Computational investigation of the impact of core sequence on immobile DNA four-way junction structure and dynamics.

Immobile four-way junctions (4WJs) are core structural motifs employed in the design of programmed DNA assemblies. Understanding the impact of sequence on their equilibrium structure and flexibility is important to informing the design of complex DNA architectures. While core junction sequence is known to impact the preferences for the two possible isomeric states that junctions reside in, previous investigations have not quantified these preferences based on molecular-level interactions. Here, we use all-atom molecular dynamics simulations to investigate base-pair level structure and dynamics of four-way junctions, using the canonical Seeman J1 junction as a reference. Comparison of J1 with equivalent single-crossover topologies and isolated nicked duplexes reveal conformational impact of the double-crossover motif. We additionally contrast J1 with a second junction core sequence termed J24, with equal thermodynamic preference for each isomeric configuration. Analyses of the base-pair degrees of freedom for each system, free energy calculations, and reduced-coordinate sampling of the 4WJ isomers reveal the significant impact base sequence has on local structure, isomer bias, and global junction dynamics.

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information.

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systematizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

Perturbations of the ZED1 pseudokinase activate plant immunity.

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.

Nasal continuous positive airway pressure levels for the prevention of morbidity and mortality in preterm infants.

BACKGROUND: Preterm infants are at risk of lung atelectasis due to various anatomical and physiological immaturities, placing them at high risk of respiratory failure and associated harms. Nasal continuous positive airway pressure (CPAP) is a positive pressure applied to the airways via the nares. It helps prevent atelectasis and supports adequate gas exchange in spontaneously breathing infants. Nasal CPAP is used in the care of preterm infants around the world. Despite its common use, the appropriate pressure levels to apply during nasal CPAP use remain uncertain. OBJECTIVES: To assess the effects of 'low' (≤ 5 cm H2O) versus 'moderate-high' (> 5 cm H2O) initial nasal CPAP pressure levels in preterm infants receiving CPAP either: 1) for initial respiratory support after birth and neonatal resuscitation or 2) following mechanical ventilation and endotracheal extubation. SEARCH METHODS: We ran a comprehensive search on 6 November 2020 in the following databases: CENTRAL via CRS Web and MEDLINE via Ovid. We also searched clinical trials databases and the reference lists of retrieved articles for randomized controlled trials (RCTs) and quasi-randomized trials. SELECTION CRITERIA: We included RCTs, quasi-RCTs, cluster-RCTs and cross-over RCTs randomizing preterm infants of gestational age < 37 weeks or birth weight < 2500 grams within the first 28 days of life to different nasal CPAP levels. DATA COLLECTION AND ANALYSIS: We used the standard methods of Cochrane Neonatal to collect and analyze data. We used the GRADE approach to assess the certainty of the evidence for the prespecified primary outcomes. MAIN RESULTS: Eleven trials met inclusion criteria of the review. Four trials were parallel-group RCTs reporting our prespecified primary or secondary outcomes. Two trials randomized 316 infants to low versus moderate-high nasal CPAP for initial respiratory support, and two trials randomized 117 infants to low versus moderate-high nasal CPAP following endotracheal extubation. The remaining seven studies were cross-over trials reporting short-term physiological outcomes. The most common potential sources of bias were absent or unclear blinding of personnel and assessors and uncertain selective reporting. Nasal CPAP for initial respiratory support after birth and neonatal resuscitation None of the six primary outcomes prespecified for inclusion in the summary of findings was eligible for meta-analysis. No trials reported on moderate-severe neurodevelopmental impairment at 18 to 26 months. The remaining five outcomes were reported in a single trial. On the basis of this trial, we are uncertain whether low or moderate-high nasal CPAP levels improve the outcomes of: death or bronchopulmonary dysplasia (BPD) at 36 weeks' postmenstrual age (PMA) (risk ratio (RR) 1.02, 95% confidence interval (CI) 0.56 to 1.85; 1 trial, 271 participants); mortality by hospital discharge (RR 1.04, 95% CI 0.51 to 2.12; 1 trial, 271 participants); BPD at 28 days of age (RR 1.10, 95% CI 0.56 to 2.17; 1 trial, 271 participants); BPD at 36 weeks' PMA (RR 0.80, 95% CI 0.25 to 2.57; 1 trial, 271 participants), and treatment failure or need for mechanical ventilation (RR 1.00, 95% CI 0.63 to 1.57; 1 trial, 271 participants). We assessed the certainty of the evidence as very low for all five outcomes due to risk of bias, a lack of consistency across multiple studies, and imprecise effect estimates. Nasal CPAP following mechanical ventilation and endotracheal extubation One of the six primary outcomes prespecified for inclusion in the summary of findings was eligible for meta-analysis. On the basis of these data, we are uncertain whether low or moderate-high nasal CPAP levels improve the outcome of treatment failure or need for mechanical ventilation (RR 1.52, 95% CI 0.92 to 2.50; 2 trials, 117 participants; I2 = 17%; risk difference 0.15, 95% CI -0.02 to 0.32; number needed to treat for an additional beneficial outcome 7, 95% CI -50 to 3). We assessed the certainty of the evidence as very low due to risk of bias, inconsistency across the studies, and imprecise effect estimates. No trials reported on moderate-severe neurodevelopmental impairment at 18 to 26 months or BPD at 28 days of age. The remaining three outcomes were reported in a single trial. On the basis of this trial, we are uncertain whether low or moderate-high nasal CPAP levels improve the outcomes of: death or BPD at 36 weeks' PMA (RR 0.87, 95% CI 0.51 to 1.49; 1 trial, 93 participants); mortality by hospital discharge (RR 2.94, 95% CI 0.12 to 70.30; 1 trial, 93 participants), and BPD at 36 weeks' PMA (RR 0.87, 95% CI 0.51 to 1.49; 1 trial, 93 participants). We assessed the certainty of the evidence as very low for all three outcomes due to risk of bias, a lack of consistency across multiple studies, and imprecise effect estimates.  AUTHORS' CONCLUSIONS: There are insufficient data from randomized trials to guide nasal CPAP level selection in preterm infants, whether provided as initial respiratory support or following extubation from invasive mechanical ventilation. We are uncertain as to whether low or moderate-high nasal CPAP levels improve morbidity and mortality in preterm infants. Well-designed trials evaluating this important aspect of a commonly used neonatal therapy are needed.

