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We have determined the heterogeneous structural ensemble of the tandem SH2 domains of the protein tyrosine phosphatase SHP2 in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings in solution.

Supplementary Figure Legends from Enhancement of Cancer Radiation Therapy by Use of Adenovirus-Mediated Secretable Glucose-Regulated Protein 94/gp96 Expression

Supplementary Figures 1-2 from Enhancement of Cancer Radiation Therapy by Use of Adenovirus-Mediated Secretable Glucose-Regulated Protein 94/gp96 Expression

Tumor-derived glucose-regulated protein 94 (GRP94/gp96) has shown great promise as a tumor vaccine. However, current protein-based approaches require the availability of large quantities of tumor tissue, which are often not possible. In addition, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of a combined GRP94/gp96-based genetic immunotherapy and radiation therapy strategy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding a modified, secretable form of GRP94 gene (AdsGRP94) was constructed and evaluated in various antitumor experiments. Lethally irradiated, virus-infected cells were used as vaccines. Adenoviral vectors were also injected directly into tumors in conjunction with tumor irradiation. Vaccination with lethally irradiated, AdsGRP94-infected 4T1 cells completely prevented subsequent tumor growth from challenge inoculations of as many as 107 cells per mouse. In established tumor models, vaccinations alone had minimal effect on local and metastatic tumor growth. However, when vaccination was combined with radiation therapy and i.t. AdsGRP94 injections, local tumor growth and pulmonary metastasis were markedly inhibited. In some cases, complete tumor regression was observed. In these cases, the mice were resistant to subsequent tumor challenge and remain tumor free up to 10 months after initial therapy. Our results indicate that combined AdsGRP94-based immunotherapy and radiation therapy may be a potentially effective strategy for cancer treatment.

Background Laser interstitial thermal therapy (LITT) in the setting of post-SRS radiation necrosis (RN) for patients with brain metastases has growing evidence for efficacy. However, questions remain regarding hospitalization, local control, symptom control, and concurrent use of therapies. Methods Demographics, intraprocedural data, safety, Karnofsky performance status (KPS), and survival data were prospectively collected and then analyzed on patients who consented between 2016–2020 and who were undergoing LITT for biopsy-proven RN at one of 14 US centers. Data were monitored for accuracy. Statistical analysis included individual variable summaries, multivariable Fine and Gray analysis, and Kaplan–Meier estimated survival. Results Ninety patients met the inclusion criteria. Four patients underwent 2 ablations on the same day. Median hospitalization time was 32.5 hours. The median time to corticosteroid cessation after LITT was 13.0 days (0.0, 1229.0) and cumulative incidence of lesional progression was 19% at 1 year. Median post-procedure overall survival was 2.55 years [1.66, infinity] and 77.1% at one year as estimated by KaplanMeier. Median KPS remained at 80 through 2-year follow-up. Seizure prevalence was 12% within 1-month post-LITT and 7.9% at 3 months; down from 34.4% within 60-day prior to procedure. Conclusions LITT for RN was not only again found to be safe with low patient morbidity but was also a highly effective treatment for RN for both local control and symptom management (including seizures). In addition to averting expected neurological death, LITT facilitates ongoing systemic therapy (in particular immunotherapy) by enabling the rapid cessation of steroids, thereby facilitating maximal possible survival for these patients.

Non-structural protein 1 (Nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major virulence factor and thus an attractive drug target. The last 33 amino acids of Nsp1 have been shown to bind within the mRNA entry tunnel of the 40S ribosomal subunit, shutting off host gene expression. Here, we report the solution-state structure of full-length Nsp1, which features an α/β fold formed by a six-stranded, capped β-barrel-like globular domain (N-terminal domain [NTD]), flanked by short N-terminal and long C-terminal flexible tails. The NTD has been found to be critical for 40S-mediated viral mRNA recognition and promotion of viral gene expression. We find that in free Nsp1, the NTD mRNA-binding surface is occluded by interactions with the acidic C-terminal tail, suggesting a mechanism of activity regulation based on the interplay between the folded NTD and the disordered C-terminal region. These results are relevant for drug-design efforts targeting Nsp1.

