bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycosylation of bioactive C13-apocarotenols inNicotiana benthamianaandMentha × piperita
Authors: Sun Guangxin , Putkaradze Natalia , Bohnacker Sina , Jonczyk Rafal , Fida Tarik , Hoffmann Thomas , Bernhardt Rita , Härtl Katja , Schwab Wilfried
Published: July 2020

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products, which perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains elusive. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected for their high transcript abundance in comparison with otherUGTsin vegetative tissues ofNicotiana benthamianaandMentha × piperita, as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the unprecedented glucosylation of C13-apocarotenols, where glucose is bound either to the cyclohexene ring or butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C) and 3-hydroxy-7,8-dihydro-β-ionol, while a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analyses of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization and the associated maintenance of carotenoid homeostasis.

One-sentence summary

We identified six transferases inNicotiana benthamianaandMentha x piperita, two rich sources of glycosylated apocarotenoids that catalyze the unprecedented glycosylation of a range of hydroxylated α- and β-ionone/ionol derivatives and were able to modify bioactivity by glucosylation.
Benchmarking full-length transcript single cell mRNA sequencing protocols
Authors: Probst Victoria , Pacheco Felix , Nielsen Finn Cilius , Bagger Frederik Otzen
Published: July 2020

Single cell mRNA sequencing technologies have transformed our understanding of cellular heterogeneity and identity. For sensitive discovery or clinical marker estimation where high transcript capture per cell is needed only plate-based techniques currently offer sufficient resolution. Here, we present a performance evaluation of three different plate-based scRNA-seq protocols. Our evaluation is aimed towards applications requiring high gene detection sensitivity, reproducibility between samples, and minimum hands-on time, as is required, for example, in clinical use. We included two commercial kits, NEBNext®Single Cell/ Low Input RNA Library Prep Kit (NEB®), SMART-seq®HT kit (Takara®), and the non-commercial protocol Genome & Transcriptome sequencing (G&T). G&T delivered the highest detection of genes per single cell, at the absolute lowest price. Takara®kit presented similar high gene detection per single cell, and high reproducibility between sample, but at the absolute highest price. NEB®delivered a lower detection of genes but remain an alternative to more expensive commercial kits.
The effect of grape juice dilution on oenological fermentation
Authors: Gardner Jennifer Margaret , Walker Michelle Elisabeth , Boss Paul Kenneth , Jiranek Vladimir
Published: July 2020

The impact of water addition to grape juice in winemaking, on both alcoholic and malolactic fermentation duration and outcome has been examined using commercial wine yeasts, Lalvin EC1118™ and Lalvin R2™ and malolactic bacteria Lalvin VP41™. As expected, dilution with water did not impede fermentation, instead resulted in shortened duration, or in the case of malolactic fermentation enabled completion in these conditions. Addition of complex organic nutrient further shortened alcoholic fermentation by Lalvin R2™ and in some conditions also reduced the duration of malolactic fermentation. In general, volatile compounds and some major yeast metabolites were present at lower concentrations at the end of fermentation where juices were diluted and the addition of organic complex nutrient also influenced the concentration of some compounds in wine. These findings are significant to commercial winemaking, highlighting that winemakers should consider potential impacts of juice dilution on processing efficiencies along with wine flavour and aroma.

Highlights: Gardner et al. The effect of grape juice dilution on fermentation

Grape juice dilution shortened both alcoholic and malolactic fermentation

In some conditions addition of commercial nutrient decreased fermentation duration

In general wine volatiles decrease with grape juice dilution

Isoamyl acetate can be decreased in wine by grape juice dilution
Molecular detection ofLeishmania donovani, Leishmania major, andTrypanosomaspp. inSergentomyia squamipleurissandflies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya
Authors: Owino Barrack O. , Mwangi Jackline Milkah , Kiplagat Steve , Mwangi Hannah Njiriku , Ingonga Johnstone M. , Chebet Alphine , Ngumbi Philip M. , Villinger Jaundouwe , Masiga Daniel K. , Matoke-Muhia Damaris
Published: July 2020

Background

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remains limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sandfly species responsible forLeishmaniatransmission in the sub-County, and their blood-meal hosts.

Methods

We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sandflies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (COI) gene.Leishmaniaparasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Bloodmeal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products.

Results

We sampled 526 sandflies consisting of eight species,Phlebotomus orientalis(1.52%; n = 8) and sevenSergentomyiaspp.Sergentomyia squamipleuriswas the most abundant sandfly species (78.71%; n = 414) followed bySergentomyia clydei(10.46%; n = 55).Leishmania major, Leishmania donovani, andTrypanosomaDNA were detected inS. squamipleurisspecimens. Humans were the main sources of sandfly bloodmeals. However, we also detected mixed bloodmeals; oneS. squamipleurisspecimen had fed on both human and mouse (Mus musculus) blood, while twoPh. orientalisspecimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood.

