bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Met25 as a Color Associated Genetic Marker inYarrowia lipolytica
Authors: Edwards Harley , Yang Zhiliang , Xu Peng
Published: April 2020

Yarrowia lipolyticaoffers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily withURA3. In this work, we investigateMET25, a locus of sulfide housekeeping within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation inMET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies ofMET25grow white. Loss ofMET25did not induce strict auxotrophic requirements for methionine inY.lipolytica, indicatingMET25deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation ofMET25deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast.MET25deficientY. lipolyticacould be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whetherMET25deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type andMET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of usingMET25as a sensitive lead sensor and the challenges of usingMET25as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis inY. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.
Determination of vancomycin exposure target and individualised dosing recommendations for neonates: model-informed precision dosing
Authors: Tang Zhe , Guan Jing , Li Jingjing , Yu Yanxia , Qian Miao , Cao Jiang , Shuai Weiwei , Jiao Zheng
Published: April 2020

Few studies incorporating population pharmacokinetic/pharmacodynamic (Pop-PK/PD) modelling have been conducted to quantify the exposure target of vancomycin in neonates. To determine this target and dosing recommendations, a retrospective observational cohort study was established in neonates (chictr.org.cn, ChiCTR1900027919). A Pop-PK model was developed to estimate PK parameters. Causalities between acute kidney injury (AKI) occurrence and vancomycin use were verified using Naranjo criteria. Thresholds of vancomycin exposure in predicting AKI or efficacy were identified via classification and regression tree analysis. Associations between exposure thresholds and clinical outcomes including AKI and efficacy, were analysed by logistic regression. Dosing recommendations were designed using Monte Carlo (MC) simulations based on the optimised exposure target. Pop-PK modelling included 182 neonates with 411 observations. In covariate analysis, neonatal physiological maturation, renal function, and concomitant use of vasoactive drugs (VAS) significantly affected vancomycin pharmacokinetics. Seven cases of vancomycin-induced AKI were detected. Area under the concentration–time curve from 0–24 h (AUC0-24) ≥ 485 mg·h/L was an independent risk factor for AKI after adjusting for VAS co-administration. Clinical efficacy of vancomycin was analysed in 42 patients with blood culture-proven staphylococcal sepsis. AUC0–24to minimum inhibitory concentration (AUC0–24/MIC) ≥ 234 was the only significant predictor of clinical effectiveness. MC simulations indicated that regimens in Neonatal Formulary 7 and Red Book (2018) were not suitable for all neonates. In summary, AUC0-24of 240-480 assuming MIC = 1 mg/L is a recommended exposure target of vancomycin in neonates. Model-informed dosing regimens are valuable in clinical practice.

Highlights

The optimised neonatal exposure target of vancomycin was uniquely quantified

Day 1 exposure of vancomycin is the predictor for clinical outcomes in neonates

Vancomycin-induced acute kidney injury is not related to high exposure alone

A lower exposure target is likely to be more effective for neonates than for adults

Monte Carlo simulation provides a more suitable dosing regimen for neonates
Why is chest CT important for early diagnosis of COVID-19? Prevalence matters
Authors: Esposito Antonio , Palmisano Anna , Scotti Giulia Maria , Morelli Marco J. , Vignale Davide , De Cobelli Francesco , Tonon Giovanni , Tacchetti Carlo
Published: April 2020

SARS-CoV-2 viral infection is a global pandemic disease (COVID-19). Reaching a swift, reliable diagnosis of COVID-19 in the emergency departments is imperative to direct patients to proper care and to prevent disease dissemination. COVID-19 diagnosis is based on the identification of viral RNA through RT-PCR from oral-nasopharyngeal swabs, which however presents suboptimal sensitivity and may require several hours in overstressed laboratories. These drawbacks have called for an additional, complementary first line approach. CT is the gold standard method for the detection of interstitial pneumonia, a hallmark feature of COVID-19, often present in the asymptomatic stage of the disease. Here, we show that CT scan presents a sensitivity of 95.48% (std.err=0.35%), vastly outperforming RT-PCR. Additionally, as diagnostic accuracy is influenced by disease prevalence, we argue that predictive values provide a more precise measure of CT reliability in the current pandemics. We generated a model showing that CT scan is endowed with a high negative predictive value (> 90%) and positive predictive value (69 - 84%), for the range of prevalence seen in countries with rampant dissemination. We conclude that CT is an expedite and reliable diagnostic tool to support first line triage of suspect COVID-19 patients in areas where the diffusion of the virus is widespread.
Development of an in vitro senescent hepatic cell model for metabolic studies in aging
Authors: Singh Brijesh Kumar , Tripathi Madhulika , Sandireddy Reddemma , Tikno Keziah , Zhou Jin , Yen Paul Michael
Published: April 2020