Graphical abstract, SHP2 is a ubiquitous protein tyrosine phosphatase, whose activity is regulated by phosphotyrosine (pY)-containing peptides generated in response to extracellular stimuli. Its crystal structure reveals a closed, auto-inhibited conformation in which the N-terminal Src homology 2 (N-SH2) domain occludes the catalytic site of the phosphatase (PTP) domain. High-affinity mono-phosphorylated peptides promote catalytic activity by binding to N-SH2 and disrupting the interaction with the PTP. The mechanism behind this process is not entirely clear, especially because N-SH2 is incapable of accommodating complete peptide binding when SHP2 is in the auto-inhibited state. Here, we show that pY performs an essential role in this process; in addition to its contribution to overall peptide-binding energy, pY-recognition leads to enhanced dynamics of the N-SH2 EF and BG loops via an allosteric communication network, which destabilizes the N-SH2–PTP interaction surface and simultaneously generates a fully accessible binding pocket for the C-terminal half of the phosphopeptide. Subsequently, full binding of the phosphopeptide is associated with the stabilization of activated SHP2. We demonstrate that this allosteric network exists only in N-SH2, which is directly involved in the regulation of SHP2 activity, while the C-terminal SH2 domain (C-SH2) functions primarily to recruit high-affinity bidentate phosphopeptides.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states., Human Histone Deacetylases (HDACs) regulate gene expression and are important drug targets. Here, the authors combine NMR measurements, enzymatic assays and molecular dynamics simulations and show that HDAC8 samples a catalytically active and an inactive state and further demonstrate that mutations and ligand binding alter the populations of the two states, which is of interest for inhibitor design.

Numerous randomized trials have revealed that hyperthermia (HT) + radiotherapy or chemotherapy improves local tumor control, progression free and overall survival vs. radiotherapy or chemotherapy alone. Despite these successes, however, some individuals fail combination therapy; not every patient will obtain maximal benefit from HT. There are many potential reasons for failure. In this paper, we focus on how HT influences tumor hypoxia, since hypoxia negatively influences radiotherapy and chemotherapy response as well as immune surveillance. Pre-clinically, it is well established that reoxygenation of tumors in response to HT is related to the time and temperature of exposure. In most pre-clinical studies, reoxygenation occurs only during or shortly after a HT treatment. If this were the case clinically, then it would be challenging to take advantage of HT induced reoxygenation. An important question, therefore, is whether HT induced reoxygenation occurs in the clinic that is of radiobiological significance. In this review, we will discuss the influence of thermal history on reoxygenation in both human and canine cancers treated with thermoradiotherapy. Results of several clinical series show that reoxygenation is observed and persists for 24–48 h after HT. Further, reoxygenation is associated with treatment outcome in thermoradiotherapy trials as assessed by: (1) a doubling of pathologic complete response (pCR) in human soft tissue sarcomas, (2) a 14 mmHg increase in pO2 of locally advanced breast cancers achieving a clinical response vs. a 9 mmHg decrease in pO2 of locally advanced breast cancers that did not respond and (3) a significant correlation between extent of reoxygenation (as assessed by pO2 probes and hypoxia marker drug immunohistochemistry) and duration of local tumor control in canine soft tissue sarcomas. The persistence of reoxygenation out to 24–48 h post HT is distinctly different from most reported rodent studies. In these clinical series, comparison of thermal data with physiologic response shows that within the same tumor, temperatures at the higher end of the temperature distribution likely kill cells, resulting in reduced oxygen consumption rate, while lower temperatures in the same tumor improve perfusion. However, reoxygenation does not occur in all subjects, leading to significant uncertainty about the thermal–physiologic relationship. This uncertainty stems from limited knowledge about the spatiotemporal characteristics of temperature and physiologic response. We conclude with recommendations for future research with emphasis on retrieving co-registered thermal and physiologic data before and after HT in order to begin to unravel complex thermophysiologic interactions that appear to occur with thermoradiotherapy.

