Analysis of large 16S rRNA Illumina data sets: Impact of singleton read filtering on microbial community description

Next‐generation sequencing technologies give access to large sets of data, which are extremely useful in the study of microbial diversity based on 16S rRNA gene. However, the production of such large data sets is not only marred by technical biases and sequencing noise but also increases computation time and disc space use. To improve the accuracy of OTU predictions and overcome both computations, storage and noise issues, recent studies and tools suggested removing all single reads and low abundant OTUs, considering them as noise. Although the effect of applying an OTU abundance threshold on α‐ and β‐diversity has been well documented, the consequences of removing single reads have been poorly studied. Here, we test the effect of singleton read filtering (SRF) on microbial community composition using in silico simulated data sets as well as sequencing data from synthetic and real communities displaying different levels of diversity and abundance profiles. Scalability to large data sets is also assessed using a complete MiSeq run. We show that SRF drastically reduces the chimera content and computational time, enabling the analysis of a complete MiSeq run in just a few minutes. Moreover, SRF accurately determines the actual community diversity: the differences in α‐ and β‐community diversity obtained with SRF and standard procedures are much smaller than the intrinsic variability of technical and biological replicates.     

Validity of Physical Activity Intensity Predictions by ActiGraph,Actical, and RT3 Accelerometers

Objective: Accelerometers are promising tools for characterizing physical activity (PA) patterns in free‐living persons. To date, validation of energy expenditure (EE) predictions from accelerometers has been restricted to short laboratory or simulated free‐living protocols. This study seeks to determine the capabilities of eight previously published regression equations for three commercially available accelerometers to predict summary measures of daily EE. Methods and Procedures: Study participants were outfitted with ActiGraph, Actical, and RT3 accelerometers, while measurements were simultaneously made during overnight stays in a room calorimeter, which provided minute‐by‐minute EE measurements, in a diverse subject population (n = 85). Regression equations for each device were used to predict the minute‐by‐minute metabolic equivalents (METs) along with the daily PA level (PAL). Results: Two RT3 regressions and one ActiGraph regression were not significantly different from calorimeter measured PAL. When data from the entire visit were divided into four intensity categories—sedentary, light, moderate, and vigorous—significant (P < 0.001) over‐ and underpredictions were detected in numerous regression equations and intensity categories. Discussion: Most EE prediction equations showed differences of <2% in the moderate and vigorous intensity categories. These differences, though small in magnitude, may limit the ability of these regressions to accurately characterize whether specific PA goals have been met in the field setting. New regression equations should be developed if more accurate prediction of the daily PAL or higher precision in determining the time spent in specific PA intensity categories is desired.     

N‐terminal autoprocessing and acetylation of multifunctional‐autoprocessing repeats‐in‐toxins (MARTX) Makes Caterpillars Floppy‐like effector is stimulated by adenosine diphosphate (ADP)‐Ribosylation Factor 1 in advance of Golgi fragmentation

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional‐autoprocessing repeats‐in‐toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)‐like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)‐Ribosylation Factor 1 (ARF1) and ADP‐Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)‐bound] form compared to the inactive [guanosine diphosphate (GDP)‐bound] form. Subsequent to auto‐cleavage, MCF is acetylated on its exposed N‐terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5‐phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin‐section electron microscopy, high‐resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post‐translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross‐kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.     

Sedentary behavior and physical activity levels in people with schizophrenia,bipolar disorder and major depressive disorder: a global systematic review and meta‐analysis

People with severe mental illness (schizophrenia, bipolar disorder or major depressive disorder) die up to 15 years prematurely due to chronic somatic comorbidities. Sedentary behavior and low physical activity are independent yet modifiable risk factors for cardiovascular disease and premature mortality in these people. A comprehensive meta‐analysis exploring these risk factors is lacking in this vulnerable population. We conducted a meta‐analysis investigating sedentary behavior and physical activity levels and their correlates in people with severe mental illness. Major electronic databases were searched from inception up to April 2017 for articles measuring sedentary behavior and/or physical activity with a self‐report questionnaire or an objective measure (e.g., accelerometer). Random effects meta‐analyses and meta‐regression analyses were conducted. Sixty‐nine studies were included (N=35,682; 39.5% male; mean age 43.0 years). People with severe mental illness spent on average 476.0 min per day (95% CI: 407.3‐545.4) being sedentary during waking hours, and were significantly more sedentary than age‐ and gender‐matched healthy controls (p=0.003). Their mean amount of moderate or vigorous physical activity was 38.4 min per day (95% CI: 32.0‐44.8), being significantly lower than that of healthy controls (p=0.002 for moderate activity, p<0.001 for vigorous activity). People with severe mental illness were significantly less likely than matched healthy controls to meet physical activity guidelines (odds ratio = 1.5; 95% CI: 1.1‐2.0, p<0.001, I²=95.8). Lower physical activity levels and non‐compliance with physical activity guidelines were associated with male gender, being single, unemployment, fewer years of education, higher body mass index, longer illness duration, antidepressant and antipsychotic medication use, lower cardiorespiratory fitness and a diagnosis of schizophrenia. People with bipolar disorder were the most physically active, yet spent most time being sedentary. Geographical differences were detected, and inpatients were more active than outpatients and those living in the community. Given the established health benefits of physical activity and its low levels in people with severe mental illness, future interventions specifically targeting the prevention of physical inactivity and sedentary behavior are warranted in this population.     

