bluuebunny/crossref_metadata_2025 huggingface dataset:
LINKAGE OF A KNOWN CHLOROPLAST GENE MUTATION TO THE UNIPARENTAL GENOME OF CHLAMYDOMONAS REINHARDII
Authors: Mets Laurens J , Geist Lois J
Published: January 2021

Data are presented that associate three new markers with the uniparental linkage group in Chlamydomonas reinhardii. One of these, mutant 10-6C, is a genetic marker for the structural gene of the large subunit of ribulose bisphosphate carboxylase. These results provide the first direct link between the uniparental gene map and the physical map of chloroplast DNA. The other two markers, Dr2 (DCMU resistant) and 8-36C (deficient in photosystem II activity), map to a single locus. The data suggest that mixing in zygotic chloroplasts may not be complete so that input genomes do not have equal opportunities to recombine. The data are not compatible with simple linear or circular maps but can be explained on the basis of the known physical structure of chloroplast DNA.
MITOCHONDRIAL DNA EVOLUTION IN MICE
Authors: Ferris Stephen D , Sage Richard D , Prager Ellen M , Ritte Uzi , Wilson Allan C
Published: January 2021

This study extends knowledge of mitochondrial DNA (mtDNA) diversity in mice to include 208 animals belonging to eight species in the subgenus Mus. Highly purified mtDNA from each has been subjected to high-resolution restriction mapping with respect to the known sequence of one mouse mtDNA. Variation attributed to base substitutions was encountered at about 200 of the 300 cleavage sites examined, and a length mutation was located in or near the displacement loop. The variability of different functional regions in this genome was as follows, from least to most: ribosomal RNA, transfer RNA, known proteins, displacement loop and unidentified reading frames.—Phylogenetic analysis confirmed the utility of the Sage and Marshall revision of mouse classification, according to which there are at least four species of commensal mice and three species of aboriginal mice in the complex that was formerly considered to be one species. The most thoroughly studied of these species is Mus domesticus, the house mouse of Western Europe and the Mediterranean region, which is the mitochondrial source of all 50 of the laboratory strains examined and of the representatives of wild house mice introduced by Europeans to North and South America during the past few hundred years.—The level of mtDNA variation among wild representatives of (M. musculus) and several other mammalian species. By contrast, among the many laboratory strains that are known or suspected to stem from the pet mouse trade, there is little interstrain variation, most strains having the "old inbred" type of domesticus mtDNA, whose frequency in the 145 wild mice examined is low, about 0.04. Also notable is the apparent homogeneity of mtDNA in domesticus races that have fixed six or more fused chromosomes and the close relationship of some of these mtDNAs to those of karyotypically normal mice.—In addition, this paper discusses fossil and other evidence for the view that in mice, as in many other mammals, the average rate of point mutational divergence in mtDNA is 2-4% per million years. From this, it is estimated that the commensal association between mice and our ancestors began more than a million years ago, i.e., at an early stage in the evolution of Homo erectus.
HOMOLOGOUS RECOMBINATION BETWEEN EPISOMAL PLASMIDS AND CHROMOSOMES IN YEAST
Authors: Falco S Carl , Rose Mark , Botstein David
Published: January 2021

We have observed genetic recombination between ura3  - mutations (among them extensive deletions) carried on "episomal" (i.e., 2μ DNA-containing) plasmids and other ura3  - alleles present at the normal chromosomal URA3 locus. The recombination frequency found was comparable to the level observed for classical mitotic recombination but was relatively insensitive to sunlamp radiation, which strongly stimulates mitotic recombination. Three equally frequent classes could be distinguished among the recombinants. Two of these are the apparent result of gene conversions (or double crossovers) which leave the URA3  + allele on the chromosome (class I) or on the plasmid (class II). The third class is apparently due to a single crossover that results in the integration of the plasmid into a chromosome. Plasmid-chromosome recombination can be useful in fine structure genetic mapping, since recombination between a chromosomal point mutation and a plasmid-borne deletion mutation only 25 base pairs distant was easily detected.
A RAPID CHROMOSOME-MAPPING METHOD FOR CLONED FRAGMENTS OF YEAST DNA
Authors: Falco S Carl , Botstein David
Published: January 2021

A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2μ plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2μ DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4.
Imaging strategies to interpret 3-D noisy audio-magnetotelluric data acquired in Gyeongju, South Korea: data processing and inversion
Authors: Uhm Janghwan , Heo Junyeong , Min Dong-Joo , Oh Seokhoon , Chung Ho-Joon
Published: January 2021

