Meckel diverticulum is the most common congenital defect of the gastrointestinal tract. It can be asymptomatic or mimic appendicitis and may be complicated by bleeding, diverticulitis, obstruction, and, rarely, neoplasia. We report the first case of extranodal marginal zone lymphoma occupying a Meckel diverticulum. A 44-year-old man with history of colonic diverticulitis presented to the emergency department for evaluation of acute abdominal pain. Radiography showed enteric obstruction, prompting diagnostic laparoscopy. Above the level of mid-ileum an intact Meckel diverticulum was identified. Microscopy showed extensive infiltration of sheets of small lymphocytes with abundant cytoplasm (monocytoid B-cells) prominently in submucosa and focally transmural involving serosal adipose tissue with multiple reactive germinal centers. The immunostains showed positivity for CD20, BCL-2, and CD43 (weak) and negativity for CD3, CD5, BCL-1, CD10, and BCL-6 in monocytoid B-cells. Fluorescence in situ hybridization studies revealed API2-MALT1 fusion signals consistent with t, which confirmed the diagnosis of extranodal marginal zone lymphoma, also known as mucosa associated lymphoid tissue lymphoma. Meckel diverticulum (MD) is the most common congenital defect of the gastrointestinal (GI) tract. It is a part of vitelline duct, which connects the growing fetus with the yolk sac. When the vitelline duct is not fully absorbed, a MD develops in the lower part of small intestine. Histologically, it is a true diverticulum, containing all tunicae of GI tract and may or may not contain ectopic gastric or pancreatic epithelium. MD can be asymptomatic or mimic appendicitis clinically or may be complicated by bleeding, diverticulitis, obstruction, perforation, and, rarely, neoplasia . Maligna In situ hybridization (ISH)-kappa and lambda showed scattered polyclonal plasma cells and no monoclonal B-cell population was detected. In addition, the proliferation index by ki-67 was less than 5%. The overall morphologic and immunophenotypic features were consistent with ENMZL, low grade. Finally, the fluorescence in situ hybridization (FISH) studies revealed MALT1 gene rearrangement signals pattern in 45.6% of the cells analyzed and was followed by positive API2-MALT1 fusion signals consistent with a reciprocal t, in 56.3% of examined cells, confirming the diagnosis  scan with contrast showed small bowel loops distention up to 4.3 cm in diameter and edema with transition point in the mid-lower abdomen concerning moderate enteric obstruction . Furtheriagnosis . The pat API2 (apoptosis inhibitor)gene on 11q21, to the MALT1 gene on 18q21. However, the frequency of API2/MALT1 fusion gene varies among MALT lymphomas originating from different anatomical sites, and little is known about the small bowel MALT lymphoma. The other common chromosomal aberrations include t, t, and t involving BCL10, MALT1, and FOXP1, respectively [ Campylobacter jejuni as a specific pathogen and most commonly involved small intestine [Marginal zone lymphoma (MZL) accounts for 5–17% of all non-Hodgkin lymphomas and are classified into three categories: (1) ENMZL of mucosa associated lymphoid tissue (MALT) type which is the most common group, (2) nodal MZL, and (3) splenic MZL . MALT lyectively . The molectively . IPSID antestine , 21. Ano tp;q32, t(1ntestine , 24. Extntestine . At presntestine , 13. Forntestine , 25, 26.


Leishmania spp. and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae, and are both obligate intracellular parasites that manipulate host signaling pathways and the innate immune system to establish infection. Mitogen-activated protein kinases (MAPKs) are serine and threonine protein kinases that are highly conserved in eukaryotes, and are involved in signal transduction pathways that modulate physiological and pathophysiological cell responses. This mini-review highlights existing knowledge concerning the mechanisms that Leishmania spp. and T. cruzi have evolved to target the host’s MAPK signaling pathways and highjack the immune response, and, in this manner, promote parasite maintenance in the host. Leishmania spp. and Trypanosomacruzi are protozoan parasites of the Trypanosomatida order  and Trypanosomatidae family . They are the etiological agents of leishmaniasis and Chagas disease, respectively, and are transmitted by the bite of infected sandflies (leishmaniasis) or through triatomine bug feces (Chagas disease). Both Leishmania spp. and T. cruzi have complex life cycles comprising diverse developing forms that alternate between the insect vector and the vertebrate host. Leishmania spp. promastigotes and amastigotes preferentially infect phagocytic cells of vertebrates, while T. cruzi metacyclic trypomastigotes, blood trypomastigotes and amastigotes are able to infect both phagocytic and non-phagocytic cells  signaling pathways—more specifically, extracellular-signal-regulated kinase (ERK), and p38 MAPK—that are highly relevant in the context of the regulation of the immune response against intracellular parasites.Although the persistence of Mitogen-activated protein kinases are protein kinases that phosphorylate their own dual serine and threonine residues (autophosphorylation), or those found on their substrates, to activate or de-activate their target . AccordiFigure 1): the ERK1/2, the c-JUN N-terminal kinase 1, 2 and 3 (JNK1/2/3), and the p38 MAPK α, β, δ, and γ pathways. ERK, JNK, and p38 isoforms are grouped according to their activation motif, structure and function .There are three well-known MAPK pathways in mammalian cells .The classical activation of ERK1 and ERK2 isoforms is initiated by the binding of a ligand to a receptor tyrosine kinase (RTK) at the plasma membrane (PM), followed by activation of the small G-protein, Ras. In turn, Ras recruits and activates the serine/threonine protein kinase, Raf, a MAP3K, which activates the MAP2K, MEK, that, in turn, phosphorylates the MAPK, ERK1/2, at both threonine and tyrosine residues within the TEY motif . The RasFigure 1B). The p38 MAPKs α, β, δ and γ, are dephosphorylated by several dual-specificity protein phosphatases, such as MPK2/4, that can also deactivate ERK. The phosphatases, MPK5/7, can also dephosphorylate JNK and p38, while MPK1 exhibits a higher specificity for p38 , factor B (gp58) and molecules that accelerate the decay of the C3 pathway. In contrast, Leishmania spp. adapt their membrane to prevent the insertion of C5b-C9. This species also produces a surface metalloproteinase, gp63, and a lipophosphoglycan (LPG) that cleave C3b, abrogating complement-mediated lysis  to replicate inside host macrophages  an intracellular stage in their life cycle, allowing them protection against humoral anti-leishmanicidal products; (ii) the suppression of the synthesis of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI); (iii) the inhibition of antigen presentation by repressing the gene expression of major histocompatibility complex (MHC) class II, interfering with antigen loading, or by sequestering/highjacking the MHC II molecule or antigen; (iv) the subversion of host cellular signaling pathways such as STAT, PI3K/AKT, and MAPK; and (v) the modulation of host cytokines to avoid T cell differentiation and thus prevent the formation of a Th1-type immune response  and IL-10 (ERK 1/2) in macrophages and dendritic cells. By this means, Leishmania spp. promote switching from IL-12 to IL-10 production, consequently altering the formation of a Th1 response to Th2, and leading to parasite prevalence in the host  and downregulation of MKP-3 (ERK1/2 high affinity phosphatase) to skew CD40 signaling toward the ERK1/2 pathway, favoring infection. Figure 2). L. amazonensis infection. L. amazonensis upregulated ERK1/2 in dendritic cells, increased IL-10 production and prevented the expression of CD40 and IL-12p40 (one of the subunits of IL-12), leading to the limited activation of dendritic cells and a deficient Th1-type response.of IL-12  by the aof IL-12 . In theiLeishmania spp., associated with parasite persistence and IL-12 down-regulation, was also seen for L. amazonensis  isolated from Mycobacterium smegmatis, activated p38 MAPK with a concomitant abrogation of ERK1/2 phosphorylation. Furthermore, the production of IL-12 , a p38 regulator .Gp63 and LPG can also modulate the host MAPK signaling pathway and the production of cytokines. Gp63 acts on both p38 and the phosphotyrosine phosphatase SHP-1. To subvert MAPK signaling, gp63 of egulator . Gp63 ofgulation . Gp63-meof IL-10 . FurtherT. cruzi evades the host’s immune response by acting on the MAPK pathway have been poorly studied and are therefore incompletely understood. However, it is well established that upon cell invasion, T. cruzi begins to subvert signaling pathways and to use host molecules to favor its entry and survival inside host cells. For instance, T. cruzi extracellular amastigotes (EAs) recruit both host protein kinase D1 (PKD1) and cortactin to induce PKD1 autophosphorylation and cortactin activation by ERK, leading to the recruitment of host actin that allows parasite entry into HeLa cells  ERK1/2, but not p38 MAPK, activation in macrophages and dendritic cells , such as TLR2, TLR4, or TLR9, in dendritic cells and macrophages (T. cruzi antigens (TcAgs) and TLR ligands induced p38 phosphorylation that was dependent on TcAg-macrophage migration inhibitory factor (MIF) synergism. This led to the enhancement of IL-12 production, thus promoting a Th1-type response -anchored mucins and trans-sialidases (TS). AgC10, a GPI-anchored mucin of 40–50 kDa, inhibits TNF and IL-12 secretion in a p38 MAPK inhibition-dependent manner, impairing the formation of a Th1 response . However, despite current knowledge concerning the strategies used by T. cruzi to subvert the host’s immune response, the precise mechanisms by which this occurs remain unknown.Other proteins related to immune modulation in response . Ropert response . T. cruztivation . RecentlLeishmania spp. skew the MAPK signaling pathway to subvert cytokine production and switch a Th1 to a Th2 response are well known compared to those for T. cruzi. Leishmania parasites often target the CD40/MAPK pathway, activating ERK1/2 to increase and decrease IL-10 and IL-12 production, respectively. In comparison, the strategy used by T. cruzi is to trigger molecules to subvert MAPK ERK1/2 and p38 pathways and thus modulate cytokine production. Further studies are required to increase our understanding of the intriguing mechanisms by which T. cruzi manipulates the host’s immune response.The MAPK signaling pathway, responsible for regulating the production of Th1-and Th2-type responses, is targeted by trypanosomatids to modulate the host’s immune response in order to favor parasite replication and survival. The mechanisms whereby MS-S, FD and GG contributed equally to the writing of the review. DB conceived and wrote the review.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.


