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.

Model-based population pharmacokinetic and exposure response analyses for safety and efficacy of nivolumab as adjuvant treatment in subjects with resected oesophageal or gastroesophageal junction cancer.

AIMS: Nivolumab is approved as adjuvant treatment in subjects with resected oesophageal or gastroesophageal junction cancer (EC/GEJC) based on results from the pivotal CheckMate 577 trial. We present a model-based clinical pharmacology profiling and benefit-risk assessment of nivolumab as adjuvant treatment in subjects with resected EC/GEJC supporting a less frequent dosing regimen. METHODS: Population pharmacokinetic (popPK) analysis was conducted to characterize nivolumab pharmacokinetics (PK) using clinical data from 1493 subjects from seven monotherapy clinical studies across multiple solid tumours. The exposure-response (E-R) analyses included data from 756 patients from CheckMate 577. E-R relationships for efficacy and safety were characterized by evaluating the relationship between nivolumab exposure and disease-free survival (DFS) for efficacy; and time to first occurrence of Grade ≥2 immune-mediated adverse events (Gr2 + IMAEs) for safety. RESULTS: Nivolumab exposure was found to be associated with both DFS and risk of Gr2 + IMAEs. However, the hazard ratios (HRs) (95% confidence interval [CI]) at the 5th and 95th percentiles of nivolumab exposure were similar for DFS and Gr2 + IMAEs, indicating flat E-R relationships within the exposure range produced by the studied regimen. Model-predicted probability of DFS and Gr2 + IMAEs were similar between the two regimens of 240 mg every 2 weeks or 480 mg every 4 weeks for 16 weeks followed by 480 mg Q4W up to 1 year. CONCLUSIONS: The analyses demonstrated a flat E-R relationship over the range of exposures produced by the studied regimen and supported the approval of an alternative dosing regimen with less frequent dosing in patients with adjuvant EC/GEJC.

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.