bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Ovarian Cancer Gene Expression Patterns Associated with Disease Progression and Mortality
Authors: Hossain Md. Ali , Islam Sheikh Muhammad Saiful , Quinn Julian , Huq Fazlul , Moni Mohammad Ali
Published: November 2018

Ovarian cancer (OC) is a common cause of death from cancer among women worldwide, so there is a pressing need to identify factors influencing mortality. Much OC patient clinical data is now publically accessible (including patient age, cancer site stage and subtype), as are large datasets of OC gene transcription profiles. These have enabled studies correlating OC patient survival with clinical variables and with gene expression but it is not well understood how these two aspects interact to influence mortality. To study this we integrated clinical and tissue transcriptome data from the same patients available from the Broad Institute Cancer Genome Atlas (TCGA) portal. We investigated OC mRNA expression levels (relative to normal patient tissue) of 26 genes already strongly implicated in OC, assessed how their expression in OC tissue predicts patient survival then employed Cox Proportional Hazard regression models to analyse both clinical factors and transcriptomic information to determine relative risk of death associated with each factor. Multivariate analysis of combined data (clinical and gene mRNA expression) found age, ovary tumour site and cancer stage IB significantly correlated with patient survival. Univariate analysis also confirmed significant differences in patient survival time when altered transcription levels of KLK6, CD36, MEF2C and SCGB2A1 were evident, while multivariate analysis that considered the 26 genes simultaneously revealed a significant relationship of mortality with KLK6, CD36 and E2F1 genes. However, analysis that considered all 26 genes with clinical variables together identified WFDC2, E2F1, BRCA1, KLK6, SCGB2A1 and SLPI genes as independently related to mortality in OC. This indicated that the latter genes affect OC patient survival, i.e., provided mechanistic and predictive information in addition to that of the clinical traits and provide strong evidence that these genes are critical markers of processes that underlie OC progression and mortality.
Evidence for absence of bilateral transfer of olfactory learned information in Apis dorsata and Apis mellifera
Authors: Vijaykumar Meenakshi , Mogily Sandhya , Dutta-Gupta Aparna , Joseph Joby
Published: November 2018

Capacity and condition under which lateral transfer of olfactory memory is possible in insects are still debated. Here we present evidence consistent with lack of ability to transfer olfactory associative memory in two species of honeybees, Apis mellifera and Apis dorsata in a PER associative conditioning paradigm where the untrained antenna is blocked by an insulating coat. We show that the olfactory system on each side of the bee can learn and retrieve independently and the retrieval using the antenna on the side contralateral to the trained one is not affected by the training. Recreating the paradigm in which the memory on the contralateral side has been reported at three hours after training we see that the memory is available on the contralateral side immediately after training and moreover, training with trained side antenna coated with insulator does not prevent learning, pointing to a possible insufficiency of block of odor stimuli in this paradigm. Bee does not learn the odor stimuli applied to one side alone as a stimulus different from odor presented to both sides. Moreover the behaviour of the bee as a whole can be predicted if the sides are assumed to learn and store independently and the organism as a whole is able to retrieve the memory if either of the sides have learned.

Summary Statement

The two halves of honeybee brain store and retrieve olfactory associative memories independently.
Strengths and potential pitfalls of hay-transfer for ecological restoration revealed by RAD-seq analysis in floodplainArabisspecies
Authors: Dittberner Hannes , Becker Christian , Jiao Wen-Biao , Schneeberger Korbinian , Hölzel Norbert , Tellier Aurélien , de Meaux Juliette
Published: November 2018

