bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic application of continuous granulation and selective laser sintering 3D printing for the development of pharmaceutical dosage forms with enhanced dissolution rates and physical properties
Authors: Thakkar Rishi , Zhang Yu , Zhang Jiaxiang , Maniruzzaman Mohammed
Published: February 2021

This study demonstrated the first case of combining novel continuous granulation with powder-based pharmaceutical 3-dimensional (3D) printing processes to enhance the dissolution rate and physical properties of a poorly water-soluble drug. Powder bed fusion (PBF) and binder jetting 3D printing processes have gained much attention in pharmaceutical dosage form manufacturing in recent times. Although powder bed-based 3D printing platforms have been known to face printing and uniformity problems due to the inherent poor flow properties of the pharmaceutical physical mixtures (feedstock). Moreover, techniques such as binder jetting currently do not provide any solubility benefits to active pharmaceutical ingredients (APIs) with poor aqueous solubility (>40% of marketed drugs). For this study, a hot-melt extrusion-based versatile granulation process equipped with UV-Vis process analytical technology (PAT) tools for the in-line monitoring of critical quality attributes (i.e., solid-state) of indomethacin was developed. The collected granules with enhanced flow properties were mixed with vinylpyrrolidone-vinyl acetate copolymer and a conductive excipient for efficient sintering. These mixtures were further characterized for their bulk properties observing an excellent flow and later subjected to a PBF-3D printing process. The physical mixtures, processed granules, and printed tablets were characterized using conventional as well as advanced solid-state characterization. These characterizations revealed the amorphous nature of the drug in the processed granules and printed tablets. Further, thein vitrorelease testing of the tablets with produced granules as a reference standard depicted a notable solubility advantage (100% drug released in 5 minutes at >pH 6.8) over the pure drug and the physical mixture. Our developed system known as DosePlus combines innovative continuous granulation and PBF-3D printing process which can potentially improve the physical properties of the bulk drug and formulations in comparison to when used in isolation. This process can further find application in continuous manufacturing of granules and additive manufacturing of pharmaceuticals to produce dosage forms with excellent uniformity and solubility advantage.

Abstract Figure
Forest loss in Indonesian New Guinea: trends, drivers, and outlook
Authors: Gaveau David L.A. , Santos Lucas , Locatelli Bruno , Salim Mohammad A. , Husnayaen Husnayaen , Meijaard Erik , Heatubun Charlie , Sheil Douglas
Published: February 2021

The rich forests of Indonesian New Guinea are threatened. We used satellite data to examine annual forest loss, road development and plantation expansion from 2001 to 2019, then developed a model to predict future deforestation in this understudied region. In 2019, 34.29 million hectares (Mha), or 83% of Indonesian New Guinea, supported old-growth forest. Over nineteen years, two percent (0.75 Mha) were cleared: 45% (0.34 Mha) converted to industrial plantations, roads, mine tailings, or other uses near cities; 55% (0.41 Mha) cleared by transient processes including selective natural timber extraction, inland water bodies-related processes, fires, and shifting agriculture. Industrial plantations expanded by 0.23 Mha, with the majority (0.21 Mha; 28% of forest loss) replacing forests and reaching 0.28 Mha in 2019 (97% oil palm; 3% pulpwood). The Trans-Papua Highway, a ~4,000 km national investment project, increased by 1,554 km. Positive correlations between highway and plantations expansion indicate these are linked processes. Plantations and roads grew rapidly after 2011, peaked in 2015/16, and declined thereafter. Indonesian government allocated 2.62 Mha of land for the development of industrial plantations (90% oil palm 10% pulpwood) of which 74% (1.95 Mha) remained forest in 2019. A spatial model predicts that an additional 4.5 Mha of forest could be cleared by 2036 if Indonesian New Guinea follows similar relationships to Indonesian Borneo. We highlight the opportunities for policy reform and the importance of working with indigenous communities, local leaders, and provincial government to protect the biological and cultural richness still embodied in this remarkable region.
Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures
Authors: Shen Zhengnan , Ratia Kiira , Cooper Laura , Kong Deyu , Lee Hyun , Kwon Youngjin , Li Yangfeng , Alqarni Saad , Huang Fei , Dubrovskyi Oleksii , Rong Lijun , Thatcher Gregory , Xiong Rui
Published: February 2021

