Real-time, single-molecule observation of biomolecular interactions inside nanophotonic zero mode waveguides.

Living cells rely on numerous protein-protein, RNA-protein and DNA-protein interactions for processes such as gene expression, biomolecular assembly, protein and RNA degradation. Single-molecule microscopy and spectroscopy are ideal tools for real-time observation and quantification of nucleic acids-protein and protein-protein interactions. One of the major drawbacks of conventional single-molecule imaging methods is low throughput. Methods such as sequencing by synthesis utilizing nanofabrication and single-molecule spectroscopy have brought high throughput into the realm of single-molecule biology. The Pacific Biosciences RS2 sequencer utilizes sequencing by synthesis within nanophotonic zero mode waveguides. A number of years ago this instrument was unlocked by Pacific Biosciences for custom use by researchers allowing them to monitor biological interactions at the single-molecule level with high throughput. In this capability letter we demonstrate the use of the RS2 sequencer for real-time observation of DNA-to-RNA transcription and RNA-protein interactions. We use a relatively complex model-transcription of structured ribosomal RNA fromE. coliand interactions of ribosomal RNA with ribosomal proteins. We also show evidence of observation of transcriptional pausing without the application of an external force (as is required for single-molecule pausing studies using optical traps). Overall, in the unlocked, custom mode, the RS2 sequencer can be used to address a wide variety of biological assembly and interaction questions at the single-molecule level with high throughput. This instrument is available for use at the Center for Integrated Nanotechnologies Gateway located at Los Alamos National Laboratory.

Rehabilitation of Watson-Jones proximal tibial avulsion injury in elite academy level football: A report of two separate cases in one season.

OBJECTIVES: Watson-Jones proximal tibial avulsion injuries occur more frequently in athletic and muscular adolescent males. However, they are rare and therefore infrequently described in the medical literature. Two of these injuries occurred in a Category 1 football academy in the same season within a six-month period. We have described the cases with the hope of better informing other clinicians should they encounter this injury. METHODS: This case report describes the injury mechanism, surgical management and rehabilitation for the two cases [Players A and B]. Outcomes measures including player speed, agility and power were compared with scores from players of the same age group at the time of injury in the Premier League academies. Risk factors are also discussed. RESULTS: Both players were managed surgically, initially. Player B had the surgical fixation removed during rehabilitation. Player A still has the fixation in situ. Post-surgery, player A returned to full play at thirty-two weeks and thirty-eight weeks for player B. No critical incidents occurred during rehabilitation. CONCLUSION: Watson-Jones avulsion fractures, although rare, can be managed successfully. Athletes can achieve a successful return to play at their previous level.

Clinical course of pain and disability following primary lumbar discectomy: systematic review and meta-analysis.

PURPOSE: To conduct a meta-analysis to describe clinical course of pain and disability in adult patients post-lumbar discectomy (PROSPERO: CRD42015020806). METHODS: Sensitive topic-based search strategy designed for individual databases was conducted. Patients (> 16 years) following first-time lumbar discectomy for sciatica/radiculopathy with no complications, investigated in inception (point of surgery) prospective cohort studies, were included. Studies including revision surgery or not published in English were excluded. Two reviewers independently searched information sources, assessed eligibility at title/abstract and full-text stages, extracted data, assessed risk of bias (modified QUIPs) and assessed GRADE. Authors were contacted to request raw data where data/variance data were missing. Meta-analyses evaluated outcomes at all available time points using the variance-weighted mean in random-effect meta-analyses. Means and 95% CIs were plotted over time for measurements reported on outcomes of leg pain, back pain and disability. RESULTS: A total of 87 studies (n = 31,034) at risk of bias (49 moderate, 38 high) were included. Clinically relevant improvements immediately following surgery (> MCID) for leg pain (0-10, mean before surgery 7.04, 50 studies, n = 14,910 participants) and disability were identified (0-100, mean before surgery 53.33, 48 studies, n = 15,037). Back pain also improved (0-10, mean before surgery 4.72, 53 studies, n = 14,877). Improvement in all outcomes was maintained (to 7 years). Meta-regression analyses to assess the relationship between outcome data and a priori potential covariates found preoperative back pain and disability predictive for outcome. CONCLUSION: Moderate-level evidence supports clinically relevant immediate improvement in leg pain and disability following lumbar discectomy with accompanying improvements in back pain. These slides can be retrieved under Electronic Supplementary Material.

Review: Molecular techniques to assess genetic variation within and between Panax ginseng and Panax quinquefolius.

