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In stretch blow molding (SBM) process, the preform growth during the stretching and blowing is critical to the thickness distribution and properties of the final bottle. Whereas the thickness distribution is one of the most important criteria in the production of bottles. So this work focused on the polyethylene terephthalate (PET) preform growth using a transparent mold, through which the instantaneous images of the preform in the stretching and blowing stage were captured. By changing the delay time of the preblow, the three preform growth types, referred to as dolphin, sandpile, and two-bubble, were observed. The longitudinal and hoop stresses acting on the preform segment during the stretching and blowing were analyzed.Two parameters, on which the longitudinal and hoop stresses depend, respectively, were defined. Then combining the geometry and sizes of the preform, the stresses and temperature distribution on it, and the stress-strain curves of the PET material used, the cause for different preform growth types was systematically analyzed. On the basis of preform growth types, the thickness distributions of the bottles obtained under different delay times of the preblow were explained.

The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA−guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA−gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.