Achieving high intraspecific genetic diversity is a critical goal in ecological restoration as it increases the adaptive potential and long-term resilience of populations. Thus, we investigated genetic diversity within and between pristine sites in a fossil floodplain and compared it to sites restored by hay-transfer between 1997 and 2014. RAD-seq genotyping revealed that the stenoecious flood-plain speciesArabis nemorensisis co-occurring with individuals that, based on ploidy, ITS-sequencing and morphology, probably belong to the close relativeArabis sagittata, which has a documented preference for dry calcareous grasslands but has not been reported in floodplain meadows. We show that hay-transfer maintains genetic diversity for both species. Additionally, inA. sagittata, transfer from multiple genetically isolated pristine sites resulted in restored sites with increased diversity and admixed local genotypes. InA. nemorensis,transfer did not create novel admixture dynamics because genetic diversity between pristine sites was less differentiated. Thus, the effects of hay-transfer on genetic diversity also depend on the genetic makeup of the donor communities of each species, especially when local material is mixed. Our results demonstrate the efficiency of hay-transfer for habitat restoration and emphasize the importance of pre-restoration characterization of micro-geographic patterns of intraspecific diversity of the community to guarantee that restoration practices reach their goal, i.e. maximize the adaptive potential of the entire restored plant community. Overlooking these patterns may alter the balance between species in the community. Additionally, our comparison of summary statistics obtained fromde novoand reference-based RAD-seq pipelines shows that the genomic impact of restoration can be reliably monitored in species lacking prior genomic knowledge.
A century of decline: loss of genetic diversity in a southern African lion-conservation stronghold
Authors: Dures Simon G. , Carbone Chris , Loveridge Andrew J. , Maude Glyn , Midlane Neil , Aschenborn Ortwin , Gottelli Dada
Published: November 2018

Aim

There is a dearth of evidence that determines the genetic diversity of populations contained within present-day protected areas compared with their historic state prior to large-scale species declines, making inferences about a species’ conservation genetic status difficult to assess. The aim of this paper is to demonstrate the use of historic specimens to assess the change in genetic diversity over a defined spatial area.

Location

Like other species, African lion populations (Panthera leo) are undergoing dramatic contractions in range and declines in numbers, motivating the identification of a number of lion conservation strongholds across East and southern Africa. We focus on one such stronghold, the Kavango-Zambezi transfrontier conservation area (KAZA) of Botswana, Namibia, Zambia and Zimbabwe.

Methods

We compare genetic diversity between historical museum specimens, collected during the late 19th and early 20th century, with samples from the modern extant population. We use 16 microsatellite markers and sequence 337 base pairs of the hypervariable control region (HVR1) of the mitochondrial genome. We use bootstrap resampling to allow for comparisons between the historic and modern data.

Results

We show that the genetic diversity of the modern population was reduced by 12% to 17%, with a reduction in allelic diversity of approximately 15%, compared to historic populations, in addition to having lost a number of mitochondrial haplotypes. We also identify reduced allelic diversity and a number of ‘ghost alleles’ in the historical samples no longer present in the extant population.

Main Conclusions

We argue a rapid decline in allelic richness after 1895 suggests the erosion of genetic diversity coincides with the rise of a European colonial presence and the outbreak of rinderpest in the region. Our results support the need to improved connectivity between protected areas in order to prevent further loss of genetic diversity in the region.
The study of hormonal metabolism of Trincadeira and Syrah cultivars indicates new roles of salicylic acid, jasmonates, ABA and IAA during grape ripening and upon infection with Botrytis cinerea
Authors: Coelho João , Almeida-Trapp Marilia , Pimentel Diana , Soares Flávio , Reis Pedro , Rego Cecília , Mithöfer Axel , Fortes Ana Margarida
Published: November 2018

Hormones play an important role in fruit ripening and in response to biotic stress. Nevertheless, analyses of hormonal profiling during plant development and defense are scarce. In this work, changes in hormonal metabolism in grapevine (Vitis vinifera) were compared between a susceptible (Trincadeira) and a tolerant (Syrah) variety during grape ripening and upon infection with Botrytis cinerea. Infection of grapes with the necrotrophic pathogen Botrytis cinerea leads to significant economic losses worldwide.

Peppercorn-sized fruits were infected in the field and mock-treated and infected berries were collected at green, veraison and harvest stages for hormone analysis and targeted qPCR analysis of genes involved in hormonal metabolism and signaling. Results indicate a substantial reprogramming of hormonal metabolism during grape ripening and in response to fungal attack. Syrah and Trincadeira presented differences in the metabolism of abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonates during grape ripening that may be connected to fruit quality. On the other hand, high basal levels of salicylic acid (SA), jasmonates and IAA at an early stage of ripening, together with activated SA, jasmonates and IAA signaling, likely enable a fast defense response leading to grape resistance/ tolerance towards B. cinerea.

