In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties

An Educational Intervention to Improve HPV Vaccination: A Cluster Randomized Trial

Background: Human Papillomavirus (HPV) infection can lead to serious health issues and remains the most common sexually transmitted infection. Despite availability of effective vaccines, HPV vaccination rates are suboptimal. Methods: In a cluster randomized trial, an intervention targeting parents of adolescents (11-17 years) eligible for a dose of HPV vaccine was tested in pediatric clinics part of an urban health system. Parents watched a digital video outlining the risks and benefits of vaccine using a tablet in the exam room. The primary outcome was change in HPV vaccine status two weeks after the clinic visit. An intention to treat analysis for the primary outcome utilized generalized estimating equations to accommodate the potential cluster effect of clinics. Results: A total of 1596 eligible adolescents were observed during the 7-month trial. One-third of adolescents visited an intervention clinic. Adolescents who attended an intervention clinic were more likely to be younger (11-12 years) than those who attended a control clinic (72.4% versus 49.8%; p<0.001). No differences in race or gender were observed. The proportion of adolescents with an observed change in vaccine status was higher for those attending an intervention clinic (64.8%) versus control clinic (50.1%; OR=1.82; 95% CI=1.47-2.25; p<0.001). Adolescents whose parents watched the video had a three times greater odds of receiving a dose of the HPV vaccine (78.0%; OR=3.07; 95% CI=1.47-6.42; p=0.003). Conclusions: Educational interventions delivered within a clinical setting hold promise to improve vaccination behaviors

An Integrated Strategy for Analyzing the Unique Developmental Programs of Different Myoblast Subtypes

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system

A Review of the Scientific Rigor, Reproducibility, and Transparency Studies Conducted by the ABRF Research Groups.

Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and thedy have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications

Compact, Miniature MMIC Receiver Modules for an MMIC Array Spectrograph

A single-pixel prototype of a W-band detector module with a digital back-end was developed to serve as a building block for large focal-plane arrays of monolithic millimeter-wave integrated circuit (MMIC) detectors. The module uses low-noise amplifiers, diode-based mixers, and a WR10 waveguide input with a coaxial local oscillator. State-of-the-art InP HEMT (high electron mobility transistor) MMIC amplifiers at the front end provide approximately 40 dB of gain. The measured noise temperature of the module, at an ambient temperature of 300 K, was found to be as low as 450 K at 95 GHz. The modules will be used to develop multiple instruments for astrophysics radio telescopes, both on the ground and in space. The prototype is being used by Stanford University to characterize noise performance at cryogenic temperatures. The goal is to achieve a 30-50 K noise temperature around 90 GHz when cooled to a 20 K ambient temperature. Further developments include characterization of the IF in-phase (I) and quadrature (Q) signals as a function of frequency to check amplitude and phase; replacing the InP low-noise amplifiers with state-of-the-art 35-nm-gate-length NGC low-noise amplifiers; interfacing the front-end module with a digital back-end spectrometer; and developing a scheme for local oscillator and IF distribution in a future array. While this MMIC is being developed for use in radio astronomy, it has the potential for use in other industries. Applications include automotive radar (both transmitters and receivers), communication links, radar systems for collision avoidance, production monitors, ground-penetrating sensors, and wireless personal networks

Characterization of the Cytochrome P450 epoxyeicosanoid pathway in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH

Pre-Training Muscle Characteristics of Subjects Who Are Obese Determine How Well Exercise Training Will Improve Their Insulin Responsiveness