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-βGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from β-oxidation that occurred in normal AML12 cells. Additionally, senescence activated secretory proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging, and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.
SMN1copy-number and sequence variant analysis from next generation sequencing data
Authors: Lopez-Lopez Daniel , Loucera Carlos , Carmona Rosario , Aquino Virginia , Salgado Josefa , Pasalodos Sara , Miranda María , Alonso Ángel , Dopazo Joaquín
Published: April 2020

Spinal Muscular Atrophy (SMA) is a severe neuromuscular autosomal recessive disorder affecting 1/10,000 live births. Most SMA patients present homozygous deletion ofSMN1, while the vast majority of SMA carriers present only a singleSMN1copy. The sequence similarity betweenSMN1andSMN2, and the complexity of the SMN locus makes the estimation of the SMN1 copy-number by next generation sequencing (NGS) very difficult Here, we present SMAca, the first python tool to detect SMA carriers and estimate the absolute SMN1 copy-number using NGS data. Moreover, SMAca takes advantage of the knowledge of certain variants specific toSMN1duplication to also identify silent carriers. This tool has been validated with a cohort of 326 samples from the Navarra 1000 Genomes project (NAGEN1000). SMAca was developed with a focus on execution speed and easy installation. This combination makes it especially suitable to be integrated into production NGS pipelines. Source code and documentation are available on Github at:www.github.com/babelomics/SMAca
Evolutionary patterns of 64 vertebrate genomes (species) revealed by phylogenomics analysis of protein-coding gene families
Authors: Song Jia , Han Xia , Lin Kui
Published: April 2020

Background

Recent studies have demonstrated that phylogenomics is an important basis for answering many fundamental evolutionary questions. With more high-quality whole genome sequences published, more efficient phylogenomics analysis workflows are required urgently.

Results

To this end and in order to capture putative differences among evolutionary histories of gene families and species, we developed a phylogenomics workflow for gene family classification, gene family tree inference, species tree inference and duplication/loss events dating. Our analysis framework is on the basis of two guiding ideas: 1) gene trees tend to be different from species trees but they influence each other in evolution; 2) different gene families have undergone different evolutionary mechanisms. It has been applied to the genomic data from 64 vertebrates and 5 out-group species. And the results showed high accuracy on species tree inference and few false-positives in duplication events dating.

Conclusions

Based on the inferred gene duplication and loss event, only 9∼16% gene families have duplication retention after a whole genome duplication (WGD) event. A large part of these families have ohnologs from two or three WGDs. Consistent with the previous study results, the gene function of these families are mainly involved in nervous system and signal transduction related biological processes. Specifically, we found that the gene families with ohnologs from the teleost-specific (TS) WGD are enriched in fat metabolism, this result implyng that the retention of such ohnologs might be associated with the environmental status of high concentration of oxygen during that period.
Measuring ontogenetic shifts in central-place foraging insects: a case study with honey bees
Authors: Requier Fabrice , Henry Mickaël , Decourtye Axel , Brun François , Aupinel Pierrick , Rebaudo François , Bretagnolle Vincent
Published: April 2020

Measuring time-activity budgets over the complete individual lifespan is now possible for many animals with the recent advances of life-long individual monitoring devices. Although analyses of changes in the patterns of time-activity budgets have revealed ontogenetic shifts in birds or mammals, no such technique has been applied to date on insects.

We tested an automated breakpoint-based procedure to detect, assess and quantify shifts in the temporal pattern of the flight activities in honey bees. We assumed that the learning and foraging stages of honey bees will differ in several respects, to detect the age at onset of foraging (AOF).