The balance among the different phytohormones seems to depend on the ripening stage and on the intra-specific genetic background and may be fundamental in providing resistance or susceptibility. In addition, this study indicated the involvement of SA and IAA in defense against necrotrophic pathogens and gains insights into possible strategies for conventional breeding and/or gene editing aiming at improving grape quality and grape resistance against Botrytis cinerea.
Red blood cell tension controlsPlasmodium falciparuminvasion and protects against severe malaria in the Dantu blood group
Authors: Kariuki Silvia N. , Marin-Menendez Alejandro , Introini Viola , Ravenhill Benjamin J. , Lin Yen-Chun , Macharia Alex , Makale Johnstone , Tendwa Metrine , Nyamu Wilfred , Kotar Jurij , Carrasquilla Manuela , Rowe J. Alexandra , Rockett Kirk , Kwiatkowski Dominic , Weekes Michael P. , Cicuta Pietro , Williams Thomas N. , Rayner Julian C.
Published: November 2018

Malaria has had a major effect on the human genome, with many protective polymorphisms such as sickle cell trait having been selected to high frequencies in malaria endemic regions1, 2. Recently, we showed that a novel blood group variant, Dantu, provides 74% protection against all forms of severe malaria in homozygous individuals3-5. This is a similar degree of protection to sickle cell trait and considerably greater than the most advanced malaria vaccine, but until now the mechanism of protection has been unknown. In the current study, we demonstrate a significant impact of Dantu on the ability ofPlasmodium falciparummerozoites to invade RBCs. The Dantu variant was associated with extensive changes to the RBC surface protein repertoire, but unexpectedly the malaria protective effect did not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we found a strong link between RBC tension and parasite invasion and, even in non-Dantu RBCs, identified a tension threshold above which RBC invasion did not occur. Dantu RBCs had higher average tension, meaning that a higher proportion of Dantu RBCs could not be invaded. These findings not only provide an explanation for the protective effect of Dantu against severe malaria, but also provide fresh insights into the essential process ofP. falciparumparasite invasion, and how invasion efficiency varies across the heterogenous populations of RBCs that are present both within and between individuals.
A phenome-wide approach to identify causal risk factors for deep vein thrombosis
Authors: Constantinescu Andrei-Emil , Bull Caroline J , Goudswaard Lucy J , Zheng Jie , Elsworth Benjamin , Timpson Nicholas J , Moore Samantha F , Hers Ingeborg , Vincent Emma E
Published: November 2018

Background

Deep vein thrombosis (DVT) is the formation of a blood clot in a deep vein. DVT can lead to a venous thromboembolism (VTE), the combined term for DVT and pulmonary embolism, a leading cause of death and disability worldwide. Despite the prevalence and associated morbidity of DVT, the underlying causes are not well understood.

Objective

To leverage publicly available genetic summary association statistics to identify causal risk factors for DVT.

Methods & Results

We conducted a Mendelian randomization phenome-wide association study (MR-PheWAS) using genetic summary association statistics for 973 exposures and DVT (6,767 cases and 330,392 controls in UK Biobank). There was evidence for a causal effect of 57 exposures on DVT risk, including previously reported risk factors (e.g. body mass index - BMI and height) and novel risk factors (e.g. hyperthyroidism, chronic obstructive pulmonary disease (COPD) and varicose veins). As the majority of identified risk factors were adiposity-related, we explored the molecular link with DVT by undertaking a two-sample MR mediation analysis of BMI-associated circulating proteins on DVT risk. Our results indicate that circulating neurogenic locus notch homolog protein 1 (NOTCH1), inhibin beta C chain (INHBC) and plasminogen activator inhibitor 1 (PAI-1) influence DVT risk, with PAI-1 mediating the BMI-DVT relationship.

Conclusion

Using a phenome-wide approach, we provide putative causal evidence that hyperthyroidism, varicose veins, COPD and BMI enhance the risk of DVT. The circulating protein PAI-1 has furthermore a causal role in DVT aetiology and is involved in mediating the BMI-DVT relationship.
On the impact of chemo-mechanically induced phenotypic transitions in gliomas
Authors: Mascheroni Pietro , Lopez Alfonso Juan Carlos , Kalli Maria , Stylianopoulos Triantafyllos , Meyer-Hermann Michael , Hatzikirou Haralampos
Published: November 2018