The growing momentum to reduce emissions from fracturing equipment has been driven by many factors: increased focus from customers and ESG-minded investors, widespread availability of natural gas, and technological improvements to fracturing equipment. Hydraulic fracturing customers now have access to many competing equipment solutions to aid in reducing the emissions of hydraulic fracturing operations. The data presented in this study provides an objective review and comparison of emissions from diesel and natural gas-powered fracturing technologies. Technologies evaluated included Tier 2 and Tier 4 diesel and dual-fuel reciprocating engines, and electric fracturing equipment powered by natural gas-fueled genset. Reported measurements included regulated and nonregulated emissions constituents associated with hydraulic fracturing operations. Initial tests were conducted in accordance with 40 CFR parts 60 and 63, consistent with previous industry studies. To further align with OEM testing methodology and EPA guidance for testing nonstationary engines operating intermittently under dynamic loads, additional tests were performed in adherence to 40 CFR parts 1039 and 1065. Due to the wide diversity of platform designs and emissions-control strategies among various nonroad engine technologies used in hydraulic fracturing equipment, it is challenging to obtain consistent in-use emissions measurements. Additionally, because non-road engines, to this point, have not been required to comply with in-use testing rules at the level of those required for on-road operation, the study team expended considerable effort to design and implement measurement systems in compliance with current road emissions-testing standards. To better elucidate overall emissions-related performance of various technologies, the study implemented redundant measurements and included nonregulated emissions. Collected data was normalized using CO2e/HHP-hour (Carbon Dioxide Equivalence). Tier 2 engines produced more air quality emissions compared to their Tier 4 engine counterparts. Displacing diesel fuel with natural gas reduced total fuel burned and employing optimized blending strategies significantly improved control of hydrocarbon emissions and fuel economy. Properly optimized hybrid technologies control emissions without sacrificing operational performance. Dedicated natural gas engines enabled near-zero particulate emissions and tighter control of methane emissions. Balance between operational sustainability and environmental stewardship can be achieved through integration of diesel and natural gas-supplemented equipment. The field data presented in this study, for the first time, reveals and assesses the emissions generated from available hydraulic fracturing technologies. Prior to this study, such assessments were in part hypothesized or determined using theoretical methods. This unique data set will enable operators, service companies, and equipment manufacturers to make impactful decisions regarding planning, utilization and reporting of emissions during petroleum and natural gas extraction for years to come.

Patients with lung cancer and brain metastases represent a markedly heterogeneous population. Accurate prognosis is essential to optimally individualize care. In prior publications, we described the graded prognostic assessment (GPA), but a GPA for patients with small cell lung cancer (SCLC) has never been reported, and in non-small cell lung cancer (NSCLC), the effect of programmed death ligand 1 (PD-L1) was unknown. The 3-fold purpose of this work is to provide the initial report of an SCLC GPA, to evaluate the effect of PD-L1 on survival in patients with NSCLC, and to update the Lung GPA accordingly.A multivariable analysis of prognostic factors and treatments associated with survival was performed on 4183 patients with lung cancer (3002 adenocarcinoma, 611 nonadenocarcinoma, 570 SCLC) with newly diagnosed brain metastases between January 1, 2015, and December 31, 2020, using a multi-institutional retrospective database. Significant variables were used to update the Lung GPA.Overall median survival for lung adenocarcinoma, SCLC, and nonadenocarcinoma was 17, 10, and 8 months, respectively, but varied widely by GPA from 2 to 52 months. In SCLC, the significant prognostic factors were age, performance status, extracranial metastases, and number of brain metastases. In NSCLC, the distribution of molecular markers among patients with lung adenocarcinoma and known primary tumor molecular status revealed alterations/expression in PD-L1 50% to 100%, PD-L1 1% to 49%, epidermal growth factor receptor, and anaplastic lymphoma kinase in 32%, 31%, 30%, and 7%, respectively. Median survival of patients with lung adenocarcinoma and brain metastases with 0, 1% to 49%, and ≥50% PD-L1 expression was 17, 19, and 24 months, respectively (P.01), confirming PD-L1 is a prognostic factor. Previously identified prognostic factors for NSCLC (epidermal growth factor receptor and anaplastic lymphoma kinase status, performance status, age, number of brain metastases, and extracranial metastases) were reaffirmed. These factors were incorporated into the updated Lung GPA with robust separation between subgroups for all histologies.Survival for patients with lung cancer and brain metastases has improved but varies widely. The initial report of a GPA for SCLC is presented. For patients with NSCLC-adenocarcinoma and brain metastases, PD-L1 is a newly identified significant prognostic factor, and the previously identified factors were reaffirmed. The updated indices establish unique criteria for SCLC, NSCLC-nonadenocarcinoma, and NSCLC-adenocarcinoma (incorporating PD-L1). The updated Lung GPA, available for free at brainmetgpa.com, provides an accurate tool to estimate survival, individualize treatment, and stratify clinical trials.

