore, the target inhibition network for the synthetic lethal target pair can ideally be represented as two nodes in parallel, similar to the topology of To initially benchmark the model performance, we generated simulated drug target data containing 100 drugs and 10 targets. The binding affinity values for the drug-target pairs were uniformly distributed in , reflecting the most challenging case where the uncertainty about the drug-target interactions is maximal. The current implementation of TIMMA assumes binary drug target profiles so that the maximization and minimization rules can be directly applied on their subsets and supersets. For the simulated data, we evaluated the model performance on the full scale of binarized drug-target binding sets, generated by gradually increasing the binding affinity threshold with a step of 0.01 from 0 to 1. The drug treatment efficacy data 50 values . The kinase targets for these 36 targeted drugs were obtained from a comprehensive competition binding assay We next applied TIMMA to the real drug sensitivity data used for evaluation of the PKIM algorithm ) values . The  vaTo further demonstrate the model performance, we applied the TIMMA model to more challenging case studies in MCF-7 breast cancer and BxPC-3 pancreatic cancer cell lines. We utilized the recently published Cancer Cell Line Encyclopedia (CCLE) collection We considered 41 kinase inhibitors for MDA-MB-231 cancer cell line, which belongs to the triple-negative breast cancer subtype (TNBC). The drug-target interaction data was again retrieved from the kinome-wide binding affinity assay as reported in 2 in a humidified incubator, cell viability was recorded by adding the Cell Titer Blue (Promega) reagent according to manufacturers' instructions and reading fluorescence at 595 nm using Pherastar FS (BMG) plate reader. The data was analysed using Dotmatics software (Dotmatics Ltd). Each plate was normalized against the positive and negative controls and the Z′-factors calculated were used to control the quality of each data set. Percent inhibition was then calculated for each siRNA combination, normalized against the positive and negative controls.siRNA against the selected genes and the positive and negative controls were purchased from Qiagen. Three different siRNAs combined against each gene were used. The final concentration of total siRNA was 6 nM for single gene knock-down. For double gene knock-down, the concentration of siRNA against each gene was 3 nM. The siRNAs were transferred on clear bottom 384-well plates (Corning #3712) using an Echo 550 acoustic dispenser (Labcyte). Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) diluted according to manufacturers' instructions. In OptiMEM (Life Technologies) was added using a Multidrop Combi nl dispenser (Thermo Scientific). The plate was incubated for 1 h at room temperature in a shaker after which MDA-MB-231 cells  were added using a Multidrop Combi dispenser (Thermo Scientific). After 96 h incubation at 37°C, in 5% CO−15), indicating a consistent readout for the same double gene knock-downs. Therefore, the inhibition percentages were averaged for each double gene knock-down, except for those where the two replicated cell inhibition percentages differ more than 15%, in which case the replicate that is located at the edge of the plate was excluded. Ten replicates  were removed due to such edge effects. To make the single and double gene knock-down results more comparable, the single gene knockdown effects were normalized by taking the average of the inhibition values of single gene knock-down and those double gene knock-down that include this gene with lower inhibition values . Since TIMMA combines the information from a drug's subsets and supersets simultaneously, its predictions are more robust to data noise and other technical factors that are inconsistent with the model assumptions, compared to PKIM, which does not consider model averaging. In particular, TIMMA gains on average 22.4% increase in the prediction accuracy especially for affinity thresholds lower than 0.8, which correspond to the promiscuous cases with, on average, more than two targets per drug  prediction accuracy, compared to PKIM, at each predefined drug-target threshold . Similarly as in simulated dataset . This demonstrates that the computationally efficient SFFS algorithm can find solutions that are not too far from the globally optimal solution.To test whether the SFFS model selection algorithm can find solutions close to the global optimal target sets, we performed an exact analysis for maximally 12 kinases, where exhaustive search can be performed at a reasonable running time. More specifically, After confirming the appropriate performance of the TIMMA model, we applied it to two practical case studies. In the first one, we systematically evaluated the predictions of the TIMMA MCF-7 model against the experimental results from an independent kinome-wide siRNA study in the MCF-7 breast cancer cells Here, we tested the essentiality of the kinases in the cancer-specific target set predicted by TIMMA using the 15 drugs targeting a total of 384 kinases. In other words, we asked the question: are the kinases selected by TIMMA as the most predictive of anticancer efficacy also highly essential individually for the cancer cell viability? The optimal target set found by TIMMA included 12 kinases {ZAK, CSF1R, GAK, MEK5, ABL2/EPHA8, ALK/LTK/PLK4/ROS1 