IDH1/NPM1/NRAS mutations. In conclusion, asparaginase-containing regimens, which are approved for treatment of acute lymphoblastic leukemia (ALL) but not AML, can be used to treat patients with AML who do not accept blood transfusion.Treatment of patients with acute myeloid leukemia (AML) who do not wish to accept blood product transfusion, including Jehovah’s Witnesses, is extremely challenging. The use of conventional chemotherapy for induction of complete remission (CR) results in profound anemia and thrombocytopenia requiring frequent transfusions of blood products, without which such treatment will be life-threatening. Finding a well tolerable, minimally myelosuppressive induction regimen for such patients with AML is a clear example of area of unmet medical need. Here, we report a successful treatment of a 52-year-old Jehovah’s Witness with newly diagnosed AML with peg-asparaginase, vincristine and methylprednisolone. The AML was characterized with normal karyotype, and mutations in isocitrate dehydrogenase 1 (IDH1-Arg132Ser), nucleophosmin 1 (NPM1-Trp289Cysfs*12) and neuroblastoma RAS viral oncogene homolog (NRAS-G1y12Va1). After one 28-day cycle of treatment, the patient achieved complete remission with incomplete count recovery (CRi) and after the second cycle, he achieved CR with full blood count recovery. The patient has never received any blood products. Notwithstanding that myeloperoxidase-induced oxidative degradation of vincristine results in its lack of activity as monotherapy in AML, its combination with corticosteroid and asparaginase has resulted in a robust remission in this patient. Diminished steroid clearance by asparaginase activity as well as reduction in serum glutamine level induced by glutaminase enzymatic activity of asparaginase may have contributed to effective killing of the myeloblasts that carry  Treatment of individuals who do not accept transfusion of blood products, including Jehovah’s Witnesses, with acute myeloid leukemia (AML) is exceptionally challenging ,2,3. ConAsparaginase products, which are approved for treatment of acute lymphoblastic leukemia (ALL) ,8,9, but9/L with 57% peripheral blast, hemoglobin 6.2 g/dL, platelet 64 × 109/L. Bone marrow aspiration and biopsy confirmed the diagnosis of AML  7.3 × 10s of AML A,B.IDH1-Arg132Ser, nucleotide change: c.394C > A, mutation level: 50%), nucleophosmin 1  and neuroblastoma RAS viral oncogene homolog . Due to religious belief, the patient did not accept transfusion of red blood cells, platelets or other blood products but wished to proceed with treatment. After a comprehensive discussion of risks and benefits with the patient and his family, a minimally myelosuppressive treatment with peg-asparaginase (2500 units/m2 intravenously on day 8), vincristine  and methylprednisolone (20 mg/m2 twice daily on days 1–28) was initiated [Immunophenotypic studies by flow cytometry on the bone marrow specimen showed 60%–70% blasts expressing CD117 (heterogeneous), CD33, CD13, CD15 , CD11b (heterogeneous), CD11c, CD38, CD64 , CD71 , and myeloperoxidase. The blasts were negative for CD34, HLA-DR, CD14, CD16, CD56, TdT, and all T- and B-lymphoid antigens. Chromosome analysis revealed 46XY in 20 metaphase cells. Mutational analysis of the DNA from bone marrow blasts revealed mutations in isocitrate dehydrogenase 1  as well as left lower extremity edema. The duplex ultrasound revealed acute thrombus in the right distal subclavian vein, right axillary vein and right brachial vein as well as acute, occlusive deep venous thrombosis (DVT) of the paired left peroneal veins  without extension proximally into the left popliteal vein or the more-proximal deep veins of the left leg. Since at this point patient had adequate platelet recovery to 142 × 109/L with 54% granulocytes, no blasts, hemoglobin 10.4 g/dL, and platelets 255 × 109/L  as an outpatient. Patient remained on anticoagulation and tolerated the chemotherapy well except for development of Grade 2 vincristine-induced peripheral neuropathy. The CBC upon completion of the second cycle showed WBC 9.2 × 10 × 109/L . His bon × 109/L E,F. The E. coli and Erwinia-derived asparaginase products including long acting pegasparaginase (polyethylene glycol L-asparaginase) possess a dual asparaginase and glutaminase enzymatic activity that diminishes both serum asparagine and glutamine levels by deamidating them to aspartate and glutamate, respectively [Both ectively . It is bectively ,14,15,16IDH1 and IDH2 have been reported in approximately 20% of de novo AML [IDH mutations confer adverse prognostic effect in patients with AML whose myeloblasts lack NPM1 mutation [13C and isotope-labeling experiments, it has been shown that the primary source for α-KG in AML cells with IDH mutation is glutamine [in vitro interruption of glutamine metabolism preferentially slows the growth of primary AML cells with mutant IDH versus wild type IDH [IDH mutations are ongoing (NCT02283190 and NCT01810705). Mutations in novo AML ,19,20,21novo AML ,24,25,26mutation . Wild-tymutation . Alteredmutation ,20. By 1lutamine . Subsequtype IDH ,30. ClinThe major adverse events of asparaginase products, reported from clinical trials involving patients with ALL, include anaphylaxis and serious allergic reactions, thrombosis, pancreatitis, glucose intolerance, coagulopathy, and hepatotoxicity. Interestingly, myelosuppression does not occur frequently and seriously after asparaginase administration. Also the myelosuppressive effect of vincristine compared to other conventional chemotherapeutic agents is minimal.The fact that asparaginase and vincristine are minimally myelosuppressive is important for patients such as Jehovah’s Witnesses that do not accept blood product transfusion; as it is well known that the use of conventional chemotherapy for remission induction in AML results in life-threatening anemia and thrombocytopenia, requiring blood product transfusions with an average of 10.8 and 8.5 units of red blood cell and platelet transfusions, respectively . We combIDH1/NPM1/NRAS AML who did not want to receive blood product transfusions. Moving forward, we plan to continue using non-myelosuppressive mono- or combination-therapies as consolidation regimens for this patient. We also plan to prospectively test the effect of this combination for similar patients with prospective measurement of serum biomarkers.A minimally myelosuppressive regimen including pegasparaginase, vincristine and steroid was used to induce durable complete remission in a patient with mutant


According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce retinal ganglion cell (RGC) degeneration. Therefore, interfering with neurotrophic signaling seems an attractive strategy to achieve neuroprotection. Indeed, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs in animal models of glaucoma; however, the neuroprotective effect was mostly insufficient for sustained RGC survival. We hypothesized that treatment at the level of neurotrophin-releasing brain areas might be beneficial, as signaling pathways activated by target-derived neurotrophins are suggested to differ from pathways that are initiated at the soma membrane. In our study, first, the spatiotemporal course of RGC degeneration was characterized in mice subjected to optic nerve crush (ONC) or laser induced ocular hypertension (OHT). Subsequently, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule, and the levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the mouse retina and superior colliculus (SC) upon ONC and OHT. Both models differentially influenced BDNF and TrkB levels. Next, we aimed for RGC protection through viral vector-mediated upregulation of collicular BDNF, thought to boost the retrograde neurotrophin delivery. Although the previously reported temporary neuroprotective effect of intravitreally delivered recombinant BDNF was confirmed, viral vector-induced BDNF overexpression in the SC did not result in protection of the RGCs in the glaucoma models used. These findings most likely relate to decreased neurotrophin responsiveness upon vector-mediated BDNF overexpression. Our results highlight important insights concerning the complexity of neurotrophic factor treatments that should surely be considered in future neuroprotective strategies. Glaucomatous optic neuropathies are characterized by degeneration of retinal ganglion cells (RGCs) and their axons, resulting in visual field defects–3. The pOne of the early events during the pathogenesis of glaucoma is axonal dystrophy within the optic nerve, perturbing axonal transport of neurotrophins, 6. The Although local administration of BDNF to the eye has repeatedly been shown to reduce loss of RGCs in various glaucoma models–14, the The overall objective of this study was to advance the knowledge of the role of neurotrophin signaling between the eye and the brain in glaucoma pathology. Therefore, two different mouse models of experimental glaucoma were used: the optic nerve crush (ONC) model, defined by mechanical trauma to the RGC axons in the optic nerve–29, and ad libitum access to food and water. The experiments were approved by the KU Leuven Animal Ethics Committee and followed the guidelines of the ARVO statement for the use of animals in ophthalmic and Vision Res.All experiments were performed on adult C56Bl/6J and CD-1 mice (10–12 weeks old at the start of the experiment), housed under a 12/12 h light/dark cycle with Surgical procedures to induce experimental glaucoma and for viral vector or recombinant BDNF administration were performed under general anaesthesia, induced by intraperitoneal (i.p.) administration of ketamine  and medetomidin . Additionally, for glaucoma induction and intravitreal (ivt) injections, a droplet of local analgesic, oxybuprocaïne hydrochloride  was applied to the eye. For intracollicular vector injections, the head region was topically analgesized by applying lidocaine hydrochloride  to the scalp. After any surgical procedure, waking was encouraged by i.p. administration of atipamezole . During recovery, tobramycin eye ointment  was applied to the cornea of both eyes to prevent desiccation and mice were placed on a heat pad to maintain body temperature.oculus sinister, OS) while the right eye  served as an internal control.In both models, experimental glaucoma was induced in the left eye  was performed in C57Bl/6J mice as described previously. BrieflyOcular hypertension (OHT) was induced in CD-1 mice through laser photocoagulation (LP) of the ocular veins as described before, 31, 35.Mice received a 2 μl ivt injection of BDNF , diluted at 1 mg/ml in 0.01 M phosphate buffered saline (PBS) containing 1% bovine serum albumin  (BSA-PBS). Ivt injections were performed using a glass capillary with a 50–70 μm outer diameter, connected to a Hamilton syringe and a Micro4 Microsyringe controller , at a rate of 500 nl/s. Control groups received an ivt injection of 2 μl vehicle (BSA-PBS). Two main treatment paradigms were used: (i) a single dose immediately after crush with sacrifice at 7 or 14 days post-injury (dpi), and (ii) repeated ivt injection at 0, 3, and 5 d with sacrifice at 7 dpi, or at 0, 5, and 10 d with sacrifice at 14 d post ONC.To optimize viral vector transduction in the mouse SC, the tropism and transduction efficiency of four rAAV serotypes  were evaluated. For more information, see supplementary methods described in 5’-TCTAGAGATGACCATCCTTTTCCTTAC-3’ and reverse primer 5’-ACTAGTCTATCTTCCCCTTTTAATGGTC-3’. Subsequently, mouse BDNF cDNA was cloned using the Zero Blunt TOPO PCR Cloning kit  and the resulting plasmid was transformed into competent Escherichia coli cells . Plasmids were isolated from positive clones by use of the PureLink Quick Plasmid Miniprep kit  and PCR was performed with forward primer 5’-AAAAAAGGATCCGCCACCATGACCATCCTTTTCCTTACTATGG-3’ and reverse primer 5’-GCCCTTACTAGTCTATCTTCCCCTTTTAATGGTCAGT-3’. Purified PCR products were ligated in the rAAV transfer plasmids[In order to enhance BDNF levels in the SC, a murine BDNF-expressing vector was developed. RNA was isolated from mouse tail tissue using TRIzol reagent  and cDNA synthesis was carried out following standard procedures. PCR amp plasmids through et al.