ADP‐ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP‐ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope‐edited Fourier transform infrared spectroscopy monitored ARF6‐induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF‐mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.     

A novel LED‐based 2D‐fluorescence spectroscopy system for in‐line bioprocess monitoring of Chinese hamster ovary cell cultivations—Part II

This study was performed in order to evaluate a new LED‐based 2D‐fluorescence spectrometer for in‐line bioprocess monitoring of Chinese hamster ovary (CHO) cell culture processes. The new spectrometer used selected excitation wavelengths of 280, 365, and 455 nm to collect spectral data from six 10‐L fed‐batch processes. The technique provides data on various fluorescent compounds from the cultivation medium as well as from cell metabolism. In addition, scattered light offers information about the cultivation status. Multivariate data analysis tools were applied to analyze the large data sets of the collected fluorescence spectra. First, principal component analysis was used to accomplish an overview of all spectral data from all six CHO cultivations. Partial least square regression models were developed to correlate 2D‐fluorescence spectral data with selected critical process variables as offline reference values. A separate independent fed‐batch process was used for model validation and prediction. An almost continuous in‐line bioprocess monitoring was realized because 2D‐fluorescence spectra were collected every 10 min during the whole cultivation. The new 2D‐fluorescence device demonstrates the significant potential for accurate prediction of the total cell count, viable cell count, and the cell viability. The results strongly indicated that the technique is particularly capable to distinguish between different cell statuses inside the bioreactor. In addition, spectral data provided information about the lactate metabolism shift and cellular respiration during the cultivation process. Overall, the 2D‐fluorescence device is a highly sensitive tool for process analytical technology applications in mammalian cell cultures.     

Evaluation of the ECOSSE model for simulating soil carbon under short rotation forestry energy crops in Britain

Understanding and predicting the effects of land‐use change to short rotation forestry (SRF) on soil carbon (C) is an important requirement for fully assessing the C mitigation potential of SRF as a bioenergy crop. There is little current knowledge of SRF in the UK and in particular a lack of consistent measured data sets on the direct impacts of land use change on soil C stocks. The ECOSSE model was developed to simulate soil C dynamics and greenhouse gas (GHG) emissions in mineral and organic soils. The ECOSSE model has already been applied spatially to simulate land‐use change impacts on soil C and GHG emissions. However, it has not been extensively evaluated under SRF. Eleven sites comprising 29 transitions in Britain, representing land‐use change from nonwoodland land uses to SRF, were selected to evaluate the performance of ECOSSE in predicting soil C and soil C change in SRF plantations. The modelled C under SRF showed a strong correlation with the soil C measurements at both 0–30 cm (R = 0.93) and 0–100 cm soil depth (R = 0.82). As for the SRF plots, the soil C at the reference sites have been accurately simulated by the model. The extremely high correlation for the reference fields (R ≥ 0.99) shows a good performance of the model spin‐up. The statistical analysis of the model performance to simulate soil C and soil C changes after land‐use change to SRF highlighted the absence of significant error between modelled and measured values as well as the absence of significant bias in the model. Overall, this evaluation reinforces previous studies on the ability of ECOSSE to simulate soil C and emphasize its accuracy to simulate soil C under SRF plantations.     

The influence of short-term sedentary behavior on circadian rhythm of heart rate and heart rate variability

The notion that sedentary behavior is harmful to human health is widespread. Little is known about the short term influence of sedentary behavior on heart rate (HR) and heart rate variability (HRV) circadian rhythms. Therefore the purpose of the present study was to examine the influence of short term sedentary behavior on the circadian rhythms of HR and HRV using cosine periodic regression analysis. Sixteen healthy young students were included in a randomized crossover study. All subjects underwent 24-h ECG Holter monitoring in two different states of physical activity, an active condition (more than 15,000 steps per day) and a sedentary condition (less than 1,000 steps per day). Hourly mean values were calculated for HR and HRV, and then were evaluated using cosine periodic regression analysis. The circadian rhythm parameters, amplitude, mesor, and acrophase for HR and HRV variables were obtained. As a result, the significance of the circadian rhythm was confirmed for all variables in each condition. The measure of fit R2 value was decreased in sedentary condition. The amplitude of the sedentary condition was significantly smaller than that of the active condition with respect to HR (7.94 ± 1.91 bpm vs. 15.4 ± 3.93 bpm, p < 0.001), natural log of the high frequency measurement (lnHF) (0.38 ± 0.21 ms2 vs. 0.80 ± 0.28 ms2, p < 0.001), and low frequency/high frequency ratio (LF/HF) (0.75 ± 0.54 vs. 1.24 ± 0.69, p = 0.008). We found that sedentary behavior not only significantly lowered the amplitude of HR and HRV variables, but also might have led to weakness of the circadian rhythm of the HR and HRV variables.     