A variety of methods have been used to examine genetic differences in P. ginseng and P. quinquefolius. They have shown genetic differences within populations of P. ginseng (within and between elite cultivars, landraces and wild accessions), within populations of P. quinquefolius (within and between wild and cultivated accessions) and between P. ginseng and P. quinquefolius as well as other Panax species. Some examples of their applications have been to show that some elite cultivars are not uniform, there are possible founder effects in certain populations, there has been the spread of cultivated types into wild populations, relative diversity differs between different populations and identification of the source and purity of commercial samples. More work in the use of molecular markers for ginseng are needed, however, particularly the use of Next Generation Sequencing. Potential applications are the use of sequence analysis for genetic selection, breeding to develop new cultivars and providing traceability from field to consumer. Research on molecular markers in ginseng has lagged compared to other crops probably because of less of an emphasis on breeding for cultivar development and relatively small areas of production. The many potential benefits for ginseng production have yet to be realized.

Exploring the disclosure decisions made by physiotherapists with a specific learning difficulty.

OBJECTIVES: To explore the disclosure decisions made in the workplace by physiotherapy staff with a specific learning difficulty (SpLD). DESIGN & SETTING: An exploratory qualitative design was used, which was informed by the social model of disability. The research was undertaken in North West England. It is presented according to the Consolidated Criteria for Reporting Qualitative Research. PARTICIPANTS: A purposive sample of eight physiotherapists recognised as having a SpLD were recruited. All participants had studied on one of two programmes at a university in England between 2004-2012. Their NHS workplace experience was from across the UK. DATA GENERATION: In-depth, semi-structured interviews were undertaken within the university setting or via telephone. Interviews lasted 40 to 70minutes and were digitally recorded. An interview guide was used to direct the interview. DATA ANALYSIS: Interview data were transcribed verbatim and analysed using thematic analysis. FINDINGS: Four participants were female. The mean number of years qualified as a physiotherapist was 4.5years (SD=2.27). Three themes were identified: 'Disclosing during the workplace application'; 'Positive about disabled people scheme'; 'Disclosing in the workplace'. CONCLUSIONS: Disclosure of dyslexia is a selective process and is a central dilemma in the lives of individuals who have a concealable stigmatised identity. As a consequence, physiotherapy staff with dyslexia may choose to conceal their disability and not disclose to their employer. In order for staff with dyslexia to get the support they need in the workplace, disclosure is recommended. A number of recommendations have been made to facilitate the disclosure process.

De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen International Expert Consensus Conference on the Primary Therapy of Early Breast Cancer 2017.

The 15th St. Gallen International Breast Cancer Conference 2017 in Vienna, Austria reviewed substantial new evidence on loco-regional and systemic therapies for early breast cancer. Treatments were assessed in light of their intensity, duration and side-effects, seeking where appropriate to escalate or de-escalate therapies based on likely benefits as predicted by tumor stage and tumor biology. The Panel favored several interventions that may reduce surgical morbidity, including acceptance of 2 mm margins for DCIS, the resection of residual cancer (but not baseline extent of cancer) in women undergoing neoadjuvant therapy, acceptance of sentinel node biopsy following neoadjuvant treatment of many patients, and the preference for neoadjuvant therapy in HER2 positive and triple-negative, stage II and III breast cancer. The Panel favored escalating radiation therapy with regional nodal irradiation in high-risk patients, while encouraging omission of boost in low-risk patients. The Panel endorsed gene expression signatures that permit avoidance of chemotherapy in many patients with ER positive breast cancer. For women with higher risk tumors, the Panel escalated recommendations for adjuvant endocrine treatment to include ovarian suppression in premenopausal women, and extended therapy for postmenopausal women. However, low-risk patients can avoid these treatments. Finally, the Panel recommended bisphosphonate use in postmenopausal women to prevent breast cancer recurrence. The Panel recognized that recommendations are not intended for all patients, but rather to address the clinical needs of the majority of common presentations. Individualization of adjuvant therapy means adjusting to the tumor characteristics, patient comorbidities and preferences, and managing constraints of treatment cost and access that may affect care in both the developed and developing world.

Radiocarbon constraints on the glacial ocean circulation and its impact on atmospheric CO2.

