Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity.

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Photosynthesis re-wired on the pico-second timescale.

Photosystems II and I (PSII, PSI) are the reaction centre-containing complexes driving the light reactions of photosynthesis; PSII performs light-driven water oxidation and PSI further photo-energizes harvested electrons. The impressive efficiencies of the photosystems have motivated extensive biological, artificial and biohybrid approaches to 're-wire' photosynthesis for higher biomass-conversion efficiencies and new reaction pathways, such as H2 evolution or CO2 fixation1,2. Previous approaches focused on charge extraction at terminal electron acceptors of the photosystems3. Electron extraction at earlier steps, perhaps immediately from photoexcited reaction centres, would enable greater thermodynamic gains; however, this was believed impossible with reaction centres buried at least 4 nm within the photosystems4,5. Here, we demonstrate, using in vivo ultrafast transient absorption (TA) spectroscopy, extraction of electrons directly from photoexcited PSI and PSII at early points (several picoseconds post-photo-excitation) with live cyanobacterial cells or isolated photosystems, and exogenous electron mediators such as 2,6-dichloro-1,4-benzoquinone (DCBQ) and methyl viologen. We postulate that these mediators oxidize peripheral chlorophyll pigments participating in highly delocalized charge-transfer states after initial photo-excitation. Our results challenge previous models that the photoexcited reaction centres are insulated within the photosystem protein scaffold, opening new avenues to study and re-wire photosynthesis for biotechnologies and semi-artificial photosynthesis.

Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2.

The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.

Adjuvant Nivolumab in Resected Esophageal or Gastroesophageal Junction Cancer.

BACKGROUND: No adjuvant treatment has been established for patients who remain at high risk for recurrence after neoadjuvant chemoradiotherapy and surgery for esophageal or gastroesophageal junction cancer. METHODS: We conducted CheckMate 577, a global, randomized, double-blind, placebo-controlled phase 3 trial to evaluate a checkpoint inhibitor as adjuvant therapy in patients with esophageal or gastroesophageal junction cancer. Adults with resected (R0) stage II or III esophageal or gastroesophageal junction cancer who had received neoadjuvant chemoradiotherapy and had residual pathological disease were randomly assigned in a 2:1 ratio to receive nivolumab (at a dose of 240 mg every 2 weeks for 16 weeks, followed by nivolumab at a dose of 480 mg every 4 weeks) or matching placebo. The maximum duration of the trial intervention period was 1 year. The primary end point was disease-free survival. RESULTS: The median follow-up was 24.4 months. Among the 532 patients who received nivolumab, the median disease-free survival was 22.4 months (95% confidence interval [CI], 16.6 to 34.0), as compared with 11.0 months (95% CI, 8.3 to 14.3) among the 262 patients who received placebo (hazard ratio for disease recurrence or death, 0.69; 96.4% CI, 0.56 to 0.86; P<0.001). Disease-free survival favored nivolumab across multiple prespecified subgroups. Grade 3 or 4 adverse events that were considered by the investigators to be related to the active drug or placebo occurred in 71 of 532 patients (13%) in the nivolumab group and 15 of 260 patients (6%) in the placebo group. The trial regimen was discontinued because of adverse events related to the active drug or placebo in 9% of the patients in the nivolumab group and 3% of those in the placebo group. CONCLUSIONS: Among patients with resected esophageal or gastroesophageal junction cancer who had received neoadjuvant chemoradiotherapy, disease-free survival was significantly longer among those who received nivolumab adjuvant therapy than among those who received placebo. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 577 ClinicalTrials.gov number, NCT02743494.).

Performance and perceived fatigability across the intensity spectrum: role of muscle mass during cycling.