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50< 10 µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a “BL2 groove” that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.
How plants minimize somatic evolution
Authors: Kiss Máté , Szöllősi Gergely J. , Derényi Imre
Published: February 2021

A remarkable property of plants is their ability to accumulate mutations at a very slow pace despite their potentially long lifespans, during which they continually form buds, each with the potential to become a new branch. Because replication errors in cell division represent an unavoidable source of mutations, minimizing mutation accumulation requires the minimization of cell divisions. Here we show that there exists a well defined theoretical minimum for the branching cost, defined as the number of cell divisions necessary for the creation of each branch. Most importantly, we also show that this theoretical minimum can be closely approached by a simple pattern of cell divisions in the meristematic tissue of apical buds during the generation of novel buds. Both the optimal pattern of cell divisions and the associated branching cost are consistent with recent experimental data, suggesting that plant evolution has led to the discovery of this mechanism.
APOBEC mutagenesis inhibits breast cancer growth through induction of a T cell-mediated antitumor immune response
Authors: DiMarco Ashley V. , Qin Xiaodi , Van Alsten Sarah , McKinney Brock , Garcia Nina Marie G. , Force Jeremy , Hanks Brent A. , Troester Melissa A. , Owzar Kouros , Xie Jichun , Alvarez James V.
Published: February 2021

The APOBEC family of cytidine deaminases is one of the most common endogenous sources of mutations in human cancer. Genomic studies of tumors have found that APOBEC mutational signatures are particularly enriched in the HER2 subtype of breast cancer and have been associated with immunotherapy response in diverse cancer types. However, the direct consequences of APOBEC mutagenesis on the tumor immune microenvironment have not been thoroughly investigated. To address this, we developed syngeneic murine mammary tumor models with inducible expression of APOBEC3B. We found that APOBEC activity induces an antitumor adaptive immune response and CD4+T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. Consistent with the observed immune infiltration in APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures as a biomarker predicting immunotherapy response in HER2-positive breast cancers.

SIGNIFICANCE

APOBEC mutational signatures are observed in many cancers, yet the consequences of these mutations on the tumor immune microenvironment are not well understood. Using a novel mouse model, we show that APOBEC activity sensitizes HER2-driven mammary tumors to checkpoint inhibition and could inform immunotherapy treatment strategies for HER2-positive breast cancer patients.
Exon 9LEPRGene SNP Polymorphism of Hybrid Chickens F2KambroCrossbreeds of ♀ F1Kambrowith ♂ F1Kambro
Authors: Mahardhika I Wayan Swarautama , Daryono Budi Setiadi
Published: February 2021

The implementation of the T-ARMS PCR method in the detection of single nucleotide polymorphisms (SNPs) in theLEPRgene in chicken DNA samples has never been conducted. This research aims to design a specific protocol for exon 9LEPRgene SNPs detection and detectLEPRgene expression orLEPRSNPs inPelungchicken samples, F1Pelung, Layer, Broiler Cobb 500, F1Kambrochicken and F2Kambrochicken using the T-ARMS PCR method. Determination ofLEPRgene correlation degree on Body Weight (BT) and Egg Productivity (PT) in F1Kambro population and F2Kambro. Qualitative phenotype parameters showed six groups of segregated phenotypes compared to F1Kambrochicken. Growth of F2Kambrochicken weight reached 753.36 ± 155.31 grams in 8 weeks was not significant for F1Kambrochicken due to inbreeding depression (Fx = 25%, IR = 4.925%) and transversion of ALEPRallele mutations. Specific protocol detection of exon 9LEPRgene SNPs using the T-ARMS PCR method can detect C127ALEPRmutations with IP: OP ratio 10:1 pmol / µM, chicken DNA template concentration of 100 ng / µL with annealing temperature of 55.7° C / 30s. The transversion mutation of C127A ofLEPRexon 9 SNP were detected in DNA samples of F1Kambrohens (80%), F2Kambroroosters (20%), Broiler Cobb 500 hens (75%). The mutations were not detected in Layer,Pelung Blirik Hitamchicken and F1Pelungpopulations.
Phylogenomic subsampling and the search for phylogenetically reliable loci
Authors: Koch Nicolás Mongiardino
Published: February 2021