Tumor microenvironment is a critical player in glioma progression and novel therapies for its targeting have been recently proposed. In particular, stress-alleviation strategies act on the tumor by reducing its stiffness, decreasing solid stresses and improving blood perfusion. However, these microenvironmental changes trigger chemo-mechanically induced cellular phenotypic transitions whose impact on therapy outcomes is not completely understood. In this work, we perform experiments to analyze the effects of mechanical compression on migration and proliferation of two glioma cell lines. From these experiments, we derive a mathematical model of glioma progression focusing on cellular phenotypic plasticity. The model reveals a trade-off between tumor infiltration and cellular content as a consequence of stress-alleviation approaches. We discuss how these findings can improve the current understanding of glioma/microenvironment interactions, and suggest strategies to improve therapeutic outcomes.
A Computational Framework for DNA Sequencing-Based Microscopy
Authors: Hoffecker Ian T. , Yang Yunshi , Bernardinelli Giulio , Orponen Pekka , Högberg Björn
Published: November 2018

Barcoded DNA polony amplification techniques provide a means to impart a unique sequence identity onto specific locations of a surface wafer or chip. We describe a method whereby micro-scale spatial information such as the relative positions of biomolecules on a surface can be transferred to a sequence-based format and reconstructed into images without the use of conventional optics. The principle is based on the pair-wise association of uniquely tagged and spatially adjacenct polonies. The network of polonies connected by shared borders forms a graph whose topology can be reconstructed from a set of edges derived from pairs of barcodes associated with each other during a polony crosslinking phase, the sequences of which could be determined by isolation of the DNA and next-gen sequencing. We developed a mathematical and computational framework for this principle called Polony Adjacency Reconstruction for Spatial Inference and Topology and show that Euclidean spatial data may be partially stored and transmitted in the form of untethered graph topology. This effect may be exploited to form images by transferring molecular information from a surface of interest, which we demonstrated in silico by reconstructing images formed from stochastic transfer of hypothetical red, green, and blue molecular markers. The theory developed here could serve as a guide for a highly automated, multiplexable, and potentially super resolution imaging method based on molecular information encoding and transmission.
The CIC-DUX4 fusion oncoprotein drives metastasis and tumor growth via distinct downstream regulatory programs and therapeutic targets in sarcoma
Authors: Okimoto Ross A. , Wu Wei , Nanjo Shigeki , Olivas Victor , Lin Yone K. , Oyama Rieko , Kondo Tadashi , Bivona Trever G.
Published: November 2018

Transcription factor fusion genes create oncoproteins that drive oncogenesis, and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. CIC-DUX4 is a transcription factor fusion that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We show that increased expression of ETV4 and CCNE1 occurs via neo-morphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We demonstrate a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4 tumors sensitive to inhibition of the CCNE-CDK2 complex, highlighting a therapeutic strategy for CIC-DUX4 tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically-aberrant transcriptional regulator, with therapeutic implications.
Variability of soybean response to rhizobia inoculant, Vermicompost, and a legume-specific fertilizer blend in Siaya County of Kenya
Authors: Mathenge Catherine , Thuita Moses , Masso Cargele , Gweyi-Onyango Joseph , Vanlauwe Bernard
Published: November 2018

Rhizobia inoculation can increase soybean yield, but its performance is influenced by soybean genotype, rhizobia strains, environment, and crop management among others. The objective of the study was to assess soybean response to rhizobia inoculation when grown in soils amended with urea or Vermicompost to improve nitrogen levels. Two greenhouse experiments and one field trial at two sites were carried out. The first greenhouse experiment included soils from sixty locations, sampled from smallholder farms in Western Kenya. The second greenhouse experiment consisted of one soil selected from soils used in the first experiment where inoculation response was poor. The soil was amended with Vermicompost or urea. In the two greenhouse experiments, Legumefix® (inoculant) + Sympal (legume fertilizer blend) were used as a standard package. Results from the second greenhouse experiment were then validated in the field. In the first greenhouse trial, soybean response to inoculation was significantly affected by soil fertility based on nodule fresh weight and shoot biomass. Soils with low nitrogen had low to no response to inoculation. After amendment, nodule fresh weight, nodule effectiveness, nodule occupancy, and shoot dry biomass were greater in the treatment amended with Vermicompost than those amended with urea (Legumefix® + Sympal + Vermicompost and Legumefix® + Sympal + urea). Under field conditions, trends were similar to the second experiment for nodulation, nodule occupancy, and nitrogen uptake resulting in significantly greater grain yields (475, 709, 856, 880, 966 kg ha−1) after application of Vermicompost at 0, 37, 74, 111, and 148 kg N ha−1, respectively. It was concluded that soybean nodulation and biological nitrogen fixation in low fertility soils would not be suppressed by organic amendments like Vermicompost up to 148 kg N ha−1.
Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens
Authors: Noirot-Gros Marie-Francoise , Forrester Sara , Malato Grace , Larsen Peter E. , Noirot Philippe
Published: November 2018