and MEK1/MEK2}, with a mean LOO prediction error of 0.1392 . The/symin vivoOn the basis of the predicted efficacy matrix based on the selected kinase targets , we deriTo visualize the combinatorial effect of the selected kinase targets, a target inhibition network was constructed by applying a threshold of 0.318 to binarize the predicted efficacy . The thrTo further show the applicability of TIMMA to such cases where combinatorial effects of kinase inhibition are considered, we utilized the results from a kinome-wide drug sensitization screen, in which the kinase siRNA-silencing was combined with the treatment of Aurora kinase inhibitors in BxPC-3 pancreatic cancer cell line We wanted to evaluate here the TIMMA model performance in predicting the experimental results in rd), MEK5 (ranked 7th) and GAK (ranked 9 th) were again found in the cancer-specific target set for BxPC-3 cells, suggesting that the synergy patterns of these kinases is common across these cancer types. In contrast, the model predicted that the combination of MEK1/MEK2 and AURKB inhibitors has least synthetic lethal capacity . Even after excluding the two essential kinases (PLK1 and AURKB), the 18 TIMMA-selected kinases showed higher cancer cell growth inhibition power in the single knock-down experiments (22% increase in cell inhibition), compared to the inhibition observed in the kinome-wide single-siRNA screen . Among these top-20 most synergistic drug combinations for the MDA-MB-231 cells, there were a number of examples, such as the two top pairs, where the efficacy of one of the drugs in the combination was relatively low, or even zero, yet the predicted and measured synergy for the drug combination was high  targets, provided there will be enough target and drug promiscuity to allow for construction of the target inhibition networks. Moreover, while the siRNA silencing screens are convenient for the drug target investigation, the perturbation effects from the siRNAs cannot fully mimic the phenotypic effects of drug treatments. RNAi has also potential limitations due to potential off-target silencing effects and variable reagent efficacy, which may also partly explain the observed discrepancies between the drug treatment-based model predictions and their siRNA-based experimental validations. Therefore, one of our future aims is to apply the TIMMA model predictions to designing potential drug combination treatments, initially in various cancer cell models In an effective combinatorial setting, one needs to modulate a set of targets to achieve maximal efficacy, while avoiding others to reduce the risk of side effects. The current TIMMA algorithm addressed the first challenge: the optimal efficacy by multi-target modulation. The different model parameters and thresholds lead to a multiple candidate target inhibition networks for combinatorial treatments. From those candidate models, clinician could then ideally choose the combination that is most feasible and results in less known adverse effects, based on prior knowledge. Although there are information sources on drug side effects scattered around in databases, such as SIDER Dataset S1Binding affinity data for the CanOS1224 cell line case study.(XLSX)Click here for additional data file.Dataset S2TIMMA prediction result for the CanOS1224 data as compared to PKIM prediction.(XLSX)Click here for additional data file.Dataset S3Anticancer efficacy measured in activity area for the 15 kinase inhibitors selected from the CCLE cancer cell line drug collection.(XLSX)Click here for additional data file.Dataset S4Binding affinity data for the MCF-7, BxPC-3 and MDA-MB-231 studies.(XLSX)Click here for additional data file.Dataset S5TIMMA prediction result for MCF-7 cancer cell.(XLSX)Click here for additional data file.Dataset S6Synthetic lethality score for the targets paired with AURKB for BxPC-3 cancer cell.(XLSX)Click here for additional data file.Dataset S7TIMMA prediction result for MDA-MB-231 cancer cell.(XLSX)Click here for additional data file.Dataset S8Single and double siRNA screen data for MDA-MB-231 cancer cell.(XLSX)Click here for additional data file.Figure S1Flowchart for TIMMA model construction.(TIF)Click here for additional data file.Figure S2Flowchart of the SFFS algorithm for TIMMA model selection.(TIF)Click here for additional data file.Figure S3Concordance between TIMMA model assumptions and the actual data. The data contains those 12 kinase inhibitors for which the quantitative binding affinity A) The binary drug-target profiles were determined using a drug-specific −15). (B) The binary drug target profiles were determined using a fixed level of (TIF)Click here for additional data file.Figure S4Synergy scores do not necessarily correlate with the single drug treatment efficacies. (A) Scatter plot between synergy scores and average single drug sensitivity scores for the selected 68 drug pairs in the MDA-MB-231 study, fitted by a loess smoothing function. (B) Synergy scores for the selected drugs when paired with dasatinib  and the corresponding individual drug sensitivity scores .(TIF)Click here for additional data file.Table S1The top most synergistic drug pairs based on the TIMMA model predictions in MCF-7 breast cancer cells.(DOCX)Click here for additional data file.Table S2Fold-changes of the average inhibition percentages between the kinase groups in the MDA-MB-231 siRNA screen.(DOCX)Click here for additional data file.