[The adeno-associated viral vector serotype 2/1 containing the mouse BDNF gene under control of a general cytomegalovirus (CMV) promoter (rAAV2/1-CMV-BDNF) and the control enhanced green fluorescent protein (eGFP)-expressing vector (rAAV2/1-CMV-eGFP) were produced by the Viral Vector Core of KU Leuven. The vector production process has been described in detail by Van der Perren et al..To induce unilateral overexpression of BDNF in the SC, 1 μl rAAV2/1-CMV-BDNF was stereotactically delivered into the right SC at 3.16E11 GC/ml, using the Nanoject II micro-injector . Control groups received 1 μl injection of rAAV2/1-CMV-eGFP vector at 3.25E11 GC/ml. Coordinates for mouse right SC were anteroposterior -3.3 mm and mediolateral +0.4 mm relative to Bregma, and dorsoventral -1.3 mm from the dura.Animals were anesthetized with sodium pentobarbital  prior to cervical dislocation. Retinas and SCi were dissected, snap-frozen, and homogenized in RIPA buffer  containing a protease and phosphatase inhibitor cocktail . Protein concentrations were determined with the Qubit Protein Assay  and samples were stored at -80°C.Levels of mBDNF, TrkB-FL, and TrkB-T1 were detected in the retina and SC samples using western blotting. Equal amounts of the sample  were loaded onto 4–12% Bis-Tris polyacrylamide gels  and transferred to polyvinylidene difluoride (PVDF) membranes . The membrane was incubated for 2 h in 5% ECL Blocking Agent  in 0.01 M tris buffered saline (TBS). Overnight incubation with primary antibody, polyclonal rabbit anti-BDNF  or polyclonal rabbit anti-TrkB , was followed by 45 min incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG . Specific protein bands were visualized using the luminol-based enhanced chemiluminescence kit . In order to normalize detected amounts of mBDNF or TrkB to the total amount of protein loaded, LavaPurple  total protein stain was performed prior to primary antibody incubation. Specific protein bands and LavaPurple staining were imaged with the ChemiDoc MP Imaging System  and optical density analyses were performed using Image Lab 4.1 software .Total BDNF levels, including both pro- and mature forms, were quantified based on standard sandwich enzyme-linked immunosorbent assay (ELISA) technology by means of the Mouse BDNF ELISA kit . Data analyses were performed with Skanit 3.1 software .Animals were anesthetized through i.p. administration of sodium pentobarbital  and transcardially perfused with 0.9% NaCl, followed by 4% phosphate buffered paraformaldehyde  for standard immunohistochemistry (IHC). Following perfusion, eyes were enucleated and fixed for 1 h in 4% PFA. Next, retinas were dissected, flat mounted, and post-fixed for 1 h in 4% PFA. Brains were dissected and post-fixed overnight in 4% PFA. For BDNF IHC, perfusion was performed using 4% phosphate buffered PFA containing 0.4% parabenzoquinone , followed by brain post-fixation for 2 h in the same solution. The SC brain region was sectioned into 50 μm coronal sections using the Micron H650 vibratome .For general IHC, free floating coronal SC sections were rinsed in PBS containing 0.1% Triton X-100  (PBST0.1) and incubated for 1 h in PBST0.1 containing 20% goat pre-immune serum , followed by incubation with primary antibodies, diluted in PBST0.1 and 10% goat pre-immune serum . After r2O2 (in methanol) for 20 min, followed by thorough rinsing. After overnight primary antibody incubation, washing, and 45 min incubation with biotinylated goat anti-rabbit IgG , tyramid signal amplification via the TSA™ Cy3 System  was used.For NeuN IHC, before incubation with goat pre-immune serum, endogenous peroxidases were blocked via incubation in 0.3% HImmunofluorescent labeling was visualized using the FV1000-D laser confocal scanning microscope , controlled by Fluoview 4.0 software.IHC labeling for Brn3a, a POU-domain transcription factor and reliable marker for RGCs, 27, 28,2) was calculated on images of the entire retinal whole-mount, using an in-house script developed for freely available Fiji software[et al., manuscript submitted to Exp Eye Res). Subsequently, RGC density data were transformed into pseudocolor isodensity maps to have a better view of the spatial differences in RGC density[RGC density  cells  were cultured in Dulbecco modified Eagle medium  supplemented with 8% fetal calf serum  and gentamicin , plated at 100,000 cells per well (24-well plates) in 500μl medium. HEK293T cells were transduced with either the rAAV2/1-CMV-BDNF or rAAV2/1-CMV-eGFP vector. After 48 h incubation with medium containing vector at 4.29E9 GC/ml, the cells were transferred to 6-well plates and medium was harvested 24 h and 72 h later. To quantify BDNF levels in the medium, the Mouse BDNF ELISA kit  was used according to the manufacturer’s guidelines. Analyses were performed with Skanit 3.1 software .+ cells.To estimate transduction efficiency, eGFP vector-transduced cells were fixed for 15 min in 4% phosphate buffered PFA and incubated for 30 min with 1 μg/ml DAPI in PBS  for nuclear staining. The DAPI and eGFP signals were visualized with the FV1000-D laser confocal scanning microscope . The percentage of eGFP-expressing cells was calculated over 400 randomly chosen DAPI™-A medium , containing 0.4% methylcellulose , 1% penicillin/streptomycin, 0.2% fungizone, 0.5% L-glutamine and 2% B-27 supplement for retina . The explant culture medium was supplemented with 1) conditioned medium from BDNF vector-transduced HEK293T cells, to a final concentration of 50 ng/ml BDNF, 2) conditioned medium from eGFP vector-transduced HEK293T cells, or 3) with recombinant BDNF in a final concentration of 50ng/ml . Every day, half of the culture medium was replaced with fresh medium, containing the same concentration of compound or vehicle.To validate the bio-activity of vector-derived BDNF, postnatal retinal explants were cultured in medium supplemented with conditioned medium derived from transduced HEK293T cells, and neurite outgrowth was studied. Retinal explants were isolated as previously described, 44. BriExplants were fixed in 4% phosphate buffered PFA and neurite outgrowth was visualized by immunostaining for β-tubulin using the monoclonal mouse anti-β-tubulin III antibody , as described, 44. β-tpost hoc tests. When criteria for parametric statistics were not fulfilled, a non-parametrical Kruskal-Wallis analysis with a Mann-Whitney U rank sum test for pairwise comparison of independent samples was applied. Additionally, Student’s t-test for pairwise comparison was used. For all tests, a probability level (α-level was set to 0.05) of <0.05 was accepted as statistically significant . All statistical analyses were performed using GraphPad Prism S5 .All data are presented as mean ± SEM. Outliers were determined via a Grubbs test and excluded from further analyses. A normal distribution was verified using the Kolmogorov-Smirnov test and parallel equal variance between groups was tested. If the test requirements were fulfilled, a parametrical ANOVA was used, followed by bonferroni Animal models of glaucoma are essential to elucidate the underlying pathophysiological mechanisms and to develop therapeutic strategies to halt or even reverse disease progression. Therefo2, N = 5, p>0.05) and 5 dpi   were similar to the naive condition . Between 5 and 21 dpi, RGC degeneration progressed, with RGC density measured at 7, 14, and 21 dpi being 1,392 ± 59.3 RGC/mm2 (N = 10), 756 ± 99.2 RGC/mm2 (N = 6), and 376 ± 12.7 RGC/mm2 (N = 3), respectively. At all time points, the contralateral eyes showed RGC densities comparable to naive retinas . ONC-induced RGC degeneration was diffuse throughout the retina . The onset of ONC-induced RGC death was situated between 4 dpi  , while ae retina .2, N = 8), 7 dpi , 14 dpi , and 21 dpi , compared to naive animals  . On top of diffusely distributed RGC death all over the retina, a pie-shaped sectorial degeneration pattern was observed , a pattern being in line with previous publications using this model, 31. RGC N = 10) . RGCs diobserved .Thus, both the ONC and OHT models elicited progressive RGC degeneration. In both models, RGC loss occurred diffusely throughout the retina, however, only in the OHT model pie-shaped sectors of severe RGC death superposed the diffuse degeneration pattern.In order to contribute to a better understanding of the role of neurotrophin signaling in glaucoma, the effect of experimental glaucoma on BDNF and TrkB levels in the glaucomatous retina and the SC contralateral to the experimental eye, was examined via ELISA for total BDNF levels and western blotting for TrkB-FL and TrkB-T1.In the ONC model, total BDNF levels in the retina increased marginally from 6 h post-ONC onwards, reaching significance at 3 dpi  to 5 dpi , and attenuating afterwards . The retFor the OHT model, patterns of BDNF and TrkB levels in the retina and SC were different from the ONC model. In the retina of OHT eyes, no changes in the total BDNF level, neither in the TrkB-FL and TrkB-T1 levels, could be detected, in comparison to naive eyes . In the Overall, retinal and collicular BDNF and TrkB levels were differentially altered after experimentally induced glaucoma in the ONC and OHT models.i.e. in the cell body, in the axon terminals, or on the dendrites)[According to the neurotrophin deprivation hypothesis, disruption of retrograde delivery of trophic support is one of the main triggers that induces apoptotic signaling in the RGCs in glaucoma, 45. Witendrites), 23, it Based on the fact that the applicability of different viral vector systems depends on the experimental setup, tissue, and species, 47, we in vivo. Unilateral delivery of the rAAV2/1-CMV-BDNF vector into the mouse SC resulted in extensive upregulation of BDNF expression, visualized through IHC  in medium harvested from BDNF vector-transduced cells, both at 24 h (298.45 ± 19.51 ng/ml) and at 72 h (264.03 ± 55.24 ng/ml) post-transduction, as compared to medium harvested from cells transduced with the control vector at 24 h (0.01 ± 0.00 ng/ml) and 72 h (0.01 ± 0.00 ng/ml) post-transduction  or with conditioned medium from BDNF vector-transduced HEK293T cells  showed enhanced neurite outgrowth compared to control explants, which were cultured in medium supplemented with equal amounts of conditioned medium from eGFP vector-transduced HEK293T cells   . Thus, vIn the following experiments, we envisioned to counteract glaucomatous RGC degeneration via brain-directed treatment, by stimulating the pro-survival colliculoretinal signaling. Thereto, the validated rAAV2/1-CMV-BDNF vector was used to upregulate BDNF in the mouse SC in an attempt to induce long-distance neurotrophic protective effects on the RGCs in both models of experimental glaucoma.2 and 1,170 ± 89.3 RGCs/mm2 measured in ONC eyes of the BDNF vector-transduced group (N = 8) and the eGFP group (N = 10), respectively. Also, for