Using an extensive dataset covering the life-long monitoring of 2,100 individuals, we compared the AOF outputs with the more conventional approaches based on arbitrary thresholds. We further evaluated the robustness of the different methods comparing the foraging time-activity budget allocations between the presumed foragers and confirmed foragers.

We revealed a clear-cut learning-foraging ontogenetic shift that differs in duration, frequency, and time of occurrence of flights. Although AOF appeared to be highly plastic among bees, the breakpoint-based procedure seems better able to detect it than arbitrary threshold-based methods that are unable to deal with inter-individual variation.

We developed theaofR-package including a broad range of examples with both simulated and empirical dataset to illustrate the simplicity of use of the procedure. This simple procedure is generic enough to be derived from any individual life-long monitoring devices recording the time-activity budgets of honey bees, and could propose new ecological applications of bio-logging to detect ontogenetic shifts in the behaviour of central-place foraging insects.
Measuring robust functional connectivity from resting-state MEG using amplitude and entropy correlation across frequency-bands and temporal scales
Authors: Godfrey Megan , Singh Krish D.
Published: April 2020

Recent studies have shown how MEG can reveal spatial patterns of functional connectivity using frequency-specific oscillatory coupling measures and that these may be modified in disease. However, there is a need to understand both how repeatable these patterns are across participants and how these measures relate to the moment-to-moment variability (or ‘irregularity’) of neural activity seen in healthy brain function. In this study, we used Multi-scale Rank-Vector Entropy (MRVE) to calculate the dynamic timecourses of signal variability over a range of temporal scales. The correlation of MRVE timecourses was then used to detect functional connections in resting state MEG recordings that were robust over 183 participants and varied with temporal scale. We then compared these MRVE connectivity patterns to those derived using more standard amplitude-amplitude coupling measures, using methods designed to quantify the consistency of these patterns across participants.

Using oscillatory amplitude envelope correlation (AEC), the most consistent connectivity patterns, across the cohort, were seen in the alpha and beta frequency bands. At fine temporal scales (corresponding to ‘scale frequencies’, fS = 30-150Hz), MRVE correlation detected mostly occipital and parietal connections and these showed high similarity with the networks identified by AEC in the alpha and beta frequency bands. The most consistent connectivity profiles between participants were given by MRVE correlation at fS = 75Hz and AEC in the beta band.

It was also found that average mid-to fine scale variability within each region (fS ∼ 10-150Hz) negatively correlated with the region’s overall connectivity strength with other brain areas, as measured by fine scale MRVE correlation (fS ∼ 30-150Hz) and by alpha and beta band AEC. These findings suggest that local activity at frequencies fS ≳ 10Hz becomes more regular when a region exhibits high levels of resting state connectivity.
The common analgesic paracetamol enhances the anti-tumour activity of decitabine through exacerbation of oxidative stress
Authors: Gleneadie Hannah J. , Baker Amy H. , Batis Nikolaos , Bryant Jennifer , Jiang Yao , Clokie Samuel J.H. , Mehanna Hisham , Garcia Paloma , Gendoo Deena M.A. , Roberts Sally , Molinolo Alfredo A. , Gutkind J. Silvio , Scheven Ben A. , Cooper Paul R. , Khanim Farhat L. , Wiench Malgorzata
Published: April 2020

The DNA demethylating agent 5-aza-2’-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2pathway, inadvertently increasing cancer cell survival, while the addition of paracetamol offsets this effect. Secondly, combined treatment leads to glutathione depletion and ROS accumulation with oxidative stress further enhanced by DAC suppressing anti-oxidant and thioredoxin responses. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.
PHARMACOLOGICAL PROTEIN INACTIVATION BY TARGETING FOLDING INTERMEDIATES
Authors: Spagnolli Giovanni , Massignan Tania , Astolfi Andrea , Biggi Silvia , Brunelli Paolo , Libergoli Michela , Ianeselli Alan , Orioli Simone , Boldrini Alberto , Terruzzi Luca , Maietta Giulia , Rigoli Marta , Lorenzo Nuria Lopez , Fernandez Leticia C. , Tosatto Laura , Linsenmeier Luise , Vignoli Beatrice , Petris Gianluca , Gasparotto Dino , Pennuto Maria , Guella Graziano , Canossa Marco , Altmeppen Hermann Clemens , Lolli Graziano , Biressi Stefano , Pastor Manuel Martin , Requena Jesús R. , Mancini Ines , Barreca Maria Letizia , Faccioli Pietro , Biasini Emiliano
Published: April 2020

