bluuebunny/crossref_metadata_2025 huggingface dataset:
Widespread selection for extremely high and low levels of secondary structure in coding sequences across all domains of life
Authors: Gebert Daniel , Jehn Julia , Rosenkranz David
Published: May 2019

Codon composition, GC content and local RNA secondary structures can have a profound effect on gene expression, and mutations affecting these parameters, even though they do not alter the protein sequence, are not neutral in terms of selection. Although evidence exists that, in some cases, selection favours more stable RNA secondary structures, we currently lack a concrete idea of how many genes are affected within a species, and whether this is a universal phenomenon in nature. We searched for signs of structural selection in a global manner, analysing a set of 1 million coding sequences from 73 species representing all domains of life, as well as viruses, by means of our newly developed software PACKEIS. We show that codon composition and amino acid identity are main determinants of RNA secondary structure. In addition, we show that the arrangement of synonymous codons within coding sequences is non-random, yielding extremely high, but also extremely low, RNA structuredness significantly more often than expected by chance. Taken together, we demonstrate that selection for high and low levels of secondary structure is a widespread phenomenon. Our results provide another line of evidence that synonymous mutations are less neutral than commonly thought, which is of importance for many evolutionary models.
Volumetric finite-element modelling of biological growth
Authors: Kennaway Richard , Coen Enrico
Published: May 2019

Differential growth is the driver of tissue morphogenesis in plants, and also plays a fundamental role in animal development. Although the contributions of growth to shape change have been captured through modelling tissue sheets or isotropic volumes, a framework for modelling both isotropic and anisotropic volumetric growth in three dimensions over large changes in size and shape has been lacking. Here, we describe an approach based on finite-element modelling of continuous volumetric structures, and apply it to a range of forms and growth patterns, providing mathematical validation for examples that admit analytic solution. We show that a major difference between sheet and bulk tissues is that the growth of bulk tissue is more constrained, reducing the possibility of tissue conflict resolution through deformations such as buckling. Tissue sheets or cylinders may be generated from bulk shapes through anisotropic specified growth, oriented by a polarity field. A second polarity field, orthogonal to the first, allows sheets with varying lengths and widths to be generated, as illustrated by the wide range of leaf shapes observed in nature. The framework we describe thus provides a key tool for developing hypotheses for plant morphogenesis and is also applicable to other tissues that deform through differential growth or contraction.
Co-Registration of Optoacoustic Tomography and Magnetic Resonance Imaging Data from Murine Tumour Models
Authors: Gehrung Marcel , Tomaszewski Michal , McIntyre Dominick , Disselhorst Jonathan , Bohndiek Sarah
Published: May 2019

As optoacoustic tomography emerges as a mainstream preclinical imaging modality, understanding the relationship between optoacoustic and other imaging biomarkers in the context of the underlying tissue biology becomes vitally important. For example, assessment of blood haemoglobin concentration and oxygenation can be achieved using OT, and also by several magnetic resonance imaging (MRI)-based techniques. To evaluate the relationship between these metrics and the relative performance of the two modalities in assessment of haemoglobin physiology, co-registration of their output imaging data is required. Unfortunately, this poses a significant challenge due to differences in the data acquisition geometries. Here, we present an integrated framework for registration of OT and MR image data in small animals. Our framework combines a novel MR animal holder, to improve animal positioning for deformable tissues, and a landmark-based software co-registration algorithm. We demonstrate that our protocol significantly improves registration of both body and tumour contours between these modalities.
Dynamics of murine brain protein synthesisin vivoidentify the hippocampus, cortex and cerebellum as highly active metabolic sites
Authors: Ng Ser Sue , Park Jung Eun , Meng Wei , Chen Christopher Li-Hsian , Kalaria Raj N. , McCarthy Neil E. , Sze Siu Kwan
Published: May 2019

Identification of proteins that are synthesizedde novoin response to specific microenvironmental cues is critical to understanding the molecular mechanisms that underpin key physiological processes and pathologies. Here we report that a brief period of pulsed SILAC diet (Stable Isotope Labelling by Amino acids in Cell culture) enables determination of biological functions corresponding to actively translating proteins in the mouse brain. Our data demonstrate that the hippocampus, cortex and cerebellum are highly active sites of protein synthesis, rapidly expressing key mediators of nutrient sensing and lipid metabolism, as well as critical regulators of synaptic function, axon guidance, and circadian entrainment. Together, these findings confirm that protein metabolic activity varies significantly between brain regionsin vivoand indicate that pSILAC-based approaches can identify specific anatomical sites and biological pathways likely to be suitable for drug targeting in neurodegenerative disorders.