The Human Gene Mutation Database (HGMD®): optimizing its use in a clinical diagnostic or research setting.

The Human Gene Mutation Database (HGMD®) constitutes a comprehensive collection of published germline mutations in nuclear genes that are thought to underlie, or are closely associated with human inherited disease. At the time of writing (June 2020), the database contains in excess of 289,000 different gene lesions identified in over 11,100 genes manually curated from 72,987 articles published in over 3100 peer-reviewed journals. There are primarily two main groups of users who utilise HGMD on a regular basis; research scientists and clinical diagnosticians. This review aims to highlight how to make the most out of HGMD data in each setting.

Nasal Intermittent Positive Pressure Ventilation Versus Nasal Continuous Positive Airway Pressure to Prevent Primary Noninvasive Ventilation Failure in Extremely Low Birthweight Infants.

Reducing the risk of primary noninvasive ventilation failure in extremely low birthweight infants is linked to reducing bronchopulmonary dysplasia. In a secondary analysis of randomized data, we identified that failure rates and time to failure were similar for nasal intermittent positive pressure ventilation vs nasal continuous positive airway pressure.

Spectrally sparse optical coherence tomography.

Swept-source optical coherence tomography (OCT) typically relies on expensive and complex swept-source lasers, the cost of which currently limits the suitability of OCT for new applications. In this work, we demonstrate spectrally sparse OCT utilizing randomly spaced low-bandwidth optical chirps, suitable for low-cost implementation with telecommunications grade devices. Micron scale distance estimation accuracy with a resolution of 40 µm at a standoff imaging distance greater than 10 cm is demonstrated using a stepped chirp approach with approximately 23% occupancy of 4 THz bandwidth. For imaging of sparse scenes, comparable performance to full bandwidth occupancy is verified for metallic targets.

Positive end-expiratory pressure for preterm infants requiring conventional mechanical ventilation for respiratory distress syndrome or bronchopulmonary dysplasia.