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Equine Facial Action Coding System for determination of pain-related facial responses in videos of horses
Authors: Rashid Maheen , Silventoinen Alina , Gleerup Karina B. , Andersen Pia H.
Published: April 2020

1

During the last decade, pain scales including facial expressions as indicators of pain have been developed for horses, mostly relying on direct observations or inspection of images. Despite differences in the research conditions and methodology the different scales focus on the same regions of the face, corresponding to moveable facial muscles related to the ears, eyes, nostrils, lips and chin. However, a detailed comparison of the facial activities occurring during pain is not possible. We used a Facial Action Coding System modified for horses to code and analyse video recordings from an earlier study of acute short-term experimental pain and from clinical cases with and without pain. We demonstrated for the first time EquiFACS based changes to pain in video of horses, using traditional statistical methods based on frequency, and novel analyses based on sliding observation windows and co-occurrence of facial actions. The most prominent differences of the experimental horses were related to the lower face actionschin raiserandnostril dilator, while less prominent, but significantly more frequent actions were related to the eye region,inner brow raiser(AU101),increased eye white(AD1),half blink(AU47), andear rotator(EAD104).Ears forward(EAD101) andeye blink(AU145) were not associated to pain. Based on this we selected the two lower face actions for analysis of the clinical videos, and found that their co-occurrence within a window of 10 to 15 second gave 100% positive predictive values, as compared to the rating from three expert pain raters. Using our developed co-occurrence analyses we were surprised to detect that the chance of identifying three or more of the facial actions related to pain in 0.04 second sequence, corresponding to one frame, was below 3%, indicating that use of randomly selected images for pain scoring may be a very insensitive method.
Screening anti-infectious molecules againstMycobacterium ulcerans: a step towards decontaminating environmental specimens
Authors: Hammoudi N , Verdot R , Delorme J , Bouam A , Drancourt M
Published: April 2020

Mycobacterium ulcerans, a non-tuberculous mycobacterium responsible for Buruli ulcer, is residing in poorly defined environmental niches in the vicinity of stagnant water points where very few isolates have been confirmed. In the perspective of culturingM. ulceransfrom such contaminated environmental specimens, we tested thein vitrosusceptibility ofM. ulceransCU001 strain co-cultivated with XTC cells to anti-infectious molecules registered in the French pharmacopoeia, using a standardised concentration, to find-out molecules inactive againstM. ulceranswhich could be incorporated in decontaminating solution. Of 116 tested molecules, 64 (55.1%] molecules including 34 (29.3%] antibiotics, 14 (12%] antivirals, 8 (6.8%] antiparasitic and 8 (6.8%] antifungals were ineffective againstM. ulceransCU001; leaving 52 molecules active againstM. ulceransCU001. Three such inactive antimicrobial molecules (oxytetracycline, polymyxin E and voriconazole] were then selected to make a decontamination solution shown to respectM. ulceransCU001 viability. These three antimicrobials could be incorporated into a decontamination solution for the tentative isolation and culture ofM. ulceransfrom environmental samples.
Mucin glycans signal through the sensor kinase RetS to inhibit virulence-associated traits inPseudomonas aeruginosa
Authors: Wang Benjamin X. , Wheeler Kelsey M. , Cady Kyle C. , Lehoux Sylvain , Cummings Richard D. , Laub Michael T. , Ribbeck Katharina
Published: April 2020

Mucus is a densely populated ecological niche that coats all epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence traits in opportunistic pathogens likePseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, activate the sensor kinase RetS via its Dismed2 domain inP. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes the downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing byP. aeruginosa. Overall, these results shed light on how mucus impactsP. aeruginosabehavior in the human host, and may inspire novel approaches for controllingP. aeruginosainfections.
Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants
Authors: Stuttmann Johannes , Barthel Karen , Martin Patrick , Ordon Jana , Erickson Jessica L. , Herr Rosalie , Ferik Filiz , Kretschmer Carola , Berner Thomas , Keilwagen Jens , Marillonnet Sylvestre , Bonas Ulla
Published: April 2020