Conclusions

Our findings implicate the potential involvement ofS. squamipleurisin the transmission ofLeishmaniaand question the dogma that human leishmaniases in the Old World are exclusively transmitted by sandflies of thePhlebotomusgenus. The presence ofTrypanosomaspp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County.Leishmania majorcauses ZCL whileL. donovaniis responsible for VL. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts ofLeishmaniaspp. in the area.
The influence of microbial mats on travertine precipitation in active hydrothermal systems (Central Italy)
Authors: Porta Giovanna Della , Reitner Joachim
Published: July 2020

The study of hydrothermal travertines contributes to the understanding of the interaction between physico-chemical processes and the role played by microbial mats and biofilms in influencing carbonate precipitation. Three active travertine sites were investigated in Central Italy to identify the types of carbonate precipitates and the associated microbial mats at varying physico-chemical parameters. Carbonate precipitated fabrics at the decimetre- to millimetre-scale and microbial mats vary with decreasing water temperature: a) at high temperature (55-44°C) calcite or aragonite crystals precipitate on microbial mats of sulphide oxidizing, sulphate reducing and anoxygenic phototrophic bacteria forming filamentous streamer fabrics, b) at intermediate temperature (44-40°C), rafts, coated gas bubbles and dendrites are associated withSpirulinacyanobacteria and other filamentous and rod-shaped cyanobacteria, c) low temperature (34-33°C) laminated crusts and oncoids in a terraced slope system are associated with diverse Oscillatoriales and Nostocales filamentous cyanobacteria, sparseSpirulinaand diatoms. At the microscale, carbonate precipitates are similar in the three sites consisting of prismatic calcite (40-100 μm long, 20-40 μm wide) or acicular aragonite crystals organized in radial spherulites, overlying or embedded within biofilm EPS (Extracellular Polymeric Substances). Microsparite and sparite crystal size decreases with decreasing temperature and clotted peloidal micrite dominates at temperatures < 40°C, also encrusting filamentous microbes. Carbonates are associated with gypsum and Ca-phosphate crystals; EPS elemental composition is enriched in Si, Al, Mg, Ca, P, S and authigenic aluminium-silicates form aggregates on EPS.

This study confirms that microbial communities in hydrothermal travertine settings vary as a function of temperature. Carbonate precipitate types at the microscale do not vary considerably, despite different microbial communities suggesting that travertine precipitation, driven by CO2degassing, is influenced by biofilm EPS acting as template for crystal nucleation (EPS-mediated mineralization) and affecting the fabric types, independently from specific microbial metabolism.
SAMHD1 promotes oncogene-induced replication stress
Authors: Zhang Si Min , Calderón-Montaño Jose M , Rudd Sean G
Published: July 2020

Oncogenes induce DNA replication stress in cancer cells. Although this was established more than a decade ago, we are still unravelling the molecular underpinnings of this phenomenon, which will be critical if we are to exploit this knowledge to improve cancer treatment. A key mediator of oncogene-induced replication stress is the availability of DNA precursors, which will limit ongoing DNA synthesis by cellular replicases. In this study, we identify a potential role for nucleotide catabolism in promoting replication stress induced by oncogenes. Specifically, we establish that the dNTPase SAMHD1 slows DNA replication fork speeds in human fibroblasts harbouring an oncogenic RAS allele, elevating levels of endogenous DNA damage, and ultimately limiting cell proliferation. We then show that oncogenic RAS-driven tumours express reduced SAMHD1 levels, suggesting they have overcome this tumour suppressor barrier, and that this correlates with worse overall survival for these patients.

Abstract Figure
Functional and genetic determinants of mutation rate variability in regulatory elements of cancer genomes
Authors: Lee Christian A. , Abd-Rabbo Diala , Reimand Jüri
Published: July 2020

Background

Cancer genomes are shaped by mutational processes with complex spatial variation at multiple scales. Entire classes of regulatory elements are affected by local variations in mutation frequency. However, the underlying mutational mechanisms with functional and genetic determinants remain poorly understood.