Bacterial biofilm formation involves multigenic signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm complex 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Genetic approaches based on gene inactivation are powerful for mechanistic studies but often are labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in P. fluorescens. We found that CRISPRi is applicable to three genetically and physiologically diverse species, SBW25, WH6 and Pf0-1 and affords extended periods of time to study complex phenotypes such as cell morphology, motility and biofilm formation. In SBW25, CRISPRi-mediated silencing of the GacA/S two-component system and genes regulated by cylic-di-GMP produced phenotypes similar to those previously described after gene inactivation in various Pseudomonas. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with biofilm architecture and extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 protein. Thus, CRISPRi is a reliable and scalable approach to interrogate gene networks in the diverse P. fluorescens group.
Toll-like receptor 3 activation increases voluntary alcohol intake in C57BL/6J male mice
Authors: Warden Anna S. , Azzam Moatasem M. , DaCosta Adriana , Mason Sonia , Blednov Yuri A. , Messing Robert O. , Mayfield R. Dayne , Harris R. Adron
Published: November 2018

Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake function are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.

Highlights

Activation of TLR3viapoly(I:C) increased alcohol intake.

Poly(I:C) and ethanol must be present together to change drinking behavior.

Increased alcohol intake due to poly(I:C) is independent of MYD88.

Increased alcohol intake due to poly(I:C) is dependent on TLR3.
Momo – Multi-Objective Metabolic mixed integer Optimization: application to yeast strain engineering
Authors: Andrade Ricardo , Doostmohammadi Mahdi , Santos João L. , Sagot Marie-France , Mira Nuno P. , Vinga Susana
Published: November 2018

In this paper, we explore the concept of multi-objective optimization in the field of metabolic engineering when both continuous and integer decision variables are involved in the model. In particular, we propose a multi-objective model that may be used to suggest reaction deletions that maximize and/or minimize several functions simultaneously. The applications may include, among others, the concurrent maximization of a bioproduct and of biomass, or maximization of a bioproduct while minimizing the formation of a given by-product, two common requirements in microbial metabolic engineering.

Production of ethanol by the widely used cell factorySaccharomyces cerevisiaewas adopted as a case study to demonstrate the usefulness of the proposed approach in identifying genetic manipulations that improve productivity and yield of this economically highly relevant bioproduct. We did anin vivovalidation and we could show that some of the predicted deletions exhibit increased ethanol levels in comparison with the wild-type strain. The multi-objective programming framework we developed, called Momo, is open-source and uses PolySCIP‡as underlying multi-objective solver. Momo is available athttp://momo-sysbio.gforge.inria.fr
Genome-wide screening reveals a novel class of carbonic anhydrase-like inorganic carbon pumps in chemoautotrophic bacteria
Authors: Desmarais John J. , Flamholz Avi I. , Blikstad Cecilia , Dugan Eli J. , Laughlin Thomas G. , Oltrogge Luke M. , Chen Allen W. , Wetmore Kelly , Diamond Spencer , Wang Joy Y. , Savage David F.
Published: November 2018

Many bacterial autotrophs rely on CO2concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, including a 200+ MDa protein organelle called the carboxysome, a systematic screen of CCM components has not been carried out. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the ɣ-proteobacteriumH. neapolitanus. Our screen revealed an operon critical for CCM function which encodes a domain of unknown function (PFAM:PF10070) and putative cation transporter subunit (PFAM:PF00361). These two proteins, which we name DabA and DabB for “DABs accumulate bicarbonate,” function as a heterodimeric, energy-coupled inorganic carbon pump inE. coli. Furthermore, DabA binds zinc and has a an active site homologous to a β-carbonic anhydrase. Based on these results, we propose that DABs function as vectorial CAs coupled to cation gradients and serve as inorganic carbon pumps throughout prokaryotic phyla.
Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast
Authors: Yukawa Masashi , Okazaki Masaki , Teratani Yasuhiro , Furuta Ken’ya , Toda Takashi
Published: November 2018

Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation towards the cell cortex in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon an NLS and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 is impaired specifically in late mitotic stages. cut7-122klp9Δ double mutant cells exhibit additive defects in spindle elongation. Together, we propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent and ensures structural integrity of spindle microtubules together with Ase1.
A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down of gene expression inClostridioides difficile
Authors: Müh Ute , Pannullo Anthony G. , Weiss David S. , Ellermeier Craig D.
Published: November 2018

Here we introduce plasmids for xylose-regulated expression and repression of genes inClostridioides difficile. The xylose-inducible expression vector allows for ~100-fold induction of anmCherryOptreporter gene. Induction is titratable and uniform from cell-to-cell. The gene repression plasmid is a CRISPR-interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of theStreptococcus pyogenesCas9 protein (dCas9) that is targeted to a gene of interest by a constitutively-expressed single guide RNA (sgRNA). Expression ofdCas9is induced by xylose, allowing investigators to control the timing and extent of gene-silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression ~100-fold). To validate the utility of CRISPRi for deciphering gene function inC. difficile, we knocked-down expression of three genes involved in biogenesis of the cell envelope: the cell division geneftsZ, the S-layer protein geneslpAand the peptidoglycan synthase genepbp-0712. CRISPRi confirmed known or expected phenotypes associated with loss of FtsZ and SlpA, and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division and protection against lysis.

Importance

Clostridioides difficilehas become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treatingC. difficileinfections. Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins inC. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.
Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast
Authors: Buchmuller Benjamin C. , Herbst Konrad , Meurer Matthias , Kirrmaier Daniel , Sass Ehud , Levy Emmanuel D. , Knop Michael
Published: November 2018

Clone collections of modified strains (‘libraries’) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ∼10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks new avenues for increased throughput in functional genomics and cell biology research.
MiR-195 regulates mitochondrial function by targeting mitofusin-2 in breast cancer cells
Authors: Purohit Paresh Kumar , Edwards Ruairidh , Tokatlidis Kostas , Saini Neeru
Published: November 2018

Mitochondrial dynamics is a highly dysregulated process in cancer. Apoptosis and mitochondrial fission are two concurrent events wherein increased mitochondrial fragmentation serves as a hallmark of apoptosis. We have shown earlier that miR-195 exerts pro-apoptotic effects in breast cancer cells. Herein, we have demonstrated miR-195 as a modulator of mitochondrial dynamics and function. Imaging experiments upon miR-195 treatment have shown that mitochondria undergo extensive fission. We validated mitofusin2 as a potential target of miR-195. Which may provide a molecular explanation for the respiratory defects induced by miR-195 over-expression in breast cancer cells? Active, but not total, mitochondrial mass, was reduced with increasing levels of miR-195. We have further shown that miR-195 enhances mitochondrial SOD-2 expression but does not affect PINK1 levels in breast cancer cells. Collectively, we have revealed that miR-195 is a modulator of mitochondrial dynamics by targeting MFN2 thereby impairing mitochondrial function. Concomitantly, it enhances the scavenger of reactive oxygen species (SOD-2) to maintain moderate levels of oxidative stress. Our findings suggest a therapeutic potential of miR-195 in both ER-positive as well as ER-negative breast cancer cells.
Balancing energy supply during photosynthesis - a theoretical perspective
Authors: Matuszyńska Anna , Saadat Nima P. , Ebenhöh Oliver
Published: November 2018

The photosynthetic electron transport chain (PETC) provides energy and redox equivalents for carbon fixation by the Calvin-Benson-Bassham (CBB) cycle. Both of these processes have been thoroughly investigated and the underlying molecular mechanisms are well known. However, it is far from understood by which mechanisms it is ensured that energy and redox supply by photosynthesis matches the demand of the downstream processes. Here, we deliver a theoretical analysis to quantitatively study the supply-demand regulation in photosynthesis. For this, we connect two previously developed models, one describing the PETC, originally developed to study non-photochemical quenching, and one providing a dynamic description of the photosynthetic carbon fixation in C3 plants, the CBB Cycle. The merged model explains how a tight regulation of supply and demand reactions leads to efficient carbon fixation. The model further illustrates that a stand-by mode is necessary in the dark to ensure that the carbon fixation cycle can be restarted after dark-light transitions, and it supports hypotheses, which reactions are responsible to generate such mode in vivo.
Physiological regulation of bud burst in grapevine
Authors: Signorelli Santiago , Shaw Jeremy , Hermawaty Dina , Wang Zi , Verboven Pieter , Considine John A. , Considine Michael J.
Published: November 2018