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA–protein complex by ssNMR using conventional 13C- and 15N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein–RNA complexes of any size.

Background Doxycycline is often used for treating COVID-19 respiratory symptoms in the community despite an absence of evidence from clinical trials to support its use. We aimed to assess the efficacy of doxycycline to treat suspected COVID-19 in the community among people at high risk of adverse outcomes. Methods We did a national, open-label, multi-arm, adaptive platform randomised trial of interventions against COVID-19 in older people (PRINCIPLE) across primary care centres in the UK. We included people aged 65 years or older, or 50 years or older with comorbidities (weakened immune system, heart disease, hypertension, asthma or lung disease, diabetes, mild hepatic impairment, stroke or neurological problem, and self-reported obesity or body-mass index of 35 kg/m2 or greater), who had been unwell (for ≤14 days) with suspected COVID-19 or a positive PCR test for SARS-CoV-2 infection in the community. Participants were randomly assigned using response adaptive randomisation to usual care only, usual care plus oral doxycycline (200 mg on day 1, then 100 mg once daily for the following 6 days), or usual care plus other interventions. The interventions reported in this manuscript are usual care plus doxycycline and usual care only; evaluations of other interventions in this platform trial are ongoing. The coprimary endpoints were time to first self-reported recovery, and hospitalisation or death related to COVID-19, both measured over 28 days from randomisation and analysed by intention to treat. This trial is ongoing and is registered with ISRCTN, 86534580. Findings The trial opened on April 2, 2020. Randomisation to doxycycline began on July 24, 2020, and was stopped on Dec 14, 2020, because the prespecified futility criterion was met; 2689 participants were enrolled and randomised between these dates. Of these, 2508 (93·3%) participants contributed follow-up data and were included in the primary analysis: 780 (31·1%) in the usual care plus doxycycline group, 948 in the usual care only group (37·8%), and 780 (31·1%) in the usual care plus other interventions group. Among the 1792 participants randomly assigned to the usual care plus doxycycline and usual care only groups, the mean age was 61·1 years (SD 7·9); 999 (55·7%) participants were female and 790 (44·1%) were male. In the primary analysis model, there was little evidence of difference in median time to first self-reported recovery between the usual care plus doxycycline group and the usual care only group (9·6 [95% Bayesian Credible Interval [BCI] 8·3 to 11·0] days vs 10·1 [8·7 to 11·7] days, hazard ratio 1·04 [95% BCI 0·93 to 1·17]). The estimated benefit in median time to first self-reported recovery was 0·5 days [95% BCI −0·99 to 2·04] and the probability of a clinically meaningful benefit (defined as ≥1·5 days) was 0·10. Hospitalisation or death related to COVID-19 occurred in 41 (crude percentage 5·3%) participants in the usual care plus doxycycline group and 43 (4·5%) in the usual care only group (estimated absolute percentage difference −0·5% [95% BCI −2·6 to 1·4]); there were five deaths (0·6%) in the usual care plus doxycycline group and two (0·2%) in the usual care only group. Interpretation In patients with suspected COVID-19 in the community in the UK, who were at high risk of adverse outcomes, treatment with doxycycline was not associated with clinically meaningful reductions in time to recovery or hospital admissions or deaths related to COVID-19, and should not be used as a routine treatment for COVID-19. Funding UK Research and Innovation, Department of Health and Social Care, National Institute for Health Research.

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates., Histone chaperones have been shown to control the activity and specificity of histone-modifying enzymes. Here the authors establish a structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4, finding that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain.

