Monitoring reactive intermediates can provide vital information in the study of synthetic reaction mechanisms, enabling the design of new catalysts and methods. Many synthetic transformations are ...centred on the alteration of oxidation states, but these redox processes frequently pass through intermediates with short life-times, making their study challenging. A variety of electroanalytical tools can be utilised to investigate these redox-active intermediates: from voltammetry to
in situ
spectroelectrochemistry and scanning electrochemical microscopy. This perspective provides an overview of these tools, with examples of both electrochemically-initiated processes and monitoring redox-active intermediates formed chemically in solution. The article is designed to introduce synthetic organic and organometallic chemists to electroanalytical techniques and their use in probing key mechanistic questions.
A range of electroanalytical tools can be applied to studying redox reactions, probing key mechanistic questions in synthetic chemistry.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Global climate change includes rising temperatures and increased pCO2 concentrations in the ocean, with potential deleterious impacts on marine organisms. In this case study we conducted a four-week ...climate change incubation experiment, and tested the independent and combined effects of increased temperature and partial pressure of carbon dioxide (pCO2), on the microbiomes of a foundation species, the giant kelp Macrocystis pyrifera, and the surrounding water column. The water and kelp microbiome responded differently to each of the climate stressors. In the water microbiome, each condition caused an increase in a distinct microbial order, whereas the kelp microbiome exhibited a reduction in the dominant kelp-associated order, Alteromondales. The water column microbiomes were most disrupted by elevated pCO2, with a 7.3 fold increase in Rhizobiales. The kelp microbiome was most influenced by elevated temperature and elevated temperature in combination with elevated pCO2. Kelp growth was negatively associated with elevated temperature, and the kelp microbiome showed a 5.3 fold increase Flavobacteriales and a 2.2 fold increase alginate degrading enzymes and sulfated polysaccharides. In contrast, kelp growth was positively associated with the combination of high temperature and high pCO2 'future conditions', with a 12.5 fold increase in Planctomycetales and 4.8 fold increase in Rhodobacteriales. Therefore, the water and kelp microbiomes acted as distinct communities, where the kelp was stabilizing the microbiome under changing pCO2 conditions, but lost control at high temperature. Under future conditions, a new equilibrium between the kelp and the microbiome was potentially reached, where the kelp grew rapidly and the commensal microbes responded to an increase in mucus production.
We studied Ulva lactuca to determine its potential for bioremediation of coastal watersheds. We cultured Ulva in orthogonal combinations of two salinities and three nutrient concentrations for six ...weeks, and then measured its growth, photosynthesis, chlorophyll fluorescence, nitrogen, carbon and phosphorus tissue concentrations, and carbon and nitrogen uptake pathways. Our findings show that Ulva was negatively affected by decreased salinity but these effects were ameliorated by the addition of nutrients to the water, such as would be expected from freshwater runoff during heavy rain events. Also, increased nutrients resulted in altered nitrogen (NH4+ vs. NO3−) and carbon (HCO3− vs. CO2) uptake pathways, which can allow Ulva to retain its bloom potential even under reduced salinities. Together, our study suggests that Ulva is an ideal species to grow for the purpose of bioremediation of coastal bays and estuaries, even during storms that freshen the surface waters and increase nutrients.
Schematic diagram of study’s main findings. Growth, fluorescence, photosynthesis, and nutrient uptake all increased under increased nutrients, while these parameters all decreased under low salinities. However, the negative effects of low salinity were ameliorated when nutrients were added. Display omitted
•Low salinity negatively affected Ulva growth, photosynthesis, and nutrient uptake.•Elevated nutrients ameliorated the negative effects of decreased salinity.•Ulva is ideal for bioremediation of polluted waterways following rain events.
Abstract
Trophic downgrading in coastal waters has occurred globally during recent decades. On temperate rocky reefs, this has resulted in widespread kelp deforestation and the formation of sea ...urchin barrens. We hypothesize that the intact kelp forest communities are more spatially variable than the downgraded urchin barren communities, and that these differences are greatest at small spatial scales where the influence of competitive and trophic interactions is strongest. To address this, benthic community surveys were done in kelp forests and urchin barrens at nine islands spanning 1230 km of the Aleutian Archipelago where the loss of predatory sea otters has resulted in the trophic downgrading of the region’s kelp forests. We found more species and greater total spatial variation in community composition within the kelp forests than in the urchin barrens. Further, the kelp forest communities were most variable at small spatial scales (within each forest) and least variable at large spatial scales (among forests on different islands), while the urchin barren communities followed the opposite pattern. This trend was consistent for different trophic guilds (primary producers, grazers, filter feeders, predators). Together, this suggests that Aleutian kelp forests create variable habitats within their boundaries, but that the communities within these forests are generally similar across the archipelago. In contrast, urchin barrens exhibit relatively low variability within their boundaries, but these communities vary substantially among different barrens across the archipelago. We propose this represents a shift from small-scale biological control to large-scale oceanographic control of these communities.
