Search

Within aerospace and automotive manufacturing, the majority of quality assurance is through inspection or tests at various steps during manufacturing and assembly. Such tests do not tend to capture or make use of process data for in-process inspection and certification at the point of manufacture. Inspection of the product during manufacturing can potentially detect defects, thus allowing consistent product quality and reducing scrappage. However, a review of the literature has revealed a lack of any significant research in the area of inspection during the manufacturing of terminations. This work utilises infrared thermal imaging and machine learning techniques for inspection of the enamel removal process on Litz wire, typically used for aerospace and automotive applications. Infrared thermal imaging was utilised to inspect bundles of Litz wire containing those with and without enamel. The temperature profiles of the wires with or without enamel were recorded and then machine learning techniques were utilised for automated inspection of enamel removal. The feasibility of various classifier models for identifying the remaining enamel on a set of enamelled copper wires was evaluated. A comparison of the performance of classifier models in terms of classification accuracy is presented. The best model for enamel classification accuracy was the Gaussian Mixture Model with expectation maximisation; it achieved a training accuracy of 85% and enamel classification accuracy of 100% with the fastest evaluation time of 1.05 s. The support vector classification model achieved both the training and enamel classification accuracy of more than 82%; however, it suffered the drawback of a higher evaluation time of 134 s.

The experiment was conducted to study the effect of supplementation of designer dietary antioxidant micronutrients on udder health, milk yield, and its quality in buffaloes under field conditions. Sixteen healthy multiparous advanced pregnant graded Murrah buffaloes (around the last 3 months of gestation), identical in body weights, parity, and feeding conditions, were selected for the study. Feed offered and residues left of an individual animal were measured and recorded for 7 consecutive days with the sampling of feeds being offered to buffaloes and analyzed for dry matter and trace minerals Zn, Cu, and Se. Carotene and vitamin E content of offered feed samples were calculated based on reported values. The calculation was made to determine the deficiency of above micronutrients, and an antioxidant micronutrient supplement was designed to take care of the deficient micronutrients and supplemented in half of the buffaloes (n = 8) considered to be a treatment, while remaining half (n = 8) is considered to be control, fed as per the existing practice of farmer without additional supplementation. This supplementation study was continued for 2 months during advanced pregnancy and thereafter for 3 months post-calving. Dry matter intake recorded weekly during the pre- and postpartum period of study remained comparable (P > 0.05) between two groups. Udder health monitored fortnightly by modified California mastitis test, milk pH and somatic cell count indicated improvement (P

Muscarinic acetylcholine receptors (mAChRs) are important therapeutic targets for several diseases of the central nervous system and periphery. However, the lack of subtype-selective ligands for these receptors is a major challenge. A novel approach involving the integration of a natural product framework with a bioactive molecule (iNPBM) by using gephyrotoxin and the isoindoline framework is demonstrated for the discovery of new and selective mAChR modulators. We established a scalable and versatile synthetic scheme to enable the synthesis of various analogues that provided the first structure-activity relationship study of this class of compounds. Pharmacological profiling of these compounds demonstrated several ligands with high affinity and selectivity for mAChRs. Specifically, RG-06 and RG-09 were found to be antagonists of M3-mAChR, whereas RG-02 was found to be an agonist at M2-mAChR. Furthermore, RG-02 exhibited salutary effects in an established pharmacological model of a cognitive deficit in mice.

An efficient and scalable synthesis of various enantiopure 1,3- disubstituted isoindolines is reported. The base catalyzed nucleophilic fragmentation of a rigid overbred template is established with various substrates to afford the corresponding 1,3-disubstituted isoindoline ester, amide, thioate, 1,3-amino alcohol and isoindolylcarboxylic acid. The crucial rigid overbred template is synthesized in an optically pure form in multigram scale by asymmetric desymmetrization of the corresponding meso compound.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is the leading cause of bacterial infectious disease mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for Mtb survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant Mtb strains. Here we present the design, synthesis, and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5′ oxygen atom with carbon as well as incorporation of a bulky group on the nucleobase to prevent the required syn-conformation necessary for proper alignment of N-...

Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.

Abstract 741 We have previously shown that a costimulation blockade-containing regimen could provide effective protection against acute GvHD (aGvHD) in a non-human primate model. We therefore designed a first-in-disease trial of in vivo CD28:CD80/86-directed costimulation blockade with CTLA4-Ig (abatacept) to prevent aGvHD after unrelated-donor HSCT for patients > 12y. (Clinical Trials.Org #NCT01012492). In this trial, 10mg/kg abatacept was administered IV on day −1, +5, +14, +28 in addition to standard prophylaxis with cyclosporine + MTX. Enrollment of all patients is complete, and the study is evaluable for feasibility, toxicity, engraftment, and the primary immunologic outcome: the incidence of Grade III-IV aGvHD by d+100. Patient Characteristics and Survival: 10 patients, with a median age of 44.5 y (17–74) were enrolled and treated. 6 patients received HLA-mismatched grafts (matched at 7/8 alleles) and 4 received 8/8 HLA-matched URD grafts. 8 received PBSCs and 2 received BM. All received high-intensity conditioning (Bu/Cy, TBI/Cy or Flu/Mel). With a median follow-up of 367 days (262–680), 7 patients are alive and in remission. 2 patients died of relapse (Day +121 and Day +147). One patient died, in remission, of multi-organ failure on day +453 post-transplant. Feasibility, Pharmacokinetics and Pharmacodynamics of Abatacept in HSCT: All 10 patients received all 4 scheduled abatacept doses, without infusion reactions. The average peak (230.9 +/− 7.4 mg/ml) and trough (45.9 +/− 2.8 mg/ml) abatacept levels, as well as the terminal T1/2 (19.6 +/− 1.9 days) were similar to that observed previously. Importantly, as has been previously established in vitro, patients receiving abatacept demonstrated significant inhibition of post-transplant CD4+ T cell proliferation (with >80% reduction in the accumulation of Ki-67+ proliferating CD4+ T cells at d+14 and +28 compared to a control cohort who received cyclosporine + methotrexate without abatacept, Figure 1). This data establishes, for the first time, the feasibility of giving abatacept to this new patient population, and that the PK and PD parameters in HSCT closely mirror those previously shown to effectively control immune-mediated diseases. Engraftment: All patients achieved neutrophil engraftment (median d+16.5) and donor engraftment (100% CD33 chimerism at d+30). Lymphocyte recovery was rapid: Day +100 mean peripheral blood counts showed ALC = 1053 +/− 259 cells/ml, total T cells = 741 +/− 208 cells/ml, and CD8+ T cells = 381 +/− 99 cells/ml. The Day +100 CD4+ T cells = 285 +/− 105 cells/ml, not significantly different from historical controls (n = 43) that received CNI/MTX aGvHD prophylaxis without abatacept (262 +/−26 cells/ml). GvHD: Patients receiving the abatacept-containing regimen had encouragingly low rates of early severe aGvHD: Two patients developed aGvHD before day +100, with one of these patients (Gr II) progressing to steroid-dependent cGvHD of the liver and one patient (Gr III) with complete resolution of aGvHD (and currently off steroids). The cumulative incidence of grade II-IV and III-IV aGvHD by day 100 was thus 20% and 10%, respectively (Figure 2). Importantly, there was no Gr IV aGvHD, no patient received salvage therapy for aGvHD, and there were no deaths from aGvHD. After day +100, one patient developed late acute GvHD (currently off all immunosuppression), and two patients developed overlap syndrome. Both had responsive disease and are currently either weaning or off corticosteroids. No other patient developed late aGvHD, and no other patient has developed moderate or severe cGvHD. Infection: No life-threatening infections occurred. 5 patients developed CMV reactivation, all responsive to antivirals. No patient developed CMV disease. No EBV viremia >1000 copies/ml occurred. One patient developed EBV+ plasmacytic hyperplasia of the tongue, which resolved without intervention. No other EBV-related disease occurred. Conclusions: This trial demonstrates, for the first time, the feasibility of adding in vivo T cell costimulation blockade with abatacept for aGvHD prevention. The decreased CD4+ T cell proliferation post-transplant and the encouragingly low rates of early, severe aGvHD suggest that costimulation blockade may be an effective agent for aGvHD prophylaxis and support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept was used in this trial. It is a costimulation blockade agent which was tested for its ability to prevent aGvHD.