Abbreviations

ApoA1: Apolipoprotein A1, ApoA4: Apolipoprotein A4, ApoE: Apolipoprotein E, ApoJ/Clu: Apolipoprotein J/Clusterin, App: Amyloid-β precursor/A4 protein: App, HDL: high density lipoprotein, Lrp1: Low density lipoprotein receptor-related protein 1, pSILAC: pulsed SILAC, pSIVOM: pulsed-SILAC in vivo labelling in mouse, SILAC: Stable Isotope Labelling by Amino acids in Cell culture)
Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri
Authors: Cenens William , Andrade Maxuel O. , Farah Chuck S.
Published: May 2019

Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized T4SS, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the entirety of structural genes of the T4SS in X. citri, is regulated by the global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri’s aggressive posture when confronted by competitors.

Author Summary

Xanthomonas citri is a member of a family of phytopathogenic bacteria that can cause substantial losses in crops. At different stages of the infection cycle, these cells will encounter other bacterial species with whom they will have to compete for space and nutrients. One mechanism which improves a cell’s chance to survive these encounters is a type IV secretion system that transfers a cocktail of antimicrobial effector proteins into other Gram-negative bacteria in a contact-dependent manner. Here, we show that this system is constitutively produced at a low basal level, even during low nutrient conditions, despite representing a significant metabolic burden to the cell. The conserved global regulator, CsrA, provides a constant, nutrient-independent, repression on the production T4SS components, thereby holding production costs to a minimum while at the same time ensuring X. citri’s competitiveness during encounters with bacterial rivals.
Biosynthesis of an Anti-Addiction Agent from the Iboga Plant
Authors: Farrow Scott C. , Kamileen Mohamed O. , Caputi Lorenzo , Bussey Kate , Mundy Julia E. A. , McAtee Rory C. , Stephenson Corey R. J. , O’Connor Sarah E.
Published: May 2019

(−)-Ibogaine and (−)-voacangine are plant derived psychoactives that show promise as effective treatments for opioid addiction. However, these compounds are produced by hard to source plants making these chemicals difficult for broad-scale use. Here we report the complete biosynthesis of (−)-voacangine, and de-esterified voacangine, which is converted to (−)-ibogaine by heating. This discovery will enable production of these compounds by synthetic biology methods. Notably, (−)-ibogaine and (−)-voacangine are of the opposite enantiomeric configuration compared to the other major alkaloids found in this natural product class. Discovery of these biosynthetic enzymes therefore demonstrates how nature generates both enantiomeric series of this medically important alkaloid scaffold using closely related enzymes, including those that catalyze enantioselective formal Diels-Alder reactions.

One Sentence Summary

Biosynthesis of iboga alkaloids with anti-addiction promise reveals enantioselectivity of enzymatic Diels-Alder reactions.
Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency
Authors: Pajares María A. , Zimmerman Tahl , Sánchez-Gómez Francisco J. , Ariza Adriana , Torres María J. , Blanca Miguel , Cañada F. Javier , Montañez María I. , Pérez-Sala Dolores
Published: May 2019

Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.
Molecular flux control encodes distinct cytoskeletal responses by specifying SRC signaling pathway usage
Authors: Kerjouan Adèle , Boyault Cyril , Oddou Christiane , Hiriart-Bryant Edwige , Grichine Alexei , Kraut Alexandra , Pezet Mylène , Balland Martial , Faurobert Eva , Bonnet Isabelle , Coute Yohann , Fourcade Bertrand , Albiges-Rizo Corinne , Destaing Olivier
Published: May 2019