To investigate epilepsy-induced changes in effective connectivity between the non-epileptic amygdalo-hippocampal complex (AHC) and the rest of the brain in patients with unilateral mesiotemporal lobe epilepsy (MTLE) associated with hippocampal sclerosis (HS).18F-Fluorodeoxyglucose positron emission tomography (FDG-PET). Right-HS patients' FDG-PET data were flipped to obtain a left–epileptic–focus–lateralized group of patients. Voxels of interest (VOI) were selected within the cytoarchitectonic probabilistic maps of the non-epileptic AHC . Patients and controls were compared using VOI metabolic activity as covariate of interest to search for epilepsy-induced changes in the contribution of the non-epileptic AHC to the level of metabolic activity in other brain areas. Age, gender, duration of epilepsy, seizure type and frequency were used as covariates of no-interest for connectivity analyses.Thirty-three patients with unilateral MTLE associated with HS  and 33 adult controls matched for age and gender underwent Significant decrease in effective connectivity was found between the non-epileptic AHC and ventral prefrontal cortical areas bilaterally, as well as with the temporal pole and the posterior cingulate cortex contralateral to HS. Significant increase in connectivity was found between the non-epileptic AHC and midline structures, such as the anterior cingulate and dorsal medial prefrontal cortices, as well as the temporo-parietal junction bilaterally. Connectivity analyses also revealed a preserved positive connectivity between the non-epileptic and the epileptic AHC in the patients' group.This study evidences epilepsy-induced changes in connectivity between the non-epileptic AHC and some limbic and default mode network areas. These changes in connectivity probably account for emotional, cognitive and decision-making impairments frequently observed in MTLE patients. The preserved neurometabolic connectivity between the non-epileptic and the epileptic AHC in MTLE patients is pivotal to explain the epilepsy-induced changes found in this study. The functional integration within and between neuronal networks is crucial for normal information processing and behaviors, but it may also ground the pathophysiological basis of long-range neuronal functional disturbances and functional networking reorganization associated with focal brain disorders Mesial temporal lobe epilepsy (MTLE) is an epileptic syndrome characterized by recurrent partial seizures arising form mesiotemporal structures 18F-Fluorodeoxyglucose (FDG-PET) performed interictally in patients with unilateral MTLE have demonstrated significant hypometabolism in the epileptic temporal regions in about 60% to 95% of the patients per sePositron emission tomography studies using Besides the occurrence of this neuronal inhibition, increasing evidence supports the existence of disease-induced plasticity mechanisms in the non-epileptic hemisphere and, more particularly, in the non-epileptic temporal lobe of patients with unilateral MTLE Taken together, all these observations indicate that the consequences of unilateral MTLE are not confined to the epileptogenic zone but rather extend to distributed networks, possibly via disease-induced changes in functional integration and plasticity phenomena. The main purpose of this study was to better characterize in a large group of patients the remote consequences of unilateral MTLE by searching for disease-induced changes in functional integration in brain areas not primarily involved in the epileptic disorder. Based on the established neuroanatomy of the amygdalo-hippocampal complex (AHC) Patients' FDG-PET data were obtained in the context of a multidisciplinary presurgical evaluation for refractory focal epilepsy. The ULB-Hôpital Erasme Ethics Committee gave approval for conducting and using these FDG-PET data in this retrospective study. The ULB-Hôpital Erasme Ethics Committee waived the requirement for obtaining patients' or parents'  informed consent  in the context of this retrospective study. The Institutional Ethics Committees gave approval for the FDG-PET investigations obtained in adult controls. Written informed consent was obtained from all control subjects.Among the population of patients studied by FDG-PET between February 2000 and May 2009 at the PET/Biomedical Cyclotron Unit of the ULB-Hôpital Erasme, we retrospectively selected 33 patients  based on the following inclusion criteria: 1) refractory focal epilepsy included in the multidisciplinary presurgical evaluation program of the ULB-Hôpital Erasme, 2) five days of scalp video-EEG suggesting unilateral MTLE, 3) unilateral HS on structural cerebral MRI, and 4) absence of neurological condition other than epilepsy. All patients underwent neuropsychological evaluation and interictal FDG-PET. During the video-EEG monitoring sessions, we recorded an average of 3.75±2.9 seizures-per-patient. In two patients, no seizure was recorded. Based on the multidisciplinary presurgical evaluation, it was concluded that the epileptic focus was unilateral and located in the mesiotemporal region in all patients. Nineteen patients had a left MTLE and fourteen had a right MTLE. Duration of epilepsy ranged from 2 to 54 