INTRODUCTION Neurocognitive dysfunction can result from radiation therapy which is the mainstay of treatment for high-grade glioma, particularly glioblastoma. Preclinical observations found that BMX-001, a novel metalloporphyrin, acts as a radioprotectant for normal CNS cells yet as a radiosensitizer to cancer cells in human GBM xenograft experiments. In a phase 1 study evaluating the safety of BMX-001 in combination with concurrent radiation therapy and temozolomide, we further studied neurocognitive function before and after concurrent radiation therapy and temozolomide in newly diagnosed high-grade glioma patients. METHODS We performed a phase 1 study of BMX-001 combined with radiation therapy (6-week total of 59.4-60 Gy) and temozolomide (75 mg/m2/day for 42 days). We administered BMX-001 as a subcutaneous injection at a loading dose before radiation therapy and temozolomide and then subsequent doses twice weekly for eight weeks. A key secondary endpoint was the evaluation of neurocognition. We performed neurocognitive testing with the computerized program CNS Vital Signsâ. This battery consists of seven tests: verbal memory, visual memory, finger tapping, symbol digit coding, the Stroop Test, a test of shifting attention, and a continuous performance test. We defined neurocognitive impairment at baseline as a z-score ≥ 1.5 SDs below the normative mean. We described improvements or declines in neurocognition at 2 and 6 months from baseline. RESULTS Fifteen patients (age 19-80 years) enrolled and underwent neurocognitive testing before and after RT. All patients had WHO grade 4 glioblastoma. Most subjects had neurocognitive impairment ranging from 46.7-to 80% on specific neurocognitive tests. At two months (N=15) and six months (N=9), most testing demonstrated improved neurocognitive performance. CONCLUSIONS Neurocognitive function is maintained and can improve after concurrent radiation therapy and temozolomide in this high-grade glioma cohort treated with BMX-001 during concurrent radiation therapy and temozolomide.

PURPOSE While stereotactic radiosurgery (SRS) is often an efficacious treatment for brain metastases, it carries a significant risk of radionecrosis (RN). Single and dual immune checkpoint inhibition (ICPI) have emerged as common treatment options for many patients, particularly those with melanoma and non-small cell lung cancer (NSCLC). While data suggest a cancer control benefit of combining SRS and ICPI, we hypothesized that concurrent receipt of dual ICPI with SRS increases the risk for RN. METHODS We performed a retrospective review of serial patients with metastatic melanoma or NSCLC treated with SRS for intact brain metastases from 2014-2020 at our single institution. Patients were stratified by receipt of dual vs. single ICPI vs. SRS alone. RN was biopsy confirmed or determined radiographically, in combination with clinical assessment and steroid use. Kaplan-Meier estimates were used to compare rates of RN between cohorts. RESULTS 673 brain lesions from 93 patients met inclusion criteria [median (Q1, Q3): 5.0 (2.0-10.0) lesions per patient]. Median follow-up of lesions was 8.1 months (95% CI: 7.3, 8.7). Most (82.8%) lesions were supratentorial and histologies included melanoma (53.5%), adenocarcinoma NSCLC (27.3%), squamous cell NSCLC (6.1%), and NSCLC NOS (6.1%). In the entire cohort, 88 lesions from 25 patients (27%) developed RN. 77 (87%) lesions were diagnosed clinico-radiographically and 11 (13%) were biopsy-proven. ICPI use was highly enriched among lesions that developed RN (85.2%) versus those that did not (19.8%). Freedom from RN at 6 months was 80% for dual ICPI, 82% for single ICPI, and 97% for SRS alone; 12 month rates were 78% in each of the ICPI cohorts and 95% with SRS alone (P=0.0002). CONCLUSIONS In a large cohort of SRS-treated brain metastases, we observed an increased risk of RN among patients who received either dual or single ICPI concurrently with SRS.

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5′ untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.

Inhibition of immune checkpoint receptor Programmed Death-1 (PD-1) via monoclonal antibodies is an established anticancer immunotherapeutic approach. This treatment has been largely successful; however, its high cost demands equally effective, more affordable alternatives. To date, the development of drugs targeting downstream players in the PD-1-dependent signaling pathway has been hampered by our poor understanding of the molecular details of the intermolecular interactions involved in the pathway. Activation of PD-1 leads to phosphorylation of two signaling motifs located in its cytoplasmic domain, the immune tyrosine inhibitory motif (ITIM) and immune tyrosine switch motif (ITSM), which recruit and activate protein tyrosine phosphatase SHP2. This interaction is mediated by the two Src homology 2 (SH2) domains of SHP2, termed N-SH2 and C-SH2, which recognize phosphotyrosines pY223 and pY248 of ITIM and ITSM, respectively. SHP2 then propagates the inhibitory signal, ultimately leading to suppression of T cell functionality. In order to facilitate mechanistic structural studies of this signaling pathway, we report the resonance assignments of the complexes formed by the signaling motifs of PD-1 and the SH2 domains of SHP2.

