Inhibitory Substrate Research Articles

CRISPR-Cas "Non-Target" Sites Inhibit On-Target Cutting Rates.

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

The stem cell marker Prom1 promotes axon regeneration by down-regulating cholesterol synthesis via Smad signaling

Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identify Prom1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program. Prom1 expression is developmentally down-regulated, and the genetic deletion of Prom1 in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role of Prom1 in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate. Prom1 overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-β receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest that Prom1 and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.

Biology of Tissue Inhibitor of Metalloproteinase 3 (TIMP3), and Its Therapeutic Implications in Cardiovascular Pathology.

Tissue inhibitor of metalloproteinase 3 (TIMP3) is unique among the four TIMPs due to its extracellular matrix (ECM)-binding property and broad range of inhibitory substrates that includes matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAM with thrombospondin motifs (ADAMTSs). In addition to its metalloproteinase-inhibitory function, TIMP3 can interact with proteins in the extracellular space resulting in its multifarious functions. TIMP3 mRNA has a long 3’ untranslated region (UTR) which is a target for numerous microRNAs. TIMP3 levels are reduced in various cardiovascular diseases, and studies have shown that TIMP3 replenishment ameliorates the disease, suggesting a therapeutic potential for TIMP3 in cardiovascular diseases. While significant efforts have been made in identifying the effector targets of TIMP3, the regulatory mechanism for the expression of this multi-functional TIMP has been less explored. Here, we provide an overview of TIMP3 gene structure, transcriptional and post-transcriptional regulators (transcription factors and microRNAs), protein structure and partners, its role in cardiovascular pathology and its application as therapy, while also drawing reference from TIMP3 function in other diseases.

Tumor Cell Membrane-Based Peptide Delivery System Targeting to Tumor Microenvironment for Cancer Immunotherapy and Diagnosis

\The development of effective delivery system for peptides targeting to the tumor microenvironment has always been a hot topic in the field of cancer diagnosis and therapy. A multifunctional delivery system by encapsulating superparamagnetic iron oxide nanoparticles (SPIO NPs) with tumor cell membrane obtained by hypotonic lysis followed by mechanical fragmentation was constructed to effectively deliver therapeutic peptides. SPIO nanoparticles were encapsulated with H460 lung cancer cell membranes (SPIO NP@M) and peptides consisted of PD-L1 inhibitory peptide (TPP-1) and MMP2 substrate peptide (PLGLLG) was conjugated to H460 membrane (SPIO NP@M-P). The abilities of homologous targeting, cytotoxicity, pharmacokinetics, and tumor targeting ability of SPIO NP@M-P were evaluated. TPP-1 peptide was delivered and released to the tumor microenvironment through the homotypic effect of tumor cell membrane and specific digestion by the tumor specific enzyme, MMP2. The newly developed delivery system (SPIO NP@M-P) for PD-L1 inhibitory peptide could effectively extend the half-life of the peptides (60 times longer) and meanwhile maintain the ability to re-activate T cell and inhibit the tumor growth in vitro and in vivo. Furthermore, SPIO NPs in the system could be used as a tumor imaging agent and thus indicate the effect of peptide treatment. The SPIO NP@M might provide a promising theranostic platform for therapeutic peptide application in cancer therapy.

Effects of conductive carbon materials on dry anaerobic digestion of sewage sludge: Process and mechanism

Dry anaerobic digestion of sewage sludge (SS-DAD) is often inhibited by excessive acidification due to low water content and high organic loading. The effects of conductive carbon materials including powdered activated carbon (PAC) and powdered graphite (PG) on SS-DAD under mesophilic condition (35℃) were investigated. The results demonstrated that the addition of PAC increased methane production of SS-DAD. The methane yield of PAC50% reactor (dosage of PAC is 50% of the volatile solids) amounted to 210 mL·gVSadded−1, which is 49% higher than that of control. PAC addition significantly enhanced the biodegradation process, as the reduction rate of total solids (TS) and volatile solids (VS) were increased by 36.4% and 34.1%, respectively, compared to the control. Inhibitory substrate adsorption experiments showed that PAC has significant adsorption (13.6 mg g−1) for VFAs, while PG showed almost no adsorption (0.81 mg g−1). Microbial community structure analysis showed hydrogenotrophic methanogens (Methanobrevibacter and Methanosphaera) were reduced in the PAC50% reactor, while methanogens (Methanobacterium) which can also use formate as electron donor were increased. PAC amendment reshaped the microbial community in the SS-DAD system which may result in shifting of the major electron carrier from hydrogen to formate and increasing electron transfer efficiency of the SS-DAD system.

Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism

Lactate oxidase (LOx), recognized to selectively catalyze the lactate oxidation in complex matrices, has been highlighted as preferable biorecognition element for the development of lactate biosensors. In a previous work, we have demonstrated that LOx crosslinking on a modified screen-printed electrode results in a dual range lactate biosensor, with one of the analysis linear range (4 to 50 mM) compatible with lactate sweat levels. It was advanced that such behavior results from an atypical substrate inhibition process. To understand such inhibition phenomena, this work relies in the study of LOx structure when submitted to increased substrate concentrations. The results found by fluorescence spectroscopy and dynamic light scattering of LOx solutions, evidenced conformational changes of the enzyme, occurring in presence of inhibitory substrate concentrations. Therefore, the inhibition behavior found at the biosensor, is an outcome of LOx structural alterations as result of a pH-dependent mechanism promoted at high substrate concentrations.

10-Phenyltriazoyl Artemisinin is a Novel P-glycoprotein Inhibitor that Suppresses the Overexpression and Function of P-glycoprotein.

The effect of drugs on ATP-binding cassette transporters, especially permeabilityglycoprotein (P-gp), is an important consideration during new anti-cancer drug development. In this context, the effects of a newly synthesized artemisinin derivative, 10-(4-phenyl-1H-1,2,3- triazol)-artemisinin (5a), were evaluated on P-gp expression and function. Reverse transcript polymerase chain reaction and immunoblotting techniques were used to determine the effect of 5a on P-gp expression in LS174T cells. In addition, the ability of 5a to work as either a substrate or an inhibitor of P-gp was investigated through different methods. The results revealed that 5a acts as a novel P-gp inhibitor that dually suppresses the overexpression and function of P-glycoprotein. Co-treatment of LS174T cell line, human colon adenocarcinoma cell line, with 5a and paclitaxel recovered the anticancer effect of paclitaxel by controlling the acquired drug resistance pathway. The overexpression of P-gp induced by rifampin and paclitaxel in a colorectal cell line was suppressed by 5a which could be a novel inhibitory substrate inhibiting the transport of paclitaxel by P-gp. The results revealed that 5a can be classified as a type B P-gp inhibitor (with both substrate and inhibitor activities) with an additional function of suppressing P-gp overexpression. The results might be clinically useful in the development of anticancer drugs against cancers with multidrug resistance.

A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons.

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.

Inhibition Mechanisms of Human Indoleamine 2,3 Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O2 binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

Selecting the Best: Evolutionary Engineering of Chemical Production in Microbes.

Microbial cell factories have proven to be an economical means of production for many bulk, specialty, and fine chemical products. However, we still lack both a holistic understanding of organism physiology and the ability to predictively tune enzyme activities in vivo, thus slowing down rational engineering of industrially relevant strains. An alternative concept to rational engineering is to use evolution as the driving force to select for desired changes, an approach often described as evolutionary engineering. In evolutionary engineering, in vivo selections for a desired phenotype are combined with either generation of spontaneous mutations or some form of targeted or random mutagenesis. Evolutionary engineering has been used to successfully engineer easily selectable phenotypes, such as utilization of a suboptimal nutrient source or tolerance to inhibitory substrates or products. In this review, we focus primarily on a more challenging problem—the use of evolutionary engineering for improving the production of chemicals in microbes directly. We describe recent developments in evolutionary engineering strategies, in general, and discuss, in detail, case studies where production of a chemical has been successfully achieved through evolutionary engineering by coupling production to cellular growth.