years with a mean duration of 25.15 years. Surgical procedures were performed in 28 patients . Outcome for seizures  showed 19 patients (68%) in Engel class I (17 IA and 2 IC), 3 in Engel class IIA, 5 in Engel class III (4 IIIA and 1 IIIB) A group of 33 healthy adult volunteers matched to obtain similar age mean and gender proportion with patients' group  was used as control population. FDG-PET data of 23 subjects were acquired at the PET/Biomedical Cyclotron Unit of the ULB-Hôpital Erasme. FDG-PET data of the other subjects were acquired at the Nuclear Medicine division of the UZ Leuven. All FDG-PET data were acquired on the same type of PET camera using the same data acquisition and reconstruction procedure.FDG-PET data were acquired using an ECAT 962 Exact HR+ camera , the characteristics of which have been previously described http://www.fil.ion.ucl.ac.uk/spm/, Wellcome Department of Imaging Neuroscience, London, UK). The PET images were spatially normalized to the Montreal Neurologic Institute template . The scans were then smoothed using a 12-mm full-width at half-maximum isotropic kernel. Global activity normalization was performed by proportional scaling FDG-PET data were analyzed using the voxel-based Statistical Parametric Mapping method . Based on MTLE pathophysiology, 2×2×2 mm3 VOIs were arbitrarily selected in the 100% probability maps For effective connectivity analyses, voxels of interest (VOIs) were selected within the non-epileptic AHC using the cytoarchitectonic probability maps of the SPM Anatomy Toolbox 1.7b (Institut für Neurowissenschaften und Medizin (INM), Jülich Forschungszentrum, a priori hypothesis of an altered effective connectivity between the non-epileptic AHC and related limbic  t-contrast analyses then searched, throughout the brain, for regions showing significantly lower or higher activity modulation with the considered VOI in the patients' group than in controls. To assess a potential effect of age, gender, main seizure type , seizure frequency and duration of epilepsy on the connectivity changes, PathoPI analyses were also performed with each of these variables used as covariates of no-interest.Pathophysiologic interactions (PathoPI) were used for effective connectivity analyses in order to search for disease-related changes in the contribution of a brain area to the level of metabolic activity in another brain area in patients compared to controls p-values were calculated using the corrcoef function of the Matlab statistical toolbox version 6.2 .Regression plots of the brain areas showing significant effective connectivity changes with the VOIs were obtained in Matlab 7.6 R2008a . After a linear interpolation of the regression plots, the associated slope coefficients and t-contrast analyses identified brain regions where glucose metabolism was significantly lower or higher in the group of patients than in the control population.Finally, in order to better understand the pathophysiological mechanisms involved in the epilepsy-induced changes in connectivity, subtractive SPM analyses were conducted at the group level to compare the PET data of the patients taken as a group with those of the control group p<0.05 corrected for multiple comparisons over the entire brain volume . For effective connectivity analyses, we also considered significant at a small volume-corrected (SVC) p<0.05  voxels located in limbic p<0.05) with the considered VOI in the control population.All results of SPM analyses were considered significant at The most consistent connectivity changes were significant decreases in connectivity between the non-epileptic AHC and ventral prefrontal cortical areas such as the orbitofrontal cortex (OFC) bilaterally , the ventral medial prefrontal cortex (VMPFC) bilaterally  and the ventral lateral prefrontal cortex  in the non-epileptic hemisphere. Altered connectivity was also found with the temporal pole  and the posterior cingulate cortex  in the epileptic hemisphere.In patients compared to controls, PathoPI analyses revealed significant increase in connectivity between the non-epileptic AHC and the anterior cingulate cortex , the dorsal medial prefrontal cortex , and the temporo-parietal junction bilaterally .t-contrast analyses searched, throughout the brain, for regions showing significant positive correlation with the considered VOI in the patients' group. These analyses did not disclose significant changes in connectivity between the non-epileptic and epileptic AHC in the patient's group.As PathoPI analyses did not show significant changes in effective connectivity between the right non-epileptic and the left epileptic AHC, separate Introducing age, gender, main seizure type , seizure frequency and duration of disease as covariates of no-interest in PathoPI analyses did not modify the results.Significant hypometabolic areas were found in the mesial and lateral temporal lobe, the VLPFC and the inferior parietal lobule ipsilateral to the epileptogenic zone as well as in the ACC bilaterally. In addition, significant hypermetabolic areas were also found in the inferior temporal gyrus contralateral to the epileptic hemisphere.The voxel-based analyses of changes in effective connectivity between the non-epileptic