Abstract 2998 Introduction: GvHD remains the most deadly complication of HSCT despite current prevention strategies. To address the unmet need for better GvHD control, we have created a non-human primate (NHP) model with which to rigorously test mechanism and efficacy of novel therapeutics. In this study, we determined whether a novel combination of mTOR inhibition (with sirolimus) and CD28:CD80/86 costimulation blockade (with belatacept) could control GvHD. Here we show for the first time that these two agents combine synergistically to prevent both the clinical and immunologic manifestations of primate aGvHD. Methods: Rhesus macaque recipients were irradiated (9.6 Gy in 2 fractions at 7cGy/min), and then transplanted with G-CSF-mobilized PBSC from a haplo-identical donor (1–5×108 TNC/kg). Recipients were treated with either sirolimus alone (n = 4, troughs targeted at 5–10 ng/mL), belatacept alone (receiving weekly doses of 20 mg/kg), or combination therapy. Clinical GvHD was monitored using our previously described NHP grading scale (Miller et al., Blood 2010), and multiparameter flow cytometric analysis was performed. Results: Untreated controls (n = 5) developed rapid, severe histopathologically-proven aGvHD and succumbed rapidly (MST = 7 days). Recipients treated with either sirolimus or belatacept alone were partially protected from the clinical manifestations of GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease (with diarrhea but no elevation of bilirubin) and had an MST of 14 days (Figure 1). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD (bilirubin rapidly rising to 6–30 × normal with histologically-confirmed lymphocytic infiltration) and an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant). We employed multiparameter flow cytometry to determine the immunologic consequences of sirolimus and belatacept on T cell proliferation (using Ki-67 expression) and cytotoxity (using granzyme B expression). We found that the clinical synergy observed with combined therapy was recapitulated immunologically. Thus, while untreated aGvHD was associated with rampant CD8+ proliferation (with 83 +/− 14% Ki-67+ CD8+ vs 4.7 +/− 0.6% pre-transplant), sirolimus or belatacept as monotherapy both partially controlled proliferation (35 +/− 3% and 65 +/− 23% Ki-67+ CD8+ with sirolimus or belatacept, respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8 +/− 3%, favorably comparing with 13% Ki-67+ CD8+ T cells using standard Calcineurin Inhibitor/Methotrexate (CNI/MTX) prophylaxis). Sirolimus and belatacept both also partially controlled GvHD-related T cell cytotoxicity. Thus, while untreated aGvHD was associated with excessive granzyme B expression in CD8+ T cells (82 +/− 2% granzyme Bvery high CD8+ cells vs 0.3 +/− 0.2% pre-transplant) sirolimus or belatacept monotherapy also partially controlled cytotoxicity (8 +/− 1% and 35 +/− 1% granzyme Bvery high with sirolimus or belatacept, respectively). Combination therapy dramatically reduced the proportion of these cells, to 1.5 +/− 0.8 % granzyme Bvery high, favorably comparing with 4% granzyme Bvery high using CNI/MTX. The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 2A, B) supporting a pathogenic role for these highly proliferative and cytotoxic cells in aGvHD pathology. Moreover, significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 2C) suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood. Implications: These results reveal a previously undiscovered synergy between sirolimus and belatacept in the control of primate aGvHD, and provide support for future clinical investigation of this novel prevention strategy. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring in either the blood or in GvHD target organs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.

SummaryThe mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development, and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent subnanomolar enzyme inhibition and antitubercular activity against multidrug resistant and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the antibacterial activity is MtBPL dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray cocrystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.