Multi-domain signaling proteins sample numerous stimuli to coordinate distinct cellular responses. Understanding the mechanisms of their pleiotropic signaling activity requires to directly manipulate their activity of decision leading to distinct cellular responses. We developed an optogenetic probe, optoSRC, to control spatio-temporally the SRC kinase, a representative example of versatile signaling node, and challenge its ability to generate different cellular responses. Genesis of different local molecular fluxes of the same optoSRC to adhesion sites, was sufficient to trigger distinct and specific acto-adhesive responses. Collectively, this study reveals how hijacking the pleiotropy of SRC signaling by modulating in space and time subcellular molecular fluxes of active SRC kinases.
Evidence that Runt Acts as a Counter-Repressor of Groucho duringDrosophila melanogasterPrimary Sex Determination
Authors: Mahadeveraju Sharvani , Erickson James W.
Published: May 2019

Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. InDrosophila melanogasterthe archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishmen promoter (Pe) of the master regulatory geneSex-lethal (Sxl). Successful activation ofSxlPerequires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activateSxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activateSxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggest that Runt activatesSxlPeby antagonizing Gro function, a conclusion consist with earlier findings that Runt is needed forSxlexpression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation activation ofSxlPe. Instead, Runt is needed to keepSxlPeactive during the subsequent period of high-levelSxltranscription suggesting that Runt helps amplfy the difference between female and male XSE signals by counterrepressing Gro in female, but not in male, embryos.
Adipocyte vesicles: ‘all-in-one’ packages that stimulate tumor mitochondrial metabolism and dynamics
Authors: Clement Emily , Lazar Ikrame , Attané Camille , Carrié Lorry , Dauvillier Stéphanie , Ducoux-Petit Manuelle , Menneteau Thomas , Moutahir Mohamed , Gonidec Sophie Le , Dalle Stéphane , Valet Philippe , Burlet-Schiltz Odile , Muller Catherine , Nieto Laurence
Published: May 2019

Extracellular vesicles are emerging key actors in adipocyte communication. Notably, small extracellular vesicles shed by adipocytes promote melanoma aggressiveness through fatty acid oxidation, with a heightened effect in obesity. However, the vesicular actors and cellular processes involved remain largely unknown. Here, we elucidate the mechanisms linking adipocyte extracellular vesicles to metabolic remodeling and cell migration. We show that adipocyte vesicles stimulate melanoma fatty acid oxidation by providing both enzymes and substrates. In obesity, the heightened effect of extracellular vesicles depends on increased transport of fatty acids, not fatty acid oxidation-related enzymes. These fatty acids, stored within lipid droplets in cancer cells, drive fatty acid oxidation after release through lipophagy. This increase in mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration in the presence of adipocyte vesicles. Our results provide key insights into the role of extracellular vesicles in the metabolic cooperation that takes place between adipocytes and tumors with particular relevance in obesity.
Novel method to determine diagnosis-defining refraction points
Authors: Yamauchi Tsuneto , Ohshima Mitsuhiro , Yamaguchi Yoko , Konishi Kazunori , Kappert Kai , Nakano Shigeru
Published: May 2019

Diagnosis of a certain disease generally relies on definitions established by professional medical societies and comprise the patient’s history, physical examination, and test results. These include physical compositions such as body mass index (BMI), and laboratory tests such as serum creatinine and albumin in urine samples. In general, laboratory tests are based on mathematical methods,e.g.defining critical values from the mean ± kσ of a population, where k is a natural number and the standard deviation is σ (“mean ± kσ-method”). In most cases k is defined as 2, leading to reference ranges defining 95% of test results as normal. However, this method mostly depends on a normal distribution of values.

Here we applied a novel method (“SoFR-method”) based on data sorting to define refraction points, which carry informative value as diagnostic criteria. Applying the SoFR-method, standard measures such as critical BMI-values are categorized by equal robustness as by the mean ± kσ-method. However, the SoFR-method showed higher validity when analyzing non-normalized values such as creatinine and albumin, as well as hepatocyte growth factor (HGF) and hemoglobin in a novel Perioscreen assay in saliva of diabetic and non-diabetic patients.