AHC and the rest of the brain in patients with unilateral MTLE associated with unilateral HS disclosed the following findings: (1) preserved connectivity between the non-epileptic and the epileptic AHC, (2) significant decrease in effective connectivity between the non-epileptic AHC and the ventral prefrontal cortex, as well as with the temporal pole and the PCC contralateral to HS, and (3) significant increase in connectivity between the non-epileptic AHC and midline structures such as the DMPFC and ACC, as well as the temporo-parietal junction bilaterally.In accordance with previous FDG-PET studies performed in patients with unilateral MTLE, we found significant relative decrease in metabolism in the epileptic temporal lobe, in extratemporal brain areas ipsilateral to the epileptic focus such as the VLPFC and the inferior parietal lobule as well as in the ACC bilaterally. We also found significant relative increase in metabolism in the temporal lobe contralateral to the epileptic focus potentially attributable, as previously proposed, to epilepsy-induced functional reorganization or plasticity phenomena The PathoPI analyses did not show any significant alterations in neurometabolic connectivity between the non-epileptic and the epileptic AHC. This finding strongly suggests that the mesiotemporal structures remain tightly connected despite the unilateral HS and the associated recurrent seizures. This preserved connectivity is pivotal to explain the functional integration changes found between the non-epileptic AHC and some specific brain areas. Indeed, by allowing the propagation of epileptiform activity towards the non-epileptic AHC, this preserved connectivity might lead to repetitive alterations in neuronal activity at the level of the non-epileptic AHC The PathoPI analyses mainly revealed a significant decrease in effective connectivity between the non-epileptic AHC and ventral prefrontal structures such as the OFC, the VMPFC and the VLPFC. These brain areas are parts of the neural networks involved in emotion processing and regulation, decision-making or reward processing Regarding the pathophysiological mechanisms that would link the changes in effective connectivity found in this study with the patients' epileptic disorder, it is noteworthy that most of the brain areas showing significant decrease in connectivity with the non-epileptic AHC did not show any substantial change in relative glucose metabolism compared to the control group. This finding suggests that the decrease in connectivity observed with the non-epileptic AHC is directly related to an epilepsy-induced alteration of the non-epileptic AHC function with subsequent decrease in normal functional integration with connected brain areas, rather than to a direct influence of the epileptic AHC on brain areas connected with the non-epileptic AHC via, for example, a mechanism of remote inhibition.The PathoPI analyses also revealed a significant increase in effective connectivity between the non-epileptic AHC and dorsal medial prefrontal areas as well as the temporo-parietal junction bilaterally. These regions are known to be part of the DMN, a network subserving an organized mode of brain function being present as a baseline or default state and attenuated during specific goal-directed tasks The epilepsy-induced increase in connectivity between the non-epileptic AHC and DMN nodes is quite challenging to explain. It might be the reflection of the reorganized contribution of the non-epileptic AHC to the multiple networks in which it normally participates. Such reorganization might result from compensatory mechanisms in the presence of unilateral HS, as proposed to explain the enhanced connectivity between the right non-epileptic medial temporal lobe and ipsilateral frontal and temporal areas during a working memory task performed by patients with MTLE from a left-sided HS We investigated the effective connectivity changes between the non-epileptic AHC and the rest of the brain that were common to patients with left and right-sided MTLE. For that purpose, we horizontally flipped the FDG-PET scans of patients with right MTLE to lateralize the epileptogenic zone on the same side (left side) in all patients. Still, functional and anatomical connectivity patterns are not integrally symmetrical in human brain hemispheres . Therefore, the patients did not undergo uniform neuropsychological investigations and it was therefore difficult to correlate our FDG-PET findings with standardized neuropsychological data. Furthermore, no MRI-based atrophy correction of the FDG-PET data was performed in this study, due to the lack of uniform MRI data acquisitions across patients. These issues should be addressed in future prospective FDG-PET studies.This study discloses preserved neurometabolic connectivity between the non-epileptic and the epileptic AHC, significant decrease in effective connectivity between the non-epileptic AHC and limbic areas, as well as significant increase in connectivity with DMN regions. These epilepsy-induced changes in functional integration might represent a neuronal correlate of the cognitive, emotional and decision-making impairments frequently found in patients with unilateral MTLE.