Results

We characterised the mutational landscape of 1.3 million gene regulatory and chromatin architectural elements in 2,419 whole cancer genomes with transcriptional and pathway activity, functional conservation and recurrent driver events. We developed RM2, a statistical model that quantifies mutational enrichment or depletion in classes of genomic elements through genetic, trinucleotide and megabase-scale effects. We report a map of localised mutational processes affecting CTCF binding sites, transcription start sites (TSS) and tissue-specific open-chromatin regions. We show that increased mutational frequency in TSSs correlates with mRNA abundance in most cancer types, while open-chromatin regions are generally enriched in mutations. We identified ∼10,000 CTCF binding sites with core DNA motifs and constitutive binding in 66 cell types that represent focal points of local mutagenesis. We detected site-specific mutational signatures, such as SBS40 in open-chromatin regions in prostate cancer and SBS17b in CTCF binding sites in gastrointestinal cancers. We also proposed candidate drivers of localised mutagenesis:BRAFmutations associate with mutational enrichments at CTCF binding sites in melanoma, andARID1Amutations with TSS-specific mutations in pancreatic cancer.

Conclusions

Our method and catalogue of localised mutational processes provide novel perspectives to cancer genome evolution, mutagenesis, DNA repair and driver discovery. Functional and genetic correlates of localised mutagenesis provide mechanistic hypotheses for future studies.
Exploring patterns of ongoing thought under naturalistic and task-based conditions
Authors: Konu Delali , Mckeown Brontë , Turnbull Adam , Ho Nerissa Siu Ping , Vanderwal Tamara , McCall Cade , Tipper Steven P. , Jefferies Elizabeth , Smallwood Jonathan
Published: July 2020

Previous research suggests that patterns of ongoing thought are heterogeneous, varying across situations and individuals. The current study investigated the influence of a wide range of tasks and individual affective style on ongoing patterns of thought. In total, we used 9 different tasks and measured ongoing thought using multidimensional experience sampling. A Principle Component Analysis of the experience sampling data revealed four patterns of ongoing thought. Linear Mixed Modelling was used to examine the contextual distribution of the thought patterns. Different thought patterns were found to relate to different types of conditions. Intrusive and negative thought pattern expression was found to be influenced by individual affective style (depression level). Overall, these data show that patterns of thought are subject to both contextual and intrinsic variation, suggesting that understanding these important features of experience across a broad range of situations will be useful in understanding their role in human experience.

Highlights

Patterns of thought vary across different task contexts

Thought pattern expression is influenced by individual affective style

There is a need to broaden the tasks used to study ongoing thought
Sero-prevalence and risk factors of leptospirosis in commercial cattle herds of Rupandehi district, Nepal
Authors: Gompo Tulsi Ram , Jyoti Sumit , Pandit Sudikchya , Sapkota Ram Chandra , Pandey Aditi
Published: July 2020

Nepal has an agrarian-based economy where the livestock sector contributes about 12 percent to the national GDP. Rupandehi district is one of the largest dairy pocket areas in the nation, but the disease, such as leptospirosis, posed a negative impact on their production and productivity. Despite the economic importance of cattle leptospirosis, the disease is concerned for the people’s occupational risk and food safety. Although leptospirosis is a priority zoonosis in Nepal, the effective implementation of the control program lacks both in animal and human health. A cross-sectional study was conducted to estimate the prevalence and identify associated risk factors of cattle leptospirosis from March 2019 to April 2020. Altogether, 206 cattle herds located in all the sixteen local levels of the district were visited. The owners were interviewed to record the cattle management system’s information and their knowledge of the zoonotic diseases. A total of 383 cattle serum samples were collected, and an indirect antibody ELISA was performed to estimate the infection status of leptospirosis in the cattle herds. All the data were analyzed by Open epi and R software for the descriptive and analytical study. Bivariate and multivariable logistic regression was applied to assess the potential herd level and animal level risk factors. Out of seven potential herd-level risk factors, the purchased cattle herds (OR: 7.2, 95% CI: 1.24-136.5, p=0.025) and cattle with herd size >10 (OR: 14.92, 95% CI: 2.61-283.38, p=0.025) were identified as significant risk factors for leptospirosis. At the animal level, the cattle taken for grazing in pastureland accessed by the community dog was a significant risk factor (OR: 4.16, 95% CI: 1.13-14.06, p=0.034). Surprisingly, none of the farmers had heard about leptospirosis before. The outcome of this research could be an epidemiological insight in control of priority zoonosis to protect the livestock economy and reduce their negative impact on public health.
A new molecular method for the exploration of hybrid zones between two toad species of conservation interest
Authors: Gál Zoltán , Kovács Tibor , Ujszegi János , Anthony Brandon P. , Vági Balázs , Hoffmann Orsolya I.
Published: July 2020