While the ocean's large-scale overturning circulation is thought to have been significantly different under the climatic conditions of the Last Glacial Maximum (LGM), the exact nature of the glacial circulation and its implications for global carbon cycling continue to be debated. Here we use a global array of ocean-atmosphere radiocarbon disequilibrium estimates to demonstrate a ∼689±53 14C-yr increase in the average residence time of carbon in the deep ocean at the LGM. A predominantly southern-sourced abyssal overturning limb that was more isolated from its shallower northern counterparts is interpreted to have extended from the Southern Ocean, producing a widespread radiocarbon age maximum at mid-depths and depriving the deep ocean of a fast escape route for accumulating respired carbon. While the exact magnitude of the resulting carbon cycle impacts remains to be confirmed, the radiocarbon data suggest an increase in the efficiency of the biological carbon pump that could have accounted for as much as half of the glacial-interglacial CO2 change.

Development of an optimised 1:1 physiotherapy intervention post first-time lumbar discectomy: a mixed-methods study.

OBJECTIVES: To develop an optimised 1:1 physiotherapy intervention that reflects best practice, with flexibility to tailor management to individual patients, thereby ensuring patient-centred practice. DESIGN: Mixed-methods combining evidence synthesis, expert review and focus groups. SETTING: Secondary care involving 5 UK specialist spinal centres. PARTICIPANTS: A purposive panel of clinical experts from the 5 spinal centres, comprising spinal surgeons, inpatient and outpatient physiotherapists, provided expert review of the draft intervention. Purposive samples of patients (n=10) and physiotherapists (n=10) (inpatient/outpatient physiotherapists managing patients with lumbar discectomy) were invited to participate in the focus groups at 1 spinal centre. METHODS: A draft intervention developed from 2 systematic reviews; a survey of current practice and research related to stratified care was circulated to the panel of clinical experts. Lead physiotherapists collaborated with physiotherapy and surgeon colleagues to provide feedback that informed the intervention presented at 2 focus groups investigating acceptability to patients and physiotherapists. The focus groups were facilitated by an experienced facilitator, recorded in written and tape-recorded forms by an observer. Tape recordings were transcribed verbatim. Data analysis, conducted by 2 independent researchers, employed an iterative and constant comparative process of (1) initial descriptive coding to identify categories and subsequent themes, and (2) deeper, interpretive coding and thematic analysis enabling concepts to emerge and overarching pattern codes to be identified. RESULTS: The intervention reflected best available evidence and provided flexibility to ensure patient-centred care. The intervention comprised up to 8 sessions of 1:1 physiotherapy over 8 weeks, starting 4 weeks postsurgery. The intervention was acceptable to patients and physiotherapists. CONCLUSIONS: A rigorous process informed an optimised 1:1 physiotherapy intervention post-lumbar discectomy that reflects best practice. The developed intervention was agreed on by the 5 spinal centres for implementation in a randomised controlled trial to evaluate its effectiveness.

Natural course of pain and disability following primary lumbar discectomy: protocol for a systematic review and meta-analysis.

INTRODUCTION: Knowledge about the natural clinical course is needed to improve understanding of recovery postsurgery as outcome is poor for some patients. Knowledge of the natural clinical course of symptoms and disability will inform optimal timing and the nature of rehabilitation intervention. The objective of this study is to provide first evidence synthesis investigating the natural clinical course of disability and pain in patients aged >16 years post primary lumbar discectomy. METHODS AND ANALYSIS: A systematic review and data synthesis will be conducted. Prospective cohorts that include a well-defined inception cohort (point of surgery) of adult participants who have undergone primary lumbar discectomy/microdiscectomy will be included. Outcomes will include measurements reported on 1 or more outcomes of disability and pain, with a baseline presurgery measurement. Following development of the search strategy, 2 reviewers will independently search information sources, assess identified studies for inclusion, extract data and assess risk of bias. A third reviewer will mediate on any disagreement at each stage. The search will employ sensitive topic-based strategies designed for each database from inception to 31 January 2016. There will be no language or geographical restrictions. Risk of bias will be assessed using a modified QUality In Prognostic Studies (QUIPS) tool . Data will be extracted for time points where follow-up was at least 80%. Means and 95% CIs will be plotted over time for pain and disability. All results will be reported in the context of study quality. ETHICS AND DISSEMINATION: This review will provide the first rigorous summary of the course of pain and disability across all published prospective cohorts. The findings will inform our understanding of when to offer and how to optimise rehabilitation following surgery. Results will be published in an open access journal. The study raises no ethical issues. PROSPERO REGISTRATION NUMBER: CRD42015020806.