The physiological constraints on bud burst in woody perennials, including the prerequisite for vascular development remain unresolved. Both light and tissue oxygen status have emerged as important cues for vascular development in other systems, however, light requirement appears to be facultative in grapevine, and the information related to the spatial variability of oxygen in buds is unclear. Here, we analysed apoplastic development at early stages of grapevine bud burst and combined molecular modelling with histochemical techniques to determine the pore size of cell walls in grapevine buds. The data demonstrate that quiescent grapevine buds were impermeable to apoplastic dyes (acid fuchsin and eosin Y) until after bud burst was established. The molecular exclusion size was calculated to be 2.1 nm, which would exclude most macromolecules except simple sugars and phytohormones.In vivoexperiments show that grapevine buds were able to resume growth even following excision from the cane, and that the outer scales of grapevine buds may participate in the biochemical repression of bud burst. Furthermore, we demonstrate that the tissue oxygen partial pressure data correlated well with structural heterogeneity within the bud and differences in tissue density. These data consolidate evidence that the meristematic core becomes rapidly oxygenated during bud burst. Taken together, and when put in the context of earlier studies, these data provide solid evidence that the physiological and biochemical events that initiate bud burst reside within the bud, and question the role of long distance signalling in this developmental transition.

Highlights

The apoplastic pore size between the grapevine bud and the mother vine is dynamically regulated in the transition to bud burst.

The molecular exclusion size of the apoplastic connection between the bud and cane is calculated 2.1 nm prior to the initiation of bud burst.

The structural heterogeneity of the bud explains the spatial variance in tissue oxygen status, and the meristematic core is oxygenated during the initiation of bud burst.

Long distance maternal signals are not a requirement for bud burst.
Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems: Biocoenosis ofBotryococcusconsortia
Authors: Blifernez-Klassen Olga , Klassen Viktor , Wibberg Daniel , Cebeci Enis , Henke Christian , Rückert Christian , Chaudhari Swapnil , Rupp Oliver , Blom Jochen , Winkler Anika , Al-Dilaimi Arwa , Goesmann Alexander , Sczyrba Alexander , Kalinowski Jörn , Bräutigam Andrea , Kruse Olaf
Published: November 2018

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host’s development, growth, health and disease. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described but owing to the complex interactions rarely functionally dissected. Multifaceted analysis of the microbial consortium of the ancient phytoplanktonBotryococcusas an attractive model food web revealed that its all abundant bacterial members belong to a distinct niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies, which adversely affect the algal cell morphology, appear evidently decimated. Synthetic rearrangement of a minimal community consisting of alga, mutualistic and parasitic bacteria underpins the model of a eukaryote that domesticates its own mutualistic bacterial “zoo” to manipulate and control its surrounding biosphere. This model of domestication of mutualistic bacteria for the defense against destruents by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction disentangles the relationships and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.
Cold-Inducible RNA-binding protein (CIRBP) adjusts clock gene expression and REM sleep recovery following sleep deprivation
Authors: Hoekstra Marieke MB , Emmenegger Yann , Franken Paul
Published: November 2018

Sleep depriving mice affects clock gene expression, suggesting that these genes partake in sleep homeostasis. The mechanisms linking wakefulness to clock gene expression are, however, not well understood. We propose CIRBP because its rhythmic expression is i) sleep-wake driven and ii) necessary for high-amplitude clock gene expressionin vitro. We therefore expectCirbpknock-out (KO) mice to exhibit attenuated sleep-deprivation (SD) induced changes in clock gene expression, and consequently to differ in their sleep homeostatic regulation. Lack of CIRBP indeed blunted the SD-incurred changes in cortical expression of the clock geneRev-erbαwhereas it amplified the changes inPer2andClock. Concerning sleep homeostasis, KO mice accrued only half the extra REM sleep wild-type (WT) littermates obtained during recovery. Unexpectedly, KO mice were more active during lights-off which was accompanied by an acceleration of theta oscillations. Thus, CIRBP adjusts cortical clock gene expression after SD and expedites REM sleep recovery.
The mouse Klf1 Nan variant impairs nuclear condensation and erythroid maturation
Authors: Cantú Ileana , Werken Harmen J.G. van de , Gillemans Nynke , Stadhouders Ralph , Heshusius Steven , Maas Alex , Ozgur Zeliha , van IJcken Wilfred F.J. , Grosveld Frank , von Lindern Marieke , Philipsen Sjaak , van Dijk Thamar B.
Published: November 2018