The role of muscle mass in modulating performance and perceived fatigability across the entire intensity spectrum during cycling remains unexplored. We hypothesized that at task failure (Tlim), muscle contractile function would decline more following single- (SL) versus double-leg (DL) cycling within severe and extreme intensities, but not moderate and heavy intensities. After DL and SL ramp-incremental tests, on separate days, 11 recreationally active males (V̇o2max: 49.5 ± 7.7 mL·kg-1·min-1) completed SL and DL cycling until Tlim within each intensity domain. Power output for SL trials was set at 60% of the corresponding DL trial. Before and immediately after Tlim, participants performed an isometric maximal voluntary contraction (MVC) coupled with one superimposed and three resting femoral nerve stimulations [100 Hz; 10 Hz; single twitch (Qtw)] to measure performance fatigability. Perceived fatigue, leg pain, dyspnea, and effort were collected during trials. Tlim within each intensity domain was not different between SL and DL (all P > 0.05). MVC declined more for SL versus DL following heavy- (-42 ± 16% vs. -30 ± 18%; P = 0.011) and severe-intensity cycling (-41 ± 12% vs. -31 ± 15%; P = 0.036). Similarly, peak Qtw force declined more for SL following heavy- (-31 ± 12% vs. -22 ± 10%; P = 0.007) and severe-intensity cycling (-49 ± 13% vs. -40 ± 7%; P = 0.048). Except for heavy intensity, voluntary activation reductions were similar between modes. Similarly, except for dyspnea, which was lower for SL versus DL across all domains, ratings of fatigue, pain, and effort were similar at Tlim between exercise modes. Thus, the amount of muscle mass modulates the extent of contractile function impairment in an intensity-dependent manner.NEW & NOTEWORTHY We investigated the modulatory role of muscle mass on performance and perceived fatigability across the entire intensity spectrum. Despite similar time-to-task failure, single-leg cycling resulted in greater impairments in muscle contractile function within the heavy- and severe-intensity domains, but not the moderate- and extreme-intensity domains. Perceived fatigue, pain, and effort were similar between cycling modes. This indicates that the modulatory role of muscle mass on the extent of performance fatigability is intensity domain-dependent.

Reliability of transcranial magnetic stimulation-evoked responses on knee extensor muscles during cycling.

Transcranial magnetic stimulation (TMS) measures the excitability and inhibition of corticomotor networks. Despite its task-specificity, few studies have used TMS during dynamic movements and the reliability of TMS paired pulses has not been assessed during cycling. This study aimed to evaluate the reliability of motor evoked potentials (MEP) and short- and long-interval intracortical inhibition (SICI and LICI) on vastus lateralis and rectus femoris muscle activity during a fatiguing single-leg cycling task. Nine healthy adults (2 female) performed two identical sessions of counterweighted single-leg cycling at 60% peak power output until failure. Five single pulses and ten paired pulses were delivered to the motor cortex, and two maximal femoral nerve stimulations (Mmax) were administered during two baseline cycling bouts (unfatigued) and every 5 min throughout cycling (fatigued). When comparing both baseline bouts within the same session, MEP·Mmax-1 and LICI (both ICC: >0.9) were rated excellent while SICI was rated good (ICC: 0.7-0.9). At baseline, between sessions, in the vastus lateralis, Mmax (ICC: >0.9) and MEP·Mmax-1 (ICC: 0.7) demonstrated good reliability; LICI was moderate (ICC: 0.5), and SICI was poor (ICC: 0.3). Across the fatiguing task, Mmax demonstrated excellent reliability (ICC > 0.8), MEP·Mmax-1 ranged good to excellent (ICC: 0.7-0.9), LICI was moderate to excellent (ICC: 0.5-0.9), and SICI remained poorly reliable (ICC: 0.3-0.6). These results corroborate the cruciality of retaining mode-specific testing measurements and suggest that during cycling, Mmax, MEP·Mmax-1, and LICI measures are reliable whereas SICI, although less reliable across days, can be reliable within the same session.

3D-printed hierarchical pillar array electrodes for high-performance semi-artificial photosynthesis.