Genome editing by RNA-guided nucleases, such asSpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well-documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 sgRNAs. We used this toolkit to explore the limits of multiplexing in two major model species, and report on isolation of transgene-free octupleNicotiana benthamianaand duodecuple (12x)Arabidopsis thalianamutant lines in a single generation (T1and T2, respectively). We developed novel counter-selection markers forN. benthamiana, most importantlySl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on production of toxic 5-fluorouracyl in presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identifiedN. benthamianamutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (~112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs inA. thaliana. Efficiency was significantly lower inA. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher order multiplexing applications. We identify a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.
Parasites as niche modifiers for the microbiome: A field test with multiple parasites
Authors: O’Keeffe Kayleigh R. , Halliday Fletcher W. , Jones Corbin D. , Carbone Ignazio , Mitchell Charles E.
Published: April 2020

Parasites can affect and be affected by the host’s microbiome, with consequences for host susceptibility, parasite transmission, and host and parasite fitness. Yet, there are two aspects of the relationship between parasite infection and the host microbiome that remain little understood: the nature of the relationship under field conditions, and how the relationship varies among parasite species. To overcome these limitations, we assayed the within-leaf fungal community in a grass population to investigate how diversity and composition of the fungal microbiome are associated with natural infection by fungal parasites with different feeding strategies. We hypothesized that parasites that more strongly modify niches available within a host will thereby alter the microbial taxa that can colonize the community and be associated with greater changes in microbiome diversity and composition. A parasite that creates necrotic tissue to extract resources (necrotrophs) may act as a particularly strong niche modifier whereas one that does not (biotrophs) may not. Barcoded amplicon sequencing of the fungal ITS region revealed that the microbiome of leaf segments that were symptomatic of necrotrophs had lower fungal diversity and distinct composition compared to segments that were asymptomatic or symptomatic of other parasites. There were no clear differences in fungal diversity or composition between leaf segments that were asymptomatic and segments that were symptomatic of other parasite feeding strategies. This supports the hypothesis that within-host niches link infection by parasites to the host’s microbiome. Together, these results highlight the importance of parasite traits in determining parasite impacts on the host’s microbiome.
Schrödinger’s phenotypes: herbarium specimens show two-dimensional images are both good and (not so) bad sources of morphological data
Authors: Borges Leonardo M. , Reis Victor Candido , Izbicki Rafael
Published: April 2020

Museum specimens are the main source of information on organisms’ morphological features. Although access to this information was commonly limited to researchers able to visit collections, it is now becoming freely available thanks to the digitization of museum specimens. With these images, we will be able to collectively build large-scale morphological datasets, but these will only be useful if the limits to this approach are well-known. To establish these limits, we used two-dimensional images of plant specimens to test the precision and accuracy of image-based data and analyses.

To test measurement precision and accuracy, we compared leaf measurements taken from specimens and images of the same specimens. Then we used legacy morphometric datasets to establish differences in the quality of datasets and multivariate analyses between specimens and images. To do so, we compared the multivariate space based on original legacy data to spaces built with datasets simulating image-based data.

We found that trait measurements made from images are as precise as those obtained directly from specimens, but as traits diminish in size, the accuracy drops as well. This decrease in accuracy, however, has a very low impact on dataset and analysis quality. The main problem with image-based datasets comes from missing observations due to image resolution or organ overlapping. Missing data lowers the accuracy of datasets and multivariate analyses. Although the effect is not strong, this decrease in accuracy suggests caution is needed when designing morphological research that will rely on digitized specimens.

As highlighted by images of plant specimens, 2D images are reliable measurement sources, even though resolution issues lower accuracy for small traits. At the same time, the impossibility of observing particular traits affects the quality of image-based datasets and, thus, of derived analyses. Despite these issues, gathering phenotypic data from two-dimensional images is valid and may support large-scale studies on the morphology and evolution of a wide diversity of organisms.
SLX4IP-mediated telomere maintenance is essential for androgen receptor-independent castration-resistant prostate cancer
Authors: Mangosh Tawna L. , Awadallah Wisam N. , Grabowska Magdalena M. , Taylor Derek J.
Published: April 2020