Diabetes mellitus is a major risk factor for peripheral arterial disease and lower extremity amputation (LEA). We evaluated the effects of intensive glucose control (IGC) on risk of LEA in patients ...with type 2 diabetes during a randomized-controlled multicenter trial.
The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial randomized patients with type 2 diabetes to IGC (HbA1c target < 6.0%) or standard glycemic control (SGC; HbA1c target 7.0% to 7.9%). Using analysis of mean HbA1c, we examined relationships between glycemic control and incident/recurrent LEA during the clinical trial/follow-up.
Mean post-randomization HbA1c over the course of the trial and post-trial follow-up was 7.3% ± 0.9% (6.8% ± 0.8% in the IGC arm, 7.7% ± 0.7% in the SGC arm). There were 124 participants who had at least 1 LEA during the study period; 73 were randomized to the SGC arm and 51 to the IGC arm (p = 0.049). Randomization to IGC was associated with decreased LEA rate (HR 0.69, 95% CI 0.483 to 0.987, p = 0.042). In multivariable models, mean HbA1c was a powerful predictor of LEA (HR 2.07 per 1% increase in HbA1c, 95% CI 1.67 to 2.57, p < 0.0001). Post-randomization mean HbA1c remained a strong predictor of LEA after controlling for other important covariates and competing risk of death (HR 1.94 per 1% increase in HbA1c, 95% CI 1.52 to 2.46, p < 0.0001).
In patients with type 2 diabetes, IGC was associated with a reduction in the risk for LEA. After 3.7 years of IGC, there was an enduring protective effect against LEA. Improved glycemic control was a strong predictor of decreased risk for subsequent LEA. This study suggests that tight glycemic control, even over a short time period, has potential to reduce risk of limb loss.
Frailty is associated with adverse events, length of stay, and nonhome discharge after vascular surgery. Frailty measures based on walking-based tests may be impractical or invalid for patients with ...walking impairment from symptoms or sequelae of vascular disease. We hypothesized that grip strength is associated with frailty, comorbidity, and cardiac risk among patients with vascular disease.
Dominant hand grip strength was measured during ambulatory clinic visits among patients with vascular disease (abdominal aortic aneurysm AAA, carotid stenosis, and peripheral artery disease PAD). Frailty prevalence was defined on the basis of the 20th percentile of community-dwelling population estimates adjusted for age, gender, and body mass index. Associations between grip strength, Charlson Comorbidity Index (CCI), Revised Cardiac Risk Index (RCRI), and sarcopenia (based on total psoas area for patients with cross-sectional abdominal imaging) were evaluated using linear and logistic regression.
Grip strength was measured in 311 participants; all had sufficient data for CCI calculation, 217 (69.8%) had sufficient data for RCRI, and 88 (28.3%) had cross-sectional imaging permitting psoas measurement. Eighty-six participants (27.7%) were categorized as frail on the basis of grip strength. Frailty was associated with CCI (odds ratio, 1.86; 95% confidence interval, 1.34-2.57; P = .0002) in the multivariable model. Frail participants also had a higher average number of RCRI components vs nonfrail patients (mean ± standard deviation, 1.8 ± 0.8 for frail vs 1.5 ± 0.7 for nonfrail; P = .018); frailty was also associated with RCRI in the adjusted multivariable model (odds ratio, 1.75; 95% confidence interval, 1.16-2.64; P = .008). Total psoas area was lower among patients categorized as frail vs nonfrail on the basis of grip strength (21.0 ± 6.6 vs 25.4 ± 7.4; P = .010). Each 10 cm2 increase in psoas area was associated with a 5.7 kg increase in grip strength in a multivariable model adjusting for age and gender (P < .0001). Adjusted least squares mean psoas diameter estimates were 25.5 ± 1.1 cm2 for participants with AAA, 26.7 ± 2.0 cm2 for participants with carotid stenosis, and 22.7 ± 0.8 cm2 for participants with PAD (P = .053 for PAD vs AAA; P = .057 for PAD vs carotid stenosis; and P = .564 for AAA vs carotid stenosis).
Grip strength is useful for identifying frailty among patients with vascular disease. Frail status based on grip strength is associated with comorbidity, cardiac risk, and sarcopenia in this population. These findings suggest that grip strength may have utility as a simple and inexpensive risk screening tool that is easily implemented in ambulatory clinics, avoids the need for imaging, and overcomes possible limitations of walking-based measures. Lower mean psoas diameters among patients with PAD vs other diagnoses may warrant consideration of specific approaches to morphomic analysis.
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