One is not enough: Two PD-1 pTyr motifs bind to SHP2 simultaneously, inducing a conformational change and phosphatase activation., In cancer, the programmed death-1 (PD-1) pathway suppresses T cell stimulation and mediates immune escape. Upon stimulation, PD-1 becomes phosphorylated at its immune receptor tyrosine–based inhibitory motif (ITIM) and immune receptor tyrosine–based switch motif (ITSM), which then bind the Src homology 2 (SH2) domains of SH2-containing phosphatase 2 (SHP2), initiating T cell inactivation. The SHP2–PD-1 complex structure and the exact functions of the two SH2 domains and phosphorylated motifs remain unknown. Here, we explain the structural basis and provide functional evidence for the mechanism of PD-1-mediated SHP2 activation. We demonstrate that full activation is obtained only upon phosphorylation of both ITIM and ITSM: ITSM binds C-SH2 with strong affinity, recruiting SHP2 to PD-1, while ITIM binds N-SH2, displacing it from the catalytic pocket and activating SHP2. This binding event requires the formation of a new inter-domain interface, offering opportunities for the development of novel immunotherapeutic approaches.

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Acetylation of histone H3 at lysine-56 by the histone acetyltransferase Rtt109 in lower eukaryotes is important for maintaining genomic integrity and is required for C. albicans pathogenicity. Rtt109 is activated by association with two different histone chaperones, Vps75 and Asf1, through an unknown mechanism. Here, we reveal that the Rtt109 C-terminus interacts directly with Asf1 and elucidate the structural basis of this interaction. In addition, we find that the H3 N-terminus can interact via the same interface on Asf1, leading to a competition between the two interaction partners. This, together with the recruitment and position of the substrate, provides an explanation of the role of the Rtt109 C-terminus in Asf1-dependent Rtt109 activation.

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson's disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosome-associated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF-RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery.

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the stalk protein bL12, and this information was used to refine a 3D structure of its C-terminal domain (CTD). Orientational constraints on the CTD alignment arising from the semiflexible linker sequence were further probed by a paramagnetic alignment strategy, and provided direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome–nascent chain complexes.

Failures in Phase III clinical trials have a major impact on escalating drug development costs. Clinical trial simulation can improve decisions made in learning Phase I and Phase II studies, leading to better designs and reduced chance of failure in high-cost, confirmatory Phase III trials. In adaptive design trials – which use early findings to modify the trial toward the most promising dose, sample size or patient population – simulation is instrumental in guiding selection of optimal design modifications. In this article, the authors present case studies demonstrating the benefits of simulations to identify maximum tolerated dose in Phase I, define dose–response relationships in Phase II, and determine the best type and timing of interim analyses in Phase III.

The aim of this study was to compare strength, power, and speed characteristics of elite junior English rugby league forwards and backs. A squad of males under 20's (n = 24; age 18.70 ± 0.90 years; body mass 86.4 ± 9.93 kg; height 178.47 ± 6.97 cm) players from a Super League team performed a range of assessments, including 10-, 20-, and 40-m sprints; vertical jump; and 1 repetition maximum (1RM) bench press and 1-RM back squat. Independent t-tests revealed no significance between body mass and height (180.13 ± 7.65 cm, 176.83 ± 6.10 cm; p > 0.05) or body mass (90.08 ± 11.72 kg, 82.75 ± 6.28 kg; p > 0.05) for the forwards and backs, respectively. Backs were significantly quicker over the 10-m sprints (1.99 ± 0.60 seconds, 2.06 ± 0.10 seconds; p = 0.011), 20-m sprints (3.26 ± 0.70 seconds, 3.39 ± 0.17 seconds; p = 0.002), and 40-m sprints (5.55 ± 0.13 seconds, 5.80 ± 0.26 seconds; p = 0.0001) compared with the forwards. No significant difference (p > 0.05) was observed for the vertical jump performances between the forwards (50.58 ± 7.06 cm) and the backs (50.60 ± 5.02 cm). In addition, forwards demonstrated a higher 1RM bench press and 1RM back squat (110.00 ± 15.8 kg and 140.21 ± 26.21 kg) compared with the backs (101.67 ± 9.13 kg and 132.71 ± 9.38 kg), although this was not statistically significant (p > 0.05); when expressed relative to body mass the differences between forwards and backs was reduced further for both bench press (1.22 ± 0.10 kg/kg, 1.23 ± 0.08 kg/kg, respectively; p > 0.05) and back squat (1.61 ± 0.13 kg/kg, 1.56 ± 0.20 kg/kg, respectively; p > 0.05). In addition, relative squat strength demonstrated moderate inverse correlations between relative squat strength sprint times (r = -0.45, -0.46, and -0.44; p < 0.01) across 10, 20, and 40 m, respectively. These findings highlight the importance of maximizing squat strength in academy rugby league athletes and highlight that differences in sprint performance between positions may be attributable to the differences in relative strength levels between positions.

