The functional mutational landscape of the lacZ gene.

The lacZ gene of Escherichia coli encodes ß-galactosidase (ß-gal), a lactose metabolism enzyme of the lactose operon. Previous chemical modification or site-directed mutagenesis experiments have identified 21 amino acids that are essential for ß-gal catalytic activity. We have assembled over 10,000 lacZ mutations from published studies that were collected using a positive selection assay to identify mutations in lacZ that disrupted ß-gal function. We analyzed 6,465 independent lacZ mutations that resulted in 2,732 missense mutations that impaired ß-gal function. Those mutations affected 492 of the 1,023 lacZ codons, including most of the 21 previously known residues critical for catalytic activity. Most missense mutations occurred near the catalytic site and in regions important for subunit tetramerization. Overall, our work provides a comprehensive and detailed map of the amino acid residues affecting the structure and catalytic activity of the ß-gal enzyme.

Remote Ischemic Conditioning and Stroke Recovery.

Remote ischemic conditioning (RIC) is a noninvasive procedure whereby several periods of ischemia are induced in a limb. Although there is growing interest in using RIC to improve stroke recovery, preclinical RIC research has focused exclusively on neuroprotection, using male animals and the intraluminal suture stroke model, and delivered RIC at times not relevant to either brain repair or behavioral recovery. In alignment with the Stroke Recovery and Rehabilitation Roundtable, we address these shortcomings. First, a standardized session (5-minute inflation/deflation, 4 repetitions) of RIC was delivered using a cuff on the contralesional hindlimb in both male and female Sprague-Dawley rats. Using the endothelin-1 stroke model, RIC was delivered once either prestroke (18 hours before, pre-RIC) or poststroke (4 hours after, post-RIC), and infarct volume was assessed at 24 hours poststroke using magnetic resonance imaging. RIC was delivered at these times to mimic the day before a surgery where clots are possible or as a treatment similar to tissue plasminogen activator, respectively. Pre-RIC reduced infarct volume by 41% compared with 29% with post-RIC. RIC was neuroprotective in both sexes, but males had a 46% reduction of infarct volume compared with 23% in females. After confirming the acute efficacy of RIC, we applied it chronically for 4 weeks, beginning 5 days poststroke. This delayed RIC failed to enhance poststroke behavioral recovery. Based on these findings, the most promising application of RIC is during the hyperacute and early acute phases of stroke, a time when other interventions such as exercise may be contraindicated.

Neuroprotection by Remote Ischemic Conditioning in Rodent Models of Focal Ischemia: a Systematic Review and Meta-Analysis.

Remote ischemic conditioning (RIC) is a promising neuroprotective therapy for ischemic stroke. Preclinical studies investigating RIC have shown RIC reduced infarct volume, but clinical trials have been equivocal. Therefore, the efficacy of RIC in reducing infarct volume and quality of current literature needs to be evaluated to identify knowledge gaps to support future clinical trials. We performed a systematic review and meta-analysis of preclinical literature involving RIC in rodent models of focal ischemia. This review was registered with PROSPERO (CRD42019145441). Eligibility criteria included rat or mice models of focal ischemia that received RIC to a limb either before, during, or after stroke. MEDLINE and Embase databases were searched from 1946 to August 2019. Risk of bias was assessed using the SYRCLE risk of bias tool along with construct validity. Seventy-two studies were included in the systematic review. RIC was shown to reduce infarct volume (SMD - 2.19; CI - 2.48 to - 1.91) when compared to stroke-only controls and no adverse events were reported with regard to RIC. Remote ischemic conditioning was shown to be most efficacious in males (SMD - 2.26; CI - 2.58 to - 1.94) and when delivered poststroke (SMD - 1.34; CI - 1.95 to - 0.73). A high risk of bias was present; thus, measures of efficacy may be exaggerated. A limitation is the poor methodological reporting of many studies, resulting in unclear construct validity. We identified several important, but under investigated topics including the efficacy of RIC in different stroke models, varied infarct sizes and location, and potential sex differences.

An RFID-based activity tracking system to monitor individual rodent behavior in environmental enrichment: Implications for post-stroke cognitive recovery.

BACKGROUND: Diminished cognitive flexibility is a common form of executive dysfunction that results from stroke in the prefrontal cortex. Potential therapies targeting this type of cognitive deficit following stroke are lacking. NEW METHOD: Here, we used environmental enrichment (EE) as a rehabilitation approach, integrated with a radio frequency identification (RFID)-based activity tracking system to evaluate the contribution of individual EE elements to promote cognitive recovery. Male and female Sprague-Dawley rats received either sham surgery or endothelin-1 (ET-1) induced focal ischemia targeting the medial prefrontal cortex (mPFC). Cognitive flexibility was assessed through an egocentric-spatial version of the Morris Water Maze (MWM) task. RESULTS: Prefrontal cortex damage resulted in impaired reversal learning using the egocentric MWM and reduced physical activity in the running wheel, while social interaction was not affected. EE exposure (2 h/day, 5 days/week, for 5 weeks) improved cognitive flexibility in reversal learning of egocentric MWM for both stroke and sham rats. COMPARISON WITH EXISTING METHOD: As changes in cognition post-stroke can be subtle and difficult to detect using conventional behavioural assessment, we suggest that the implementation of individualized automated animal tracking as used herein will ultimately help decipher whether individual components of EE are important for promoting cognitive recovery post-stroke. CONCLUSION: This study represents an attempt to better align preclinical and clinical implementations of EE and facilitate the uptake of this intervention in the clinical setting.