α-Tubulin Acetyltransferase Is a Novel Target Mediating Neurite Growth Inhibitory Effects of Chondroitin Sulfate Proteoglycans and Myelin-Associated Glycoprotein.

Damage to the CNS results in neuronal and axonal degeneration, and subsequent neurological dysfunction. Endogenous repair in the CNS is impeded by inhibitory chemical and physical barriers, such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated glycoprotein (MAG), which prevent axon regeneration. Previously, it has been demonstrated that the inhibition of axonal histone deacetylase-6 (HDAC6) can promote microtubule α-tubulin acetylation and restore the growth of CSPGs- and MAG-inhibited axons. Since the acetylation of α-tubulin is regulated by two opposing enzymes, HDAC6 (deacetylation) and α-tubulin acetyltransferase-1 (αTAT1; acetylation), we have investigated the regulation of these enzymes downstream of a growth inhibitory signal. Our findings show that exposure of primary mouse cortical neurons to soluble CSPGs and MAG substrates cause an acute and RhoA-kinase-dependent reduction in α-tubulin acetylation and αTAT1 protein levels, without changes to either HDAC6 levels or HDAC6 activity. The CSPGs- and MAG-induced reduction in αTAT1 occurs primarily in the distal and middle regions of neurites and reconstitution of αTAT1, either by Rho-associated kinase (ROCK) inhibition or lentiviral-mediated αTAT1 overexpression, can restore neurite growth. Lastly, we demonstrate that CSPGs and MAG signaling decreases αTAT1 levels posttranscriptionally via a ROCK-dependent increase in αTAT1 protein turnover. Together, these findings define αTAT1 as a novel potential therapeutic target for ameliorating CNS injury characterized by growth inhibitory substrates that are prohibitive to axonal regeneration.

Protamine neutralizes chondroitin sulfate proteoglycan-mediated inhibition of oligodendrocyte differentiation.

Chondroitin sulfate proteoglycans (CSPGs), which are enriched in demyelinating plaques in neurodegenerative diseases, such as multiple sclerosis (MS), impair remyelination by inhibiting the migration and differentiation of oligodendrocyte precursor cells (OPCs) in the central nervous system (CNS). We herein show that protamine (PRM, also known as a heparin antagonist) effectively neutralizes the inhibitory activities of CSPGs, thereby enhancing OPC differentiation and (re)myelination in mice. Cell-based assays using mouse OPC-like OL1 cells revealed that the PRM treatment exerted masking effects on extracellular CSPGs and improved oligodendrocyte differentiation on inhibitory CSPG-coated substrates. PRM also bound to the extracellular region of protein tyrosine phosphatase receptor type Z (PTPRZ), a membrane-spanning CSPG predominantly expressed in OPCs, and functioned as a ligand mimetic of PTPRZ, thereby suppressing its negative regulatory activity on oligodendrocyte differentiation. In primary cultures, the differentiation of OPCs from wild-type and Ptprz-deficient mice was equally enhanced by PRM. Moreover, the intranasal administration of PRM accelerated myelination in the developing mouse brain, and its intracerebroventricular administration stimulated remyelination after cuprizone-induced demyelination. These results indicate that PRM has CSPG-neutralizing activity which promotes oligodendrocyte differentiation under developmental and morbid conditions.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

BackgroundThere is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.Methodology/Principal findingsTo determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3’UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Conclusion/SignificanceSome intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Biodegradation of anthracene by a newly isolated bacterial strain, Bacillus thuringiensis AT.ISM.1, isolated from a fly ash deposition site.

Polycyclic aromatic hydrocarbons (PAHs) are recognized as significant health risks and consequently listed as priority pollutants by environmental protection agencies across the globe. The aim of the present study was to degrade one of the important PAHs, anthracene, by a newly isolated Bacillus thuringiensis strain. This is the first report of anthracene degradation by B. thuringiensis. This is also the very first growth kinetic study of a bacteria in an anthracene-containing medium. Some diphenol metabolites were found for the first time as anthracene biodegradation by-products, which can be an indication towards a new pathway.