The recent development of multifocal stereotactic radiosurgery (SRS) using a single isocenter volumetric modulated arc theory (VMAT) technique warrants a re-examination of the quality assurance (QA) tolerances for routine mechanical QA recommended by the American Association of Physicists in Medicine Task Group Report Number 142. Multifocal SRS can result in targets with small volumes being at a large off-axis distance from the treatment isocenter. Consequently, angular errors in the collimator, patient support assembly (PSA), or gantry could have an increased impact on target coverage.The authors performed a retrospective analysis of dose deviations caused by systematic errors in PSA, collimator, and gantry angle at the tolerance level for routine linear accelerator QA as recommended by TG-142. Dosimetric deviations from multifocal SRS plans (N = 10) were compared to traditional single target SRS using dynamic conformal arcs (N = 10). The chief dosimetric quantities used in determining clinical impact were VInduced errors at tolerance levels showed the greatest change in multifocal SRS target coverage for collimator rotations (±1.0°) with the average changes to VThis study indicates that institutions utilizing a single isocenter VMAT technique for multifocal disease should pay careful attention to the angular mechanical tolerances in designing a robust and complete QA program.

Summary In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions., Highlights ► An α1-antitrypsin deficiency mutant is a forme fruste model for the Z variant ► NMR spectroscopic and ion-mobility mass spectrometric characterization of the mutant ► Residue-specific discrimination of disease-relevant and denaturant-induced ensembles ► A “clasp” motif caps a network of stabilizing interactions in α1-antitrypsin

Alpha1-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of α1-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of α1-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods. Electronic supplementary material The online version of this article (doi:10.1007/s12104-011-9345-y) contains supplementary material, which is available to authorized users.

Summary Piwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3′-end of the RNA, which in the case of piRNAs is invariably modified with a 2′- O -methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand β7 and helix α3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3′ ends from a variety of RNA substrates.

The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.

Ribonucleoprotein (RNP) complexes are involved in several cellular processes, including RNA processing, transcription and translation. RNP structures are often dynamic in nature, undergoing significant remodeling during the course of their function. Visualization of the three-dimensional arrangement of single components in the complex and characterization of the intermolecular interactions are essential for understanding the mechanisms of operation. Crystallization either is not always achievable for these highly dynamic RNP particles or requires trimming the complex to a stable, well-structured core that lacks the flexible, regulatory domains. Alternative techniques that can provide structural information for complexes in solution under native conditions, where they retain their natural dynamic properties, are needed. In this study, we explored the possibility of using a combination of NMR, biochemical data and molecular modeling to generate an accurate high-resolution model of RNP complexes. We applied this strategy to the ternary hPrp31 (human Prp31)-15.5K-U4 5'-SL (stem-loop) spliceosomal complex, which, due to its large size and instability and because of the difficulty in obtaining isotopically labeled hPrp31, is not amenable to complete structure determination by NMR. We designed a protocol where the protein-protein interaction surface is defined for 15.5K by NMR data, while the relative orientations of the U4 RNA and the hPrp31 protein are described by mutational and cross-linking data. Using these data in a restrained ensemble docking protocol, we obtained a model for the ternary complex that reveals a novel rationale for the hierarchical assembly of the complex. Comparison of the docking model with the crystal structure recently obtained for a trimmed version of the complex reveals the high accuracy of the docking model, even down to an atomic level. This work shows that the architecture of large RNP complexes is within reach by NMR investigation in solution even for those cases where a traditional structural determination cannot be performed.