BACKGROUND: Conventional mechanical ventilation (CMV) is a common therapy for neonatal respiratory failure. While CMV facilitates gas exchange, it may simultaneously injure the lungs. Positive end-expiratory pressure (PEEP) has received less attention than other ventilation parameters when considering this benefit-risk balance. While an appropriate PEEP level may result in clinical benefits, both inappropriately low or high levels may cause harm. An appropriate PEEP level may also be best achieved by an individualized approach. OBJECTIVES: 1. To compare the effects of PEEP levels in preterm infants requiring CMV for respiratory distress syndrome (RDS). We compare both: zero end-expiratory pressure (ZEEP) (0 cm H2O) versus any PEEP and low (< 5 cm H2O) vs high (≥ 5 cm H2O) PEEP.2. To compare the effects of PEEP levels in preterm infants requiring CMV for bronchopulmonary dysplasia (BPD). We compare both: ZEEP (0 cm H2O) vs any PEEP and low (< 5 cm H2O) versus high (≥ 5 cm H2O) PEEP.3. To compare the effects of different methods for individualizing PEEP to an optimal level in preterm newborn infants requiring CMV for RDS. SEARCH METHODS: We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials, MEDLINE via PubMed, Embase, and CINAHL to 14 February 2018. We also searched clinical trials databases, conference proceedings, and the reference lists of retrieved articles for randomized controlled trials and quasi-randomized trials. SELECTION CRITERIA: We included all randomized or quasi-randomized controlled trials studying preterm infants born at less than 37 weeks' gestational age, requiring CMV and undergoing randomization to either different PEEP levels (RDS or BPD); or, two or more alternative methods for individualizing PEEP levels (RDS only). We included cross-over trials but limited outcomes to those from the first cross-over period. DATA COLLECTION AND ANALYSIS: We performed data collection and analysis according to the recommendations of the Cochrane Neonatal Review Group. We used the GRADE approach to assess the quality of evidence for prespecified key clinically relevant outcomes. MAIN RESULTS: Four trials met the inclusion criteria. Two cross-over trials with 28 participants compared different PEEP levels in infants with RDS. Meta-analysis was limited to short-term measures of pulmonary gas exchange and showed no differences between low and high PEEP.We identified no trials comparing PEEP levels in infants with BPD.Two trials enrolling 44 participants compared different methods for individualizing PEEP in infants with RDS. Both trials compared an oxygenation-guided lung-recruitment maneuver (LRM) with gradual PEEP level titrations for individualizing PEEP to routine care (control). Meta-analysis showed no difference between LRM and control on mortality by hospital discharge (risk ratio (RR) 1.00, 95% confidence interval (CI) 0.17 to 5.77); there was no statistically significant difference on BPD, with an effect estimate favoring LRM (RR 0.25, 95% CI 0.03 to 2.07); and a statistically significant difference favoring LRM for the outcome of duration of ventilatory support (mean difference -1.06 days, 95% CI -1.85 to -0.26; moderate heterogeneity, I2 = 67%). Short-term oxygenation measures also favored LRM. We graded the quality of the evidence as low for all key outcomes due to risk of bias and imprecision of the effect estimates. AUTHORS' CONCLUSIONS: There continues to be insufficient evidence to guide PEEP level selection for preterm infants on CMV for RDS or BPD. Low-quality data suggests that selecting PEEP levels through the application of an oxygenation-guided LRM may result in clinical benefit. Well-conducted randomized trials, particularly to further evaluate the potential benefits of oxygenation-guided LRMs, are needed.

A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

Variation in Positive End-Expiratory Pressure Levels for Mechanically Ventilated Extremely Low Birth Weight Infants.

OBJECTIVE: To test the hypothesis that significant positive end-expiratory pressure (PEEP) level variation exists between neonatal centers. STUDY DESIGN: We performed a secondary analysis cohort study of the Nasal Intermittent Positive-Pressure Ventilation trial. Our study population was extremely low birth weight infants requiring mechanical ventilation within 28 days of life. The exposure was neonatal center; 34 international centers participated in the trial. Subjects from centers with fewer than 5 eligible cases were excluded. The main outcome was the maximal PEEP level used during the first course of mechanical ventilation. Infant characteristics judged a priori to directly influence clinical PEEP level selection and all characteristics associated with PEEP at P <.05 in bivariable analyses were included with and without center in multivariable linear regression models. Variation in PEEP level use between centers following adjustment for infant characteristics was assessed. RESULTS: A total of 278 extremely low birth weight infants from 17 centers were included. Maximal PEEP ranged from 3 to 9 cm H2O, mean = 5.7 (SD = 0.9). Significant variation between centers remained despite adjustment for infant characteristics (P < .0001). Further, center alone explained a greater proportion of the PEEP level variation than all infant characteristics combined. CONCLUSIONS: Marked variation in PEEP levels for extremely low birth weight infants exists between neonatal centers. Research providing evidence-based guidance for this important aspect of respiratory care in preterm infants at high risk of lung injury is needed. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00433212.

Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency.

Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRß sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21(lo) B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease.

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor ß2AR.

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human ß2-adrenergic receptor (ß2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

A trial comparing noninvasive ventilation strategies in preterm infants.

BACKGROUND: To reduce the risk of bronchopulmonary dysplasia in extremely-low-birth-weight infants, clinicians attempt to minimize the use of endotracheal intubation by the early introduction of less invasive forms of positive airway pressure. METHODS: We randomly assigned 1009 infants with a birth weight of less than 1000 g and a gestational age of less than 30 weeks to one of two forms of noninvasive respiratory support--nasal intermittent positive-pressure ventilation (IPPV) or nasal continuous positive airway pressure (CPAP)--at the time of the first use of noninvasive respiratory support during the first 28 days of life. The primary outcome was death before 36 weeks of postmenstrual age or survival with bronchopulmonary dysplasia. RESULTS: Of the 497 infants assigned to nasal IPPV for whom adequate data were available, 191 died or survived with bronchopulmonary dysplasia (38.4%), as compared with 180 of 490 infants assigned to nasal CPAP (36.7%) (adjusted odds ratio, 1.09; 95% confidence interval, 0.83 to 1.43; P=0.56). The frequencies of air leaks and necrotizing enterocolitis, the duration of respiratory support, and the time to full feedings did not differ significantly between treatment groups. CONCLUSIONS: Among extremely-low-birth-weight infants, the rate of survival to 36 weeks of postmenstrual age without bronchopulmonary dysplasia did not differ significantly after noninvasive respiratory support with nasal IPPV as compared with nasal CPAP. (Funded by the Canadian Institutes of Health Research; NIPPV ClinicalTrials.gov number, NCT00433212; Controlled-Trials.com number, ISRCTN15233270.).

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

Pathogenetic mechanisms of amyloid A amyloidosis.

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy.

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.

High-dimensional modulation for coherent optical communications systems.

In this paper, we examine the performance of several modulation formats in more than four dimensions for coherent optical communications systems. We compare two high-dimensional modulation design methodologies based on spherical cutting of lattices and block coding of a 'base constellation' of binary phase shift keying (BPSK) on each dimension. The performances of modulation formats generated with these methodologies is analyzed in the asymptotic signal-to-noise ratio regime and for an additive white Gaussian noise (AWGN) channel. We then study the application of both types of high-dimensional modulation formats to standard single-mode fiber (SSMF) transmission systems. For modulation with spectral efficiencies comparable to dual-polarization (DP-) BPSK, polarization-switched quaternary phase shift keying (PS-QPSK) and DP-QPSK, we demonstrate SNR gains of up to 3 dB, 0.9 dB and 1 dB respectively, at a BER of 10(-3).

Noninvasive respiratory support for neonates.

PURPOSE OF THE REVIEW: Noninvasive respiratory support for neonates is growing in popularity as clinicians increasingly recognize the dangers of prolonged invasive ventilation. The purpose of this review is to critically evaluate the existing evidence for safety and efficacy of these modes of respiratory support in neonates. RECENT FINDINGS: In recent years, multiple randomized controlled trials (RCTs) have evaluated several modes of noninvasive support, most importantly nasal intermittent positive pressure ventilation and high flow nasal cannulae, in comparison to the standard therapy of continuous positive airway pressure (CPAP). The three largest RCTs were recently published in 2013. One demonstrated no difference in death or survival with bronchopulmonary dysplasia between nasal intermittent positive pressure ventilation and CPAP, both when used as primary support and as postextubation support. Two others demonstrated that high flow nasal cannulae are noninferior to or no better than CPAP when used to support preterm infants after extubation. These trials showed no serious safety concerns with current modalities. SUMMARY: The optimal forms of noninvasive respiratory support for neonates remain to be determined. Continued evaluation of these technologies with large, well-designed RCTs is warranted.

Stealth transgenes enable CAR-T cells to evade host immune responses.

BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

Dynamics of site switching in DNA polymerase.

DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3' end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3'-5' exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.