Markers of nitric oxide are associated with sepsis severity: an observational study

BackgroundNitric oxide (NO) regulates processes involved in sepsis progression, including vascular function and pathogen defense. Direct NO measurement in patients is unfeasible because of its short half-life. Surrogate markers for NO bioavailability are substrates of NO generating synthase (NOS): L-arginine (lArg) and homoarginine (hArg) together with the inhibitory competitive substrate asymmetric dimethylarginine (ADMA). In immune cells ADMA is cleaved by dimethylarginine-dimethylaminohydrolase-2 (DDAH2). The aim of this study was to investigate whether concentrations of surrogate markers for NO bioavailability are associated with sepsis severity.MethodThis single-center, prospective study involved 25 controls and 100 patients with surgical trauma (n = 20), sepsis (n = 63), or septic shock (n = 17) according to the Sepsis-3 definition. Plasma lArg, hArg, and ADMA concentrations were measured by mass spectrometry and peripheral blood mononuclear cells (PBMCs) were analyzed for DDAH2 expression.ResultslArg concentrations did not differ between groups. Median (IQR) hArg concentrations were significantly lower in patient groups than controls, being 1.89 (1.30–2.29) μmol/L (P < 0.01), with the greatest difference in the septic shock group, being 0.74 (0.36–1.44) μmol/L. In contrast median ADMA concentrations were significantly higher in patient groups compared to controls, being 0.57 (0.46–0.65) μmol/L (P < 0.01), with the highest levels in the septic shock group, being 0.89 (0.56–1.39) μmol/L. The ratio of hArg:ADMA was inversely correlated with disease severity as determined by the Sequential Organ Failure Assessment (SOFA) score. Receiver-operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for the hArg:ADMA ratio compared to the SOFA score. DDAH2 expression was lower in patients than controls and lowest in the subgroup of patients with increasing SOFA.ConclusionsIn patients with sepsis, plasma hArg concentrations are decreased and ADMA concentrations are increased. Both metabolites affect NO metabolism and our findings suggest reduced NO bioavailability in sepsis. In addition, reduced expression of DDAH2 in immune cells was observed and may not only contribute to blunted NO signaling but also to subsequent impaired pathogen defense.

58. Focusing on the Significance of, and the Mechanisms Underlying, Cannabis-Induced Psychosis

Overall Abstract: Dr Marta Di Forti Cannabis use across Europe and its interaction with the polygenic risk score for schizophrenia. Dr Di Forti examined the pattern of cannabis use across 16 European sites as part of the EU-GEI study. She will show striking differences in incidence of psychosis (high in North and low in South), and how differences in pattern of cannabis use result in differences in the proportion of new cases of psychosis attributable to cannabis use across Europe (highest in London and Amsterdam). Dr Di Forti will examine the effects of cannabis use on incidence of psychosis, and show how cannabis use interacts with the Schizophrenia Polygenic Risk Score (in 1200 cases and similar number of controls). Dr David Lewis Adolescent Cannabis Use, Cortical Circuitry and Schizophrenia Dr Lewis studied both adolescent monkeys and boys. In adolescent monkeys, repeated exposure to Δ9-tetrahydrocannabinol (THC) blunted the trajectory of accuracy improvements in spatial working memory. These effects were more marked and more persistent in male than in female monkeys. In a longitudinal human study, 1006 boys were followed regularly through their adolescence. For each year that they engaged in regular marijuana use, their expected odds of experiencing subsequent subclinical paranoia or hallucinations rose by 133% and 92%, respectively. Dr Steve Laviolette The Molecular and Neuronal Effects of Adolescent THC Exposure in Mesocorticolimbic Circuitry. Dr Laviolette has discovered that neurodevelopmental exposure to THC dramatically dysregulates dopamine transmission in the mesocorticolimbic circuit, inducing a hyper-DAergic phenotype into adulthood; schizophrenia-like abnormalities are found in affective and cognitive processes and schizophrenia-like molecular adaptations in the prefrontal cortex (PFC) and nucleus accumbens involving the GSK-3 pathway. Developmental THC exposure leads to deficits in GABAergic inhibitory substrates within the PFC and a loss of control over subcortical DAergic activity states. Dr Anissa Abi Dargham Chronic cannabis use is associated with abnormal dopaminergic function. Dr Abi Dargham measured amphetamine induced dopamine release with PET and D2 radiotracers in striatal subdivisions in multiple cohorts of patients with schizophrenia, patients with schizophrenia and comorbid substance use disorders, in patients with cannabis use, and in controls matched to each cohort. Her studies show that substance use disorders, and cannabis use, result in blunting of dopamine release. In addition, patients with schizophrenia exposed to chronic substance use show excess responsivity of the D2 system associated with psychosis, despite low presynaptic storage capacity. These findings will be discussed and compared to address the contribution of cannabis use to the dopaminergic dysfunction in schizophrenia.

