Investigating the New Zealand Off-Site Manufacturing Industry’s Readiness for Automated Compliance Checking

Numerous automated compliance checking (ACC) approaches have been developed over the last half of the twentieth century. However, little is known as to how well the ACC technology has served the off-site manufacturing (OSM) industry from the end users’ perspective. This paper aims to measure the New Zealand (NZ) OSM industry’s awareness and readiness for ACC and explore a pathway toward wider ACC adoption. It first reports on a survey study in NZ with 44 valid survey responses. It then proposes a high-level roadmap with key actions that can facilitate wider ACC adoption through 16 interviews with international ACC experts and a focus group with nine local OSM stakeholders. The results show that although there is a high demand for automating compliance processes, the OSM industry, especially small and medium enterprises, are not ready to adopt the ACC technology. Suggestions to address this include (1) establish the foundation for broad ACC adoption; (2) boost the development of the ACC technology to expedite its maturity, (3) test the ACC technology under different scenarios and customize it for the NZ context; (4) encourage the government to provide funding and policy support; and (5) promote education and training of both building information modeling (BIM) and ACC to OSM stakeholders. The results can provide software vendors with valuable information about user expectations and requirements to develop ACC products that can better serve NZ OSM projects, and help OSM stakeholders in NZ and countries with similar economic and regulatory structures to understand the technological and nontechnological gaps to better prepare for the ACC technology adoption

Structure-function study of maize ribosome-inactivating protein: implications for the internal inactivation region and the sole glutamate in the active site

Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 Å, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long α-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. © 2007 The Author(s).published_or_final_versio

Maize ribosome-inactivating protein uses Lys158-lys161 to interact with ribosomal protein P2 and the strength of interaction is correlated to the biological activities.

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158-Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the 'FGLFD' motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K(D)) of their interaction, which are 1037.50 ± 65.75 µM, 611.70 ± 28.13 µM and 194.84 ± 9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity

MOD can be crosslinked to rat liver ribosome and P2.

<p>Crosslinking reactions were carried with individual proteins (lanes 1 and 3, 4 and 6) or mixtures of two proteins (lanes 2 and 5) and subject for western analysis. Protein bands were detected by anti-myc, anti-MOD and anti-P antibodies.</p

Comparison of Five TRIzol-Based Protein Preparation Methods for 2-DE Production From Challenging Marine Dinoflagellate Samples: A Case Study on Two Benthic Prorocentrum Species

Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method&rsquo;s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples

Interaction between RIPs and P2.

<p><b>A) Interaction of MOD, TCS and StxA with P2 at different ionic strengths.</b> Pull-down assay was conducted on RIPs under various ionic strengths to compare their strength of interaction with P2. Input indicates the same amount of purified RIPs was loaded to the P2-sepharose column and proteins were eluted using buffer with 1M NaCl. <b>B) Interactions between RIPs and C-terminal truncated P2.</b> C-terminal truncated P2 variants were subject to pull down assay with RIPs. The C-terminal amino acid sequences of P2, P2 [ΔC5] and P2 [ΔC10] are: AEEKKDEKKEESEE<b>SDDDMGFGLFD</b>, AEEKKDEKKEESEE<b>SDDDMG</b> and AEEKKDEKKEESEE<b>S</b> respectively. The bold letters refer to the conserved residues in P-proteins.</p

Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.

<p>Relative <i>N</i>-glycosidase activities of maize RIP and its variants on 28S rRNA and rat liver ribosome.</p

Residues on MOD that correspond to the C11-P interacting residues on TCS.

<p><b>A) Superimposed structures of MOD (pdb: 2PQI) and TCS-C11-P complex (pdb: 2JDL).</b> The beta strands β7 and β8 in TCS (wheat) are replaced by the helix αI in MOD (cyan) while helices αG and αJ are conserved. P2-binding residues on MOD and TCS are distant apart as indicated in red and green respectively. <b>B) Comparison of C11-P interacting residues on TCS and the corresponding residues on MOD.</b> Structures of MOD and TCS are superimposed to locate the residues on MOD (colored in cyan and labelled in black and italic) corresponding to the C11-P interacting residues on TCS (colored in wheat and labelled in red). Many residues in these two RIPs are different, especially those at the hydrophobic patch of TCS (Phe-166, Val-223, Ile-225, Gly-231, Val-232 and Asn-236), suggesting MOD may interact with C11-P at a different site. <b>C) Stereo image zooming in the hydrophobic pocket of MOD (colored in cyan) with C11-P (colored in yellow).</b> The model reveals Arg-275 on MOD may crash with Leu-9 on C11-P while Glu-272 confronts directly Asp-11. Residues on TCS that interact with C11-P are highlighted in wheat for reference.</p

Screening of basic residues on MOD that are responsible for ribosome binding.

<p>The indicated residues were mutated to alanine and screened for their abilities to bind ribosome. W and E denote last wash and elution respectively.</p

Lys158–Lys161 on MOD are responsible for the interaction between maize RIP and P2.

<p><b>A) MOD does not bind to P2 when Lys158–Lys161 are mutated to alanine.</b> SDS-PAGE of the last wash (W) and elution (E) obtained from the pull-down assay of maize RIP and P2. Input indicates the purified proteins loaded to the column. <b>B) Sensorgram showing MOD but not pro-RIP and MOD [K158A-K161A] interact with sensor chip-immobilized P2.</b> 500 nM of maize RIP variants or running buffer were injected to the sensor chip for three minutes for association, followed by a dissociation using running buffer. Response unit (RU) was monitored in a real-time manner.</p