Taken together, we defined a novel method based on data sorting of test results from patients to effectively define refraction points which might guide more accurately clinical diagnoses and define relevant thresholds.
Pyrenophora teresandRhynchosporium secalisinfections in malt barley as influenced by genotype, spatial and temporal effects and nitrogen fertilization
Authors: Vahamidis Petros , Stefopoulou Angeliki , Lagogianni Christina S. , Economou Garyfalia , Dercas Nicholas , Kotoulas Vassilis , Kalivas Dionissios , Tsitsigiannis Dimitrios I.
Published: May 2019

Net form net blotch (NFNB) and barley leaf scald are among the most important barley diseases worldwide and particularly in Greece. Their occurrence in malt barley can exert a significant negative effect on malt barley grain yield and quality. An experimental trial across two growing seasons was implemented in Greece in order i) to assess the epidemiology of NFNB and leaf scald in a barley disease free area when the initial inoculation of field occurs through infected seeds, and ii) to further explore the relationship among nitrogen rate, grain yield, quality variables (i.e. grain protein content and grain size) and disease severity and epidemiology. It was demonstrated that both NFNB and leaf scald can be carried over from one season to the next on infected seed under Mediterranean conditions. However, disease severity was more pronounced after barley tillering phase when soil had been successfully inoculated first. When nitrogen rate and genotype were the main sources of variation the epidemiology assessment was implemented with hotspot and Anselin Local Moran’s I analysis. It was found that the location of hotspots was modified during growing season. Soil and plant variables were assessed for the explanation of this variability. According to commonality analysis the effect of distance from the locations with the highest disease infections was a better predictor of disease severity (for both diseases) compared to nitrogen rate during pre-anthesis period. However, disease severity after anthesis was best explained by nitrogen rate only for the most susceptible cultivars to NFNB. The effect of disease infections on yield, grain size and grain protein content varied in relation to genotype, pathogen and stage of crop development. The importance of crop residues on the evolution of both diseases was also highlighted.
Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Authors: Trobaugh Derek W. , Sun Chengqun , Bhalla Nishank , Gardner Christina L. , Dunn Matthew , Klimstra William B.
Published: May 2019

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3’ untranslated region (3’ UTR) of the virus. Three of the sites are “canonical” with all 8 seed sequence residues complimentary to miR-142-3p while one is “non-canonical” and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.

Author Summary

Eastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in myeloid cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3’ untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3’ UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo. These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.
From cells to tissue: How cell scale heterogeneity impacts glioblastoma growth and treatment response
Authors: Gallaher Jill A. , Massey Susan C. , Hawkins-Daarud Andrea , Noticewala Sonal S. , Rockne Russell C. , Johnston Sandra K. , Gonzalez-Cuyar Luis , Juliano Joseph , Gil Orlando , Swanson Kristin R. , Canoll Peter , Anderson Alexander R. A.
Published: May 2019

Glioblastomas are aggressive primary brain tumors known for their inter- and intratumor heterogeneity. This disease is uniformly fatal, with intratumor heterogeneity the major reason for treatment failure and recurrence. Just like the nature vs nurture debate, heterogeneity can arise from heritable or environmental influences. Whilst it is impossible to clinically separate observed behavior of cells from their environmental context, using a mathematical framework combined with multiscale data gives us insight into the relative roles of variation from inherited and environmental sources.

To better understand the implications of intratumor heterogeneity on therapeutic outcomes, we created a hybrid agent-based mathematical model that captures both the overall tumor kinetics and the individual cellular behavior. We track single cells as agents, cell density on a coarser scale, and growth factor diffusion and dynamics on a finer scale over time and space. Our model parameters were fit utilizing serial MRI imaging and cell tracking data from ex vivo tissue slices acquired from a growth-factor driven glioblastoma murine model.