Phylogenomic subsampling is a procedure by which small sets of loci are selected from large genome-scale datasets and used for phylogenetic inference. This step is often motivated by either computational limitations associated with the use of complex inference methods, or as a means of testing the robustness of phylogenetic results by discarding loci that are deemed potentially misleading. Although many alternative methods of phylogenomic subsampling have been proposed, little effort has gone into comparing their behavior across different datasets. Here, I calculate multiple gene properties for a range of phylogenomic datasets spanning animal, fungal and plant clades, uncovering a remarkable predictability in their patterns of covariance. I also show how these patterns provide a means for ordering loci by both their rate of evolution and their relative phylogenetic usefulness. This method of retrieving phylogenetically useful loci is found to be among the top performing when compared to alternative subsampling protocols. Relatively common approaches such as minimizing potential sources of systematic bias or increasing the clock-likeness of the data are found to fare worse than selecting loci at random. Likewise, the general utility of rate-based subsampling is found to be limited: loci evolving at both low and high rates are among the least effective, and even those evolving at optimal rates can still widely differ in usefulness. This study shows that many common subsampling approaches introduce unintended effects in off-target gene properties, and proposes an alternative multivariate method that simultaneously optimizes phylogenetic signal while controlling for known sources of bias.
Separate functional subnetworks of excitatory neurons show preference to periodic and random sound structures
Authors: Mehra Muneshwar , Mukesh Adarsh , Bandyopadhyay Sharba
Published: February 2021

Auditory cortex (ACX) neurons are sensitive to spectro-temporal sound patterns and violations in patterns induced by rare stimuli embedded within streams of sounds. We investigate the auditory cortical representation of repeated presentations of sequences of sounds with standard stimuli (common) with an embedded deviant (rare) stimulus in two conditions – Periodic (Fixed deviant position) or Random (Random deviant position), using extracellular single-unit and 2-photon Ca+2imaging recordings in Layer 2/3 neurons of the mouse ACX. In the population average, responses increased over repetitions in the Random-condition and were suppressed or did not change in the Periodic-condition, showing irregularity preference. A subset of neurons also showed the opposite behavior, indicating regularity preference. Pairwise noise correlations were higher in Random-condition over Periodic-condition, suggesting the role of recurrent connections. 2-photon Ca+2imaging of excitatory (EX) and parvalbumin-positive (PV) and somatostatin-positive (SOM) inhibitory neurons, showed different categories of adaptation or change in response over repetitions (categorized by the sign of the slope of change) as observed with single units. However, the examination of functional connectivity between pairs of neurons of different categories showed that EX-PV connections behaved opposite to the EX-EX and EX-SOM pairs that show more functional connections outside category in Random-condition than Periodic-condition. Finally considering Regularity preference, Irregularity preference and no preference categories, showed that EX-EX and EX-SOM connections to be in largely separate functional subnetworks with the different preferences, while EX-PV connections were more spread. Thus separate subnetworks could underly the coding of periodic and random sound sequences.