The largest earthquake recorded in South Korea occurred in Gyeongju on 2016 September 12. Since then, understanding the geological structures near the Gyeongju earthquake's epicentre has been demanded. As a preliminary step to understand the geological structures around the epicentre, a 3-D audio-magnetotelluric (AMT) survey was conducted. These 3-D AMT data were imaged through data processing and inversion. However, because the AMT data acquired in Gyeongju were very noisy, conventional imaging procedures did not yield reliable results. To obtain a reliable model inverted from such noisy data, we propose various imaging strategies: an additional data processing technique using the Nyquist diagram after conventional data processing and several inversion strategies related to the selection of data, the weighting of the data, the constraints on the model parameter vector and the Lagrange multiplier used for the regularization. By applying the additional data processing step and several inversion strategies, we were able to successfully invert the noisy field data. The inversion results verify that the data-screening procedure applying the Nyquist diagram remarkably improves the results compared with those obtained using only conventional data processing. The imaging strategies proposed in this case study can be used to image noisy MT/AMT field data for other regions.
NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4
Authors: Chaudhary Bharat P , Zoetewey David L , McCullagh Martin J , Mohanty Smita
Published: January 2021

Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix–helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.
A home visit-based early childhood stimulation programme in Brazil—a randomized controlled trial
Authors: Brentani Alexandra , Walker Susan , Chang-Lopez Susan , Grisi Sandra , Powell Christine , Fink Günther
Published: January 2021

Home visiting programmes are increasingly recognized as one of the most effective interventions to improve child health and development in low-income settings. However, the best platforms to deliver such programmes remain unclear. We conducted a randomized controlled trial to test the relative effectiveness of child development agents (CDAs) and community health workers (CHWs) as two possible delivery platforms for early childhood development (ECD) focused home visiting intervention in São Paulo, Brazil. A total of 900 children aged 9–15 months were screened for potential study inclusion between January and March 2015. Children who did not attend crèches at enrolment were included in the trial. Children were randomly assigned to control or to receive biweekly home visits either through a CHW in the areas covered by the Brazilian Family Health Strategy (FHS) or by a newly hired cadre of CDAs in the areas not covered by the FHS. The primary study outcome was children’s development (cognition, motor, language and social emotional skills) assessed after 12 months of intervention with the PRIDI and Caregiver-Reported Early Development Instruments tools. A total of 826 mother-child dyads were enrolled in the trial. In intention-to-treat analysis, neither intervention arm improved study outcomes. In per-protocol (PP) analysis, the CDA programme resulted in a 0.22 standard deviation increase in children’s development (95% confidence interval [0.01–0.43]). The results presented in this study suggest that home visiting programmes have the potential to improve child development among poor urban families in Brazil. However, delivering home visiting interventions through already active CHWs may not be feasible in the Brazilian context and coordination across sectors is essential to effective ECD policies.
Health Promotion Glossary 2021
Authors: Nutbeam Don , Muscat Danielle M
Published: January 2021

Lay summary

The Health Promotion Glossary 2021 is designed to help clarify the meaning and relationship between terms commonly used in health promotion. This is the first full review and revision of the Glossary in over 20 years. It reflects the continued evolution and development of concepts since the 1998 Glossary, providing an updated overview of the many ideas which are central to contemporary health promotion.

The aim of the Glossary is to facilitate communication among the professions and sectors working in health promotion. The list of terms in the Glossary is not intended to be either exhaustive or exclusive, and draws upon the wide range of disciplines from which health promotion has its origins. The definitions should not be regarded as ‘the final word’ on the terms included. Definitions by their very nature are restrictive, often representing summaries of complex ideas and actions. The use of terms will often be context-specific, and influenced by different social, cultural and economic conditions in countries and communities. Despite these obvious restrictions, the glossary has been assembled to enable as wide an audience as possible to understand the basic ideas and concepts which are central to the development of health promotion.
Modeling the immune response toSalmonelladuring typhoid
Authors: Dhingra Divy , Marathe Sandhya Amol , Sharma Nandita , Marathe Amol , Chakravortty Dipshikha
Published: January 2021

Several facets of the host immune response to Salmonella infection have been studied independently at great depths to understand the progress and pathogenesis of Salmonella infection. The circumstances under which a Salmonella-infected individual succumbs to an active disease, evolves as a persister or clears the infection are not understood in detail. We have adopted a system-level approach to develop a continuous-time mechanistic model. We considered key interactions of the immune system state variables with Salmonella in the mesenteric lymph node to determine the final disease outcome deterministically and exclusively temporally. The model accurately predicts the disease outcomes and immune response trajectories operational during typhoid. The results of the simulation confirm the role of anti-inflammatory (M2) macrophages as a site for persistence and relapsing infection. Global sensitivity analysis highlights the importance of both bacterial and host attributes in influencing the disease outcome. It also illustrates the importance of robust phagocytic and anti-microbial potential of M1 macrophages and dendritic cells (DCs) in controlling the disease. Finally, we propose therapeutic strategies for both antibiotic-sensitive and antibiotic-resistant strains (such as IFN-γ therapy, DC transfer and phagocytic potential stimulation). We also suggest prevention strategies such as improving the humoral response and macrophage carrying capacity, which could complement current vaccination schemes for enhanced efficiency.
Embodied Theodicy: From Conceptual to Bodily Engagements with Suffering
Authors: Winfield Taylor Paige
Published: January 2021