When fitting our model to serial imaging only, there was a spectrum of equally-good parameter fits corresponding to a wide range of phenotypic behaviors. This wide spectrum of in silico tumors also had a wide variety of responses to an application of an antiproliferative treatment. Recurrent tumors were generally less proliferative than pre-treatment tumors as measured via the model simulations and validated from human GBM patient histology. When fitting our model using imaging and cell scale data, we determined that heritable heterogeneity is required to capture the observed migration behavior. Further, we found that all tumors increased in size after an anti-migratory treatment, and some tumors were larger after a combination treatment than with an anti-proliferative treatment alone. Together our results emphasize the need to understand the underlying phenotypes and tumor heterogeneity in designing therapeutic regimens.
Structural and Functional Responses of Soil Microbial Communities to Biodegradable Plastic Film Mulching in Two Agroecosystems
Authors: Bandopadhyay Sreejata , Sintim Henry Y. , DeBruyn Jennifer M.
Published: May 2019

Polyethylene (PE) plastic mulch films are used globally in crop production but incur considerable disposal and environmental pollution issues. Biodegradable plastic mulch films (BDMs), an alternative to PE-based films, are designed to be tilled into the soil where they are expected to be mineralized to carbon dioxide, water and microbial biomass. However inadequate research regarding the impacts of repeated incorporation of BDMs on soil microbial communities has partly contributed to limited adoption of BDMs. In this study, we evaluated the effects of BDM incorporation on soil microbial community structure and function over two years in two geographical locations: Knoxville, TN, and in Mount Vernon, WA. Treatments included four plastic BDMs, a completely biodegradable cellulose mulch, a non-biodegradable PE mulch and a no mulch plot. Bacterial community structure determined using 16S rRNA amplicon sequencing revealed significant differences by location and season. Differences in bacterial communities by mulch treatment were not significant for any season in either location, except for Fall 2015 in WA where differences were observed between BDMs and no-mulch plots. Extracellular enzyme rate assays were used to characterize communities functionally, revealing significant differences by location and sampling season in both TN and WA but minimal differences between BDMs and PE treatments. Limited effects of BDM incorporation on soil bacterial community structure and soil enzyme activities when compared to PE suggest that BDMs have comparable influences on soil microbial communities, and therefore could be considered an alternative to PE.

Importance

Plastic film mulches increase crop yields and improve fruit quality. Most plastic mulches are made of polyethylene (PE), which is poorly degradable, resulting in undesirable end-of-life outcomes. Biodegradable mulches (BDMs) may be a sustainable alternative to PE. BDMs are made of polymers which can be degraded by soil microbial enzymes, and are meant to be tilled into soil after use. However, uncertainty about impacts of tilled-in BDMs on soil health has restricted adoption of BDMs. Our previous research showed BDMs did not have a major effect on a wide range of soil quality indicators (Sintim et al. 2019); here we focus on soil microbial communities, showing that BDMs do not have detectable effects on soil microbial communities and their functions, at least over the short term. This informs growers and regulators about use of BDMs in crop production, paving a way for an agricultural practice that reduces environmental plastic pollution.
Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs
Authors: Sarink Maarten J. , Verbon Annelies , Tielens Aloysius G.M. , van Hellemond Jaap J.
Published: May 2019

Primary Amoebic Meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoebaNaegleria fowleri. The disease mostly affects healthy children and young adults after contaminated water enters the nose, generally during recreational water activities. The amoeba migrate along the olfactory nerve to the brain, resulting in seizures, coma and eventually death. Previous research has shown thatNaegleria gruberi, a close relative ofN. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine and valproic acid inhibited growth ofN. gruberi. Furthermore, additive effects were seen when drugs were combined. Both thioridazine and valproic acid inhibit in vitro growth ofN. gruberiin concentrations that can be obtained at the site of infection, which is doubtful with the currently used drugs amphotericin B and miltefosine. Both thioridazine and valproic acid have already been used for other diseases. As the development of new drugs and randomized controlled trials for this rare disease is nearly impossible, repurposing drugs is the most promising way to obtain additional drugs to combat PAM. Thioridazine and valproic acid are available drugs without major side-effects and can, therefore, be used as new complementary options in PAM therapy.
Expression of 9-O- and 7,9-O-acetyl modified sialic acid in cells and their effects on influenza viruses
Authors: Barnard Karen N. , Wasik Brian R. , LaClair Justin R. , Weichert Wendy S. , Lawrence Brynn K. , Parrish Colin R.
Published: May 2019

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C7, C8, and C9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. As Sia or their variant forms are receptors for influenza A and influenza C viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. While they were expressed on the cell surface of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1-2% of total Sia in these cell lines. We knocked out and over-expressed the sialate O-acetyltransferase gene (CasD1), and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, over-expression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C infection, but had no obvious effect on influenza A infection.