Descriptive analysis of a 1:1 physiotherapy outpatient intervention post primary lumbar discectomy: one arm of a small-scale parallel randomised controlled trial across two UK sites.

OBJECTIVE: There is a lack of high-quality evidence for physiotherapy post lumbar discectomy. Substantial heterogeneity in treatment effects may be explained by variation in quality, administration and components of interventions. An optimised physiotherapy intervention may reduce heterogeneity and improve patient benefit. The objective was to describe, analyse and evaluate an optimised 1:1 physiotherapy outpatient intervention for patients following primary lumbar discectomy, to provide preliminary insights. DESIGN: A descriptive analysis of the intervention embedded within an external pilot and feasibility trial. SETTING: Two UK spinal centres. PARTICIPANTS: Participants aged ≥18; post primary, single level, lumbar discectomy were recruited. INTERVENTION: The intervention encompassed education, advice, mobility and core stability exercises, progressive exercise, and encouragement of early return to work/activity. Patients received ≤8 sessions for ≤8 weeks, starting 4 weeks post surgery (baseline). OUTCOMES: Blinded outcome assessment at baseline and 12 weeks (post intervention) included the Roland Morris Disability Questionnaire. STarT Back data were collected at baseline. Statistical analyses summarised participant characteristics and preplanned descriptive analyses. Thematic analysis grouped related data. FINDINGS: Twenty-two of 29 allocated participants received the intervention. STarT Back categorised n=16 (55%) participants 'not at low risk'. Physiotherapists identified reasons for caution for 8 (36%) participants, commonly risk of overdoing activity (n=4, 18%). There was no relationship between STarT Back and physiotherapists' evaluation of caution. Physiotherapists identified 154 problems (mean (SD) 5.36 (2.63)). Those 'not at low risk', and/or requiring caution presented with more problems, and required more sessions (mean (SD) 3.14 (1.16)). CONCLUSIONS: Patients present differently and therefore require tailored interventions. These differences may be identified using clinical reasoning and outcome data. TRIAL REGISTRATION NUMBER: ISRCTN33808269; post results.

Evidence-based development of a post-surgical lumbar discectomy leaflet intervention: a Delphi consensus study.

OBJECTIVE: To produce free, expert-informed postoperative information for lumbar discectomy patients, satisfying UK National Health Service Information Standards. DESIGN: A mixed methods approach utilising the Delphi technique and focus groups. SETTING: Five spinal centres across the UK. PARTICIPANTS: Panel members included 23 physiotherapists, 11 patients and 17 spinal surgeons. INTERVENTION: Three rounds of questionnaires including open and closed questions and attendance at a clinician/patient focus group. RESULTS: Response rates of 85%, 26% and 35% were achieved for the Delphi rounds. Ten clinicians and six patients participated in the focus groups. Consensus for leaflet sections was achieved in round 1 and content in round 3. The focus groups informed further revisions. CONCLUSIONS: A consensually agreed, Information Standard compliant, patient lumbar discectomy leaflet was produced containing: (1) normal spine anatomy; (2) anatomy disc herniation and surgery; (3) back protection strategies and (4) frequently asked questions. Illustrations of exercises enable tailoring to the individual patient.

Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging.

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.

Interferometric three-dimensional single molecule localization microscopy using a single high-numerical-aperture objective.

Interferometric detection of the fluorescence emission from a single molecule [interferometric photoactivated localization microscopy (iPALM)] enables a localization accuracy of nanometers in axial localization for 3D superresolution imaging. However, iPALM uses two high-numerical-aperture (NA) objectives in juxtaposition for fluorescence collection (a 4Pi microscope geometry), increasing expense and limiting samples that can be studied. Here, we propose an interferometric single molecule localization microscopy method using a single high-NA objective. The axial position of single molecules can be unambiguously determined from the phase-shifted interference signals with nanometer precision and over a range of 2λ. The use of only one objective simplifies the system configuration and sample mounting. In addition, due to the use of wavefront-splitting interference in our approach, the two parts of the wavefront that eventually merge and interfere with each other travel along nearly equivalent optical paths, which should minimize the effect of drift for long-term 3D superresolution imaging.

Confocal line scanning of a Bessel beam for fast 3D imaging.