The rewiring of photosynthetic biomachineries to electrodes is a forward-looking semi-artificial route for sustainable bio-electricity and fuel generation. Currently, it is unclear how the electrode and biomaterial interface can be designed to meet the complex requirements for high biophotoelectrochemical performance. Here we developed an aerosol jet printing method for generating hierarchical electrode structures using indium tin oxide nanoparticles. We printed libraries of micropillar array electrodes varying in height and submicrometre surface features, and studied the energy/electron transfer processes across the bio-electrode interfaces. When wired to the cyanobacterium Synechocystis sp. PCC 6803, micropillar array electrodes with microbranches exhibited favourable biocatalyst loading, light utilization and electron flux output, ultimately almost doubling the photocurrent of state-of-the-art porous structures of the same height. When the micropillars' heights were increased to 600 µm, milestone mediated photocurrent densities of 245 µA cm-2 (the closest thus far to theoretical predictions) and external quantum efficiencies of up to 29% could be reached. This study demonstrates how bio-energy from photosynthesis could be more efficiently harnessed in the future and provide new tools for three-dimensional electrode design.

Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa.

Bacteria deploy multiple defenses to prevent mobile genetic element (MGEs) invasion. CRISPR-Cas immune systems use RNA-guided nucleases to target MGEs, which counter with anti-CRISPR (Acr) proteins. Our understanding of the biology and co-evolutionary dynamics of the common Type I-C CRISPR-Cas subtype has lagged because it lacks an in vivo phage-host model system. Here, we show the anti-phage function of a Pseudomonas aeruginosa Type I-C CRISPR-Cas system encoded on a conjugative pKLC102 island, and its Acr-mediated inhibition by distinct MGEs. Seven genes with anti-Type I-C function (acrIC genes) were identified, many with highly acidic amino acid content, including previously described DNA mimic AcrIF2. Four of the acr genes were broad spectrum, also inhibiting I-E or I-F P. aeruginosa CRISPR-Cas subtypes. Dual inhibition comes at a cost, however, as simultaneous expression of Type I-C and I-F systems renders phages expressing the dual inhibitor AcrIF2 more sensitive to targeting. Mutagenesis of numerous acidic residues in AcrIF2 did not impair anti-I-C or anti-I-F function per se but did exacerbate inhibition defects during competition, suggesting that excess negative charge may buffer DNA mimics against competition. Like AcrIF2, five of the Acr proteins block Cascade from binding DNA, while two function downstream, likely preventing Cas3 recruitment or activity. One such inhibitor, AcrIC3, is found in an 'anti-Cas3' cluster within conjugative elements, encoded alongside bona fide Cas3 inhibitors AcrIF3 and AcrIE1. Our findings demonstrate an active battle between an MGE-encoded CRISPR-Cas system and its diverse MGE targets.

Intermittent blood flow occlusion modulates neuromuscular, perceptual, and cardiorespiratory determinants of exercise tolerance during cycling.

PURPOSE: Constant blood flow occlusion (BFO) superimposed on aerobic exercise can impair muscle function and exercise tolerance; however, no study has investigated the effect of intermittent BFO on the associated responses. Fourteen participants (n = 7 females) were recruited to compare neuromuscular, perceptual, and cardiorespiratory responses to shorter (5:15s, occlusion-to-release) and longer (10:30s) BFO applied during cycling to task failure. METHODS: In randomized order, participants cycled to task failure (task failure 1) at 70% of peak power output with (i) shorter BFO, (ii) longer BFO, and (iii) no BFO (Control). Upon task failure in the BFO conditions, BFO was removed, and participants continued cycling until a second task failure (task failure 2). Maximum voluntary isometric knee contractions (MVC) and femoral nerve stimuli were performed along with perceptual measures at baseline, task failure 1, and task failure 2. Cardiorespiratory measures were recorded continuously across the exercises. RESULTS: Task failure 1 was longer in Control than 5:15s and 10:30s (P < 0.001), with no differences between the BFO conditions. At task failure 1, 10:30s elicited a greater decline in twitch force compared to 5:15s and Control (P < 0.001). At task failure 2, twitch force remained lower in 10:30s than Control (P = 0.002). Low-frequency fatigue developed to a greater extent in 10:30s compared to Control and 5:15s (P < 0.047). Dyspnea and Fatigue were greater for Control than 5:15s and 10:30s at the end of task failure 1 (P < 0.002). CONCLUSION: Exercise tolerance during BFO is primarily dictated by the decline in muscle contractility and accelerated development of effort and pain.