IMPORTANCE

Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.
Environmentally induced lipidome adaptation in the bacterial model organismM. extorquens
Authors: Chwastek Grzegorz , Surma Michal A. , Rizk Sandra , Grosser Daniel , Lavrynenko Oksana , Rucińska Magdalena , Jambor Helena , Sáenz James
Published: May 2019

Cells, from microbes to man, adapt their membranes in response to the environment to maintain their properties and functions. To adapt, lipid composition is homeostatically regulated to conserve optimal membrane properties. Global patterns of lipidome remodelling are poorly understood, particularly in model organisms with simple lipid compositions that can provide insight into fundamental principles underlying membrane adaptation. Using shotgun lipidomics, we examined the simple yet adaptive lipidome of the plant-associated Gram-negative bacteriumMethylobacterium extorquensover varying temperature, hyperosmotic and detergent stress, carbon sources, and cell density. We observed that as few as ten lipids account for 90% of the total changes, thus constraining the upper limit of variable lipids required for an adaptive living membrane. Across all conditions, the highest degree of lipidomic variability was observed for changing growth temperature. We also revealed that variations in lipid structural features are not monotonic over a given range of conditions and are heterogeneous across lipid classes. Interestingly, phosphotidylcholine showed the most extreme acyl chain remodeling among all lipid classes, suggesting a new link to its importance in bacterial-host interactions and pathogenicity. These patterns in lipidomic remodeling suggest a highly adaptive mechanism with many degrees of freedom and constrain the lipidomic requirements for an adaptive membrane.
Trochodendron aralioides, the first chromosome-level draft genome in Trochodendrales and a valuable resource for basal eudicot research
Authors: Strijk Joeri S. , Hinsinger Damien D. , Zhang Feng-Ping , Cao KunFang
Published: May 2019

Background

The wheel tree (Trochodendron aralioides) is one of only two species in the basal eudicot order Trochodendrales. Together with Tetracentron sinense, the family is unique in having secondary xylem without vessel elements, long considered to be a primitive character also found in Amborella and Winteraceae. Recent studies however have shown that Trochodendraceae belong to basal eudicots and demonstrate this represents an evolutionary reversal for the group. Trochodendron aralioides is widespread in cultivation and popular for use in gardens and parks.

Findings

We assembled the T. aralioides genome using a total of 679.56 Gb of clean reads that were generated using both PacBio and Illumina short-reads in combination with 10XGenomics and Hi-C data. Nineteen scaffolds corresponding to 19 chromosomes were assembled to a final size of 1.614 Gb with a scaffold N50 of 73.37 Mb in addition to 1,534 contigs. Repeat sequences accounted for 64.226% of the genome, and 35,328 protein-coding genes with an average of 5.09 exons per gene were annotated using de novo, RNA-seq, and homology-based approaches. According to a phylogenetic analysis of protein-coding genes, T. aralioides diverged in a basal position relatively to core eudicots, approximately 121.8-125.8 million years ago.

Conclusions

Trochodendron aralioides is the first chromosome-scale genome assembled in the order Trochodendrales. It represents the largest genome assembled to date in the basal eudicot grade, as well as the closest order relative to the core-eudicots, as the position of Buxales remains unresolved. This genome will support further studies of wood morphology and floral evolution, and will be an essential resource for understanding rapid changes that took place at the base of the Eudicot tree. Finally, it can serve as a valuable source to aid both the acceleration of genome-assisted improvement for cultivation and conservation efforts of the wheel tree.
Molecular detection and characterization of mutation on 23S rRNA gene associated with clarithromycin resistant in Helicobacter pylori
Authors: Abd albagi Sahar Obi , aldayem Altayeb Hisham Nour , Khalil Abuzeid Nadir Musa
Published: May 2019

Background

Helicobacter pylori consider as pathogenic resistant bacterium was colonized mainly in stomach and causing a prolonged gastritis with gastric ulcers were progressing to gastric carcinoma. Also its resistance to antibiotic considered as the main reason for failure to eradicate of this pathogen has been difficult when this resistance occurred as mutant on protein binding site in 23s ribosomal RNA. The highest cure rates have required multidrug antimicrobial therapies including combinations of omeprazole, clarithromycin and metronidazole.