Analyzing hybrid zones between previously isolated lineages allows insight into processes determining the fate of re-encounter of these taxa. The distributions of Fire-bellied (Bombina bombina) and Yellow-bellied toads (B. variegata) meet in the Carpathian Basin resulting in a narrow contact zone in the foothill regions, where hybrids often appear. Our aim was to explore a transient zone betweenB. bombinaandB. variegatawithin the Carpathian Basin along a transect including the Börzsöny Hills in Hungary and Krupinská Planina in Slovakia. We visited 28 locations in these areas and collected altogether 230 specimens, photographed and sampled them using buccal swabs. In order to distinguish between the two species and determine hybrid individuals, we used mitochondrial markers and designed a novel technique based on the restriction of the Ncx-1 gene. The geographical distribution pattern of these two species delivered unexpected results, as Börzsöny Hills was completely colonised byB. bombinaincluding locations which can be classified as typical habitats ofB. variegata. Moreover, in Krupinská Planina many locations were also colonised byB. bombina, including high altitude ponds. The most remote sites still harbourB. variegatapopulations, but seven of these were found with hybrid individuals. This pattern may indicate the northward and altitudinal range expansion ofB. bombinaand the colonisation of habitats of its sister species in these areas. Our results warrant enhanced attention to hybrid zones, where introgression and changes in population composition may reflect recent rapid environmental alterations and redirect conservation focus.
Cutaneous inputs from perineal region facilitates and modulates spinal locomotor activity and reduces cutaneous reflexes from the foot in spinal cats
Authors: Merlet Angèle N , Harnie Jonathan , Macovei Madalina , Doelman Adam , Gaudreault Nathaly , Frigon Alain
Published: July 2020

It is well known that mechanically stimulating the perineal region potently facilitates hindlimb locomotion and weight support in mammals with a spinal transection (spinal mammals). However, how perineal stimulation mediates this excitatory effect is poorly understood. We evaluated the effect of mechanically stimulating (vibration or pinch) the perineal region on ipsilateral (9-14 ms onset) and contralateral (14-18 ms onset) short-latency cutaneous reflex responses evoked by electrically stimulating the superficial peroneal or distal tibial nerve in seven adult spinal cats where hindlimb movement was restrained. Cutaneous reflexes were evoked before, during, and after mechanical stimulation of the perineal region. We found that vibration or pinch of the perineal region effectively triggered rhythmic activity, unilateral and bilateral to nerve stimulation. When electrically stimulating nerves, adding perineal stimulation modulated rhythmic activity by decreasing cycle and burst durations and by increasing the amplitude of flexors and extensors. Perineal stimulation also disrupted the timing of the ipsilateral rhythm, which had been entrained by nerve stimulation. Mechanically stimulating the perineal region decreased ipsilateral and contralateral short-latency reflex responses evoked by cutaneous inputs, a phenomenon we observed in muscles crossing different joints and located in different limbs. The results suggest that the excitatory effect of perineal stimulation on locomotion and weight support is not mediated by increasing cutaneous reflex gain and instead points to an excitation of central pattern-generating circuitry. Our results are consistent with a state-dependent modulation of reflexes by spinal interneuronal circuits.

Significance Statement

Mechanically stimulating the skin of the perineal region strongly facilitates hindlimb locomotion in mammals following a complete spinal cord injury (SCI). Despite its remarkable effectiveness in promoting hindlimb locomotion in spinal cord-injured mammals, we do not know how this is mediated. The present study provides data on how inputs from the perineal region interact with neuronal circuits that generate locomotor-like activity and reflexes from the foot. A better understanding of how inputs from the perineal region interact with neuronal circuits of the spinal cord could lead to non-invasive approaches to restore walking in people with SCI.
The genetic variant analyses of SARS-CoV-2 strains; circulating in Bangladesh
Authors: Mohmmad Mahmud Abu Sayeed , Taznin Tarannum , Hasan Sarkar Md. Murshed , Uzzaman Mohammad Samir , Osman Eshrar , Habib Md. Ahasan , Akter Shahina , Banu Tanjina Akhter , Goswami Barna , Jahan Iffat , Hossain Md. Saddam , Khan Md. Salim
Published: July 2020