Multimodal Intervention Assessing the Appropriateness of Acid Suppression Therapy is Associated With Reduced Prescriptions at the Time of Discharge for Hospitalized Inpatients.

Background: Acid suppression therapy (AST), including proton pump inhibitors and histamine 2 receptor antagonists, are an overused class of medications. When used inappropriately, AST leads to polypharmacy, increased healthcare costs, and possible negative health consequences. Objective: To assess whether an intervention including prescriber education combined with a pharmacist-driven protocol was effective in reducing the percentage of patients who were discharged with inappropriate AST. Methods: This was a prospective pre-post study of adult patients who were prescribed AST before or during their admission to an internal medicine teaching service. All internal medicine resident physicians received education on appropriate AST prescribing. During the 4-week intervention period, dedicated pharmacists assessed the appropriateness of AST and made recommendations regarding deprescribing if no appropriate indication was identified. Results: During the study period, there were 14 166 admissions during which patients were prescribed AST. Out of the 1143 admissions during the intervention period, appropriateness of AST was assessed by a pharmacist for 163 patients. AST was determined to be inappropriate for 52.8% (n = 86) of patients and discontinuation or de-escalate of therapy occurred in 79.1% (n = 68) of these cases. The percentage of patients discharged on AST decreased from 42.5% before the intervention to 39.9% after the intervention (P = .007). Conclusion: This study suggests that a multimodal deprescribing intervention reduced prescriptions for AST without an appropriate indication at the time of discharge. To increase the efficiency of the pharmacist assessment several workflow improvements were identified. Further study is necessary to understand the long-term outcomes of this intervention.

Determining the association between the type of intervention for ischaemic heart disease and mortality and morbidity in patients with chronic kidney disease.

BACKGROUND: Association between chronic kidney disease (CKD) and ischaemic heart disease (IHD) is well known. Clinically, because of the use of intra-arterial contrast, coronary angiograms are sometimes not performed to avoid further deterioration in kidney function among CKD patients. AIMS: To identify whether intervention for non-ST elevation myocardial infarction (NSTEMI) is associated with increased mortality or further renal deterioration. METHODS: A retrospective observational cohort study involving 144 patients with a diagnosis of IHD in the CKD.QLD registry from May 2011 to August 2017, with a minimum of 2-years follow up, was undertaken. Patients were divided into two groups based on whether they obtained an interventional or medical management for NSTEMI. RESULTS: Fifty-nine patients had medically managed and 85 patients had intervention for IHD. Patients in the medically managed group were observed to be significantly older (median: 78 vs 69 years; P < 0.05) with worse baseline renal function (median: 31 vs 36 mL/min/1.73 m2 ; P <0.05) and higher serum urate level (median: 0.5 vs 0.4 mmol/L; P = 0.2). The interventional group had lower prevalence of diabetes, dyslipidaemia, cerebrovascular disease and peripheral vascular disease. Although this was not significant, Kaplan-Meier analysis revealed a significant decrease in mean survival of medically managed group compared with the interventional group. Furthermore, post adjustment for age and above comorbidities, the medically managed group and higher age were associated with significantly higher mortality. However, the patients in the medically managed and interventional groups had no significant difference in delta estimated glomerular filtration rate. CONCLUSIONS: In this observational study, intervention for IHD was associated with increased survival with no change in renal disease progression in comparison with medically managed patients.

Exercising muscle mass influences neuromuscular, cardiorespiratory, and perceptual responses during and following ramp-incremental cycling to task failure.