HFE gene, an atypical MHC class I molecule. Recent studies classified HFE/Hfe-HH as a liver disease with the primarily failure in the production of the liver iron hormone hepcidin in hepatocytes. Inadequate hepcidin expression signals for excessive iron absorption from the diet and iron deposition in tissues causing multiple organ damage and failure. This review focuses on the molecular actions of the HFE/Hfe and hepcidin in maintaining systemic iron homeostasis and approaches undertaken so far to combat iron overload in HFE/Hfe-HH. In the light of the recent investigations, novel roles of extra-hepatocytic Hfe are discussed raising a question to the relevance of the multipurpose functions of Hfe for the understanding of HH-associated pathologies.Iron-overload disorders owing to genetic misregulation of iron acquisition are referred to as hereditary hemochromatosis (HH). The most prevalent genetic iron overload disorder in Caucasians is caused by mutations in the  Iron overload disorders comprise a wide range of inherited and acquired disorders of iron metabolism. Hereditary hemochromatosis (HH) encompasses a heterogenous group of inherited iron overload disorders with distinct underlying molecular defects and varying clinical symptoms . HH begiHFE, Transferrin receptor 2  gene (HFE gene was first identified in 1996 as a major histocompatibility (MHC) class I-like gene in which homozygosity for a missense mutation that results in cystein-to-tyrosine substitution at amino acid 282 of human HFE protein (C282Y), was found in vast majority of HH patients (The most common type of hereditary HH is associated with the mutations in the Fe) gene . This isFe) gene . The HFEpatients . Approxipatients . It is lpatients .HFE gene and the identification of the mutations associated with the HFE-HH were spectacular. The wealth of scientific and clinical data led towards more accurate diagnosis of HFE-HH, improved family screening and provided the first insights into the regulation of iron homeostasis by the HFE.The discovery of the HFE gene is a non-classical MHC class I-like protein which contains a signal sequence, peptide-binding extracellular domains, a transmembrane region, and a small cytoplasmic portion. Within the alpha-2 and alpha-3 extracellular domains are the four cysteine residues that form disulfide bridges representing one of the most conserved structural features of MHC class I molecules. HFE interacts with beta2-microglobulin and this association enables efficient transport of HFE to the cell surface where it interacts with TfR1. C282Y mutation disrupts the disulfide bridges in the extracellular domains of the HFE protein thereby preventing the association of HFE with beta2-microglobulin and TfR1. The lack of HFE interaction with TfR1 increases the affinity of TfR1 for the transferrin-bound iron thereby modulating iron absorption. In contrast to the C282Y mutation, mutant H63D HFE formed stable complexes with TfR1 being in line with the clinical data that H63D HFE mutations marginally affect iron absorption and rarely lead to HH.The protein encoded by the Hfe in the duodenal enterocytes    co-receptor Hjv    .It is proposed that Hfe is sequestered by TfR1 protein in an iron-sensing complex located in the hepatocyte cell membrane (igure 1) . The cloigure 1) . In vitrigure 1) . Howevert manner . Importat manner . The Bmpigure 1) . This inigure 1) . The lacoverload . Further or Alk3 , or Smadpression , or for pression  present pression  as its dGiven the fact that hepcidin levels are low in Hfe-HH and that Hfe is involved in optimizing iron-response via Bmp/Smad signaling activity in the liver, it would be expected that by modulating hepcidin levels and/or the activity of the Bmp/Smad pathway, the iron homeostatic parameters in HFE-HH patients may be normalized. In contrast to phlebotomy therapies that manipulate the excess of iron, few pilot approaches were conducted with the aim to target the defective molecular mechanisms underlying the HFE-HH.Hfe-deficient mice resulted in successful reconstitution of hepcidin expression to the levels present in wild type mice. Furthermore, high plasma iron levels present in Hfe-/- mice were significantly reduced by hepcidin treatments, without affecting hepatic iron load which remained inappropriately high in regard to hepcidin levels . Noteworthy, both Hfe and hepcidin are expressed in several extra-hepatic tissues  but so far, little is known whether Hfe-hepcidin regulation is operating in these cells and how it may impact on cellular and/or systemic iron homeostasis . For example, general Hfe deficiency was associated with better tolerance of Hfe-/- mice to severe blood loss in regard to wild type mice or animals kept on an iron-rich diet . However a direct role of Hfe in erythroid cells and its contribution to cellular and overall iron homeostasis remains still to be confirmed.The above data raise a critical question to whether Hfe-HH is an exclusive consequence of defective Bmp/Smad signaling and low hepcidin expression, or whether HH-associated pathologies may be intensified or refractory to iron-depletion strategies or hepcidin substitution if Hfe exerts distinct, ich diet . During eostasis  . Transplantation of Hfe+/+ donor liver into Hfe-/- recipient mice improved hepatic hepcidin expression and reduced the liver iron burden in Hfe-/- mice, being in line with the initial clinical transplantation results (Hfe (Furthermore, Hfe may be involved in controlling iron homeostasis in a n uptake . On the n uptake , suggestpression  and thatse cells  (Figure  results . Surprislts (Hfe .These observations bring up a tantalizing thought whether Hfe is only required for the up regulation of hepcidin in response to elevated iron levels and thus for the maintenance of cellular and systemic iron pool, or the functions of this MHC-class I like molecule may be uncoupled from the control of iron homeostasis. Understanding distinct pathways that Hfe employs beyond the hepatocytes and beyond hepcidin regulation may leap translational medicine research in that some symptoms assigned to HH as a consequence of iron overload may be recognized appropriately and may better be treated by organ-specific therapies rather than systemic iron-depletion.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.