Genomic mutation of the virus may impact the viral adaptation to the local environment, their transmission, disease manifestation, and the effectiveness of existing treatment and vaccination. The objectives of this study were to characterize genomic variations, non-synonymous amino acid substitutions, especially in target proteins, mutation events per samples, mutation rate, and overall scenario of coronaviruses across the country. To investigate the genetic diversity, a total of 184 genomes of virus strains sampled from different divisions of Bangladesh with sampling dates between the 10th of May 2020 and the 27thof June 2020 were analyzed. To date, a total of 634 mutations located along the entire genome resulting in non-synonymous 274 amino acid substitutions in 22 different proteins were detected with nucleotide mutation rate estimated to be 23.715 substitutions per year. The highest non-synonymous amino acid substitutions were observed at 48 different positions of the papain-like protease (nsp3). Although no mutations were found in nsp7, nsp9, nsp10, and nsp11, yet orf1ab accounts for 56% of total mutations. Among the structural proteins, the highest non-synonymous amino acid substitution (at 36 positions) observed in spike proteins, in which 9 unique locations were detected relative to the global strains, including 516E>Q in the boundary of the ACE2 binding region. The most dominated variant G614 (95%) based in spike protein is circulating across the country with co-evolving other variants including L323 (94%) in RNA dependent RNA polymerase (RdRp), K203 (82%) and R204 (82%) in nucleocapsid, and F120 (78%) in NSP2. These variants are mostly seen as linked mutations and are part of a haplotype observed in Europe. Data suggest effective containment of clade G strains (4.8%) with sub-clusters GR 82.4%, and GH clade 6.4%.

Highlights

We have sequenced 137 and analyzed 184 whole-genomes sequences of SARS-CoV-2 strains from different divisions of Bangladesh.

A total of 634 mutation sites across the SARS-CoV-2 genome and 274 non-synonymous amino acid substitutions were detected.

The mutation rate of SARS-CoV-2 estimated to be 23.715 nucleotide substitutions per year.

Nine unique variants were detected based on non-anonymous amino acid substitutions in spike protein relative to the global SARS-CoV-2 strains.
Dynamic Configuration of Coactive Micropatterns in the Default Mode Network during Wakefulness and Sleep
Authors: Cui Yan , Li Min , Biswal Bharat , Jing Wei , Zhou Changsong , Liu Huixiao , Guo Daqing , Xia Yang , Yao Dezhong
Published: July 2020

The default mode network (DMN) is a prominent intrinsic network that is observable in many mammalian brains. However, few studies have investigated the temporal dynamics of this network based on direct physiological recordings. Herein, we addressed this issue by characterizing the dynamics of local field potentials (LFPs) from the rat DMN during wakefulness and sleep with an exploratory analysis. We constructed a novel coactive micropattern (CAMP) algorithm to evaluate the configurations of rat DMN dynamics and further revealed the relationship between DMN dynamics with different wakefulness and alertness levels. From the gamma activity (40-80 Hz) in the DMN across wakefulness and sleep, three spatially stable CAMPs were detected: a common low-activity level micropattern (cDMN), an anterior high-activity level micropattern (aDMN) and a posterior high-activity level micropattern (pDMN). A dynamic balance across CAMPs emerged during wakefulness and was disrupted in sleep stages. In the slow-wave sleep (SWS) stage, cDMN became the primary activity pattern, whereas aDMN and pDMN were the major activity patterns in the rapid eye movement sleep (REM) stage. Additionally, further investigation revealed phasic relationships between CAMPs and the up-down states of the slow DMN activity in the SWS stage. Our study revealed that the dynamic configurations of CAMPs were highly associated with different stages of wakefulness and provided a potential three-state model to describe the DMN dynamics for wakefulness and alertness.

Impact Statement

In the current study, a novel coactive micropattern method (CAMP) was developed to elucidate fast DMN dynamics during wakefulness and sleep. Our findings demonstrated that the dynamic configurations of DMN activity are specific to different wakefulness stages and provided a three-state DMN CAMP model to depict wakefulness levels, thus revealing a potentially new neurophysiological representation of alertness levels. This work could elucidate the DMN dynamics underlying different stages of wakefulness and have important implications for the theoretical understanding of the neural mechanism of wakefulness and alertness.
On the Validity of the Saccharum Complex and the Saccharinae Subtribe: A Re-assesment
Authors: Evans Dyfed Lloyd , Joshi Shailesh Vinay
Published: July 2020

The ‘Saccharum Complex’ represents an hypothetical collective of species that were supposedly responsible, through interbreeding, for the origins of sugarcane. Though recent phylogenetic studies have cast doubt on the veracity of this hypothesis, it has cast a long shadow over the taxonomics of the Andropogoneae and the Saccharinae subtribe. Though evidence suggests that Saccharum s.s. is comprised of only three true species, according to Kew’s GrassBase there are as many as 34 species inSaccharum s.l. Our recent work has shown that many of these species are millions of years divergent from Saccharum. As the Saccharum complex represents the species that sugarcane breeders attempt to introgress into sugarcane, and as the Saccharinae, in its current form, covers almst 12 million years of Andropogoneae evolution an update on the extents of the Taxonomic and customary groupings is much needed. Based on the latest sequence based phylogenies and the inclusion of traditional taxonomics we develop an integrated view of the Saccharinae + Saccharum complex species in the context of the major groupings within the Andropogoneae. We use this phylogeny to re-circumscribe the limits of both the Saccharinae subtribe and the Saccharum complex group of interbreeding species.
Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays
Authors: El-Baba Tarick J. , Lutomski Corinne A. , Kantsadi Anastassia L. , Malla Tika R. , John Tobias , Mikhailov Victor , Bolla Jani R. , Schofield Christopher J. , Zitzmann Nicole , Vakonakis Ioannis , Robinson Carol V.
Published: July 2020

Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mprocleaves at eleven sites to release non-structural proteins required for viral replication. MProis an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant ofKd= 0.14 ± 0.03 μM, revealing MProhas a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively “flash-frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MProactivity.
Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation
Authors: Esposito CL , Autiero I , Basal MA , Sandomenico A , Ummarino S , Borchiellini M , Ruvo M , Catuogno S , Ebralidze AK , de Franciscis V , Di Ruscio A
Published: July 2020

DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of toxic, unspecific demethylating drugs. Aptamers are novel high affinity targeting ligand molecules. By conjugating the inherent DNMT1 inhibiting capabilities of RNA to an aptamer platform, we generated a first-of-its kind aptamer approach that can target and neutralize DNMT1 function – the aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This novel RNA bio-drug blocks DNA methylation and impairs cancer cell viability.

Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
Structures of the germline-specific Deadhead and Thioredoxin T proteins fromDrosophila melanogasterreveal unique features among Thioredoxins
Authors: Freier Regina , Aragón Eric , Bagiński Błażej , Pluta Radoslaw , Martin-Malpartida Pau , Ruiz Lidia , Condeminas Miriam , Gonzalez Cayetano , Macias Maria J.
Published: July 2020

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. InDrosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome.

Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein.

The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development inDrosophilaand may help in the design of molecular inhibitors through a structure-based approach.

Highlights

We have determined the first structures of the germline-specific Trxs Dhd and TrxT.

Dhd has a highly positively charged surface that facilitates its approximation to DNA and protamine oligomers, to promote chromatin remodeling.

TrxT contains a C-terminal extension, highly unusual in canonical Trxs, mostly unstructured and highly flexible.

The TrxT C-terminal extension partially covers the catalytic site and modulates the redox activity of the protein.

The differences observed in Thioredoxins can help in fine-tuning specific molecules to be active against selected insect species.
An endometrial organoid model ofChlamydia-epithelial and immune cell interactions
Authors: Dolat Lee , Valdivia Raphael H.
Published: July 2020

Our understanding of how the obligate intracellular bacteriumChlamydia trachomatisreprograms the cell biology of host cells in the upper genital tract is largely based on observations made in cell culture with transformed epithelial cell lines. Here we describe a primary spherical organoid system derived from endometrial tissue to recapitulate epithelial cell diversity, polarity, and ensuing responses toChlamydiainfection. Using high-resolution and time-lapse microscopy, we catalogue the infection process in organoids from invasion to egress, including the reorganization of the cytoskeleton and positioning of intracellular organelles. We show this model is amenable to screeningC. trachomatismutants for defects in the fusion of pathogenic vacuoles, the recruitment of intracellular organelles, and inhibition of cell death. Moreover, we reconstructed a primary immune cell response by co-culturing infected organoids with neutrophils, and determined that the effector TepP limits the recruitment of neutrophils to infected organoids. Collectively, our model details a system to study the cell biology ofChlamydiainfections in three dimensional structures that better reflect the diversity of cell types and polarity encountered byChlamydiaupon infection of their animal hosts.

Summary statement

3D endometrial organoids to modelChlamydiainfection and the role of secreted virulence factors in reprogramming host epithelial cells and immune cell recruitment
XLF acts as a flexible connector during non-homologous end joining
Authors: Carney Sean M. , Moreno Andrew T. , Piatt Sadie C. , Cisneros-Aguirre Metztli , Lopezcolorado Felicia Wednesday , Stark Jeremy M. , Loparo Joseph J.
Published: July 2020

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
Identification and characterization of CD4+ T cell epitopes after Shingrix vaccination
Authors: Voic Hannah , de Vries Rory D. , Sidney John , Rubiro Paul , Moore Erin , Phillips Elizabeth , Mallal Simon , Schwan Brittany , Weiskopf Daniela , Sette Alessandro , Grifoni Alba
Published: July 2020