Neuromuscular (NM), cardiorespiratory, and perceptual responses to maximal-graded exercise using different amounts of active muscle mass remain unclear. We hypothesized that during dynamic exercise, peripheral NM fatigue (declined twitch force) and muscle pain would be greater using smaller muscle mass, whereas central fatigue (declined voluntary activation) and ventilatory variables would be greater using larger muscle mass. Twelve males (29.8 ± 4.7 years) performed two ramp-incremental cycling tests until task failure: 1) single-leg (SL) with 10 W·min-1 ramp and 2) double-leg (DL) with 20 W·min-1 ramp. NM fatigue was assessed at baseline, task failure (post), and after 1, 4, and 8 min of recovery. Cardiorespiratory and perceptual variables [i.e., ratings of perceived exertion (RPE), pain, and dyspnea] were measured throughout cycling. Exercise duration was similar between sessions (SL: 857.7 ± 263.6 s; DL: 855.0 ± 218.8 s; P = 0.923), and higher absolute peak power output was attained in DL (SL: 163.2 ± 43.8 W; DL: 307.0 ± 72.0 W; P < 0.001). Although central fatigue did not differ between conditions (SL: -6.6 ± 6.5%; DL: -3.5 ± 4.8%; P = 0.091), maximal voluntary contraction (SL: -41.6 ± 10.9%; DL: -33.7 ± 8.5%; P = 0.032) and single twitch forces (SL: -59.4 ± 18.8%; DL: -46.2 ± 16.2%; P = 0.003) declined more following SL. DL elicited higher peak oxygen uptake (SL: 42.1 ± 10.0 mL·kg-1·min-1; DL: 50.3 ± 9.3 mL·kg-1·min-1; P < 0.001), ventilation (SL: 137.1 ± 38.1 L·min-1; DL: 171.5 ± 33.2 L·min-1; P < 0.001), and heart rate (SL: 167 ± 21 bpm; DL: 187 ± 8 bpm; P = 0.005). Dyspnea (P = 0.025) was higher in DL; however, RPE (P = 0.005) and pain (P < 0.001) were higher in SL. These results suggest that interplay between NM, cardiorespiratory, and perceptual determinants of exercise performance during ramp-incremental cycling to task failure is muscle mass dependent.

Video or Telephone? A Natural Experiment on the Added Value of Video Communication in Community Paramedic Responses.

STUDY OBJECTIVE: The objective of this study was to determine the effect of video versus telephonic communication between community paramedics and online medical control physicians on odds of patient transport to a hospital emergency department (ED). METHODS: This was a retrospective analysis of data from a telemedicine-capable community paramedicine program operating within an advanced illness management program that provides home-based primary care to approximately 2,000 housebound patients per year who have advanced medical illness, multiple chronic conditions, activities of daily living dependencies, and past-year hospitalizations. Primary outcome was difference in odds of ED transport between community paramedicine responses with video communication versus those with telephonic communication. Secondary outcomes were physicians' perception of whether video enhanced clinical evaluation and whether perceived enhancement affected ED transport. RESULTS: Of 1,707 community paramedicine responses between 2015 and 2017, 899 (53%) successfully used video; 808 (47%) used telephonic communication. Overall, 290 patients (17%) were transported to a hospital ED. In the adjusted regression model, video availability was not associated with a significant difference in the odds of ED transport (odds ratio 0.80; 95% confidence interval 0.62 to 1.03). Online medical control physicians reported that video enhanced clinical evaluation 85% of the time, but this perception was not associated with odds of ED transport. CONCLUSION: We found support that video is considered an enhancement by physicians overseeing a community paramedicine response, but is not associated with a statistically significant difference in transport to the ED compared with telephonic communication in this nonrandom sample. These results have implications for new models of out-of-hospital care that allow patients to be evaluated and treated in the home.

Advanced bioinformatics rapidly identifies existing therapeutics for patients with coronavirus disease-2019 (COVID-19).