To assess the impact of a simple computer-based decision-support system (computer help) on the quality of nutrition support orders and patients' outcome in Intensive-Care Unit (ICU).TM) to guide physicians' prescriptions according to guidelines. This computer help was displayed in computers previously used for medical orders. Physicians were informed but no systematic protocol was implemented. Patients included during the first (control group) and second period (computer help group) were compared for achievement of nutrition goals and ICU outcomes.This quality-improvement study was carried out in a 16-bed medical-surgical ICU in a French university hospital. All consecutive patients who stayed in ICU more than 10 days with non-oral feeding for more than 5 days were retrospectively included during two 12-month periods. Prescriptions of nutrition support were collected and compared to French national guidelines as a quality-improvement process. A computer help was constructed using a simple Excel-sheet  and nitrogen intake . Incidence of nosocomial infections decreased significantly between the two groups . Mortality did not significantly differ between control (21%) and computer help groups .Use of a widespread inexpensive computer help is associated with significant improvements in nutrition support orders and decreased nosocomial infections in ICU patients. This computer-help is provided in electronic supplement. Malnutrition may concern more than 40% of Intensive-Care-Unit (ICU) patients Compared to French national consensus recommendations TM) providing a simple tool available to all physicians on the workstations dedicated to computer-assisted ordering. This computer help is provided in electronic supplement  approved the design of the study and written consent was waived as for quality-improvement retrospective audit aimed to implement national guidelines.This quality-improvement study took place in the 16-bed medical-surgical ICU of the St Eloi Hospital, a 660-bed teaching and referral facility of the University Hospital of Montpellier in France. This medical-surgical ICU is specialized in gastro-intestinal diseases. All consecutive patients >18 years who stayed more than 10 days in the ICU and had fasted  more than 5 consecutive days were retrospectively included for a quality-improvement audit in nutrition support in two 12-month periods. There were no exclusion criteria. The control period was from June 2004 to May 2005 and the post-quality improvement intervention period was from January to December 2007.TM) was created based on French guidelines for nutrition support of critically ill patients Although a qualified nutritionist is present in the ICU upon request, nutrition support was ordered routinely by the attending physician or by the resident upon supervision by the attending physician. Nutrition support was defined as enteral and/or parenteral nutrition. To help physicians for nutritional calculations, an Excel spreadsheet  The amounts of calories and nitrogen ordered were recorded each day from the first day of stay in ICU (Day-1) until Day-15 in absence of oral feeding, or until the day oral feeding was started without any nutrition support ordering. The Duration of Nutrition Support (DNS) was calculated as the number of days when an enteral, parenteral or mix nutrition support was ordered. For each patient, daily mean non-nitrogen calorie ordering was calculated as the total ordered non-nitrogen calories, i.e. carbohydrate and lipid calories, divided by DNS and by the patient's real weight .The calculation was made separately for enteral and parenteral non-nitrogen ordered calories. Total non-nitrogen ordered calories were calculated as the sum of both enteral and parenteral calories. The same calculation was made for daily mean nitrogen ordering. The mean carbohydrate ratio was calculated as carbohydrate calories divided by total non-nitrogen calories for each day and then expressed as a mean over the DNS for each patient. Data were compared to the French consensus recommendations for nutrition of critically ill patients Contraindications for enteral nutrition (see above) were collected from medical charts from Day-1 to Day-15, as well as any use of a nasogastric tube with postpyloric access or feeding jejunostomy.Organ failure treatments and patients' outcomes were recorded throughout the ICU stay: renal failure as defined by the SOFA score criteria for renal dysfunction or failure 3 or >12000/mm3, fever, worsening oxygenation and a positive culture from bronchoalveolar lavage or protected distal sampling (>104cfu/ml) or from endotracheal aspirate (>106cfu/ml). Bloodstream infection was defined as a recognized pathogen cultured from 1 or more blood cultures (2 or more blood cultures for common skin contaminant). Urinary tract infection was defined as the association of a positive urine culture (>105cfu/ml) and at least one sign with no other cause . Those definitions of infections acquired in the ICU had not been changed throughout the study. Data were independently and prospectively collected by the medical team as part as a regional continuous survey on ICU nosocomial infections  Sud-Est, http://cclin-sudest.chu-lyon.fr).The diagnosis of an ICU-acquired infection was defined as an infection occurring at least 48 h after admission to the ICU Patients included during the first period (control group) were compared to patients included during the second period (computer help group). The primary endpoints were the proportion of patients whose calorie and nitrogen ordering were below 80% of recommended intake. Secondary endpoints were the proportion of patients whose calorie and nitrogen ordering were above 120% of recommended intake, median daily amount of calorie and nitrogen intake ordering, proportion of patients receiving enteral nutrition, carbohydrate ratio, incidence rate of ICU-acquired infections, proportion of patients receiving a RRT and the duration of RRT, length of ICU stay and mortality. The sample size was calculated based on our first audit Qualitative data are expressed as number of events (percent) and continuous data as median and interquartile range. Chi-square or Fisher's tests were used for categorical variables and non-parametric