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We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows—channel flow and cross-flow—in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics. Zhang and Lyden describe a protocol for asymmetric-flow field-flow fractionation (AF4) to separate and characterize extracellular nanoparticles for investigation of their biogenesis, function and potential in molecular diagnostics and therapeutics.

Bayesian stable isotope mixing models are widely used in geochemical and ecological studies for partitioning sources that contribute to various mixtures. However, none of the existing tools allows accounting for the influence of processes other than mixing, especially stable isotope fractionation. Bridging this gap, new software for the stable isotope Fractionation And Mixing Evaluation (FRAME) has been developed with a user-friendly graphical interface ( malewick.github.io/frame ). This calculation tool allows simultaneous sources partitioning and fractionation progress determination based on the stable isotope composition of sources/substrates and mixture/products. The mathematical algorithm applies the Markov-Chain Monte Carlo model to estimate the contribution of individual sources and processes, as well as the probability distributions of the calculated results. The performance of FRAME was comprehensively tested and practical applications of this modelling tool are presented with simple theoretical examples and stable isotope case studies for nitrates, nitrites, water and nitrous oxide. The open mathematical design, featuring custom distributions of source isotope signatures, allows for the implementation of additional processes that alternate the characteristics of the final mixture and its application for various range of studies.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N -glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations. Lyden and colleagues use asymmetric flow field-flow fractionation to classify nanoparticles derived from cell lines and human samples, including previously uncharacterized large, Exo-L and small, Exo-S, exosome subsets.

Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization–tandem mass spectrometry (MALDI–MS/MS), nanoelectrospray–MS/MS (NanoES-MS/MS) or liquid chromatography–MS/MS (LC–MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.

The Meta-Analysis of Radiotherapy in squamous cell Carcinomas of Head and neck (MARCH) showed that altered fractionation radiotherapy is associated with improved overall and progression-free survival compared with conventional radiotherapy, with hyperfractionated radiotherapy showing the greatest benefit. This update aims to confirm and explain the superiority of hyperfractionated radiotherapy over other altered fractionation radiotherapy regimens and to assess the benefit of altered fractionation within the context of concomitant chemotherapy with the inclusion of new trials. For this updated meta-analysis, we searched bibliography databases, trials registries, and meeting proceedings for published or unpublished randomised trials done between Jan 1, 2009, and July 15, 2015, comparing primary or postoperative conventional fractionation radiotherapy versus altered fractionation radiotherapy (comparison 1) or conventional fractionation radiotherapy plus concomitant chemotherapy versus altered fractionation radiotherapy alone (comparison 2). Eligible trials had to start randomisation on or after Jan 1, 1970, and completed accrual before Dec 31, 2010; had to have been randomised in a way that precluded prior knowledge of treatment assignment; and had to include patients with non-metastatic squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx undergoing first-line curative treatment. Trials including a non-conventional radiotherapy control group, investigating hypofractionated radiotherapy, or including mostly nasopharyngeal carcinomas were excluded. Trials were grouped in three types of altered fractionation: hyperfractionated, moderately accelerated, and very accelerated. Individual patient data were collected and combined with a fixed-effects model based on the intention-to-treat principle. The primary endpoint was overall survival. Comparison 1 (conventional fractionation radiotherapy vs altered fractionation radiotherapy) included 33 trials and 11 423 patients. Altered fractionation radiotherapy was associated with a significant benefit on overall survival (hazard ratio [HR] 0·94, 95% CI 0·90–0·98; p=0·0033), with an absolute difference at 5 years of 3·1% (95% CI 1·3–4·9) and at 10 years of 1·2% (−0·8 to 3·2). We found a significant interaction (p=0·051) between type of fractionation and treatment effect, the overall survival benefit being restricted to the hyperfractionated group (HR 0·83, 0·74–0·92), with absolute differences at 5 years of 8·1% (3·4 to 12·8) and at 10 years of 3·9% (−0·6 to 8·4). Comparison 2 (conventional fractionation radiotherapy plus concomitant chemotherapy versus altered fractionation radiotherapy alone) included five trials and 986 patients. Overall survival was significantly worse with altered fractionation radiotherapy compared with concomitant chemoradiotherapy (HR 1·22, 1·05–1·42; p=0·0098), with absolute differences at 5 years of −5·8% (−11·9 to 0·3) and at 10 years of −5·1% (−13·0 to 2·8). This update confirms, with more patients and a longer follow-up than the first version of MARCH, that hyperfractionated radiotherapy is, along with concomitant chemoradiotherapy, a standard of care for the treatment of locally advanced head and neck squamous cell cancers. The comparison between hyperfractionated radiotherapy and concomitant chemoradiotherapy remains to be specifically tested. Institut National du Cancer; and Ligue Nationale Contre le Cancer.