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >65 KLF1 variants, causing different erythroid phenotypes, have been described. The Klf1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the ~780 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We conclude that reduced expression of XPO7 is partially responsible for the erythroid defects observed in Nan erythroid cells.
Astroglial dysfunctions drive aberrant synaptogenesis in developing brain with lengthy general anesthesia
Authors: Zhou Bin , Chen Lingmin , Liao Ping , Huang Lu , Chen Zhuo , Yu Guoqiang , Wang Li , Zhang Jianguo , Zuo Yunxia , Liu Jin , Jiang Ruotian
Published: November 2018

Lengthy use of general anesthetics (GAs) causes cognitive deficits in developing brain, which has raised significant clinical concerns such that FDA is warning on the use of GAs in children younger than 3 years. However, the molecular and cellular mechanisms for GAs-induced neurotoxicity remain largely unknown. Here we report that sevoflurane, a commonly used GA in pediatrics, causes compromised astrocyte morphogenesis, spatiotemporally correlated to the synaptic overgrowth with reduced synaptic function in developing cortex in a regional-, exposure-length- and age-specific manner. Sevoflurane disrupts astrocyte Ca2+ homeostasis both acutely and chronically, which leads to the down regulation of Ezrin, an actin-binding membrane protein, which we found is critically involved in astrocyte morphogenesis in vivo. Importantly, in normal developing brain, the genetic intervention of astrocyte morphogenesis is sufficient to produce the aberrant synaptic structure and function virtually identical to the ones induced by lengthy sevoflurane exposure. Our data uncover that astrocytes are unexpectedly central targets for GAs to exert toxic effects, and that astrocyte morphological integrity is crucial for synaptogenesis in the developing brain.
Small GTPase patterning: How to stabilise cluster coexistence
Authors: Jacobs Bas , Molenaar Jaap , Deinum Eva E.
Published: November 2018

Many biological processes have to occur at specific locations on the cell membrane. These locations are often specified by the localised activity of small GTPase proteins. Some processes require the formation of a single cluster of active GTPase, also called unipolar polarisation (here “polarisation”), whereas others need multiple coexisting clusters. Moreover, sometimes the pattern of GTPase clusters is dynamically regulated after its formation. This raises the question how the same interacting protein components can produce such a rich variety of naturally occurring patterns. Most currently used models for GTPase-based patterning inherently yield polarisation. Such models may at best yield transient coexistence of at most a few clusters, and hence fail to explain several important biological phenomena. These existing models are all based on mass conservation of total GTPase and some form of direct or indirect positive feedback. Here, we show that either of two biologically plausible modifications can yield stable coexistence: including explicit GTPase turnover, i.e., breaking mass conservation, or negative feedback by activation of an inhibitor like a GAP. Since we start from two different polarising models our findings seem independent of the precise self-activation mechanism. By studying the net GTPase flows among clusters, we provide insight into how these mechanisms operate. Our coexistence models also allow for dynamical regulation of the final pattern, which we illustrate with examples of pollen tube growth and the branching of fungal hyphae. Together, these results provide a better understanding of how cells can tune a single system to generate a wide variety of biologically relevant patterns.

Author summary

Where to form a bud? Where to reinforce the cell wall? In which direction to move? These are all important decisions a cell may have to make. Proper patterning of the cell membrane is a critical part of such decisions. These patterns are often specified by the local activity of proteins called small GTPases. Mathematical models have been an important tool in understanding the mechanisms behind small GTPase-based patterning. Most of these models, however, only allow for the formation of a single cluster of active GTPase and thus cannot explain patterns of multiple coexisting GTPase clusters. A previously proposed mechanism for such coexistence can only explain a temporary, unstable coexistence, and fails to explain several key biological phenomena. In this manuscript, we investigate two mechanisms that can produce patterns of many stably coexisting GTPase clusters. Using a combination of modelling techniques, we show why these mechanisms work. We also show that these mechanisms allow for the addition of new clusters to an existing pattern, as is observed for example during the branching of fungal hyphae. With our results, we now have handles to explain the full range of naturally occurring small GTPase patterns.