Significance Statement

Studying how the ACX neurons respond to streams of sound sequences help us understand the importance of changes in dynamic acoustic noisy scenes around us. Humans and animals are sensitive to regularity and its violations in sound sequences. Psychophysical tasks in humans show that auditory brain differentially responds to periodic and random structures, independent of the listener’s attentional states. Here we show that mouse ACX L2/3 neurons detect a change and respond differentially to changing patterns over long-time scales. The differential functional connectivity profile obtained in response to two different sound contexts, suggest the stronger role of recurrent connections in the auditory cortical network. Furthermore, the excitatory-inhibitory neuronal interactions can contribute to detecting the changing sound patterns.
Newly developedad hocmolecular assays show how eDNA can witness and anticipate the monk seal recolonization of central Mediterranean
Authors: Valsecchi Elena , Coppola Emanuele , Pires Rosa , Parmegiani Andrea , Casiraghi Maurizio , Galli Paolo , Bruno Antonia
Published: February 2021

The monk sealMonachus monachusis the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness, and records are biased towards occasional, mostly coastal, encounters. Nowadays molecular techniques allow us to detect and quantify minute amounts of DNA traces released in the environment (eDNA) by any organism. We present three qPCR-assays targeting the monk seal mitogenome. The assays were soundly tested on an extensive and diversified sample set (n=73), including positive controls from Madeira breeding population collected during the peak of abundance, and two opportunistic Mediterranean eDNA-sample collections (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% and 66.7% of the samples collected in the Tyrrhenian from a ferry platform (2018-2019) and in the Pelagie archipelago -Strait of Sicily- (2020) respectively, anticipating (up to 2 year) visual observations occurred subsequently in proximity of the sampled areas. In the Tyrrhenian, detection occurrence increased between 2018 and 2019. Monk seal DNA recoveries were commoner in night-time ferry-samples, suggesting nocturnal predatory activity in pelagic waters. The proposed technique provides a non-invasive and yet highly-sensitive tool for defining the monk seal actual distribution and home range, its recovery rate and pinpoint coastal/offshore localities where prioritizing conservation, research, citizen science and education initiatives.
Characterizing white matter alterations in drug-naïve de novo Parkinson’s disease with diffusion MRI
Authors: Xiao Yiming , Peters Terry M. , Khan Ali R.
Published: February 2021

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by a range of motor and non-motor symptoms, often with the motor dysfunction initiated unilaterally. Knowledge regarding disease-related alterations in white matter pathways can effectively help improve the understanding of the disease and propose targeted treatment strategies. Microstructural imaging techniques, including diffusion tensor imaging (DTI), allows inspection of white matter integrity to study the pathogenesis of various neurological conditions. Previous voxel-based analyses with DTI measures, such as fractional anisotropy and mean diffusivity have uncovered changes in brain regions that are associated with PD, but the conclusions were inconsistent, partially due to small patient cohorts and the lack of consideration for clinical laterality onset, particularly in early PD. Fixel-based analysis (FBA) is a recent framework that offers tract-specific insights regarding white matter health, but very few FBA studies on PD exist. We present a study that reveals strengthened and weakened white matter integrity that is subject to symptom laterality in a large drug-naïvede novoPD cohort using complementary DTI and FBA measures. The findings suggest that the disease gives rise to both functional degeneration and the creation of compensatory networks in the early stage.
Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases
Authors: Mindrebo Jeffrey T. , Chen Aochiu , Kim Woojoo E. , Re Rebecca N. , Davis Tony D. , Noel Joseph P. , Burkart Michael D.
Published: February 2021

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures ofEscherichia colielongating type II FAS KSs, FabF and FabB, in complex withE. coliACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.
Constructing local Cell Sepcific Networks from Single Cell Data
Authors: Wang Xuran , Choi David , Roeder Kathryn
Published: February 2021

Single-cell RNA sequencing facilitates investigations of gene co-expression networks at the cellular level potentially yielding unique biological insights, but the estimation problem is challenging. We develop a non-parametric approach to estimate cell-specific networks for each cell and cell type and propose novel downstream analyses. The individual networks preserve the cellular heterogeneity and facilitate testing for differences between cell groups. To further our understanding of autism spectrum disorder, we examine the evolution of gene networks in fetal brain cells and compare the gene networks of cells sampled from case and control subjects to reveal patterns in gene co-expression.
Influence of warming temperatures on coregonine embryogenesis within and among species
Authors: Stewart Taylor R. , Mäkinen Mikko , Goulon Chloé , Guillard Jean , Marjomäki Timo J. , Lasne Emilien , Karjalainen Juha , Stockwell Jason D.
Published: February 2021