the OHT model, in which IOP patterns were very similar between groups (2 for the BDNF group (N = 6) and 2,473 ± 192.2 RGCs/mm2 for the eGFP group (N = 6).Two weeks after intracollicular vector delivery to induce chronic BDNF or eGFP expression, mice received unilateral ONC or OHT induction and were sacrificed 7 d later. First, BDNF and eGFP expression was validated via IHC in three randomly chosen animals of each group. For each of these mice, a robust transgene expression was observed throughout the SC . However, the protective effect was transient, as at 14 dpi, there was no longer a difference in Brn3a+ RGC counts between BDNF- (700 ± 65.8 RGCs/mm2) and vehicle-treated (710 ± 93.2 RGCs/mm2) ONC eyes (N = 5).A single ivt administration of recombinant mBDNF at the time of ONC resulted in 10% neuroprotection at 7 d after ONC . Brn3a+ 2, N = 7) at 7 dpi, as compared to vehicle-injected eyes . However, ivt BDNF injections at 0, 5, and 10 dpi did slightly prolong the neuroprotective effect, with 4.5% enhanced survival in the ONC eyes of the BDNF (N = 8) versus vehicle (N = 4) groups at 14 dpi. RGC densities in ONC eyes were 532 ± 20.2 RGCs/mm2 in BDNF-treated groups versus 389 ± 28.4 RGCs/mm2 in the vehicle group.Strikingly, triple BDNF administration at 0, 3, and 5 dpi was not found to enhance the neuroprotective effect seen at 7 dpi after a single dose . Using tThus, our results confirmed the temporary RGC protective effect of ivt delivered recombinant BDNF in the ONC model. Despite its ability to prevent RGC degeneration in the short-term, the long-term effectiveness of ivt BDNF administration was limited, even when provided as multiple injections.BDNF = 4, NeGFP = 5, Then why did the delivery of BDNF to the SC not have a similar neuroprotective outcome? To be able to support RGC survival, BDNF, released and activated within the SC, needs to reach the retina via retrograde transport through the RGC axons. Unfortunately, both ELISA and western blot analyses of retinal samples (N = 5), dissected at 18 d post collicular BDNF vector injection, revealed no change in total and mBDNF levels in the retina despite a clear BDNF overexpression in the SC . PossiblThus, our data indicated that in response to vector-based BDNF upregulation in the SC, TrkB-FL levels were downregulated in the transduced SC, thereby possibly reducing complex formation between BDNF and TrkB-FL and hence colliculoretinal transport of BDNF.The main objective of the performed experiments was to study the contribution of BDNF, the most commonly studied neurotrophin, to glaucoma disease progression, using two experimental mouse glaucoma models, commonly applied to mimic glaucoma pathology.We first characterized the RGC degeneration pattern for both models. ONC-triggered retinal neurodegeneration had an onset between 4 and 5 dpi, progressively evolving towards a survival ratio of 12% at 21 dpi. RGC loss elicited by ONC appeared diffuse and affected the whole retina, without any evidence of a sectorial pattern, which is in line with literature, 49. DifWithin the OHT model, characterized by a temporary elevation of the IOP, RGC loss occurred diffusely throughout the retina with pie-shaped sectors of severe RGC death superposing the diffuse degeneration pattern, as clearly demonstrated by the isodensity maps. This pattern reflects a typical glaucomatous retinal degeneration pattern described in human patients and seveet al., describing reduced TrkB levels in rat RGCs after optic nerve transection (ONT)[The current study, investigating levels of BDNF together with its main receptor TrkB in the retina and SC in two mouse models of experimentally induced glaucoma, firstly demonstrated that retinal BDNF levels were elevated after ONC . ONC indion (ONT). Howeverion (ONT).Although both BDNF and TrkB concentrations increased in the retina after ONC, this did not protect RGCs from dying, possibly because the TrkB upregulating effect was the highest for the TrkB-T1 isoform. The expected neuroprotective role of the extra BDNF might be attenuated because of sequestration by TrkB-T1 expressing glia or becauIn contrast to ONC, BDNF and TrkB levels in the retina were not affected by OHT in our study . AlthougLiterature describing the effect of OHT on retinal BDNF signaling is confusing and far from complete, with various studies, using a number of rodent OHT models, showing assorted effects of elevated IOP on BDNF and TrkB mRNA and protein levels in the retina and ONH 13, 60–6–660–63. In the SC, total BDNF was significantly enhanced as soon as 6 h post-ONC, slowly decreasing to baseline levels afterwards . CollicuIn the OHT model, no significant effect on the BDNF concentration in the SC was measured . In compThus, BDNF-TrkB signaling is regulated by spatial and temporal changes in protein localization, gene expression, the mBDNF/pro-BDNF ratio, the TrkB-FL/TrkB-T1 ratio, and downstream signaling molecules. Defining a specific role for BDNF signaling within glaucoma pathology remains difficult, as various studies, using a number of animal glaucoma models, have culminated in a variety of unique results, the preBased on the neurotrophin deprivation hypothesis, neurotrophins gained wide acceptance as suitable candidates for neuroprotective drug usage. Indeed, upon experimentally induced glaucoma, RGCs have been described to survive longer after ivt administration of various neurotrophic factors, 10, 68.et al. have demonstrated that combined application of BDNF to the eye and the central visual pathway enhanced RGC survival and function relative to treatment of the eye alone, in an ONC model in cats[in vitro, and to be transformed into the mature form in vivo. In addition, viral vector-derived BDNF was demonstrated to be bio-active, since it stimulated neurite outgrowth in an ex vivo retinal explant culture. Notably, as viral vector-based BDNF upregulation in the SC elicited effects on the availability of its receptor (discussed in detail below), our findings clearly indicate that transgenically expressed BDNF was secreted into the extracellular space by transduced collicular neurons in vivo as well.Since local BDNF administration to the eye appears insufficient for sustained RGC survival, we hypothesized that treatment at the level of extraretinal neurotrophin sources in the brain might be favorable. Indeed, Weber l in cats, 26. Baset al. postulated that the enhanced survival achieved in their dual treatment paradigm was the result of the contribution from both the eye and visual brain center treatment[In line with our data, based on unpublished results showing that treatment of the visual cortex alone was insufficient to provide RGC neuroprotection, Weber treatment, 26. Thetreatment, 31, thetreatment. Howevertreatment. Of notetreatment, 58, 72.treatment or by entreatment.i.e. ONC and LP-induced OHT. Furthermore, endogenous BDNF and TrkB expression patterns in the retina and SC were shown to be differentially affected upon ONC or OHT induction. Using the ONC model, the neuroprotective capacity of ivt BDNF administration was confirmed. Unfortunately, viral vector-induced overexpression of BDNF in the SC did not result in enhanced RGC survival in none of the two glaucoma models. These negative results seemed mainly attributed to reduced neurotrophin responsiveness as a response to vector-mediated BDNF overexpression and secretion. Overall, our data provide important information for the design of additional research on the neurotrophin-deprivation-hypothesis in glaucoma.The temporal course of RGC degeneration was characterized in two mouse models of experimentally induced glaucoma, S1 Fig(A) Two weeks after intracollicular delivery of different viral vectors , expressing the eGFP gene under control of the general CMV promoter, astroglial reactivity, defined by upregulated GFAP labeling, remains locally around the injection site (± 300 μm) for each vector and the difference between rAAV vector-injected SC and sham controls is limited. Scale bars: 100 μm. (B) Unilateral transduction with rAAV2/1, rAAV2/7, rAAV2/8, and rAAV2/9, visualized through CMV promoter-regulated eGFP expression, spreads throughout the entire SC in anteroposterior and lateromedial direction. 200 μm spaced serial sections between Bregma -3.30 mm and -4.50 mm are depicted. Scale bars: 200 μm. (C) In contrast to rAAV2/1, unilateral injection of rAAV2/7, rAAV2/8, and rAAV2/9 vectors into the mouse SC results in retrograde transduction and eGFP expression in RGCs, identified through Brn3a IHC, in the eye contralateral to the injected SC. Arrowheads point towards double-labeled cells, showing green cytoplasm and a red nucleus. Scale bars: 20 μm. Key: GFAP, glial fibrillary acidic protein; IHC, immunohistochemistry; SC, superior colliculus; RGC; retinal ganglion cell; eGFP, enhanced green fluorescent protein; rAAV2/x, recombinant adeno-associated viral vector serotype 2/x; CMV, cytomegalovirus promoter.(TIF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The absence of alkyl catechols from the modern Western diet suggests serious negative consequences for Nrf2 cell defense, resulting in reduced protection against multiple chronic diseases associated with oxidative stress.The Nrf2 (NFE2L2) cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both  In all mammals, oxygen is critical for cellular metabolism and survival. However, oxygen is also a toxic gas . OxidatiIn mammalian cells, the master regulator of oxidant defense is the Nrf2 (NFE2L2) pathway , 24. TheCompelling evidence for the importance of protection provided by the Nrf2 pathway comes from numerous studies with mice lacking the Nrf2 gene. Although viable and fertile, Nrf2 null mice are considerably more sensitive to chemical carcinogens, with increased incidence of cancers demonstrated in skin, stomach, colon, bladder, and mammary gland , 35–39. Although oxidative stress is sufficient for activation of the Nrf2 pathway and induction of Nrf2 target genes, activation by oxidative stress is greatly enhanced by the presence of chemical compounds with specific redox cycling properties. Originally, before the discovery of Nrf2, a variety of small chemical compounds were observed to protect rodents from chemically induced carcinogenesis . Remarkain vitro and in vivo. Moreover, our findings identify important and previously unrecognized connections between activation of the Nrf2 pathway, probiotic bacteria, fermented foods, traditional diets, and traditional methods of food preservation and suggest that alkyl catechols were common in ancient diets. However, because of extensive changes in food preservation and preparation, the alkyl catechols mostly have been lost from modern Western foods. The absence of alkyl catechols from modern Western diets suggests serious negative consequences for maintenance of Nrf2 cell defense, resulting in reduced protection against chronic diseases associated with oxidative stress.In contrast to sulforaphane and curcumin, the naturally occurring redox-sensitive alkyl catechols, 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol, have not received attention as potential activators of the Nrf2 pathway. Structurally similar to catechol, these alkyl catechols are unrelated to sulforaphane and curcumin . Also, iThe following antibodies  were used for western blotting: heme oxygenase-1 , Nrf2 , CD31 . Secondary antibody used for western blotting was polyclonal goat anti-rabbit (H+L), conjugated to horseradish peroxidase . The following antibody was used for immunohistochemical