In advanced prostate cancer, resistance to androgen deprivation therapy is achieved through numerous mechanisms, including loss of the androgen receptor (AR) allowing for AR-independent growth. Therapeutic options are limited for AR-independent castration-resistant prostate cancer, and defining mechanisms critical for its survival is of utmost importance for targeting this lethal disease. Our studies have focused on defining the telomere maintenance mechanism (TMM) required for castration-resistant prostate cancer (CRPC) cell survival. TMMs are responsible for telomere elongation to instill replicative immortality and prevent senescence, with the two TMM pathways available being telomerase and alternative lengthening of telomeres (ALT). Here, we show that AR-independent CRPC exhibits ALT hallmarks and limited telomerase expression and activity, whereas AR-dependent models use telomerase for telomere maintenance. AR-independent CRPC exhibited elevated levels of SLX4IP, a protein implicated in TMM switching. SLX4IP overexpression in AR-dependent CRPC C4-2B cells promoted ALT hallmarksin vitro. SLX4IP knockdown in AR-independent CRPC cells (DU145 and PC-3) led to the loss of ALT hallmarks, dramatic telomere shortening, induction of senescence, and reduced tumor volume. Using anin vitromodel of CRPC progression, induction of neuroendocrine differentiation in AR-dependent CRPC cells promoted ALT hallmarks in an SLX4IP-dependent manner. Lack of sufficient SLX4IP expression prevented ALT hallmarks rendering a TMM deficient environment, thus inducing senescence. This study demonstrates a unique reliance of AR-independent CRPC on SLX4IP-mediated ALT. Furthermore, ALT hallmark inhibition via SLX4IP induces senescence, thereby abolishing the replicative immortality of AR-independent CRPC.
Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyaseGtCPES expands its substrate specificity
Authors: Tomazic Natascha , Overkamp Kristina E. , Aras Marco , Pierik Antonio J. , Hofmann Eckhard , Frankenberg-Dinkel Nicole
Published: April 2020

Cryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins).Guillardia thetautilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyaseGtCPES fromG. theta. We showGtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82of the acceptor PBP β-subunit (PmCpeB) ofProchlorococcus marinusMED4. Based on the previously solved crystal structure, theGtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. VariantGtCPES_M67A binds both PEB and PCB forming a stable, colorful complexin vitroandin vivoproduced inEscherichia coli.GtCPES_M67A is able to mediate PCB transfer to Cys82ofPmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.
Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within theP. syringaespecies complex
Authors: Jayaraman Jay , Jones William T. , Harvey Dawn , Hemara Lauren M. , McCann Honour C. , Yoon Minsoo , Warring Suzanne L. , Fineran Peter C. , Mesarich Carl H. , Templeton Matthew D.
Published: April 2020

The common polysaccharide antigen (CPA) from the lipopolysaccharide (LPS) component of cell walls from the species complexPseudomonas syringaeis highly variable both in structure and immunological specificity, but the genetic basis for this is not well understood. We have characterised the CPA locus fromP. syringaepv.actinidiae(Psa). This locus has a modular structure with genes for both L- and D- rhamnose (Rha) biosynthesis and that of an unknown sugar. It also contains an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an O-methyltransferase. This operon is predicted to have a role intransport,elongation andtermination of the Rha backbone of the CPA oligosaccharide and is referred to as the TET operon. This is the first report of the identification of this operon inP. syringae. Two alleles of the TET operon were present amongst the different biovars ofPsaand lineages of the closely related pathovarP. syringaepv.actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from biovar 1 and replacement with that from biovar 3, demonstrated the link between the genetic locus and the electrophoretic and immunogenic properties of the LPS molecules inPsa. Sequence analysis of the TET operon from a wide range ofP. syringaeandP. viridiflavaisolates displayed a phylogenetic history which is incongruent with core gene phylogeny, but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.
HbtR, a heterofunctional homolog of the virulence regulator TcpP, facilitates the transition between symbiotic and planktonic lifestyles inVibrio fischeri
Authors: Bennett Brittany D. , Essock-Burns Tara , Ruby Edward G.
Published: April 2020

The bioluminescent bacteriumVibrio fischeriforms a mutually beneficial symbiosis with the Hawaiian bobtail squid,Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching,E. scolopesjuveniles acquireV. fischerifrom the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient ofN-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ,V. fischeritransitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression oflitRand luminescence are repressed by a homolog of theV. choleraevirulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production.