The greatest known global response of lakes to climate change has been an increase in water temperatures. The responses of many lake fishes to warming water temperatures are projected to be inadequate to counter the speed and magnitude of climate change. We experimentally evaluated the responses of embryos from a group of cold, stenothermic fishes (Salmonidae Coregoninae) to increased incubation temperatures. Study groups included cisco (Coregonus artedi) from lakes Superior and Ontario (USA), and vendace (C. albula) and European whitefish (C. lavaretus) from Lake Southern Konnevesi (Finland). Embryos from artificial crossings were incubated at water temperatures of 2.0, 4.5, 7.0, and 9.0°C, and their responses were quantified for developmental and morphological traits. Embryo survival, incubation period, and length-at- hatch were inversely related to incubation temperature whereas yolk-sac volume increased with incubation temperature within study groups. However, varying magnitudes of responses among study groups suggested differential levels of developmental plasticity to climate change. Differential levels of parental effects indicate genetic diversity may enable all study groups to adapt to cope with some degree of changing environmental conditions. Our results suggest that the coregonines sampled within and among systems may have a wide range of embryo responses to warming incubation conditions.
Systematic beach monitoring as a health assessment tool: cetacean morbillivirus under non-epizootic circumstances in stranded dolphins
Authors: Marutani Victor H. B. , Miyabe Flávia , Alfieri Alice F. , Domit Camila , de Matos Andressa M. R. N. , Filho Mário R. C. M. , Bracarense Ana P. F. R. L.
Published: February 2021

Cetacean morbillivirus (CeMV) was identified as the etiologic agent of several epizootic episodes worldwide. Most of these studies are based on unusual mortality events or identification of new viral strains. We investigated the occurrence of CeMV under non-epizootic circumstances at a world heritage in Southern Brazil by a combination of pathologic, immunohistochemical and molecular assays. From 325 stranded cetaceans, 40 were included. Guiana dolphin (Sotalia guianensis) was the species most frequent. Interstitial pneumonia and non-suppurative encephalitis were the main pathologic findings associated to CeMV infection. Intracytoplasmic immunolabeling anti-CeMV was observed mainly in lungs and lymph nodes. All samples were negative in RT-PCR assay. Diagnosis of CeMV is challenging in areas where epizootic episodes have not been recorded and due topostmortemchanges. We observed a CeMV prevalence of 27.5%. The results described here increase the knowledge about CeMV under non-epizootic conditions in Brazil.

Article Summary Line

We observed a prevalence of 27.5% of CeMV in a World Heritage site of Paraná’s coast. The results indicate an increase in the prevalence of CeMV at this region and, possibly, a degradation of marine ecosystem. Marine mammals are sentinels of marine environment and the ocean health is inextricably linked to human health on a global scale.
StrainGE: A toolkit to track and characterize low-abundance strains in complex microbial communities
Authors: van Dijk Lucas R. , Walker Bruce J. , Straub Timothy J. , Worby Colin J. , Grote Alexandra , Schreiber Henry L. , Anyansi Christine , Pickering Amy J. , Hultgren Scott J. , Manson Abigail L. , Abeel Thomas , Earl Ashlee M.
Published: February 2021

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify nearest known references and find variants for multiple conspecific strains within a sample at relative abundances below 0.1% in typical metagenomic datasets.
Structure of an infectious mammalian prion
Authors: Kraus Allison , Hoyt Forrest , Schwartz Cindi L. , Hansen Bryan , Hughson Andrew G. , Artikis Efrosini , Race Brent , Caughey Byron
Published: February 2021