staining of Nrf2 in cells : Nrf2  contains a vinyl group see  it polymPrimary human dermal microvascular endothelial cells, human umbilical vein endothelial cells, human astrocytes, and human dermal keratinocytes were purchased from Lonza Inc, Walkersville MD, and cultured in Lonza media as follows: EGM-2MV for all endothelial cells, KGM-Gold for keratinocytes, AGM for astrocytes. Keratinocytes were used for experiments at passage 3; astrocytes at passage 4, and endothelial cells at passage 6 or less. Cell viability was measured with the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells from Molecular Probes. This method uses calcein AM (green fluorescence) to mark live cells and ethidium homodimer-1 (red fluorescence) to stain the nuclei of cells with damaged membranes. For each data point, at least four random microscopic fields containing at least 250 cells/field were photographed and enumerated.For RNA, cells were cultured in 6 well plates, and RNA isolated with the Qiagen RNeasy Plus Mini Kit that provides on-column removal of genomic DNA. For immunohistochemical staining of Nrf2, cells were fixed and stained exactly as described . In addi6 copies of 18S rRNA that is superior to using housekeeping genes for normalizing across multiple cell types [6 copies 18S rRNA, and (ii) direct comparisons between mRNA copy number for genes of interest vs. relevant housekeeping genes, each expressed as a function of mRNA copy number/106 copies 18S rRNA.Multi-gene transcriptional profiling with quantitative RT-PCR was performed with SYBR Green I and the Applied Biosystems 7500 Fast Real-Time PCR System, as described . cDNA wall types , 69, 70.All data are presented as mean ± S.D. Statistical analyses were performed with GraphPad software using the unpaired t-test. In all cases, an individual experimental group was compared with the appropriate vehicle control group; and calculated p-values are based on direct comparisons between the two groups. For assignment of statistical significance based on p values, standard threshold limits were used: p < 0.001 extremely significant, p = 0.001 to < 0.01 very significant, p = 0.01 to < 0.05 significant, p ≥ 0.05 not significant.Cell lysates subjected to SDS polyacrylamide electrophoresis on pre-cast 4–20% gradient gels (GenScript) and separated proteins transferred to PVDF immunoblotting membrane (Bio-Rad). PVDF membrane was blocked in 5% w/v skim milk , and stained with primary antibodies and secondary horseradish peroxidase-conjugated antibody . Signal was developed with ECL western blotting substrate  and captured on x-ray film. For re-probing with additional antibodies, blots were stripped  and blocked again with skim milk, as above.Selective inhibition of Nrf2 (NFE2L2) expression with small interfering RNAs (siRNAs) was accomplished with a predesigned human NFE2L2 TriFECTa RNAi Kit containing Dicer-substrate 27mer siRNA duplexes from Integrated DNA Technologies (cat. ID: HSC.RNAI.N001145413.12). For these experiments we used human umbilical vein endothelial cells (see above) because they are particularly well suited for transfection that was performed with the Lipofectamine RNAiMAX Reagent in Opti-MEM Reduced Serum Medium (both from Life Technologies) according to manufacturer’s instructions. Two Nrf2 (NFE2L2) siRNA duplexes were highly effective: NM_001145423 duplex 1 and NM_001145413 duplex 2, each at a final concentration of 1 nM. Another duplex, DS NC1 (provided with the kit) served as negative control.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee (IACUC) specifically approved this study (protocol #045–2012). All efforts were made to minimize suffering; euthanasia was performed according to National Institutes of Health guidelines.Retired breeder male BALB/c mice  were used for these experiments. Mice were placed on the purified rodent diet AIN 76A  for 3–4 weeks prior to administration of alkyl catechols, as described in figure legends. Kidney and lung were minced and submerged in RNAlater and RNA isolation performed with a Polytron tissue homogenizer and the RNeasy Lipid Tissue Mini Kit (Qiagen) that employs on-column removal of DNA with DNAse digestion (RNase-free DNAse set from Qiagen).L. plantarum (ATCC 8014), L. brevis (ATCC 8287), L. collinoides (ATCC 27611), L. reuteri MM4-1A (ATCC PTA-6475), L. ruminus (ATCC 27780), and L. paracaseii (ATCC 25302). All were grown in Lactobacilli MRS broth (Difco) according to the specific instructions provided.All lactobacillus strains were purchased from the American Type Culture Collection (ATCC), Rockville MD. Strain designations are as follows: 8 bacterial cells/ml with the various solutions, including PBS-glucose control, on a rocker for 24 hours at room temperature. Supernatants were harvested by centrifugation and filter-sterilized before adding to endothelial cells for Nrf2 assays with RT-PCR and western blotting.Solutions of chlorogenic acid, caffeic acid, and 3,4-dihydroxybenzoic acid were prepared to a final concentration of 6 mM by dissolving in PBS containing 10 mg/ml D-glucose (PBS-glucose). Each was filter sterilized with Steriflip 0.22 micron filter units (EMD-Millipore). Lactobacilli were concentrated by centrifugation, washed twice with sterile PBS-glucose, and incubated at a final density of ~ 8 x 10Reversed-phase high-pressure liquid chromatography (HPLC) was performed with a Phenomenex Luna 5 micron C18 column (100x4.6mm) using a 20 minute gradient consisting of 10%-100% acetonitrile (in 0.1% formic acid). Standards were dissolved in methanol (1 mg/mL) and volume injected was 10 μL.i.e., HO-1, NQO1, G6PD) depicted in the Nrf2 is a transcription factor, and as summarized in the Introduction, activation of the Nrf2 pathway requires: (i) stabilization of Nrf2 protein, (ii) transport of Nrf2 protein to the cell nucleus, and (iii) transcriptional activation of Nrf2 target genes. Consequently, measure of Nrf2 target gene expression is required for determination of Nrf2 pathway activation, and analyses of nuclear concentration of Nrf2 can provide complementary information. Therefore, as described here, we employed multi-faceted strategy for analyzing Nrf2 pathway activation by alkyl catechols, including: (i) real-time PCR (RT-PCR) of mRNAs encoding three prominent Nrf2 target genes: heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase1 (NQO1), and glucose-6-phosphate dehydrogenase (G6PD), (ii) western blotting for protein expression of the prominent Nrf2 target gene HO-1, (iii) immunohistochemical analyses of Nrf2 protein to visualize nuclear localization, and (iv) transfection with Nrf2 siRNA to demonstrate Nrf2-dependence of target gene induction. The three Nrf2 target genes chosen for investigation here, HO-1, NQO1, and G6PD, each serve critically important functions. Mice lacking HO-1 are more susceptible to myocardial infarction  and atheIn contrast to Nrf2 target genes HO-1, NQO1, and G6PD, we observed a very modest but still statistically significant induction of Nrf2 mRNA by 4-methylcatechol and, in some cases, by 4-vinylcatechol and 4-ethylcatechol . This reNext, with western blotting of human endothelial cells, we examined induction of HO-1 protein expression by each of the alkyl catechols, catechol, sulforaphane (positive control), and 4-ethylphenol (negative control). Cells were harvested 24 hours after addition of each of the compounds to 30 μM final concentration, except 20 μM for sulforaphane (SF) that we found is the maximum tolerated dose of this compound for endothelial cells see . As showIn addition to HO-1 protein, the alkyl catechols, catechol, and sulforaphane also consistently increased Nrf2 protein > 2.5-fold . Finallyi.e., 4-ethylphenol (4EP), 2-methoxy-4-methylphenol (2M4M), and caffeic acid (CFA), are shown in meta position (e.g. orcinol) are also inactive (Group 2). Finally, a more diverse group of compounds, containing individual catechol moieties appended either to electron-withdrawing or bulky side groups, were inactive in our assays (Group 3). Thus, collectively, our findings underscore the special importance of alkyl catechols and catechol, in comparison with a variety of related compounds.Additional support for a direct link between alkyl catechols and activation of the Nrf2 pathway comes from immunohistochemical staining for Nrf2. We observed marked increases in nuclear localization of Nrf2 24 hours after addition of alkyl catechols and catechol . IncreasNotably, we did not find evidence with our assays that the flavonoids quercetin or luteolin activated the Nrf2 pathway , S3 Fig.Next, we performed a series of experiments to compare the potency of alkyl catechols and catechol with sulforaphane, which is a well established and much studied activator of the Nrf2 pathway –62, 85. The Nrf2 pathway and its target genes are designed to mitigate the deleterious consequences of oxidative stress, and Nrf2 pathway activation is tightly regulated for this purpose , 24. Indin vitro, these alkyl catechols also induce Nrf2 target gene expression in live mice, and induction is readily demonstrable in two major organs (kidney and lung). These experiments also demonstrate that 4-ethylcatechol is potently active when administered orally; and therefore we presume that it enters the bloodstream, in active form, following absorption by the gastrointestinal tract. Finally, we did not investigate 4-vinylcatechol or catechol in these experiments, but based on in vitro data  injection, and in some experiments 4-ethylcatechol was administered orally (by gavage). The total dose chosen for each compound  in all of these experiments was less than an oral dose of sulforaphane  shown previously to induce HO-1 and NQO1 mRNA expression in rat mammary gland . As showFindings described above, raise important questions about possible dietary sources of alkyl catechols and catechol. Interestingly, some but not all species of lactobacilli, particularly those associated with traditional fermentation of vegetables and fruits, express the microbial enzyme phenolic acid decarboxylase (PAD) that removes carboxyl groups from phenolic acids –93. In pLactobacillus plantarum and Lactobacillus brevis, both of which are found in traditional vegetable, fruit, and malt whiskey fermentations and both of which express PAD [L. plantarum and L. brevis with caffeic acid that is a common hydroxycinnamic acid found in all plants and in various foods including fruits, vegetables, nuts, grains, spices, and coffee [L. plantarum or L. brevis had no significant effect on induction of Nrf2 target genes HO-1, NQO1, or G6PD. In contrast, supernatants from incubations containing L. plantarum + caffeic acid and L. brevis + caffeic acid strongly induced all three Nrf2 target genes. Thus, as predicted by the model presented in L. plantarum and L. brevis to a compound with chromatographic retention time identical to 4-vinylcatechol , we examined biotransformation of 3,4-dihydroxybenzoic acid , by  berries . 