Infections with varicella zoster virus (VZV), a member of the Herpesviridae family, are associated with a range of clinical manifestations. Primary infection with VZV causes chicken pox, and due to the virus’s capacity to remain latent in neurons, it can reactivate later in life causing herpes zoster (HZ), also known as shingles. Two different vaccines have been developed to prevent HZ, one based on a live attenuated VZV strain (Zostavax) and the other on adjuvanted gE recombinant protein (Shingrix). While Zostavax efficacy wanes with age, Shingrix protection retains its efficacy in elderly subjects (80 years of age and beyond). In this context, it is of much interest to understand if there is a role for T cell immunity in differential clinical outcome, and if there is a correlate of protection between T cell immunity and Shingrix efficacy. In this study, we characterized Shingrix specific ex vivo CD4 T cell responses in the context of natural exposure and HZ vaccination using pools of predicted epitopes. We show that T cell reactivity following natural infection and Zostavax vaccination dominantly targets non-structural proteins (NS), while Shingrix vaccination redirects dominant reactivity to target gE. We mapped the gE-specific responses following Shingrix vaccination to 89 different gE epitopes, 34 of which accounted for 80% of the response. Using antigen presentation assays and single HLA molecule transfected lines, we experimentally determined HLA restrictions for 94 different donor/peptide combinations. Finally, we used our results as a training set to assess strategies to predict restrictions based on measured or predicted HLA binding and the corresponding HLA types of responding subjects.

Importance

Understanding the T cell profile associated with the protection observed in elderly vaccinees following Shingrix vaccination is relevant to the general definition of correlates of vaccine efficacy. Our study enables these future studies by clarifying patterns of immunodominance associated with Shingrix vaccination, as opposed to natural infection or Zostavax vaccination. Identification of epitopes recognized by Shingrix-induced CD4 T cells and their associated HLA restrictions enables the generation of tetrameric staining reagents and, more broadly, the capability to characterize specificity, magnitude and phenotype of VZV specific T cells.
A spatially-tracked single cell transcriptomics map of neuronal networks in the intrinsic cardiac nervous system
Authors: Moss Alison , Robbins Shaina , Achanta Sirisha , Kuttippurathu Lakshmi , Turick Scott , Nieves Sean , Hanna Peter , Smith Elizabeth H. , Hoover Donald B. , Chen Jin , Cheng Zixi (Jack) , Ardell Jeffrey L. , Shivkumar Kalyanam , Schwaber James S. , Vadigepalli Rajanikanth
Published: July 2020

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion - the right atrial ganglionic plexus (RAGP) that is a critical mediator of vagal control of the sinoatrial node (SAN) activity. We developed a 3D representation of RAGP with extensive mapping of neurons and used neuronal tracing to identify the spatial distribution of the subset of neurons that project to the SAN. RNAseq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to CNS neuronal subtypes. High-throughput qPCR of hundreds of laser capture microdissected single neurons led to a surprising finding that cholinergic and catecholaminergic neuronal markersThandChatwere correlated, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. Interestingly, no single gene or module was an exclusive marker of RAGP neuronal connectivity to SAN. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP, informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.
Non-parametric modelling of temporal and spatial counts data from RNA-seq experiments
Authors: BinTayyash Nuha , Georgaka Sokratia , John ST , Ahmed Sumon , Boukouvalas Alexis , Hensman James , Rattray Magnus
Published: July 2020

Abstract

Motivation

The negative binomial distribution has been shown to be a good model for counts data from both bulk and single-cell RNA-sequencing (RNA-seq). Gaussian process (GP) regression provides a useful non-parametric approach for modeling temporal or spatial changes in gene expression. However, currently available GP regression methods that implement negative binomial likelihood models do not scale to the increasingly large datasets being produced by single-cell and spatial transcriptomics.

Results

The GPcounts package implements GP regression methods for modelling counts data using a negative binomial likelihood function. Computational efficiency is achieved through the use of variational Bayesian inference. The GP function models changes in the mean of the negative binomial likelihood through a logarithmic link function and the dispersion parameter is fitted by maximum likelihood. We validate the method on simulated time course data, showing that it is better able to identify changes in over-dispersed counts data than methods based on Gaussian or Poisson likelihoods. To demonstrate temporal inference, we apply GPcounts to single-cell RNA-seq datasets after pseudotime and branching inference. To demonstrate spatial inference, we apply GPcounts to data from the mouse olfactory bulb to identify spatially variable genes and compare to two published GP methods. We also provide the option of modelling additional dropout using a zero-inflated negative binomial. Our results show that GPcounts can be used to model temporal and spatial counts data in cases where simpler Gaussian and Poisson likelihoods are unrealistic.

Availability

GPcounts is implemented using the GPflow library in Python and is available athttps://github.com/ManchesterBioinference/GPcountsalong with the data, code and notebooks required to reproduce the results presented here.