Importance

TcpP homologs are widespread throughout theVibriogenus; however, the only protein in this family described thus far is aV. choleraevirulence regulator. Here we show that HbtR, the TcpP homolog inV. fischeri, has both a biological role and regulatory pathway completely unlike that inV. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of theE. scolopeslight organ. While LitR becomes activated within the crypts, and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion ofV. fischericells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival ofV. fischerioutside of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.
Selective Cell Propagation via Micropatterning of Thermal-activated Hydrogel
Authors: Chiu Jeffrey C.Y. , Teodoro Joyce A. , Lee Jeong Hyun , Matthews Kerryn , Duffy Simon P. , Ma Hongshen
Published: April 2020

The ability to selectively propagate specific cells is fundamentally important to the development of clonal cell populations. Current methods rely on techniques such as limiting dilution, colony picking, and flow cytometry to transfer single cells into single wells, resulting in workflows that are low-throughput, slowed by propagation kinetics, and susceptible to contamination. Here, we developed a method, called selective laser gelation (SLG), to micropattern hydrogels in cell culture media in order to encapsulate specific cells to selectively arrest their growth. This process relies on the inverse gelation of methylcellulose, which forms a hydrogel when heated rather than cooled. Local heating using an infrared laser enables hydrogel micropatterning, while phase transition hysteresis retains the hydrogel after laser excitation. As a demonstration, we used this approach to selectively propagate transgenic CHO cells with increased antibody productivity. More generally, hydrogel micropatterning provides a simple and non-contact method to selective propagation of cells based on features identified by imaging.

One Sentence Summary

Inverse gelation of methylcellulose enables hydrogel micropatterning to selectively propagate cells identified by microscopy.
Structure and mechanism of the RNA polymerase II transcription machinery
Authors: Schier Allison C. , Taatjes Dylan J.
Published: April 2020

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.
Blunting senescence boosts liver regeneration
Authors: Birch Jodie , Gil Jesus
Published: April 2020

The mammalian liver possesses a unique capacity for regeneration. However, this regenerative potential declines with age due to unknown mechanisms. In this issue of Genes & Development, Ritschka and colleagues (pp. 489–494). compare liver regeneration upon partial hepatectomy in young and adult mice. Partial hepatectomy causes a transient increase in p21 in a subpopulation of hepatocytes that persists in adult mice. Remarkably, treatment with the BCL-2 family inhibitor ABT-737 blunts p21 expression, enhancing liver regeneration.
BRAF V600E-mutated metastatic pediatric Wilms tumor with complete response to targeted RAF/MEK inhibition
Authors: Obasaju Patience , Shahab Shubin , Dunn Emily , Rhee Daniel S. , Jiang LiQun , Dome Jeffrey S. , Friedman Alan D. , Argani Pedram , Pratilas Christine A.
Published: April 2020

Wilms tumor (WT) is the most common renal malignancy of childhood and accounts for 6% of all childhood malignancies. With current therapies, the 5-yr overall survival (OS) for children with unilateral favorable histology WT is greater than 85%. The prognosis is worse, however, for the roughly 15% of patients who relapse, with only 50%–80% OS reported in those with recurrence. Herein, we describe the extended and detailed clinical course of a rare case of a child with recurrent, pulmonary metastatic, favorable histology WT harboring a BRAF V600E mutation. The BRAF V600E mutation, commonly found in melanoma and other cancers, and previously undescribed in WT, has recently been reported by our group in a subset of epithelial-predominant WT. This patient, who was included in that series, presented with unilateral, stage 1, favorable histology WT and was treated with standard chemotherapy. Following the completion of therapy, the patient relapsed with pulmonary metastatic disease, that then again recurred despite an initial response to salvage chemotherapy and radiation. Next-generation sequencing (NGS) on the metastatic pulmonary nodule revealed a BRAF V600E mutation. After weighing the therapeutic options, a novel approach with dual BRAF/MEK inhibitor combination therapy was initiated. Complete radiographic response was observed following 4 months of therapy with dabrafenib and trametinib. At 12 months following the start of BRAF/MEK combination treatment, the patient continues with a complete response and has experienced minimal treatment-related side effects. This represents the first case, to our knowledge, of effective treatment with BRAF/MEK molecularly targeted therapy in a pediatric Wilms tumor patient.
Labeling Antibodies Using Colloidal Gold
Authors: Berg Eric A. , Fishman Jordan B.
Published: April 2020