C3 transferase gene therapy for continuous conditional RhoA inhibition

Regrowth inhibitory molecules prevent axon regeneration in the adult mammalian central nervous system (CNS). RhoA, a small GTPase in the Rho family, is a key intracellular switch that mediates the effects of these extracellular regrowth inhibitors. The bacterial enzyme C3-ADP ribosyltransferase (C3) selectively and irreversibly inhibits the activation of RhoA and stimulates axon outgrowth and regeneration. However, effective intracellular delivery of the C3 protein in vivo is limited by poor cell permeability and a short duration of action. To address this, we have developed a gene therapy approach using viral vectors to introduce the C3 gene into neurons or neuronal progenitors. Our vectors deliver C3 in a cell-autonomous (endogenous) or a cell-nonautonomous (secretable/permeable) fashion and promote in vitro process outgrowth on inhibitory chondroitin sulfate proteoglycan substrate. Further conditional control of our vectors was achieved via the addition of a Tet-On system, which allows for transcriptional control with doxycycline administration. These vectors will be crucial tools for promoting continued axonal regeneration after CNS injuries or neurodegenerative diseases.

Numerical modelling of anaerobic digestion reactor dynamics with a delay

Anaerobic digestion of municipal and animal wastes provides a potentially important means of producing energy sustainably. The reliable production of methane from anaerobic digestion requires that bacteria metabolise municipal and agricultural wastes to produce mainly carbon dioxide and methane. Our model uses Monod based kinetics and is complex since the biomass concentration growth is inhibited with the increase in substrate. The production of methane depends on the number of methanogenic bacteria; there is evidence from previous chemostats experiments that there is a time delay in population number of these cells to respond to changes in substrate concentrations. In this article we focus on determining the dynamical response of the methane production rate under different dynamics of feed conditions and temperatures. Since there is a delay in the methane bacteria mass response, we include the effects of the delay on the dynamics of the system based on modification of kinetics in a simple fermentation anaerobic model. We compare the dynamics of the system with and without delay to show the contribution of delay to the overall stability of the system. References O. Bernard, Z. Hadj-Sadok, D. Dochain, A. Genovesi, J. P. Steyer. Dynamical model development and parameter identification for an anaerobic wastewater treament process. Biotechnol. Bioeng. 75:424–438, 2001. doi:10.1002/bit.10036 T. F. Thingstad and T. I. Langeland. Dynamics of chemostat culture: The effect of a delay in cell response. J. Theor. Biol. , 48:149–159, 1974. doi:10.1016/0022-5193(74)90186-6 J. R. Beddington and R. M. May. Time delays are not nessarily destabilizing. Math. Biosci. 27:109–119, 1975. doi:10.1016/0025-5564(75)90028-0 K. Y. San and G. Stephanopoulos. The effect of growth rate delays in substrate-inhibited kinetics on the optimal profile of fed-batch reactors. J. Theor. Biol. 28:356–361, 1986. doi:10.1002/bit.260280308 G. Stephanopoulos and K. Y. San. Studies on-line bioreactor identification. I. Theory. Biotechnol. Bioeng. 