Contact

nuha.bintayyash@manchester.ac.ukormagnus.rattray@manchester.ac.uk
Polygenic Risk Score for Smoking is associated with Externalizing Psychopathology and Disinhibited Personality Traits but not Internalizing Psychopathology in Adolescence
Authors: Hicks Brian M. , Clark D. Angus , Deak Joseph D. , Liu Mengzhen , Durbin C. Emily , Schaefer Jonathan D. , Wilson Sylia , Iacono William G. , McGue Matt , Vrieze Scott I.
Published: July 2020

Importance

Large consortia of genome wide association studies have yielded more accurate polygenic risk scores (PRS) that aggregate the small effects of many genetic variants to characterize the genetic architecture of disorders and provide a personalized measure of genetic risk.

Objective

We examined whether a PRS for smoking measured genetic risk for general behavioral disinhibition by estimating its associations with externalizing and internalizing psychopathology and related personality traits. We examined these associations at multiple time points in adolescence using more refined phenotypes defined by stable characteristics across time and at young ages, which reduced potential confounds associated with cumulative exposure to substances and reverse causality.

Methods

Random intercept panel models were fit to symptoms of conduct disorder, oppositional defiant disorder, major depressive disorder (MDD), and teacher ratings of externalizing and internalizing problems and personality traits at ages 11, 14, and 17 years-old in the Minnesota Twin Family Study (N= 3225).

Results

The smoking PRS had strong associations with the random intercept factors for all the externalizing measures (mean standardizedβ= .27), agreeableness (β=−.22, 95% CI: −.28, −.16), and conscientiousness (β=−.19, 95% CI: −.24, −.13), but was not significantly associated with the internalizing measures (meanβ= .06) or extraversion (β=.01, 95% CI: −.05, .07). After controlling for smoking at age 17, the associations with the externalizing measures (meanβ= .13) and personality traits related to behavioral control (meanβ= −.10) remained statistically significant.

Conclusions and Relevance

The smoking PRS measures genetic influences that contribute to a spectrum of phenotypes related to behavioral disinhibition including externalizing psychopathology and normal-range personality traits related to behavioral control, but not internalizing psychopathology. Continuing to identify the correlates and delineate the mechanisms of the genetic influences associated with disinhibition could have substantial impact in mitigating a variety of public health problems (e.g., mental health, academic achievement, criminality).

Key Points

Question

Does a polygenic risk scores (PRS) for smoking measure genetic risk for behavioral disinhibition in general?

Findings

The smoking PRS was associated with externalizing psychopathology and personality traits related to behavioral control, but not internalizing psychopathology and extraversion during adolescence, even after controlling for smoking status.

Meaning

The smoking PRS measures genetic influences on behavioral disinhibition in general which is associated with a variety of important outcomes including mental health, academic success, and criminality.
Genetically Determined Strength of Natural Killer Cells is Enhanced by Adaptive HLA class I Admixture in East Asians
Authors: Deng Zhihui , Zhen Jianxin , Harrison Genelle F. , Zhang Guobin , Chen Rui , Sun Ge , Yu Qiong , Nemat-Gorgani Neda , Guethlein Lisbeth A. , He Liumei , Tang Mingzhong , Gao Xiaojiang , Cai Siqi , Shortt Jonathan A. , Gignoux Christopher R. , Carrington Mary , Zou Hongyan , Parham Peter , Hong Wenxu , Norman Paul J.
Published: July 2020

Human natural killer (NK) cells are essential for controlling infection, cancer and fetal development. NK cell functions are modulated by interactions between polymorphic inhibitory killer cell immunoglobulin-like receptors (KIR) and polymorphic HLA-A, -B and -C ligands expressed on tissue cells. AllHLA-Calleles encode a KIR ligand and contribute to reproduction and immunity. In contrast, only someHLA-Aand-Balleles encode KIR ligands and they focus on immunity. By high-resolution analysis ofKIRandHLA-A,-Band-Cgenes, we show that the Chinese Southern Han are significantly enriched for interactions between inhibitory KIR and HLA-A and -B. This enrichment has had substantial input through population admixture with neighboring populations, who contributedHLA class Ihaplotypes expressing the KIR ligands B*46:01 and B*58:01, which subsequently rose to high frequency by natural selection. Consequently, over 80% of Southern HanHLAhaplotypes encode more than one KIR ligand. Complementing the high number of KIR ligands, the Chinese Southern HanKIRlocus combines a high frequency of genes expressing potent inhibitory KIR, with a low frequency of those expressing activating KIR. The Southern Han centromericKIRregion encodes strong, conserved, inhibitory HLA-C specific receptors, and the telomeric region provides a high number and diversity of inhibitory HLA-A and -B specific receptors. In all these characteristics, the Southern Han represent other East Asians, whose NK cell repertoires are thus enhanced in quantity, diversity and effector strength, likely through natural selection for resistance to endemic viral infections.