Ensemble clustering for step data via binning

This paper considers the clustering problem of physical step count data recorded on wearable devices. Clustering step data give an insight into an individual's activity status and further provide the groundwork for health‐related policies. However, classical methods, such as K‐means clustering and hierarchical clustering, are not suitable for step count data that are typically high‐dimensional and zero‐inflated. This paper presents a new clustering method for step data based on a novel combination of ensemble clustering and binning. We first construct multiple sets of binned data by changing the size and starting position of the bin, and then merge the clustering results from the binned data using a voting method. The advantage of binning, as a critical component, is that it substantially reduces the dimension of the original data while preserving the essential characteristics of the data. As a result, combining clustering results from multiple binned data can provide an improved clustering result that reflects both local and global structures of the data. Simulation studies and real data analysis were carried out to evaluate the empirical performance of the proposed method and demonstrate its general utility.     

Centaurin-alpha1 is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent GTPase-activating protein for ARF6 that is involved in actin cytoskeleton organization

The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6GDP. Centaurin-alpha1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we show that centaurin-alpha1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-alpha1 also inhibited cortical actin formation in a PIP3-dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-alpha1. An artificially plasma membrane-targeted centaurin-alpha1 bypasses the requirement of PIP3 for its involvement in ARF6 inactivation, suggesting that PIP3 is required for recruitment of centaurin-alpha1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-alpha1 negatively regulates ARF6 activity by functioning as an in vivo PIP3-dependent ARF6 GAP.     

Textile‐Based Electrochemical Energy Storage Devices

In the past few years, insensitive attentions have been drawn to wearable and flexible energy storage devices/systems along with the emergence of wearable electronics. Much progress has been achieved in developing flexible electrochemical energy storage devices with high end‐use performance. However, challenges still remain in well balancing the electrochemical properties, mechanical properties, and the processing technologies. In this review, a specific perspective on the development of textile‐based electrochemical energy storage devices (TEESDs), in which textile components and technologies are utilized to enhance the energy storage ability and mechanical properties of wearable electronic devices, is provided. The discussion focuses on the material preparation and characteristics, electrode and device fabrication strategies, electrochemical performance and metrics, wearable compatibility, and fabrication scalability of TEESDs including textile‐based supercapacitors and lithium‐ion batteries.     

AGD5 is a GTPase-activating protein at the trans-Golgi network

ARF‐GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs), which facilitate the activation or inactivation of ARF‐GTPases, respectively. There are 15 predicted proteins that contain an ARF‐GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF‐GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF‐GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans‐Golgi network (TGN), where it co‐localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post‐Golgi structures of unknown nature. Taking advantage of the in vivo AGD5–ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF‐GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post‐Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN‐localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane‐localised ADP ribosylation factor B (ARFB), confirming that ARF‐GAP specificity for ARF‐GTPases within the cell environment may be spatially regulated.     

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.     

Distinct Recycling of Active and Inactive β1 Integrins

Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin‐ and dynamin‐dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6‐ and early endosome antigen 1‐positive compartment in an Rab 4a‐ and actin‐dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic.     

Unity Convoluted Design of Solid Li‐Ion Battery and Triboelectric Nanogenerator for Self‐Powered Wearable Electronics

Wearable electronics suffer from severe power shortage due to limited working time of Li‐ion batteries, and there is a desperate need to build a hybrid device including energy scavenging and storing units. However, previous attempts to integrate the two units are mainly based on simple external connections and assembly, so that maintaining small volume and low manufacturing cost becomes increasingly challenging. Here a convoluted power device is presented by hybridizing internally a solid Li‐ion battery (SLB) and a triboelectric nanogenerator (TENG), so that the two units are one inseparable entity. The fabricated device acts as a TENG that can deliver a peak output power of 7.4 mW under a loading resistance of 7 MΩ, while the device also acts as an SLB to store the obtained electric energy. The device can be mounted on a human shoe to sustainably operate a green light‐emitting diode, thus demonstrating potential for self‐powered wearable electronics.     

Regulation of the INK4a/ARF locus by histone deacetylase inhibitors

Despite the importance of the INK4a/ARF locus in tumor suppression, its modulation by histone deacetylase inhibitors (HDACis) remains to be characterized. Here, we have shown that the levels of p16INK4a are decreased in human and murine fibroblasts upon exposure to relatively high concentrations of trichostatin A and sodium butyrate. Interestingly, the levels of p19ARF are strongly upregulated in murine cells even at low concentrations of HDACis. Using ARF-deficient cells, we have demonstrated that p19ARF plays an active role in HDACi-triggered cytostasis and the contribution of p19ARF to this arrest is of higher magnitude than that of the well established HDACi target p21Waf1/Cip. Moreover, chemically induced fibrosarcomas in ARF-null mice are more resistant to the therapeutic effect of HDACis than similar tumors in wild type or p21Waf1/Cip-null mice. Together, our results have established the tumor suppressor ARF as a relevant target for HDACi chemotherapy.