BACKGROUND: The recent global pandemic has placed a high priority on identifying drugs to prevent or lessen clinical infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused by Coronavirus disease-2019 (COVID-19). METHODS: We applied two computational approaches to identify potential therapeutics. First, we sought to identify existing FDA approved drugs that could block coronaviruses from entering cells by binding to ACE2 or TMPRSS2 using a high-throughput AI-based binding affinity prediction platform. Second, we sought to identify FDA approved drugs that could attenuate the gene expression patterns induced by coronaviruses, using our Disease Cancelling Technology (DCT) platform. RESULTS: Top results for ACE2 binding iincluded several ACE inhibitors, a beta-lactam antibiotic, two antiviral agents (Fosamprenavir and Emricasan) and glutathione. The platform also assessed specificity for ACE2 over ACE1, important for avoiding counterregulatory effects. Further studies are needed to weigh the benefit of blocking virus entry against potential counterregulatory effects and possible protective effects of ACE2. However, the data herein suggest readily available drugs that warrant experimental evaluation to assess potential benefit. DCT was run on an animal model of SARS-CoV, and ranked compounds by their ability to induce gene expression signals that counteract disease-associated signals. Top hits included Vitamin E, ruxolitinib, and glutamine. Glutathione and its precursor glutamine were highly ranked by two independent methods, suggesting both warrant further investigation for potential benefit against SARS-CoV-2. CONCLUSIONS: While these findings are not yet ready for clinical translation, this report highlights the potential use of two bioinformatics technologies to rapidly discover existing therapeutic agents that warrant further investigation for established and emerging disease processes.

Advancing Techniques for Investigating the Enzyme-Electrode Interface.

Enzymes are the essential catalytic components of biology and adsorbing redox-active enzymes on electrode surfaces enables the direct probing of their function. Through standard electrochemical measurements, catalytic activity, reversibility and stability, potentials of redox-active cofactors, and interfacial electron transfer rates can be readily measured. Mechanistic investigations on the high electrocatalytic rates and selectivity of enzymes may yield inspiration for the design of synthetic molecular and heterogeneous electrocatalysts. Electrochemical investigations of enzymes also aid in our understanding of their activity within their biological environment and why they evolved in their present structure and function. However, the conventional array of electrochemical techniques (e.g., voltammetry and chronoamperometry) alone offers a limited picture of the enzyme-electrode interface. How many enzymes are loaded onto an electrode? In which orientation(s) are they bound? What fraction is active, and are single or multilayers formed? Does this static picture change over time, applied voltage, or chemical environment? How does charge transfer through various intraprotein cofactors contribute to the overall performance and catalytic bias? What is the distribution of individual enzyme activities within an ensemble of active protein films? These are central questions for the understanding of the enzyme-electrode interface, and a multidisciplinary approach is required to deliver insightful answers. Complementing standard electrochemical experiments with an orthogonal set of techniques has recently allowed to provide a more complete picture of enzyme-electrode systems. Within this framework, we first discuss a brief history of achievements and challenges in enzyme electrochemistry. We subsequently describe how the aforementioned challenges can be overcome by applying advanced electrochemical techniques, quartz-crystal microbalance measurements, and spectroscopic, namely, resonance Raman and infrared, analysis. For example, rotating ring disk electrochemistry permits the simultaneous determination of reaction kinetics and quantification of generated products. In addition, recording changes in frequency and dissipation in a quartz crystal microbalance allows to shed light into enzyme loading, relative orientation, clustering, and denaturation at the electrode surface. Resonance Raman spectroscopy yields information on ligation and redox state of enzyme cofactors, whereas infrared spectroscopy provides insights into active site states and the protein secondary and tertiary structure. The development of these emerging methods for the analysis of the enzyme-electrode interface is the primary focus of this Account. We also take a critical look at the remaining gaps in our understanding and challenges lying ahead toward attaining a complete mechanistic picture of the enzyme-electrode interface.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

Efficacy and Acceptance of a Lombard-response Device for Hypophonia in Parkinson's Disease.