Classical mammalian prions are assemblies of prion protein molecules that are extraordinarily transmissible, with a microgram of protein containing up to 108lethal doses of infectivity1,2. Unlike most other pathologic and amyloidogenic proteins, prions typically contain glycolipid anchors3and abundant asparagine‐linked glycans4‐6. The infectious nature, complexity, and biophysical properties of prions have complicated structural analyses and stymied any prior elucidation of 3D conformation at the polypeptide backbone level7. Here we have determined the structure of the core of a fully infectious, brain‐derived prion by cryo‐electron microscopy with ∼3.1 Å resolution. The purified prions are amyloid fibrils comprised of monomers assembled with parallel in‐register intermolecular beta sheets and connecting chains. Residues ∼95‐227 of each monomer provide one rung of the ordered fibril core, with the glycans and glycolipid anchor projecting from the lateral surfaces of the fibril. The fibril ends, where prion growth occurs, are formed by single monomers in an extended serpentine combination of β‐ arches, a Greek key, and loops that presumably template the refolding of incoming monomers. Our results describe an atomic model to underpin detailed molecular hypotheses of how pathologic prion proteins can propagate as infectious agents, and how such propagation and associated pathogenesis might be impeded.
In vitroefficacy of Artemisia extracts against SARS-CoV-2
Authors: Nie Chuanxiong , Trimpert Jakob , Moon Sooyeon , Haag Rainer , Gilmore Kerry , Kaufer Benedikt B. , Seeberger Peter H.
Published: February 2021

Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts ofArtemisia annuaandArtemisia afrahave been widely used in Africa in efforts to prevent and fight COVID-19 infections. We sought to study the ability of differentA. annuaandA. afraextracts and the Covid-Organics drink produced in Madagascar to inhibit SARS-CoV-2 and feline coronavirus (FCoV) replicationin vitro.Several extracts as well as Covid-Organics inhibit SARS-CoV-2 and FCoV replication at concentrations that did not affect cell viability. It remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral replication following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment in patients.
Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10kb C-to-T splicing mutation
Authors: Oren Yifat S. , Sinai Michal Irony-Tur , Golec Anita , Barchad-Avitzur Ofra , Mutyam Venkateshwar , Li Yao , Hong Jeong , Ozeri-Galai Efrat , Hatton Aurélie , Reiter Joel , Sorscher Eric J. , Wilton Steve D. , Kerem Eitan , Rowe Steven M. , Sermet-Gaudelus Isabelle , Kerem Batsheva
Published: February 2021

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2′-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients’ HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43±8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2’-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.
Subcellular dynamics studies reveal how tissue-specific distribution patterns of iron are established in developing wheat grains
Authors: Sheraz Sadia , Wan Yongfang , Venter Eudri , Verma Shailender K , Xiong Qing , Waites Joshua , Connorton James M , Shewry Peter R , Moore Katie L , Balk Janneke
Published: February 2021

Understanding iron trafficking in plants is key to enhancing the nutritional quality of crops. Due to the difficulty of imaging iron in transit, little is known about iron translocation and distribution in developing seeds. A novel approach, combining57Fe isotope labelling and NanoSIMS, was used to visualize iron translocation dynamics at the subcellular level in wheat grain,Triticum aestivumL. We were able to track the main route of iron from maternal tissues to the embryo through different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, as confirmed by histological staining and TEM-EDS. Virtually all iron was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Aleurone cells contained a new type of iron body, highly enriched in57Fe, and most likely represents iron-nicotianamine being delivered to phytate globoids. Correlation with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification efforts.
SUMOylation of the chromodomain factor MRG-1 inC. elegansaffects chromatin-regulatory dynamics
Authors: Baytek Gülkiz , Blume Alexander , Demirel Funda Gerceker , Bulut Selman , Mertins Philipp , Tursun Baris
Published: February 2021