Radiotherapy reduces the risk of local recurrence in rectal cancer. However, the optimal radiotherapy fractionation and interval between radiotherapy and surgery is still under debate. We aimed to study recurrence in patients randomised between three different radiotherapy regimens with respect to fractionation and time to surgery. In this multicentre, randomised, non-blinded, phase 3, non-inferiority trial (Stockholm III), all patients with a biopsy-proven adenocarcinoma of the rectum, without signs of non-resectability or distant metastases, without severe cardiovascular comorbidity, and planned for an abdominal resection from 18 Swedish hospitals were eligible. Participants were randomly assigned with permuted blocks, stratified by participating centre, to receive either 5 × 5 Gy radiation dose with surgery within 1 week (short-course radiotherapy) or after 4–8 weeks (short-course radiotherapy with delay) or 25 × 2 Gy radiation dose with surgery after 4–8 weeks (long-course radiotherapy with delay). After a protocol amendment, randomisation could include all three treatments or just the two short-course radiotherapy treatments, per hospital preference. The primary endpoint was time to local recurrence calculated from the date of randomisation to the date of local recurrence. Comparisons between treatment groups were deemed non-inferior if the upper limit of a double-sided 90% CI for the hazard ratio (HR) did not exceed 1·7. Patients were analysed according to intention to treat for all endpoints. This study is registered with ClinicalTrials.gov, number NCT00904813. Between Oct 5, 1998, and Jan 31, 2013, 840 patients were recruited and randomised; 385 patients in the three-arm randomisation, of whom 129 patients were randomly assigned to short-course radiotherapy, 128 to short-course radiotherapy with delay, and 128 to long-course radiotherapy with delay, and 455 patients in the two-arm randomisation, of whom 228 were randomly assigned to short-course radiotherapy and 227 to short-course radiotherapy with delay. In patients with any local recurrence, median time from date of randomisation to local recurrence in the pooled short-course radiotherapy comparison was 33·4 months (range 18·2–62·2) in the short-course radiotherapy group and 19·3 months (8·5–39·5) in the short-course radiotherapy with delay group. Median time to local recurrence in the long-course radiotherapy with delay group was 33·3 months (range 17·8–114·3). Cumulative incidence of local recurrence in the whole trial was eight of 357 patients who received short-course radiotherapy, ten of 355 who received short-course radiotherapy with delay, and seven of 128 who received long-course radiotherapy (HR vs short-course radiotherapy: short-course radiotherapy with delay 1·44 [95% CI 0·41–5·11]; long-course radiotherapy with delay 2·24 [0·71–7·10]; p=0·48; both deemed non-inferior). Acute radiation-induced toxicity was recorded in one patient (<1%) of 357 after short-course radiotherapy, 23 (7%) of 355 after short-course radiotherapy with delay, and six (5%) of 128 patients after long-course radiotherapy with delay. Frequency of postoperative complications was similar between all arms when the three-arm randomisation was analysed (65 [50%] of 129 patients in the short-course radiotherapy group; 48 [38%] of 128 patients in the short-course radiotherapy with delay group; 50 [39%] of 128 patients in the long-course radiotherapy with delay group; odds ratio [OR] vs short-course radiotherapy: short-course radiotherapy with delay 0·59 [95% CI 0·36–0·97], long-course radiotherapy with delay 0·63 [0·38–1·04], p=0·075). However, in a pooled analysis of the two short-course radiotherapy regimens, the risk of postoperative complications was significantly lower after short-course radiotherapy with delay than after short-course radiotherapy (144 [53%] of 355 vs 188 [41%] of 357; OR 0·61 [95% CI 0·45–0·83] p=0·001). Delaying surgery after short-course radiotherapy gives similar oncological results compared with short-course radiotherapy with immediate surgery. Long-course radiotherapy with delay is similar to both short-course radiotherapy regimens, but prolongs the treatment time substantially. Although radiation-induced toxicity was seen after short-course radiotherapy with delay, postoperative complications were significantly reduced compared with short-course radiotherapy. Based on these findings, we suggest that short-course radiotherapy with delay to surgery is a useful alternative to conventional short-course radiotherapy with immediate surgery. Swedish Research Council, Swedish Cancer Society, Stockholm Cancer Society, and the Regional Agreement on Medical Training and Clinical Research in Stockholm.