OBJECTIVE: The purpose of this study was to examine the effectiveness, satisfaction, and acceptance of a low-cost Lombard-response (LR) device in a group of individuals with Parkinson's disease (IWPD) and their communication partners (CPs). METHOD: Sixteen IWPD and hypophonia and their CPs participated in the study. The IWPD wore a LR device that included a small MP3 player (Sony Walkman) and headphones playing a multi-talker noise audio file at 80 dB during lab-based speech tasks and during their daily conversational speech over a 2-week device trial period. Outcome measures included average conversational speech intensity and scores on a questionnaire related to speech impairment, communication effectiveness, and device satisfaction. RESULTS: Conversational speech intensity of the IWPD is increased by 7 to 10 dB with the LR device. Following a 2-week trial period, eight of the IWPD (50%) gave the LR device moderate-to-high satisfaction and effectiveness ratings and decided to purchase the device for long-term daily use. At the 4-month follow-up, none of the IWPDs were still using the LR device. Device rejection was related to discomfort (loudness), headaches, interference with cognition, and difficulty controlling device. CONCLUSION: Short-term acceptance and satisfaction with the LR device was moderate, but long-term acceptance, beyond 4 months, was absent. Future studies are required to determine if other types of low-cost LR devices can be developed that improve long-term efficacy and device acceptance in IWPD and hypophonia.

Structure-Activity Relationships of Hierarchical Three-Dimensional Electrodes with Photosystem II for Semiartificial Photosynthesis.

Semiartificial photosynthesis integrates photosynthetic enzymes with artificial electronics, which is an emerging approach to reroute the natural photoelectrogenetic pathways for sustainable fuel and chemical synthesis. However, the reduced catalytic activity of enzymes in bioelectrodes limits the overall performance and further applications in fuel production. Here, we show new insights into factors that affect the photoelectrogenesis in a model system consisting of photosystem II and three-dimensional indium tin oxide and graphene electrodes. Confocal fluorescence microscopy and in situ surface-sensitive infrared spectroscopy are employed to probe the enzyme distribution and penetration within electrode scaffolds of different structures, which is further correlated with protein film-photoelectrochemistry to establish relationships between the electrode architecture and enzyme activity. We find that the hierarchical structure of electrodes mainly influences the protein loading but not the enzyme activity. Photoactivity is more limited by light intensity and electronic communication at the biointerface. This study provides guidelines for maximizing the performance of semiartificial photosynthesis and also presents a set of methodologies to probe the photoactive biofilms in three-dimensional electrodes.

Modulating the Cellular Uptake of Fluorescently Tagged Substrates of Prostate-Specific Antigen before and after Enzymatic Activation.

A series of peptides based on the prostate-specific antigen (PSA)-specific sequence histidine-serine-serine-lysine-leucine-glutamine were functionalized with an anthraquinone fluorophore at the C-terminal residue side chain using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The effect of incorporating a negatively charged N-terminal tetra-glutamic acid group into the substrate and the effect of masking the negatively charged C-terminal carboxylic acid functionality of the substrate were investigated using confocal fluorescence microscopy in two cell lines, DLD-1 and LnCaP. The addition of a tetra-glutamic acid group to the N-terminus of the intact sequence was shown to reduce cellular uptake of the intact substrate prior to activation by PSA. In contrast, masking the C-terminal carboxylic acid group of the substrate as a methyl ester was shown to improve cellular uptake of the peptide fragment after activation by PSA. The synthesized C-terminal methyl ester substrates with the anthraquinone attached to the side chain were confirmed to be cleaved by PSA in LC-MS analysis, and the cytotoxicity of the substrates was shown to increase in the presence of PSA, consistent with cleavage and uptake of the C-terminal fragment. The results indicate that C- and N-terminal functionalization of peptide substrates targeting PSA can be used to modulate the cellular uptake of peptides before and after enzymatic activation, which may thus be an important consideration in the design of tumor-activated prodrugs.