We have developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples with inexpensive visible lasers and a single galvo mirror (GM). The light-sheet is created by raster scanning a Bessel beam with a GM, with this same GM also being used to rescan the fluorescence across a chip of a camera to construct an image in real time. A slit is used to reject out-of-focus fluorescence such that the image formed in real time has minimal contribution from the sidelobes of the Bessel beam. Compared with two-photon Bessel beam excitation or other confocal line-scanning approaches, our method is of lower cost, is simpler, and does not require calibration and synchronization of multiple GMs. We demonstrated the optical sectioning and out-of-focus background rejection capabilities of this microscope by imaging fluorescently labeled actin filaments in fixed 3T3 cells.

Fast, super resolution imaging via Bessel-beam stimulated emission depletion microscopy.

A substantial advantage of stimulated emission depletion (STED) microscopy over other super-resolution methods is that images can be acquired in real-time without any post-processing. However imaging speed and photodamage are two major concerns for STED imaging of whole cells. Here we propose a new microscopy method we have termed Bessel-Beam STED (or BB-STED) that overcomes both of these limitations of conventional STED microscopy. In the proposed method, rather than exciting a single STED spot in the sample, an entire line of the sample is illuminated. This line-scanning technique dramatically increases the speed of STED. In addition, plane-illumination by scanning of the line across the focal plane of a detection objective limits the light to a thin layer of the sample and thus significantly reduces photobleaching and photodamage above and below the focal plane compared to epi-illumination. Using the organic dye Atto647N as an example, we calculated the STED power required to break the diffraction limit. The results presented here will be used to guide future experimental designs.

A randomized double-blind placebo-controlled cross-over trial of the impact on quality of life of continuing dexamethasone beyond 24 h following adjuvant chemotherapy for breast cancer.

Uncertainty remains about the optimal anti-emetic regimen for control of delayed nausea and vomiting after adjuvant chemotherapy for breast cancer. Many patients receive dexamethasone but complain of insomnia, anxiety/agitation, and indigestion. The aim was to determine if patients receiving chemotherapy for breast cancer prefer treatment with dexamethasone or placebo for prophylaxis against delayed nausea and vomiting, and to compare quality of life (QOL) between the two treatments. In this randomized, double-blind, cross-over trial, we compared oral dexamethasone (4 mg twice daily for 2 days) versus placebo for chemotherapy-naïve patients with breast cancer. All patients received intravenous granisetron and dexamethasone pre-chemotherapy and oral granisetron on day 2. Primary endpoints were: (i) patient preference; (ii) difference between cycles in change of QOL from days 1 to 8. Median age of the 94 women was 51 years (range 27-76): 79 received fluorouracil/epirubicin/cyclophosphamide and 15 received doxorubicin/cyclophosphamide. Thirteen withdrew pre-cycle 2 with no differences between arms. Of 80 patients stating a preference, 31 preferred placebo (39 %, 95 % CI: 28-50 %) and 37 (46 %, 95 % CI: 35-58 %) preferred dexamethasone; 12 had no preference. There were no differences in intensity of vomiting, nausea, or time to onset of vomiting. There was greater decrease in global QOL (p = 0.06) when patients received dexamethasone. No other symptom/QOL domains differed significantly. In conclusion, no significant difference was found in patient preference, QOL, or symptoms regardless of whether dexamethasone or placebo was used after adjuvant chemotherapy.

The role of a putative peroxisomal-targeted epoxide hydrolase of Nicotiana benthamiana in interactions with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci.

Motif analysis among 30 EH1 and EH2 epoxide hydrolases from Solanaceaeous plants showed differences primarily in the lid region around the catalytic site. Based on in silico models of 3D structures, EH1 proteins lack a catalytic triad because of the orientation of one of the conserved lid tyrosines, while the orientation of that tyrosine in EH2 proteins fomed a catalytic triad inside a hydrophobic tunnel. Two similar EH2 protein genes from Nicotiana benthamiana, NbEH2.1 and NbEH2.2, have a predicted peroxisomal targeting sequence, catalytic triad, and structural similarities to a potato cutin monomer-synthesizing epoxide hydrolase. NbEH2.1 expression increased with infections by the hemibiotrophs, Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci only during their biotrophic phases, while there was only a slight increase during the hypersensitive response to P. syringae pv. tabaci (avrPto). In contrast, among the four pathogens, NbEH2.2 expression increased only in response to P. syringae pv. tabaci. Virus-induced gene silencing of NbEH2.1 significantly affected only the interaction with C. destructivum, resulting in a delay in the appearance of necrosis that may be related to its biotrophic phase being restricted to single epidermal cells, which is unique among these pathogens. These results differed from that of a previously reported EH1 gene of N. benthamiana for these interactions, demonstrating specialization among EH genes in basal resistance.