3,4-DHB berries , 98. As catechol . Thus, tL. plantarum + caffeic acid (CFA) strongly induced HO-1 protein whereas CFA alone and supernatants from L. plantarum alone were without effect. Although less potent than supernatants from L. plantarum + caffeic acid, supernatants from L. plantarum + 3,4-DHBA also induced HO-1 protein whereas 3,4-DHBA alone did not induce HO-1 detectably above baseline. In addition to increasing HO-1 protein, incubations from L. plantarum + CFA and L. plantarum + 3,4-DHBA also consistently increased Nrf2 protein, as quantified with Image J . Similar findings of increased Nrf2 protein were made with experiments presented in L. plantarum + CFA and L. plantarum + 3,4-DHBA. Also, as shown in L. brevis + CFA demonstrated findings comparable to those made with L. plantarum + CFA, consistent with the RT-PCR data and HPLC analyses presented in Figs in vivo by supernatants from incubations containing L. plantarum + CFA or L. brevis + CFA, and also by diets containing CFA together with L. plantarum or L. brevis.In additional experiments, we independently confirmed these findings with western blot analyses of HO-1 protein expression. As shown in Lactobacillus collinoides [L. plantarum and L. brevis, L. collinoides also has been shown to express PAD [L. collinoides also expresses cinnamoyl esterase [L. collinoides also expresses phenolic acid reductase (vinylphenol reductase) [L. collinoides enzymes PAD, cinnamoyl esterase, and phenolic acid reductase have the potential in combination to generate the Nrf2 activator 4-ethylcatechol from both chlorogenic acid and caffeic acid. To test this possibility, we performed incubations with L. collinoides and chlorogenic acid and caffeic acid, comparable to incubations performed with L. plantarum and L. brevis (above). As shown in L. collinoides alone showed no significant induction of Nrf2 target genes HO-1, NQO1, or G6PD. In contrast, supernatants from incubations of L. collinoides + chlorogenic acid and L. collinoides + caffeic acid strongly induced all three Nrf2 target genes comparably to 4-ethylcatechol (4EC). Consistent with these findings, HPLC demonstrated nearly complete conversion of chlorogenic acid by L. collinoides to a compound with chromatographic retention time identical to 4-ethylcatechol but not 4-vinylcatechol   that cancatechol . Similarcatechol . Thus, tL. plantarum, L. brevis, and L. collinoides described above, we found in identical experiments that three other strains of lactobacillus did not biotransform caffeic acid to Nrf2 activator, as measured with RT-PCR for Nrf2 target genes HO-1, NQO1, and G6PD (L. reuteri MM4-1A isolated from human mother’s milk (ATCC PTA-6475), L. ruminus isolated from bovine rumen (ATCC 27780), and L. paracaseii (ATCC 25302), associated with fermentation of dairy products. Presumably, these strains of lactobacilli did not biotransform caffeic acid because they do not express PAD significantly; however, we did not investigate PAD directly. Interestingly, none of these strains are typically associated with plant sources [L. plantarum, L. brevis, and L. collinoides, all of which express PAD, are typically associated with plant fermentations [In contrast to our findings with and G6PD . These s sources –105. In ntations , 106, suThe Nrf2 cell defense pathway provides natural protection against oxidative stress and chemical toxicity through the induction of numerous target genes , 24, 27.Although catechol was among the first compounds recognized as a cancer-protective compound , 4-ethylAn important and unresolved question is whether or not the Nrf2 pathway evolved independently of exogenous “inducers” or “activators” or, instead, evolved in the presence of ubiquitously available, redox-cycling, electrochemicals that “collaborate” with ROS to activate Nrf2. If the latter is true, exogenous Nrf2 “inducers” should be considered more appropriately as co-factors that are essential to support proper function of the Nrf2 pathway. From both evolutionary and biochemical perspectives, it seems likely that the Nrf2 pathway evolved in the presence of electrochemical co-factors. However, the identity of such co-factors has remained obscure. Few natural compounds truly satisfy the structural criteria for “oxidation-reduction lability” , 78. AmoBrettanomyces/Dekkera species found in traditional ciders and beers also express PAD [Brettanomyces yeasts [As illustrated in Figs ress PAD , 113–115ress PAD , 116, caress PAD , and 4-vs yeasts . To our s yeasts .L. collinoides, found in traditional ciders [L. collinoides.As illustrated in l ciders , performl ciders , is an iL. colloinoides, other lactobacilli also express cinnamoyl esterase including L. helveticus [L. reuteri [L. johnsonii [L. fermentum and L. reuteri [L. reuteri [L. plantarum [L. collinoides. Such “collaborative” bioconversions by multiple strains are entirely plausible and even probable.In addition to lveticus , L. reut reuteri , and L. ohnsonii . Other l reuteri . Interes reuteri , 121), olantarum , 95). CoL. plantarum is reportedly unable to demethylate ferulic acid to form caffeic acid [It should be noted that caffeic acid, 3,4-dihydroxybenzoic acid, and chlorogenic acid are different from most common dietary phenolic acids with regard to the benzene ring. For example, the benzene ring of ferulic acid, another common phenolic acid, contains two hydroxyls similar to caffeic acid, however one of the hydroxyls is methylated. Coumaric acid, another common phenolic acid, has only one hydroxyl group, whereas cinnamic acid has no hydroxyls. Sinapic acid and syringic acid each have three hydroxyls on the benzene ring, but two are methylated. Consequently, decarboxylation of either ferulic acid, coumaric acid, cinnamic acid, sinapic acid or syringic acid is not expected to yield compounds with oxidation-reduction lability similar to the catechols or quinones . Also L.eic acid . TherefoL. plantarum, are commonly associated with plants in the wild; consequently, a diet consisting of raw vegetables and fruits provides both precursor molecules and PAD-expressing bacteria for generation of catechols directly within the digestive tract. Interestingly, 4-methylcatechol and catechol have been detected in the urine of rats fed crude vegetable diets [in vitro by bacteria of the human gastrointestinal tract, mixed cultures of fecal bacteria were found to convert caffeic acid to 4-ethylcatechol [For plant eating mammals, including early hominids, it seems likely that the earliest source of alkyl catechols and catechol for supporting the Nrf2 pathway was provided by microbial metabolism of dietary precursors directly within the digestive tract. Mammals do not express PAD; therefore generation of 4-vinlycatechol from caffeic acid and catechol from 3,4-dihydroxybenzoic acid Figs –14, is ele diets . Also, wle diets . In studcatechol .Lactobacillus acidophilus, a popular commercially available probiotic, has been sequenced [L. plantarum, which is also a commercially available probiotic, does indeed express PAD [Foods fermented with PAD-expressing lactobacilli  likely pequenced ; and it ress PAD   [Lactobacillus plantarum and Brettanomyces are ubiquitous in nature. However, modern sterilization techniques and fermentation technology have allowed for stricter control of microbial content and thereby provided the means for eliminating PAD-expressing microbes from the fermentation process.In modern times, a variety of changes in food technology have resulted in a precipitous decline in traditional dietary sources of alkyl catechols and catechol. Cooking with wood fire and preservation of meat and fish with wood smoke are relatively rare. Similarly, alkyl catechols and catechol, together with PAD-expressing lactobacilli, have been lost from modern Western foods. Technological advances in canning and refrigeration have caused a marked shift away from foods fermented with traditional methods, particularly those fermented with “wild” lactobacilli and yeasts that express PAD. Undoubtedly, this shift is partly due to the convenience of modern refrigeration and the advantages of food production on an industrial scale. However, the modern perception of undesirable “off-flavors” associated with compounds generated by PAD, such as alkyl catechols and also alkyl phenols, has driven the elimination of PAD-expressing microbes from commercially fermented foods and beverages. For example, 4-ethylcatechol is considered an undesirable off-flavor defect of commercial French ciders that is attributable to the conversion of caffeic acid by two PAD-expressing microbes,  (yeast) , 101. Si (yeast) , 138–140Thus, several aspects of modern food technology, including the elimination of cooking with wood fire, the shift to food preservation by canning and refrigeration, and the brewing of alcoholic beverages under strictly controlled conditions, all have contributed to the elimination PAD-expressing microbes, together with elimination of alkyl catechols and catechol, from the modern Western diet. Collectively, these large-scale dietary changes have potentially serious negative consequences for Nrf2 defense and health among persons relying exclusively on modern Western foods. In contrast, Asian, Middle Eastern, and African diets still contain many foods prepared and preserved by traditional methods through fermentation with PAD-expressing lactobacilli (reviewed in –145) andin vitro and in vivo. The alkyl catechols and also catechol, another Nrf2 co-factor, are widely available from plant sources but must be generated from these sources either by microbial digestion or heat. Nonetheless, the general availability of these Nrf2 co-factors from numerous natural sources suggests that they are integral to proper functioning of the Nrf2 defense. Until recent times, dietary sources of akyl catechols and catechol were common, but now virtually all are rare in modern commercially available foods due to changes in food preservation and processing. Consequently, findings here illustrate important distinctions between traditional and modern diets, specifically as they relate to Nrf2 defense against disease. Findings here also describe previously undocumented connections between specific compounds found in fruits and vegetables and specific bacteria that convert these compounds to Nrf2 activators. Although there is a general consensus that diets rich in fruits and vegetables are beneficial for health ; aiewed in , 149), mS1 FigKey: Ctrl = control, Cat = catechol, 4MC = 4-methylcatechol, 4VC = 4-vinylcatechol, 4EC = 4-ethylcatechol, SF = sulforaphane. Error bars = +/- S.D; n ≥ 4 for each data point. Viability was not compromised by catechol or the alkyl catechols in either cell type. However, sulforaphane, at a concentration of 30 μM, particularly reduced endothelial cell and astrocyte viability ~ 25%. Statistical significance: extremely significant for individual comparisons between control and 30 μM SF (p < 0.001); no statistically significant differences between control and the other compounds or control and 20 μM SF. Because sulforaphane at 20 μM did not compromise viability detectably in either cell type, we used this concentration of sulforaphane in our experiments.Human microvascular endothelial cells, astrocytes, and keratinocytes were incubated with the indicated compounds at the indicated doses in complete medium for 24 hours, and cell viability was measured as described in Materials and Methods. (TIF)Click here for additional data file.S2 Fig2O2) was added to a final concentration of 100 μM for 16 hours and cells assayed for viability as described in Materials and Methods. Error bars = +/- S.D; n ≥ 4 for each data point. Catechol and the akyl catechols each strongly protected against H2O2-induced cell death by ~ 50–80%. Statistical significance: Individual comparisons between control cells to which H2O2 was added versus corresponding cells pre-incubated with the individual catechols prior to addition of H2O2 indicated that protection provided by each of the compounds was extremely significant (p < 0.0001).Human microvascular endothelial cells were pre-incubated