This article leverages ethnographic research on spiritual journeys to expand traditional definitions of theodicy. Embodied theodicy builds upon embodiment literature to demonstrate how bodily experiences have real emotional consequences—they have the potential to change how individuals make sense of their suffering. Whereas in some cases the bodily experience of pain leads to suffering, in others, pain provides individuals with perspective on their suffering and helps them overcome it. Data from two spiritual journeys, El Camino de Santiago and a Vipassana meditation retreat, introduce three models of embodied theodicy: pain as purifier, pain as teacher, and pain as solidarity. Embodied theodicy bridges the meaning-making concerns of classical and neoclassical literature with the embodiment theories of poststructuralists and contemporary scholars.
Baseline integrase drug resistance mutations and conserved regions across HIV-1 clades in Cameroon: implications for transition to dolutegravir in resource-limited settings
Authors: Semengue Ezechiel Ngoufack Jagni , Armenia Daniele , Inzaule Seth , Santoro Maria Mercedes , Dambaya Béatrice , Takou Désiré , Teto Georges , Nka Alex Durand , Yagai Bouba , Fabeni Lavinia , Chenwi Collins , Angong Beloumou Grâce , Djupsa Ndjeyep Sandrine Claire , Colizzi Vittorio , Perno Carlo-Federico , Ceccherini-Silberstein Francesca , Fokam Joseph
Published: January 2021

Background

Transition to dolutegravir-based regimens in resource-limited settings (RLS) requires prior understanding of HIV-1 integrase variants and conserved regions. Therefore, we evaluated integrase drug resistance mutations (DRMs) and conserved regions amongst integrase strand transfer inhibitor (INSTI)-naive patients harbouring diverse HIV-1 clades in Cameroon.

Methods

A cross-sectional study was conducted amongst 918 INSTI-naive patients from Cameroon (89 ART-naive and 829 ART-experienced patients). HIV-1 sequences were interpreted regarding INSTI-DRMs using the Stanford HIVdb v8.9-1 and the 2019 IAS–USA list. Amino acid positions with <1% variability were considered as highly conserved. Subtyping was performed by phylogeny.

Results

Overall prevalence (95% CI) of INSTI-DRMs was 0.8% (0.4–1.7), with 0.0% (0.0–4.0) amongst ART-naive versus 0.9% (0.5–1.9) amongst ART-experienced patients; P = 0.44. Accessory mutations (95% CI) were found in 33.8% (30.9–37.0), with 38.2% (28.1–49.1) amongst ART-naive versus 33.4% (30.4–36.7) amongst ART-experienced patients; P = 0.21. Of 288 HIV-1 integrase amino acid positions, 58.3% were highly conserved across subtypes in the following major regions: V75–G82, E85–P90, H114–G118, K127–W132, E138–G149, Q168–L172, T174–V180, W235–A239 and L241–D253. Wide genetic diversity was found (37 clades), including groups M (92.3%), N (1.4%), O (6.2%) and P (0.1%). Amongst group M, CRF02_AG was predominant (47.4%), with a significantly higher frequency (95% CI) of accessory mutations compared with non-AG [41.4% (36.8–46.0) versus 27.1% (23.3–31.2) respectively; P < 0.001].

Conclusions

The low baseline of INSTI-DRMs (<1%) in Cameroon suggests effectiveness of dolutegravir-based regimens. In spite of high conservation across clades, the variability of accessory mutations between major circulating strains underscores the need for monitoring the selection of INSTI-DRMs while scaling up dolutegravir-based regimens in RLS.
Prevalence of Vibrio mimicus in Fish, Fishery Products, and Environment of South West Coast of Kerala, India
Authors: Nilavan Ezhil , Vaiyapuri Murugadas , Sadanandan Sheela Greeshma , Nadella Ranjit Kumar , Thandapani Muthulakshmi , Kumar Abhay , Mothadaka Mukteswar Prasad
Published: January 2021

Background

Vibrio mimicus is a seafood-borne bacterium involved in incidences of human infections following consumption of raw or undercooked seafood. Regular monitoring of seafood for V.mimicus is necessary for risk assessment and to establish mitigation measures.