Mann-Whitney test was used for continuous variables. Changes in linear trends of targeted calories and nitrogen intake ordering over time, before and after implementation of the computer help, were estimated by interrupted time-series analysis in units of one month ordering, as previously described Among the 519 patients admitted to the ICU during the first period of the study, 71 met the inclusion criteria, representing 875 study days of nutrition support ordering. Among the 645 patients admitted during the second period of the study, 95 met the inclusion criteria representing 1151 days of nutrition support ordering. There was no significant difference between the control group and the computer-help group regarding patients' characteristics at admission to the ICU . PatientCharacteristics of nutrition support from Day-1 to Day-15 are shown in There was a significant increase between the two groups in calorie intake from 1508 1315–1646] kcal/day to 1793 [1710–2058] kcal/day (p<0.01) and in nitrogen intake from 8 6 kcal/daThe computer help was associated with a significant increase in patients prescribed more than 120% of recommended calorie intake . The 11 patients who were prescribed more than 120% of recommended calorie intake in the computer help group were prescribed a median excess of 549 [478–595] calories or 34 Nutrition ordering was improved over time with the implementation of the computer help. The proportion of orders with a glucose-lipid ratio between 50 and 70% of non protein energy as recommended was significantly higher in the computer help group .Clinical outcome at ICU discharge is showed in The main result of the present study is that a simple widespread and inexpensive computer assistance based on common sense can improve nutrition support prescription, with a 2.5-fold decrease in the proportion of patients who prescribed less than 80% of recommended calorie intake and a 1.5-fold decrease in the proportion of patients who prescribed less than 80% of recommended nitrogen intake. This improvement in nutritional support is associated with a reduction in ICU-acquired infections.There are ample data suggesting that nutrition support recommendations and management protocols improve the achievement of target intakes, associated outcome and cost Computer-assisted prescription has been shown to reduce workload Our system has the advantage of being very simple and does not need specific software, Excel being widespread. Furthermore the file is compatible with the Open Office series spreadsheet  which is free. The recent widespread use of smartphones by primary care team Enteral nutrition delivery was increased in the computer help group even if it remained lower than parenteral nutrition. Two thirds of patients admitted to our unit presented with a digestive pathology  and many had a contraindication to full enteral feeding  or digestive intolerance. Several meta-analyses have compared enteral and parenteral nutrition in critically ill patients In our study an improvement in nutrition prescriptions was associated with fewer cases of ventilator acquired pneumonia and of bacteremia. ICU-acquired infections in general were significantly less frequent, which could be explained by increased calorie intake. In a study of 48 surgical ICU patients, a negative energy balance was associated with more infectious complications, greater length of stay and of mechanical ventilation, from the first week of hospitalization in ICU This study has several limits. First of all, with the “before/after” design, improved nutritional support could have been linked to greater awareness regarding nutrition support in the “after” group. However, this possible Hawthorne effect should be lessened because of the retrospective design of the study. Moreover, the quality of ordering improved over a large period (one year) suggesting that the computer help has been successfully implemented during the 1.5-year period of study interphase. Duration of this interphase was necessary to collect and analyze data from the first baseline phase, as well as to construct the computer help, test, adjust and implement it in a routine use. Secondly, determining caloric targets using predictive equations is imperfect compared to indirect calorimetry Prescribed artificial nutrition intakes are often under recommended goals in critically ill patients. Complexity of prescription formulas could partly explain this observation. This study, conducted in a medical-surgical ICU with an orientation towards digestive pathologies, showed that a simple, widespread and inexpensive program could dramatically increase prescribed patients' energy and protein intake and improve the application of recommendations for artificial nutrition of critically ill patients. This improvement of prescriptions was associated with a decrease in infectious complications.File S1Computer-assisted prescription aid for nutrition. Excel spreadsheet  created to help physicians for nutritional calculations.(XLS)Click here for additional data file.


The modeling of interactions among transcription factors (TFs) and their respective target genes (TGs) into transcriptional regulatory networks is important for the complete understanding of regulation of biological processes. In the case of human TF-TG interactions, there is no database at present that explicitly provides such information even though many databases containing human TF-TG interaction data have been available, such as TRANSFAC  and TREDThe HTRIdb is implemented as a relational database PostgreSQL that is connected to a web interface via the JBOSS AS application server that dynamically generates user-friendly HTML front-end queries using the Apache Tomcat web server. For the visualization of TF-TG interactions, we embedded in the HTRIdb the Cytoscape Web.The HTRIdb offers several mechanisms of data query and extraction, such as download in spreadsheet or text format and the visualization of TF-TG interactions. There is an update mechanism that allows scientists to send new data. The HTRIdb currently holds a collection of 2,114 unique transcriptional regulation interactions among 163 TFs and 1,034 TGs.HTRIdb is a powerful user-friendly tool from which human TF-TG interactions can be easily extracted.