Redirecting Metabolic Flux via Combinatorial Multiplex CRISPRi-Mediated Repression for Isopentenol Production in Escherichia coli.

CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18-24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.

Higher Ventricular Premature Complex Burden is Associated with Lower Systolic Blood Pressure Response.

BACKGROUND: Ventricular premature complexes (VPCs) with a burden higher than 10% to 20% of total daily heart beats can cause VPC-induced cardiomyopathy. The systolic blood pressure response (SBPR) is the difference between the SBP during maximal exercise and rest. A low SBPR was recently identified to be a marker of cardiomyopathy. The aim of this manuscript was to clarify the association between VPC burden and SBPR. METHODS: From January to December 2015, all patients with a VPC burden larger than 240 beats/day on Holter recordings and treadmill exercise tests were enrolled. The patients with a heart rhythm other than sinus rhythm, coronary artery disease, and severe cardiomyopathy were excluded. The SBPR was measured during a treadmill test. The basic characteristics and echocardiographic findings were collected. RESULTS: All patients were classified into three groups: Group 1; 240-1,000 VPCs/day (n = 78), Group 2; 1,000-10,000 VPCs/day (n = 54), and Group 3; > 10,000 VPCs/day (n = 21). Group 1 had a higher SBPR than the other groups. Multivariate analysis revealed that only VPC burden was associated with SBPR. Receiver operating characteristic curve analysis showed that a VPC burden > 1,055 beats/day predicted a SBPR < 40 mmHg. The results were consistent in all subgroups. There were no significant differences in echocardiographic findings among the groups. CONCLUSIONS: AVPC burden higher than 1,055 beats/day was associated with a reduced SBPR.

Correction: Relationship of mechanical impact magnitude to neurologic dysfunction severity in a rat traumatic brain injury model.

[This corrects the article DOI: 10.1371/journal.pone.0178186.].

Relationship of mechanical impact magnitude to neurologic dysfunction severity in a rat traumatic brain injury model.

OBJECTIVE: Traumatic brain injury (TBI) is a major brain injury type commonly caused by traffic accidents, falls, violence, or sports injuries. To obtain mechanistic insights about TBI, experimental animal models such as weight-drop-induced TBI in rats have been developed to mimic closed-head injury in humans. However, the relationship between the mechanical impact level and neurological severity following weight-drop-induced TBI remains uncertain. In this study, we comprehensively investigated the relationship between physical impact and graded severity at various weight-drop heights. APPROACH: The acceleration, impact force, and displacement during the impact were accurately measured using an accelerometer, a pressure sensor, and a high-speed camera, respectively. In addition, the longitudinal changes in neurological deficits and balance function were investigated at 1, 4, and 7 days post TBI lesion. The inflammatory expression markers tested by Western blot analysis, including glial fibrillary acidic protein, beta-amyloid precursor protein, and bone marrow tyrosine kinase gene in chromosome X, in the frontal cortex, hippocampus, and corpus callosum were investigated at 1 and 7 days post-lesion. RESULTS: Gradations in impact pressure produced progressive degrees of injury severity in the neurological score and balance function. Western blot analysis demonstrated that all inflammatory expression markers were increased at 1 and 7 days post-impact injury when compared to the sham control rats. The severity of neurologic dysfunction and induction in inflammatory markers strongly correlated with the graded mechanical impact levels. CONCLUSIONS: We conclude that the weight-drop-induced TBI model can produce graded brain injury and induction of neurobehavioral deficits and may have translational relevance to developing therapeutic strategies for TBI.