Colloidal gold–antibody conjugates are easy to prepare and are an excellent choice for microscopic applications. Colloidal gold is an aqueous suspension of nanometer-sized particles of gold. Typically, chloroauric acid, HAuCl4, is reduced with dilute solutions of sodium citrate, as described here. This will cause the gold to form small aggregates that will associate with proteins. Gold particles of specific sizes can be isolated and differentiated microscopically, allowing these particles to be used for multiple-label experiments. Colloidal gold-labeled antibodies are widely used in electron microscopy (EM), and can be used for light microscopy but require additional steps (silver enhancement).
Optimally scheduling and loading tow trains of in-plant milk-run delivery for mixed-model assembly lines
Authors: Zhou Binghai , Zhu Zhexin
Published: April 2020

Purpose

This paper aims to investigate the scheduling and loading problems of tow trains for mixed-model assembly lines (MMALs). An in-plant milk-run delivery model has been formulated to minimize total line-side inventory for all stations over the planning horizon by specifying the departure time, parts quantity of each delivery and the destination station.

Design/methodology/approach

An immune clonal selection algorithm (ICSA) combined with neighborhood search (NS) and simulated annealing (SA) operators, which is called the NSICSA algorithm, is developed, possessing the global search ability of ICSA, the ability of SA for escaping local optimum and the deep search ability of NS to get better solutions.

Findings

The modifications have overcome the deficiency of insufficient local search and deepened the search depth of the original metaheuristic. Meanwhile, good approximate solutions are obtained in small-, medium- and large-scale instances. Furthermore, inventory peaks are in control according to computational results, proving the effectiveness of the mathematical model.

Research limitations/implications

This study works out only if there is no breakdown of tow trains. The current work contributes to the in-plant milk-run delivery scheduling for MMALs, and it can be modified to deal with similar part feeding problems.

Originality/value

The capacity limit of line-side inventory for workstations as well as no stock-outs rules are taken into account, and the scheduling and loading problems are solved satisfactorily for the part distribution of MMALs.
Determinants and outcome of Islamic corporate social responsibility (ICSR) adoption in Islamic banking industry of Pakistan
Authors: Bukhari Syed Asim Ali , Hashim Fathyah , Amran Azlan Bin
Published: April 2020

Purpose

The purpose of this study is to empirically examine the determinants and outcomes of Islamic corporate social responsibility (ICSR) adoption in Islamic bank branches in Pakistan. The research framework examines the influence of stakeholder’s pressure on ICSR adoption. It also analyses the relationship between ICSR adoption and intangible outcomes achieved by the Islamic bank branch.

Design/methodology/approach

A total of 400 questionnaires were distributed through a mail survey to Islamic bank branches in Pakistan. The respondents of the study were the branch manager of Islamic bank branches in Pakistan. A simple random sampling technique was used and resulted in the collection of 293 usable questionnaires. SMART PLS was used to statistically analyse the data using partial least squares structural equation modelling approach. The measurement and structural models were analysed.

Findings

The results indicate a significant and positive relationship between Shariah supervisory board pressure, competitor pressure and ICSR adoption in Islamic bank branches. A moderate strength positive relationship was found out between top management pressure and ICSR adoption. Results reveal that customer pressure and community pressure have an insignificant influence on ICSR adoption. Data analysis shows that the adoption of ICSR practices have a significant and positive influence on an Islamic bank branch’s Intangible outcomes.

Research limitations/implications

The sample size was relatively small because of the limited time duration.

Originality/value

The construct of ICSR has not been extensively researched upon especially through empirical studies. Limited research exists in the area of factors than can influence Islamic bank branches to adopt ICSR practices and currently no empirical research has focussed on the intangible outcomes that can be achieved through ICSR adoption by an Islamic bank branch. The limited study exists in the Pakistan context as well, which is a rapidly growing Islamic banking industry.