in complete medium with 30 μM of catechol (Cat), 4-methylcatechol (4MC), 4-vinylcatechol (4VC), or 4-ethylcatechol (4EC) for 24 hours. (Ctrl) = control without added compound. Next, where indicated, hydrogen peroxide (H(TIF)Click here for additional data file.S3 Fig6 18S rRNA copies). Nrf2 target genes = heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Control mRNA = CD31 (PECAM-1). Error bars = ± standard deviation (S.D.); n ≥ 3 for each data point. Key: (Ctrl) control, (Guaiacol) guaiacol,  1,2-dimethoxybenze, (2M4M) 2-methoxy-4-methylphenol, (4EG) 4-ethylguaiacol, (4EP) 4-ethylphenol, (Orcinol) orcinol,  2,3-dihydroxybenzoic acid,  3,4-dihydroxyphenylacetic acid, (Quercetin) quercetin, (Luteolin) luteolin, (4EC) 4-ethylcatechol = positive control. All compounds were added to a final concentration of 30 μM with the exception of quercetin and luteolin that were added to a final concentration of 20 μM (the maximum tolerated dose). Statistical significance: For HO-1 and NQO1 data panels, individual comparisons between Ctrl and all of the other samples (apart from 4EC positive control) indicated that there are no statistically significant differences. In contrast, individual comparisons between 4EC and control and 4EC and each of the other compounds indicated differences that are all extremely statistically significant (p < 0.0001). For CD31: no statistically significant differences.Test compounds were added to human microvascular endothelial cells and RNA isolated 24 hours later for analyses with RT-PCR. Y-axis = (mRNA copies)/(10(TIF)Click here for additional data file.S4 Fig6 18S rRNA copies). Human microvascular endothelial cells, 24 hours after addition of test samples: Ctrl = control, CFA = caffeic acid, (L reut + CFA) = supernatant from L. reuteri  incubated with CFA in PBS-glucose and filter-sterilized, (L rum + CFA) = supernatant from L. ruminus (ATCC 27780) incubated with CFA in PBS-glucose and filter-sterilized, (L para + CFA) = supernatant from L. paracaseii (ATCC 25302) incubated with CFA in PBS-glucose and filter-sterilized. CFA and lactobacillus incubations with CFA were added to a final concentration corresponding to 30 μM CFA starting material  indicated that there are no statistically significant differences. In contrast, individual comparisons between 4EC and Ctrl and between 4EC and each of the other samples indicated differences that are extremely statistically significant (p< 0.0002). For CD31: no statistically significant differences.Y-axis = (mRNA copies)/Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Escherichia coli (STEC) comprise E. coli strains capable of producing toxins named Shiga toxin type 1 (Stx1), type 2 (Stx2), or both, encoded by stx1 and stx2 genes, respectively. Stx2 is the most heterogeneous group possessing subtypes that differ in their association with human disease , a plasmid-encoded enterohemolysin and, in strains lacking eae, an autoagglutinating adhesin (Saa)  in children, STEC O157:H7 being the most notorious agent of the group. However, there are more than 400 non-O157 serotypes that have been involved in human disease and isolated from reservoirs of infection . These enrichment samples could have individual bacteria or mixture of different bacteria harboring stx, eae, and serogroups that should be confirmed with isolation.During 2012, Food Safety and Inspection Service (FSIS) determined the zero-tolerance policy for STEC O157:H7 including the top 6 non-O157 STEC serogroups in raw, nonintact beef products  , A large outbreak that ocurred in 2011 in Germany was caused by a strain O104:H4 harboring stx1, stx2, eae, ehxA, and O157 followed by a second GeneDisc assay targeting eight STEC serogroup: O26, O45, O91, O103, O111, O113, O121, O145 in ground beef. Notably, these authors have incorporated two important STEC serogroups: O91 and O113 that lack the eae gene but carry ehxA. Both serogroups have been related to HUS cases.In order to evaluate a new PCR-real time technology based on simultaneous detection of multiple targets, Fratamico and Bagi  have usestx1, stx2, and eae. These authors have described the use of Rainbow Agar O157 for isolation of non-O157 because non-O157 strains produce colonies of different colors according to serogroup.In this study two enrichment media were compared, buffered peptone water (BPW) recommended by GeneDisc and mTSB for STEC detection in the ground beef artificially inoculated. With both media, PCR results were similar and target genes were correctly identified with similar Ct values. Immunomagnetic separation (IMS) for serogroups O26, O45, O103, O111, O121, O145, O157, and plating onto Rainbow agar O157 were used and isolates were confirmed as the correct STEC serogroup. Serogroups O91 and O113 were not subjected to IMS and were more difficult to isolate on Rainbow Agar O157. All isolates were confirmed by conventional PCR targeting Fratamico and Bagi  demonstrThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atherosclerosis is one of the common health threats all over the world. It is a complex heritable disease that affects arterial blood vessels. Chronic inflammatory response plays an important role in atherogenesis. There has been little success in fully identifying functionally important genes in the pathogenesis of atherosclerosis.p < 0.05) as overrepresented terms, indicating that atherosclerosis invokes many genes with a wide range of different functions. Pathway analysis demonstrated that the most highly enriched pathway is the Toll-like receptor signaling pathway. Finally, through gene network analysis, we prioritized 48 genes using the hub gene method.In the present study, we performed a systematic analysis of atherosclerosis-related genes using text mining. We identified a total of 1312 genes. Gene ontology (GO) analysis revealed that a total of 35 terms exhibited significance  contains supplementary material, which is available to authorized users. Atherosclerosis is a complex heritable disease involving multiple cell types and the interactions of many different molecular pathways . AtherosRecent genome-wide association studies (GWAS), involving hundreds of thousands of individuals, have identified numerous loci contributing to atherosclerotic traits and to risk factors such as blood lipoprotein levels and blood pressure . Plasma Recently, the text mining methodology has been implemented, providing a necessary means to retrieve disease-related genes in an automated way . Here, wWe ran a key word search in the PubMed database for articles related to atherosclerosis and obtained 45,304 entries as a result (from January 1980 to April 2016). Abstracts of these articles were downloaded and processed through a text mining pipeline shown in Fig. p < 0.05) as overrepresented terms. In the biological process category, response to stimulus, cell communication, regulation of biological process, cellular process, behavior, multicellular organismal development, cell motility, cell death, metabolic process, cell differentiation, enzyme regulator activity, transcription regulator activity, electron carrier activity, secretion, catabolic process, transport, macromolecule metabolic process, and unspecific monooxygenase activity were found to be significantly enriched. GO terms related to extracellular region, extracellular space, cell surface, cytoplasm, membrane, proteinaceous extracellular matrix, and cell were overrepresented under the cellular component category. The overrepresented GO terms in the molecular function category were protein binding, binding, signal transducer activity, receptor activity, antioxidant activity, oxidoreductase activity, catalytic activity, hydrolase activity, kinase activity, and transferase activity  annotation terms using the BiNGO program package. Enrichment analysis revealed that a total of 35 terms exhibited significance (p < 0.05)  network was constructed by merging up-to-date protein-protein interactions available in IntAct , BioGRIDIn the present study, we attempted to compile a complete list of genes involved in atherosclerosis. In recent years, high-throughput transcriptomic and proteomic approaches make it possible for studying the expression levels of thousands of genes and proteins simultaneously. However, these data suffer from high technical variability and high dimension size , 16. On p value, the most highly overrepresented pathway went to the Toll-like receptor (TLR) signaling pathway. The Toll-like receptor signaling pathway is known to play an important role during atherosclerosis in both immune and inflammatory response. The disruptions of cellular or organismal cholesterol homeostasis that occur as a risk factor of atherosclerosis may lead to an augmentation of inflammatory responses via enhanced TLR signaling or inflammasome activation [We found that 1312 genes were associated with atherosclerosis. Based on GO analysis, 35 GO terms were significantly enriched. Additionally, our study also revealed 20 enriched pathways. Based on enrichment tivation . TLR acttivation .A genome-wide gene network was constructed by using up-to-date interaction data available in the PINA2 database . We obtaThe top 20 hub genes are the following: APP (amyloid beta A4 precursor protein), HSP90AA1 , GRB2 (growth factor receptor-bound protein 2), SRC , TP53 (tumor protein p53), ESR1 (estrogen receptor 1), FN1 (fibronectin 1), TRAF6 (TNF receptor-associated factor 6), EGFR , SUMO1 (SMT3 suppressor of mif two 3 homolog 1), YWHAZ (14-3-3 zeta), MYC , CDK2 (cyclin-dependent kinase 2), HSPA8 (heat shock 70-kDa protein 8), MAPK1 (mitogen-activated protein kinase 1), AKT1 , COPS5 (COP9 constitutive photomorphogenic homolog subunit 5), MDM2 (Mdm2 p53 binding protein homolog), RELA , and HSPA4 (heat shock 70-kDa protein 4). APP is present in advanced human carotid plaques, in proximity to activated macrophages and platelets , and lacA limitation for text mining-based strategies is that there is no chance to discover new genes. In order to solve this problem, we enlarged the network by inducing new genes that are not reported to be involved in atherosclerosis. According to the rule of “guilty-by-association,” these new genes may be potential susceptibility genes. Finally, we made a list of 50 new genes, all of which have more than 44 connections with known genes . Text mining was performed at sentence level.Gene mention recognition was performed using two different gene mention taggers, the hidden Markov model (HMM) tagger implemented in LingPipe and the ABNER tagger based on a machine learning system of conditional random fields (CRF) . Gene mep value <0.01 was used as significance threshold to identify enriched categories.GO enrichment analysis was performed by using BiNGO 2.3 with GOslim dataset . To testp values and Benjamini-Hochberg adjusted p values through the DAVID bioinformatics resource 6.7 [To rank overall importance of pathways involved in atherosclerosis, we calculated Fisher’s exact test urce 6.7 . The sigurce 6.7 , 51.The genes associated with atherosclerosis were cross-referenced with the PINA2 database to create the PPI network . The PIN


Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major  and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come. Anopheles, Aedes and Culex are vectors of, e.g., malaria, yellow fever, dengue fever, hemorrhagic fever and lymphatic filariasis [Anopheles gambiae) and West Nile virus (Culex pipiens) emerged over 25 years ago in Africa, America and Europe and it is frequently due to loss of sensitivity of the insect's acetylcholinesterase to organophosphates and carbamates [Mosquitoes are an enormous public health menace in transmitting various tropical diseases and generally as a nuisance . Many splariasis ,3,4. Deslariasis . Chemicarbamates . Alternarbamates .Bacillus thuringiensis subsp. israelensis (Bti) is the first subspecies of B. thuringiensis (Bt) found to be toxic to dipteran larvae. This gram-positive spore-forming subspecies is the most powerful and environmental-friendly biological alternative component in integrated programs to control disease vectors [Bti forms a crystalline parasporal body composed of protein protoxins (δ-endotoxins)   that ared midges ,9. Bti ild usage ,12,13,14ld usage ,16,17,18ld usage . Thus, lld usage . The lacd Cyt1Aa ,21,22. Bti includes the following species: Tabanus triceps (Diptera: Tabanidae) [Anastrepha ludens and Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) [Tipula paludosa (Diptera: Nematocera) [Bradysia coprophila and Bradysia impatiens (Diptera: Sciaridae) [Rivellia angulata (Diptera: Platystomatidae) [Acyrthosiphon pisum (Hemiptera: Aphidoidea) [Macrosiphum euphorbiae (Homoptera: Aphididae) [Anthonomus grandis (Coleoptera: Tenebrionidae) [Chrysomela scripta (Coleoptera: Chrysomelidae) [Spodoptera frugiperda (Lepidoptera: Noctuidae) [Plutella xylostella (Lepidoptera: Plutellidae) [Meloidogyne incognita on barley [Schistosoma mansoni and Trichobilharzia szidati (Trematoda: Schistosomatidae) [In addition to mosquitoes, black flies  and chirbanidae) , Mexicanritidae) ,28, Tipuatocera) , fungus iaridae) ,31, nodumatidae) , pea aphidoidea) , potato hididae) , cotton ionidae) , leaf bemelidae) , fall arctuidae) , diamondellidae) , root-knn barley  and tremmatidae) .Cx. pipiens larvae [Bti seems able to reproduce and persist under natural conditions [Bti  settles within 24–48 h at the bottom of mosquito breeding sites. Ingested spores germinate and recycle in carcasses of Bti-killed mosquito larvae [Originally isolated from a temporary pond with dying s larvae , Bti seenditions ,43,44. So larvae ,46,47 anBti multiplication in nature, e.g., the ciliate protozoan Tetrahymena pyriformis [T. pyriformis during the digestion process; the spores germinate in excreted food vacuoles and complete a full growth and sporulation cycle in them [-endotoxin components (Cry4 > Cry11 > Cyt) differs from that of the Bti parasporal body crystals [Other organisms coexisting in mosquito breeding sites may support riformis . Spores  in them ,50. In t in them , in whiccrystals .Bti harbors eight circular plasmids ranging in size between 5 and 210 kb and a linear replicon of approximately 16 kb [Bti resides in at least four major crystal protoxins, of 134, 128, 72 and 27 kDa, encoded by cry4Aa, cry4Ba, cry11Aa and cyt1Aa respectively, all mapped on the 128 kb plasmid known as pBtoxis [cry10Aa, cyt2Ba and cyt1Ca: Cry10Aa and Cyt2Ba accumulate in small amounts in the parasporal body and seem to contribute to the overall toxicity of Bti [cry11Aa is organized in an operon together with p19 and p20 [Escherichia coli [Pseudomonas putida [Bt [The original isolate of ly 16 kb ,52. The  pBtoxis ,54,55. Iy of Bti ,57,58. Ty of Bti ,60, whery of Bti ,62,63. T and p20 ,65. The hia coli ,67, Pseutida [Bt ,69 by intida [Bt ,62,63. The Cry and Cyt toxins are membrane-perforating proteins although unrelated structurally and differ in their requirement of essential membrane components; the Cry’s bind to membrane receptors ,73,74,75Culex and less to Anopheles and Aedes [Anopheles and Aedes but very low against Culex [Aedes and Culex but lower against Anopheles [Cry4Aa, encoded by a sequence of 3543 bp (1180 amino acids), is highly toxic to larvae of nd Aedes ,70,78,79st Culex ,70,78,79st Culex . Cry11Aanopheles ,21,82.in vitro to cells of certain vertebrates and invertebrates [ in vivo, implying a specific mode of action [Bti’s Cry proteins [The larvicidal activity of Cyt1Aa, encoded by a sequence of 744 bp (248 amino acids), is low against all three mosquito genera ,83,84. Itebrates  and highf action . The cytf action  but the proteins ,88,89,90Bti crystals [cry10Aa is arranged in an operon 48 bp upstream orf2 [cry10Aa and orf2 is a variant of the cry4-type genes. Toxicity of Cry10Aa is comparable to those of the other Cry4 toxins and is synergistic with Cyt1Aa against Aedes aegypti larvae [C. pipiens larvae [Cry10Aa, encoded by a sequence of 2025 bp (675 amino acids), accumulates to minor amounts in crystals ,91 and deam orf2 . Orf2 isi larvae  and withs larvae .Bti crystals [in vitro [Culex, Aedes and Anopheles than Cyt1Aa [Bt subsp. medellin [B sphaericus binary toxin [Bti.Cyt2Ba, encoded by a sequence of 789 bp (263 amino acids), is found at very low concentrations in crystals . Proteolin vitro ,95 and en Cyt1Aa  but highmedellin . Cyt2Ba medellin ,98 and wry toxin ; it may β-trefoil carbohydrate-binding motif, similar to the receptor binding domain of ricin-B lectin type found in several ricin-like toxins [cyt1Ca were detected, but Cyt1Ca has not been found [Cyt1Ca is encoded by sequence of 1575 bp (525 amino acids) ,99. Its en found ; the reaen found ,99. The en found .In vitro and in vivo processing yields two fragments, of 45 and 20 kDa for Cry4Aa and 45 and 18 kDa for Cry4Ba [Basic studies of the structures and modes of action of δ-endotoxins and their receptors are important for future development of biopesticides that will not be prone to insect resistance . The levr Cry4Ba ,109,110.r Cry4Ba ,112,113.r Cry4Ba .in vitro to the apical brush border of midgut cells in the gastric caecae and posterior stomach of An. gambiae larvae [Ae. aegypti [Cx. pipiens bind Cry4Aa specifically (both in vitro and in vivo) [Cx. pipiens, but together they display significant toxicity through association with each other to form an active complex of apparently 60 kDa [Subsequent steps involve toxin binding to receptors ,115,116,e larvae  and to t aegypti . The samin vivo) . Each ofy 60 kDa ,110.Aedes, Anopheles and Culex [Anopheles and Aedes [Ae. aegypti larval midgut were identified as the receptors of Cry4Ba [Anopheles albimanus [Bt in their receptor binding mechanism(s) [Different mosquito larvicidal activity spectra of both activated Cry4Aa and Cry4Ba likely stem from the structural differences found within domain II and distinct sites binding to the host receptors ,81,105. nd Culex , whereasnd Aedes ,105,122.f Cry4Ba ,124, andlbimanus . Cry4Aa anism(s) .α4-α5 hairpins into the membrane [in vitro can also form pre-pore oligomers that are proficient in perforation and formation of stable ion channels even without support of the receptors [Pre-pore trimeric structures of either Cry4Aa or Cry4Ba seem to form in aqueous solution and in lipid monolayer, which may facilitate insertion of their three membrane ,127,128.eceptors .in silico model was obtained based on that of Cry2Aa [Cx. pipiens and Culex quinquefasciatus [An. stephensi and Tipula oleracea respectively [The three-dimensional structure of Cry11Aa has still not been solved but an f Cry2Aa . The patasciatus ,112,113.ectively . Its putectively ,130,131.ectively . Loop α-ectively .Btkyushuensis [The crystal structure of the proteolytically activated, monomeric forms of Cyt2Ba and Cyt1Aa were solved to 1.8 Å and 2.2 Å resolutions, respectively ,132. Theshuensis  and the shuensis , suggestBased on its structure, toxicity of Cyt1Aa is correlated with ability to undergo conformational changes that must occur prior to membrane insertion and perforation ,132. TheIn vitro processing of Cyt1Aa protoxin yields single active 22–25 kDa fragment [Cyt1Aa do not bind specific receptors but have strong binding affinity to the unsaturated fatty acids that compose the membrane of midgut epithelial cells of dipterans ,135. In fragment ,137 thatfragment ,139.An. gambiae larvae; this may be related to the ability of the toxin to perforate cell membranes without participation of any specific receptor [in vivo. This implies a specific mode of action that is different to those of Bti Cry’s, but an insect-specific receptor may still be essential for the specificity of the Cyt toxins [Cyt1Aa binds to the apical brush border of midgut cells, to the gastric caecae and to stomach cells of receptor  by a mect toxins ,140.Two different models were proposed for the mode of action of Cyt toxins: pore-forming ,142 and Bti δ-endotoxins are attributed to complex interactions between six proteins, Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa and Cyt2Ba, differing in toxicity levels and against different species of mosquitoes  preserves cell viability [E. coli causes instant arrest in biomass growth and decreased viability [Expression of  ability ,162; the ability ,164. The ability . Co-expriability ,165. Antiability .Bti, are antibacterial also exogenously, against E. coli and Gram-positive species  [E. coli, whereas it is bacteriostatic for S. aureus as reflected in morphological changes and ion balance alteration [Cyt1Aa, Cry4Ba and Cry11Aa, as well as two proteins (of 36 and 34 kDa) isolated from  aureus) ,166,167.teration . Cyt1Aa teration .Bt subspecies  are toxic to the anaerobic Gram-positive bacteria Clostridium butyricum and Clostridium acetobutylicum and the archaea Methanosarcina barkeri [Cry toxins from other  barkeri .Cyt1Aa and Cyt2Ba display anti-cancer activities as well; conjugation of activated Cyt1Aa to a peptide carrier molecule is toxic against murine hybridoma cells  whereas Bti for mosquito control is limited by short residual activity of current preparations under field conditions [B. sphaericus in Bti [Cx. quinquefasciatus and Cx. tarsalis, even to lines selected for resistance to the binary toxin. Higher toxicity against fourth-instar Cx. quinquefasciatus was achieved in recombinant acrystalliferous Bti strain that produces the combination of B. sphaericus binary toxin together with Cyt1Aa of Bti and Cry11Ba from Bt subsp. jegathesan [Applying nditions  due to: nditions ,9. Best s in Bti . The recgathesan .cry4Aa and cry11Aa alone or with cyt1Aa were expressed from the dual constitutive and efficient promoters psbAP and A1P in the filamentous, nitrogen-fixing cyanobacterium Anabaena PCC 7120 [50 values of these clones against third and fourth instar Ae. aegypti larvae were 8.3 × 104 and 3.5 × 104 cells mL−1 respectively, the lowest reported values for engineered cyanobacterial cells with Bti toxin genes. Toxicity of the Anabaena clone expressing constitutively cry4Aa and cry11Aa with p20 is retained following irradiation by high doses of UV-B, doses that partially inactivate Bti toxicity [An. merus, An. arabiensis and An. gambiae, but very weak activity against An. funestus [Several attempts have been made during the last two decades to produce transgenic mosquito larvicidal cyanobacteria ,174. MosPCC 7120 ,178,179.toxicity . This laBti is environmentally friendly and a safe alternative means to control mosquitoes and blackflies. Emergence of resistant variants has not been found despite three decades of extensive use, likely due to the complex and diverse δ-endotoxins composition of its crystal. To overcome or prevent theoretical, future emergence of resistance, recombinant microorganisms can be engineered to co-express toxins with different modes of action or chimeric toxins with improved efficacy [Bti’s mosquito larvicidal activity can be achieved by totally different mechanisms that wane larval survival, e.g., chitinase (damaging the peritrophic membrane) [efficacy . Enhanciembrane)  and Trypembrane) ,183.