Epigenetic mechanisms to control chromatin accessibility and structure is important for gene expression in eukaryotic cells. Chromatin regulation ensures proper development and cell fate specification but is also essential later in life. Modifications of histone proteins as an integral component of chromatin can promote either gene expression or repression, respectively. Proteins containing specific domains such as the chromodomain recognize mono-, di- or tri-methylated lysine residues on histone H3. The chromodomain protein MRG-1 inCaenorhabditis elegansis the ortholog of mammalian MRG15, which belongs to theMORF4RelatedGene (MRG) family in humans. InC. elegansMRG-1 predominantly binds methylated histone H3 lysine residues at position 36 (H3K36me3). MRG-1 is important during germline maturation and for safeguarding the germ cell identity. However, it lacks enzymatic activity and depends on protein-protein interaction to cooperate with other factors to regulate chromatin. To elucidate the variety of MRG-1 interaction partners we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with mass-spectrometry. Besides previously described and novel interactions with other proteins, we also detected a strong association with the Small Ubiquitin-like Modifier (SUMO). Since SUMO is known to be attached to proteins in order to modulate the target proteins activity we assessed whether MRG-1 is post-translationally modified by SUMOylation. Notably, we provide evidence that MRG-1 is indeed SUMOylated and that this post-translational modification influences the chromatin-binding profile of MRG-1 in theC. elegansgenome. Our presented study hints towards an important role of SUMOylation in the context of epigenetic regulation via the chromodomain protein MRG-1, which may be a conserved phenomenon also in mammalian species.
CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors
Authors: Arora Reety , Gupta Komal , Vijaykumar Anjali
Published: February 2021

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration (∼80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together byin vitrocleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially deliveredin vivofor advancing MCPyV positive MCC treatment in the future.
Close to optimal cell sensing ensures the robustness of tissue differentiation process: the avian photoreceptor mosaic case
Authors: Barua Arnab , Beygi Alireza , Hatzikirou Haralampos
Published: February 2021

The way that progenitor cell fate decisions and the associated environmental sensing are regulated to ensure the robustness of the spatial and temporal order in which cells are generated towards a fully differentiating tissue still remains elusive. Here, we investigate how cells regulate their sensing intensity and radius to guarantee the required thermodynamic robustness of a differentiated tissue. In particular, we are interested in finding the conditions where dedifferentiation at cell level is possible (microscopic reversibility) but tissue maintains its spatial order and differentiation integrity (macroscopic irreversibility). In order to tackle this, we exploit the recently postulated Least microEnvironmental Uncertainty Principle (LEUP) to develop a theory of stochastic thermodynamics for cell differentiation. To assess the predictive and explanatory power of our theory, we challenge it against the avian photoreceptor mosaic data. By calibrating a single parameter, the LEUP can predict the cone color spatial distribution in the avian retina and, at the same time, suggest that such a spatial pattern is associated with quasi-optimal cell sensing. By means of the stochastic thermodynamics formalism, we find out that thermodynamic robustness of differentiated tissues depends on cell metabolism and cell sensing properties. In turn, we calculate the limits of the cell sensing radius that ensure the robustness of differentiated tissue spatial order. Finally, we further constrain our model predictions to the avian photoreceptor mosaic.
Fast and Accurate Distance-based Phylogenetic Placement using Divide and Conquer
Authors: Balaban Metin , Jiang Yueyu , Roush Daniel , Zhu Qiyun , Mirarab Siavash
Published: February 2021

Phylogenetic placement of query samples on an existing phylogeny is increasingly used in molecular ecology, including sample identification and microbiome environmental sampling. As the size of available reference trees used in these analyses continues to grow, there is a growing need for methods that place sequences on ultra-large trees with high accuracy. Distance-based placement methods have recently emerged as a path to provide such scalability while allowing flexibility to analyze both assembled and unassembled environmental samples. In this paper, we introduce a distance-based phylogenetic placement method, APPLES-2, that is more accurate and scalable than existing distance-based methods and even some of the leading maximum likelihood methods. This scalability is owed to a divide-and-conquer technique that limits distance calculation and phylogenetic placement to parts of the tree most relevant to each query. The increased scalability and accuracy enables us to study the effectiveness of APPLES-2 for placing microbial genomes on a data set of 10,575 microbial species using subsets of 381 marker genes. APPLES-2 has very high accuracy in this setting, placing 97% of query genomes within three branches of the optimal position in the species tree using 50 marker genes. Our proof of concept results show that APPLES-2 can quickly place metagenomic scaffolds on ultra-large backbone trees with high accuracy as long as a scaffold includes tens of marker genes. These results pave the path for a more scalable and widespread use of distance-based placement in various areas of molecular ecology.
ExpandingN-Glycopeptide Identifications by Fragmentation Prediction and Glycome Network Smoothing
Authors: Klein Joshua , Carvalho Luis , Zaia Joseph
Published: February 2021

Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a layered approach to glycopeptide fragmentation modeling that improvesN-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further. We demonstrate our techniques on a pair of previously published datasets, showing the performance gains at each stage of optimization, as well as its flexibility in glycome definition and search space complexity. These techniques are provided in the open-source glycomics and glycoproteomics platform GlycReSoft available athttps://github.com/mobiusklein/glycresoft.
Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription
Authors: Asfaha Jonathan B. , Örd Mihkel , Carlson Christopher R. , Faustova Ilona , Loog Mart , Morgan David O.
Published: February 2021

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates1–3. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle4–7, together with variations in substrate docking to sites on associated cyclin and Cks subunits3, 6, 8–10. Many substrates are modified at multiple sites to provide more complex regulation9, 11–14. Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of genes required for mitotic progression15, 16. As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback17–20. Consistent with these findings, we show that low Cdk1 activity promotesCLB2expression at mitotic entry. We also find, however, thatCLB2expression is inhibited by high levels of Cdk1 activity in a mitotic arrest. Inhibition is accompanied by Ndd1 degradation, and we present evidence that high mitotic Cdk1-Clb2 activity generates phosphodegrons on Ndd1, leading to its degradation. Complete Ndd1 phosphorylation by the Clb2-Cdk1-Cks1 complex requires the phosphothreonine-binding site of Cks1, as well as a novel phosphate-binding pocket on the cyclin Clb221. We therefore propose that initial phosphorylation by Cdk1 primes the protein for secondary phosphorylation at phosphodegrons, resulting in degradation only at high Cdk1 activity. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.
Dynamin-2 controls complement receptor 3- mediated phagocytosis completion and closure
Authors: Mularski Anna , Wimmer Ryszard , Arbaretaz Floriane , Le Goff Gabriel , Niedergang Florence
Published: February 2021

Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.
Closeness or opportunistic behavior? Mediating the business ecosystem governance mechanisms and coordination relationship
Authors: Pomegbe Wisdom Wise Kwabla , Li Wenyuan , Dogbe Courage Simon Kofi , Otoo Charles Oduro Acheampong
Published: February 2021

Purpose

Business ecosystem is described as a web of loose relationships, thereby posing some challenges to coordination. In attempt to ensure coordination in business relationships, scholars have proposed relational and contractual governance mechanisms. Hence, this study aims to examine the effects of governance mechanisms on coordination in the pharmaceutical business ecosystem (BE), with the potential mediating effects of closeness and opportunistic behavior.

Design/methodology/approach

The study adopted quantitative approach with cross-sectional survey research design to collect data from the business ecosystem of pharmaceutical company X in Ghana. A structured questionnaire based on a five-point Likert scale was employed to collect data. Analysis was based on 173 institutions (both local and international) selected from pharmaceutical BE. Various validity and reliability checks were conducted before the presentation of the actual analysis, which was conducted using ordinary least squares (OLS) regression in Statistical Product and Service Solutions (SPSS).

Findings

The results of the study revealed that both relational and contractual governance have a direct positive effect on coordination between pharmaceutical BE members and the focal firm. The effect of contractual governance on coordination is however, partially mediated by opportunistic behavior. Finally, closeness also fully mediated the effect of relational governance on coordination.

Originality/value

The study builds on the governance mechanisms in exchange relationship, which was hitherto largely limited to the dyadic and triadic business network, partnerships and strategic alliances studies. The contribution to BE literature provides further understanding into transaction cost economics and relational exchange theory, which was dominantly applied in dyadic relationships ties such as partnerships, alliances and networks.