Appendix-1,2 – Supplemental material for Influence of respondent type on relationships between safety climate and safety performance in manufacturing firm

<p>Supplemental material, Appendix-1,2 for Influence of respondent type on relationships between safety climate and safety performance in manufacturing firm by Vedant Singh and Anita Verma in Proceedings of the Institution of Mechanical Engineers, Part O: Journal of Risk and Reliability</p

Synthesis of dopamine in <i>E. coli</i> using plasmid-based expression system and its marked effect on host growth profiles

<p>L-Dopa and dopamine are important pathway intermediates toward the synthesis of catecholamine such as epinephrine and norepinephrine from amino acid L-tyrosine. Dopamine, secreted from dopaminergic nerve cells, serves as an important neurotransmitter. We report the synthesis of dopamine by extending the aromatic amino acid pathway of <i>Escherichia coli</i> DH5α by the expression of 4-hydroxyphenylacetate-3-hydrolase (HpaBC) from <i>E. coli</i> and an engineered dopa decarboxylase (DDC) from pig kidney cell. The activity of HpaBC and DDC require 200 µM iron supplementation and 50 µM vitamin B6, respectively as additives to the growth media. The maximum concentration of L-dopa and dopamine obtained from the broth was around 26 and 27 mg/L after 24 hr of separate shake flask studies. We observed that in the presence of dopamine synthesized <i>in vivo</i> host growth was remarkably enhanced. These observations lead us to an interesting finding about the role of these catecholamines on bacterial growth. It is clear that synthesis of dopamine <i>in vivo</i> actually promotes growth much efficiently as compared to when dopamine is added to the system from outside. From HPLC and GC–MS data it was further observed that L-dopa was stable within the observable time of experiments whereas dopamine actually was subjected to degradation via oxidation and host consumption.</p

Metabolic engineering of <i>Escherichia coli</i> W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol

<p>L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in <i>Escherichia coli</i> is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.</p

Multiple Amino acid Sequence alignment of BmCRT with parasites and human CRT.

<p>Six C1q binding sites (green shading), C terminal ER targeting sequences (Grey shading), Putative nuclear localization signal site (Red shading) and CRT signature motifs (yellow shading) are indicated.</p

BmCRT and Human C1q interaction details showing number of hydrogen bonds and non bonded contacts.

<p>BmCRT and Human C1q interaction details showing number of hydrogen bonds and non bonded contacts.</p

Validation of Protein Models.

<p>(<b>A</b>) Graphical representation of ANOLEA, QMEAN and GROMOS analysis of modeled BmCRT protein structure. (<b>B</b>) ProSA analysis indicated that the overall interaction energy of the model was measure in Z-Score -6.86 kcal/mol and template was −6.41 kcal/mol.</p

<i>In Silico</i> and <i>In Vitro</i> Studies on the Protein-Protein Interactions between <i>Brugia malayi</i> Immunomodulatory Protein Calreticulin and Human C1q

<div><p>Filarial parasites modulate effective immune response of their host by releasing a variety of immunomodulatory molecules, which help in the long persistence of the parasite within the host. The present study was aimed to characterize an immunomodulatory protein of <i>Brugia malayi</i> and its interaction with the host immune component at the structural and functional level. Our findings showed that <i>Brugia malayi</i> Calreticulin (BmCRT) is responsible for the prevention of classical complement pathway activation via its interaction with the first component C1q of the human host. This was confirmed by inhibition of C1q dependent lysis of immunoglobulin-sensitized Red Blood Cells (S-RBCs). This is possibly the first report which predicts CRT-C1q interaction on the structural content of proteins to explain how BmCRT inhibits this pathway. The molecular docking of BmCRT-C1q complex indicated that C1qB chain (IgG/M and CRP binding sites on C1q) played a major role in the interaction with conserved and non-conserved regions of N and P domain of BmCRT. Out of 37 amino acids of BmCRT involved in the interaction, nine amino acids (Pro¹²⁶, Glu¹³², His¹⁴⁷, Arg¹⁵¹, His¹⁵³, Met¹⁵⁴, Lys¹⁵⁶, Ala¹⁹⁶ and Lys²¹²) are absent in human CRT. Both ELISA and <i>in silico</i> analysis showed the significant role of Ca⁺² in BmCRT-HuC1q complex formation and deactivation of C1r₂–C1s₂. Molecular dynamics studies of BmCRT-HuC1q complex showed a deviation from ∼0.4 nm to ∼1.0 nm. CD analyses indicated that BmCRT is composed of 49.6% α helix, 9.6% β sheet and 43.6% random coil. These findings provided valuable information on the architecture and chemistry of BmCRT-C1q interaction and supported the hypothesis that BmCRT binds with huC1q at their targets (IgG/M, CRP) binding sites. This interaction enables the parasite to interfere with the initial stage of host complement activation, which might be helpful in parasites establishment. These results might be utilized for help in blocking the C1q/CRT interaction and preventing parasite infection.</p></div

BmCRT prevent C1q-dependent haemolysis of Ig-sensitized erythrocytes.

<p>Recombinant BmCRT inhibits C1q-dependent haemolysis while BSA (control) shows no effect on this when incubated with C1q deficient serum together with C1q as described in material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106413#s2" target="_blank">method</a> section. Percentage of cell lysis was calculated with reference to 100% lysis of the cells in water. The significant were analyzed by two-way ANOVA (***P<0.001). Assay was performed in triplicates. Bar represent the standard deviations of the mean.</p

Dose dependent interaction between BmCRT and HuC1q by solide-phase binding assay.

<p>Microtiter plate was coated with 0–30 µg of rBmCRT (<b>A</b>) or BSA (<b>B</b>) in carbonate buffer. After blocking with 5% skimmed milk the plate was incubated with human C1q and rabbit anti-human IgG Abs were used to detect the interaction of both protein as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106413#s2" target="_blank">method</a> section. No binding of C1q was observed with BSA (control). The significant between BSA (or 0.0 concentration of C1q) and different concentration of C1q were analyzed by two-way ANOVA (***P<0.001). Assay was performed in triplicates. Bar represent the standard deviations of the mean.</p

Predicted binding site region of BmCRT showing pink colored surface located in N and P domains, and structure of Human C1q, showing pink colored surface in the head region located in top portion of all three chains.

<p>Predicted binding site region of BmCRT showing pink colored surface located in N and P domains, and structure of Human C1q, showing pink colored surface in the head region located in top portion of all three chains.</p