Result

Bacterial DNA sequence from gastritis patients with confirmed previous positive ICT samples (Stool and Bloo) were used to obtain co-related between phenotypic & genotypic variant outcome have been observed as SNPs carried on nucleotides which could be altered protein prediction as result of that caused chronic gastritis incline to gastric carcinoma due to abnormal consequence on genetic level in H. species (23s rRNA) was referred to clarithromycin resistance, was achieved on this cross-sectional studies by running two different primers were amplify in PCR machine, first one for urease producing gene (Glm as universal primer) and second one for 23s rRNA as specific primer (rp1/fp1). Two samples out of Four samples were amplified as final isolate in the first cycle and have a specific band in 23s rRNA (NO.11, NO.24) as further DNA samples investigation were sent to get our target sequence.

Conclusion

Bioinformatics tools used to confirm a specific types of mutations give specific position responsible for bacteriostatic activity of macrolides such as clarithromycin depends on capacity to inhibit protein synthesis by binding to the 23S ribosomal subunit (23S rRNA) as resistant gene. The detection tools as MSA (multiple sequence alignment) for our nucleotides sequence to (11&24) samples with Genbank accession number 24_MK208582 and 11_MK208583. One type of mutation has been observed in nucleotide sequence (sample-24) in position 2516 (1344 _complementary) sequence result compared with reference sequence standard reference strain (H. pylori U27270) was confirmed which consider it as novel mutation in database for 23S rRNA Gene of H. pylori associated with Clarithromycin Resistance gene in Sudanese patients.
A comparative assessment of aging-related NADPH diaphorase positivity in the spinal cord and medullary oblongata between pigeon and murine
Authors: Jia Yunge , Hou Wei , Li Yinhua , Wen Xiaoxin , Rao Chenxu , Wei Zichun , Zhang Tianyi , Wang Xinghang , Li Xiuyuan , bai Lu , Zhang Weijin , Wang Pan , Bi Jing , Guo Anchen , Wang Jie , Tan Huibing
Published: May 2019

NADPH diaphorase (N-d) positive neurons has been examined in many animals. N-d neurodegenerative neurites were detected in some animal models. However, detailed information of N-d positivity and aging related changes was still lack in the spinal cord and medulla oblongata of pigeons. In this study, we evaluated the N-d positivity and aging alterations in the spinal cord and medullary oblongata of the pigeon compared with rat and mouse. In pigeons, N-d neurons were more numerous in the dorsal horn, around the central canal and in the column of Terni in the thoracic and lumbar segments and scattered neurons occurred in the ventral horn of spinal segments. N-d neurons also occurred in the white matter of spinal cord. Morphometrical analysis demonstrated in the lumbosacral, cervical and thoracic regions. Compared with young pigeons, the size of N-d soma was significantly altered in aged pigeons. Meanwhile, the dramatic morphological changes occurred in the lumbar to sacral segments. The most important findings of this study were aging-related N-d positive bodies (ANB) in aged pigeons, mainly in the nucleus cuneatus externus (CuE), occasionally in the nuclei gracilis et cuneatus. ANBs were identified in the gracile nuclei in spinal cord in the aged rats and mice. ANBs were also detected in the CuE spinal nucleus in the aged rats. Immunohistochemistry also showed that the aging changes occurred in the cell types and neuropeptides in aged animals. The results suggested the weak inflammation and neuronal dysfunction in the spinal cord in aged pigeons. Our results suggested that the ANB could be considered as aging marker in the central nervous system.
Gated electron transport in rhodopsin and its relevance to GPCR activation
Authors: Gehrckens Angela S , Horsfield Andrew P , Skoulakis Efthimios M C , Turin Luca
Published: May 2019