Pre-training muscle characteristics of subjects who are obese determine how well exercise training will improve their insulin responsiveness. J Strength Cond Res 31(3): 798–808, 2017—Only half of prediabetic subjects who are obese who underwent exercise training without weight loss increased their insulin responsiveness. We hypothesized that those who improved their insulin responsiveness might have pretraining characteristics favoring a positive response to exercise training. Thirty nondiabetic subjects who were obese volunteered for 8 weeks of either strength training or endurance training. During training, subjects increased their caloric intake to prevent weight loss. Insulin responsiveness by euglycemic clamps and muscle fiber composition, and expression of muscle key biochemical pathways were quantified. Positive responders initially had 52% higher intermediate muscle fibers (fiber type IIa) with 27% lower slow-twitch fibers (type I) and 23% lower expression of muscle insulin receptors. Whether after weight training or stationary bike training, positive responders\u27 fiber type shifted away from type I and type IIa fibers to an increased proportion of type IIx fibers (fast twitch). Muscle insulin receptor expression and glucose transporter type 4 (GLUT4) expression increased in all trained subjects, but these moderate changes did not consistently translate to improvement in whole-body insulin responsiveness. Exercise training of previously sedentary subjects who are obese can result in muscle remodeling and increased expression of key elements of the insulin pathway, but in the absence of weight loss, insulin sensitivity improvement was modest and limited to about half of the participants. Our data suggest rather than responders being more fit, they may have been less fit, only catching up to the other half of subjects who are obese whose insulin responsiveness did not increase beyond their pretraining baseline

High-Order SNP Combinations Associated with Complex Diseases: Efficient Discovery, Statistical Power and Functional Interactions

There has been increased interest in discovering combinations of single-nucleotide polymorphisms (SNPs) that are strongly associated with a phenotype even if each SNP has little individual effect. Efficient approaches have been proposed for searching two-locus combinations from genome-wide datasets. However, for high-order combinations, existing methods either adopt a brute-force search which only handles a small number of SNPs (up to few hundreds), or use heuristic search that may miss informative combinations. In addition, existing approaches lack statistical power because of the use of statistics with high degrees-of-freedom and the huge number of hypotheses tested during combinatorial search. Due to these challenges, functional interactions in high-order combinations have not been systematically explored. We leverage discriminative-pattern-mining algorithms from the data-mining community to search for high-order combinations in case-control datasets. The substantially improved efficiency and scalability demonstrated on synthetic and real datasets with several thousands of SNPs allows the study of several important mathematical and statistical properties of SNP combinations with order as high as eleven. We further explore functional interactions in high-order combinations and reveal a general connection between the increase in discriminative power of a combination over its subsets and the functional coherence among the genes comprising the combination, supported by multiple datasets. Finally, we study several significant high-order combinations discovered from a lung-cancer dataset and a kidney-transplant-rejection dataset in detail to provide novel insights on the complex diseases. Interestingly, many of these associations involve combinations of common variations that occur in small fractions of population. Thus, our approach is an alternative methodology for exploring the genetics of rare diseases for which the current focus is on individually rare variations

Heat stored in the Earth system 1960–2020: where does the energy go?

The Earth climate system is out of energy balance, and heat has accumulated continuously over the past decades, warming the ocean, the land, the cryosphere, and the atmosphere. According to the Sixth Assessment Report by Working Group I of the Intergovernmental Panel on Climate Change, this planetary warming over multiple decades is human-driven and results in unprecedented and committed changes to the Earth system, with adverse impacts for ecosystems and human systems. The Earth heat inventory provides a measure of the Earth energy imbalance (EEI) and allows for quantifying how much heat has accumulated in the Earth system, as well as where the heat is stored. Here we show that the Earth system has continued to accumulate heat, with 381±61 ZJ accumulated from 1971 to 2020. This is equivalent to a heating rate (i.e., the EEI) of 0.48±0.1 W m−2. The majority, about 89 %, of this heat is stored in the ocean, followed by about 6 % on land, 1 % in the atmosphere, and about 4 % available for melting the cryosphere. Over the most recent period (2006–2020), the EEI amounts to 0.76±0.2 W m−2. The Earth energy imbalance is the most fundamental global climate indicator that the scientific community and the public can use as the measure of how well the world is doing in the task of bringing anthropogenic climate change under control. Moreover, this indicator is highly complementary to other established ones like global mean surface temperature as it represents a robust measure of the rate of climate change and its future commitment. We call for an implementation of the Earth energy imbalance into the Paris Agreement's Global Stocktake based on best available science. The Earth heat inventory in this study, updated from von Schuckmann et al. (2020), is underpinned by worldwide multidisciplinary collaboration and demonstrates the critical importance of concerted international efforts for climate change monitoring and community-based recommendations and we also call for urgently needed actions for enabling continuity, archiving, rescuing, and calibrating efforts to assure improved and long-term monitoring capacity of the global climate observing system. The data for the Earth heat inventory are publicly available, and more details are provided in Table 4

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d