26:1176–1188, 1984. doi:10.1002/bit.260261006 A. A. Khan, A. J. Stacey and J. J. Shepherd. Optimization of methane output for an anaerobic waste digester. ANZIAM J. 54:C523–C539, 2013. http://journal.austms.org.au/ojs/index.php/ANZIAMJ/article/view/6322 J. F. Andrews. A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates. Biotechnol. Bioeng. 10:707–723, 1968. doi:10.1002/bit.260100602 D. J. Batstone, J. Keller, I. Angelidaki, S. V. Kalyuzzhni, S. G. Pavlostathis, A. Rozzi, W. T. M. Sanders, H. Siegrist, and V. A. Vavilin. Anaerobic digestion model No. 1 (ADM1). Water Sci. Technol. 45(10):65–73, 2002 http://wst.iwaponline.com/content/45/10/65 K. J. Beers. Numerical methods for chemical engineering: Applications in Matlab . Cambridge, New York, USA, 2007. http://www.cambridge.org/us/academic/subjects/engineering/chemical-engineering/numerical-methods-chemical-engineering-applications-matlab J. F. Andrews. The development of a dynamic model and control strategies for the anaerobic digestion process. In Mathematical Models in Water Pollution Control , A. James (ed). Wiley, 1978. A. W. Bush and A. E. Cook. The effect of time delay and growth rate inhibition in the bacterial treatment of wastewater J. Theor. Biol. 63:385–395, 1976. doi:10.1016/0022-5193(76)90041-2 L. F. Shampine, I. Gladwell and S. Thompson. Solving ODEs with Matlab . Cambridge, New York, USA, 2003. http://www.cambridge.org/us/academic/subjects/mathematics/numerical-analysis/solving-odes-matlab?format=PB

Lentiviral Delivery of miR-133b Improves Functional Recovery After Spinal Cord Injury in Mice.

Based on the observation that microRNA (miRNA) 133b enhances regeneration after spinal cord injury in the adult zebrafish, we investigated whether this miRNA would be beneficial in a mammalian system in vitro and in vivo. We found that infection of cultured neurons with miR-133b promotes neurite outgrowth in vitro on an inhibitory substrate consisting of mixed chondroitin sulfate proteoglycans, when compared to infection with green fluorescent protein (GFP) for control. In vivo, viral infection of the injured adult mouse spinal cord at the time of injury at and in the vicinity of the lesion site enhanced expression of miR-133b. Measurements of locomotor recovery by Basso Mouse Scale (BMS) showed improvement of recovery starting at 4weeks after injury and virus injection. This improvement was associated with downregulation of the expression levels of Ras homolog gene family member A (RhoA), chondroitin sulfate proteoglycans, and microglia/macrophage marker in the spinal cord as assayed 6weeks after injury. Potential inhibitory molecules carrying consensus sequences for binding of miR-133b were identified in silico and verified in a reporter assay in vitro showing reductions in expression of RhoA, xylosyltransferase 1 (Xylt1), ephrin receptor A7 (Epha7), and purinergic receptor P2X ligand-gated ion channel 4 (P2RX4). These results encourage targeting miR-133 for therapy.

Expressing Constitutively Active Rheb in Adult Dorsal Root Ganglion Neurons Enhances the Integration of Sensory Axons that Regenerate Across a Chondroitinase-Treated Dorsal Root Entry Zone Following Dorsal Root Crush.

While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the injection. ChABC-mediated regeneration is functional, as stimulation of ipsilateral median and ulnar nerves induced neuronal c-Fos expression in deafferented dorsal horn in both ChABC groups. Interestingly, caRheb + ChABC animals had significantly more c-Fos+ nuclei indicating that caRheb expression in DRGs promoted functional synaptogenesis of their axons that regenerated beyond a ChABC-treated DREZ.