We identify, by density-functional theory calculations, an electron donor-bridge-acceptor (DBA) complex within the highest resolution X-ray diffraction structures of rhodopsin. The donor is a conserved tryptophan, the acceptor is a zinc ion surrounded by a tryptophan, a histidine and a conserved glutamate. The unusual environment of the zinc ion confers high electron affinity on the zinc site. The bridge is the retinal which can exist either in the neutral aldimine (Schiff’s base) or aldiminium (protonated) state. When the retinal is unprotonated, no electron transfer occurs. Upon protonation of the aldimine, the DBA complex conducts and a full electron charge is transferred from donor tryptophan to the zinc complex. This gated electron transfer creates the molecular equivalent of a tunnel triode. Since rhodopsin is the ancestor of GPCRs, we discuss the possible relevance of this gated electron transport to other GPCRs, in particular to olfactory receptors which have been proposed to use an electron tunneling mechanism to detect molecular vibrations.
Next-generation in situ conservation and educational outreach in Madagascar using a mobile genetics lab
Authors: Blanco Marina B. , Greene Lydia K. , Williams Rachel C. , Andrianandrasana Lanto , Yoder Anne D. , Larsen Peter A.
Published: May 2019

Madagascar is a biodiversity hotspot that is facing rapid rates of deforestation, habitat destruction and poverty. Urgent action is required to document the status of biodiversity to facilitate efficacious conservation plans. Within country, new generations of Malagasy scientists and conservationists are taking on leadership roles, although many lack access to modern genetic sequencing and are underrepresented in academic publications, when compared to international counterparts.

With the recent advent of portable and affordable genetic technologies, it is now possible to tackle logistical considerations. Mobile genetics labs, with the capacity for in situ DNA extraction, amplification and sequencing, can produce scientifically reproducible data under field conditions, minimizing the time between sample collection and data analysis. Additionally, mobile labs offer powerful training opportunities for in-country scientists that enable local students and researchers to actively participate and contribute fully to the research enterprise, and that further empower these communities to contribute to the conservation dialog.

Here, we show “proof of concept” by deploying a miniaturized thermal cycler alongside the Oxford Nanopore MinION DNA sequencer in Madagascar, including in the newly established Anjajavy Protected Area in northwestern Madagascar. We successfully extracted DNA from tissue samples collected using minimally-invasive techniques, amplified and sequenced a phylogenetically informative mitochondrial gene (cytochrome-b; cytb), and thereby confirmed the presence of Danfoss’ mouse lemur (M. danfossi) within the Anjajavy Reserve.

To demonstrate the reproducibility of our methods, we successfully performed our established molecular and analytical pipeline at two additional locations in Madagascar, where we also conducted two-day workshops at local higher-education Institutions to demonstrate the process from tissue samples to DNA sequencing. Ultimately, we show that a mobile genetics lab can provide reliable and expeditious results, become a powerful educational tool, and allow scientists to conduct genetic analyses, potentially allowing for rapid interventions under emergency conditions in situ.
Exploiting codon usage identifies RpS21 as anin vivosignal strength-dependent Ras/MAPK regulator
Authors: Sawyer Jessica K. , Kabiri Zahra , Montague Ruth A. , Paramore Sarah V. , Cohen Erez , Zaribafzadeh Hamed , Counter Christopher M. , Fox Donald T.
Published: May 2019

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened theDrosophilagenome for modifiers specific to either weak or strong Ras-driven eye phenotypes. We mapped the underlying gene from one modifier to the ribosomal geneRpS21. RpS21 preferentially influences weak Ras/MAPK signaling outputs, and negatively regulates Ras/MAPK in multiple cell/tissue and signaling settings. In turn, MAPK signaling may regulate its own negative feedback by promoting RpS21 expression. These data show that codon usage manipulation can identify output-specific signaling regulators, and identify RpS21 as anin vivoRas/MAPK phenotypic regulator.
Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters
Authors: Matsuda Mitsuhiro , Hayashi Hanako , Garcia-Ojalvo Jordi , Yoshioka-Kobayashi Kumiko , Kageyama Ryoichiro , Yamanaka Yoshihiro , Ikeya Makoto , Toguchida Junya , Alev Cantas , Ebisuya Miki
Published: May 2019

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system1. Since murine and human embryos differ in their sizes, geometries, and nutrients, we usein vitrodifferentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments2–4. In this way we recapitulate the murine and human segmentation clocksin vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock5,6, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.