The profitability and sustainability of future biorefineries are dependent on efficient feedstock use. Therefore, it is essential to valorize lignin when using wood. We have developed an integrated biorefinery that converts 78 weight % (wt %) of birch into xylochemicals. Reductive catalytic fractionation of the wood produces a carbohydrate pulp amenable to bioethanol production and a lignin oil. After extraction of the lignin oil, the crude, unseparated mixture of phenolic monomers is catalytically funneled into 20 wt % of phenol and 9 wt % of propylene (on the basis of lignin weight) by gas-phase hydroprocessing and dealkylation; the residual phenolic oligomers (30 wt %) are used in printing ink as replacements for controversial para-nonylphenol. A techno-economic analysis predicts an economically competitive production process, and a life-cycle assessment estimates a lower carbon dioxide footprint relative to that of fossil-based production.

We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO₂. Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (-34[per thousand] to-27[per thousand]) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (<-12[per thousand]), and the kinetic isotope effect at 122°C and 40 MPa was -9.4[per thousand], which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.

Several studies have reported a low α to β ratio for prostate cancer, suggesting that hypofractionation could enhance the biological tumour dose without increasing genitourinary and gastrointestinal toxicity. We tested this theory in the phase 3 HYPRO trial for patients with intermediate-risk and high-risk prostate cancer. We have previously reported acute incidence of genitourinary and gastrointestinal toxicity; here we report data for late genitourinary and gastrointestinal toxicity. In this randomised non-inferiority phase 3 trial, done in seven radiotherapy centres in the Netherlands, we enrolled intermediate-risk or high-risk patients aged between 44 and 85 years with histologically confirmed stage T1b–T4 NX–0MX–0 prostate cancer, a prostate-specific antigen concentration of 60 ng/mL or lower, and WHO performance status of 0–2. A web-based application was used to randomly assign (1:1) patients to receive either standard fractionation with 39 fractions of 2 Gy in 8 weeks (five fractions per week) or hypofractionation with 19 fractions of 3·4 Gy in 6·5 weeks (three fractions per week). Randomisation was done with the minimisation procedure, stratified by treatment centre and risk group. The primary endpoint was to detect a 10% enhancement in 5-year relapse-free survival with hypofractionation. A key additional endpoint was non-inferiority of hypofractionation in cumulative incidence of grade 2 or worse acute and late genitourinary and gastrointestinal toxicity. We planned to reject inferiority of hypofractionation for late genitourinary toxicity if the estimated hazard ratio (HR) was less than 1·11 and for gastrointestinal toxicity was less than 1·13. We scored toxicity with the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer (RTOG/EORTC) criteria from both physicians' records (clinical record form) and patients' self-assessment questionnaires. Analyses were done in the intention-to-treat population. Patient recruitment for the HYPRO trial was completed in 2010. The trial was registered with www.controlled-trials.com, number ISRCTN85138529. Between March 19, 2007, and Dec 3, 2010, 820 patients (410 in both groups) were randomly assigned. Analyses for late toxicity included 387 assessable patients in the standard fractionation group and 395 in the hypofractionation group. The median follow-up was 60 months (IQR 51·2–67·3). The database for all analyses (both groups and both genitourinary and gastrointestinal toxicities) was locked on March 26, 2015. The incidence of grade 2 or worse genitourinary toxicity at 3 years was 39·0% (95% CI 34·2–44·1) in the standard fractionation group and 41·3% (36·6–46·4) in the hypofractionation group. The estimated HR for the cumulative incidence of grade 2 or worse late genitourinary toxicity was 1·16 (90% CI 0·98–1·38), suggesting that non-inferiority could not be shown. The incidence of grade 2 or worse gastrointestinal toxicity at 3 years was 17·7% (14·1–21·9) in standard fractionation and 21·9% (18·1–26·4) hypofractionation. With an estimated HR of 1·19 (90% CI 0·93–1·52) for the cumulative incidence of grade 2 or worse late gastrointestinal toxicity, we could not confirm non-inferiority of hypofractionation for cumulative late gastrointestinal toxicity. Cumulative grade 3 or worse late genitourinary toxicity was significantly higher in the hypofractionation group than in the standard fractionation group (19·0% [95% CI 15·2–23·2] vs 12·9% [9·7–16·7], respectively; p=0·021), but there was no significant difference between cumulative grade 3 or worse late gastrointestinal toxicity (2·6% [95% CI 1·2–4·7]) in the standard fractionation group and 3·3% [1·7–5·6] in the hypofractionation group; p=0·55). Our data could not confirm that hypofractionation was non-inferior for cumulative late genitourinary and gastrointestinal toxicity compared with standard fractionation. Before final conclusions can be made about the utility of hypofractionation, efficacy outcomes need to be reported. The Dutch Cancer Society.