Method

During the period 2017–2020, a total of 250 samples comprising finfish, shellfish, water, ice, and sediment samples were collected from fish markets, fish landing centers, and fish farms in the Ernakulum district on the Southwest coast of Kerala, India. V. mimicus was isolated using enrichment in alkaline peptone water for 18 h followed by plating on thiosulfate citrate bile salts sucrose agar and then incubated at 37°C for 18–24 h. The presumptive V. mimicus isolates were confirmed by biochemical characterization and molecularly with vmh gene-specific for V. mimicus.

Results

The study revealed that the prevalence of V. mimicus is 5.6% in the total of samples screened. The highest occurrence was observed in brackish water fish (19%) followed by freshwater fish (18%) and marine fish (2%) samples. The study points out the risk of brackish water fishes as potential carriers of this pathogen. This requires preventive measures to mitigate health hazards associated with V. mimicus entering into the seafood production chain.
Positive Learning or Deviant Interviewing? Mechanisms of Experience on Interviewer Behavior
Authors: Kosyakova Yuliya , Olbrich Lukas , Sakshaug Joseph W , Schwanhäuser Silvia
Published: January 2021

Interviewer (mis)behavior has been shown to change with interviewers’ professional experience (general experience) and experience gained during the field period (survey experience). We extend this study by using both types of experiences to analyze interviewer effects on a core quality indicator: interview duration. To understand whether the effect of interviewer experience on duration is driven by increased efficiency or deviant behavior—both mechanisms of shorter interview durations—we additionally examine the triggering rate of filter questions to avoid burdensome follow-up questions and response differentiation over the field period. Using multilevel models and data from a large-scale survey on a special and difficult-to-interview population of refugees in Germany, we find that interview duration decreases with increasing survey experience, particularly among the generally inexperienced interviewers. However, this effect is not found for the triggering rate and response differentiation. The results are robust to different sample and model specifications. We conclude that the underlying mechanism driving interview duration is related to increasing efficiency, and not deviant behavior.
Ethanol modulates the effector functions of human monocyte-derived macrophages in response toParacoccidioides brasiliensisyeast cells
Authors: de Castro Lívia Furquim , de Araújo Mathias Kamila , Nunes Júlia Vieira , Galastri Ana Lúcia Bergamasco , da Silva Dennis Henrique Leandro , Longhi Larissa Nara Alegrini , de Souza Lima Blotta Maria Heloisa , Mamoni Ronei Luciano
Published: January 2021

We aimed to investigate the effects of ethanol and its metabolites (β-hydroxybutyrate and sodium acetate) in the effector functions of macrophages in response to Paracoccidioides brasiliensis yeast cells and to determine their influence in the development of the adaptive response. Purified peripheral blood monocytes were differentiated into macrophages and were treated with ethanol, β-hydroxybutyrate, and sodium acetate, and stimulated with P. brasiliensis yeast cells and evaluated for their phenotypic characteristics, functional activity, and capability to induce T cells activation/differentiation. We found that the ethanol treatment diminished the expression of HLA-AB, HLA-DR, CD80, and CD86, modulating the expression of dectin-1, as well as Syk phosphorylation. The ethanol treatment increased the phagocytic activity, expression of CD206, and IL-10 production; however, reduced ROS production, fungicidal activity, caspase-1 cleavage, and IL-1β and IL-6 production. Our data also showed that the presence of ethanol reduced the differentiation of Th1 and Th17 cells and increased the frequency of Th2 cells. Our results indicated that ethanol exposure could suppress effector function of macrophages, possibly leading to the polarization of M2 macrophages. The ethanol modulates the expression of costimulatory and antigen-presentation molecules and interferes with the NLRP3 inflammasome. Altogether, these alterations affect the development of the adaptive response, decreasing the frequency of IL-17, IL-22, and IFN- γ producing cells, and increasing the frequency of IL-4 producing cells. Therefore, exposure to ethanol can impair the capability of macrophages to exert their effector functions and activate the acquired response related to resistance to P. brasiliensis infection.
The bursty star formation history of the Fornax dwarf spheroidal galaxy revealed with the HST
Authors: Rusakov V , Monelli M , Gallart C , Fritz T K , Ruiz-Lara T , Bernard E J , Cassisi. S
Published: January 2021

We present a new derivation of the star formation history (SFH) of the dSph galaxy Fornax in two central regions, characterized by unprecedented precision and age resolution. It reveals that star formation has proceeded in sharp bursts separated by periods of low level or quiescent activity. The SFH was derived through colour–magnitude diagram (CMD) fitting of two extremely deep Hubble Space Telescope CMDs, sampling the centre and one core radius. The attained age resolution allowed us to single out a major star formation episode at early times, a second strong burst 4.6 ± 0.4 Gyr ago and recent intermittent episodes ∼2–0.2 Gyr ago. Detailed testing with mock stellar populations was used to estimate the duration of the main bursts and study the occurrence of low-level star formation between them. The SFHs in both regions show common features, with activity at the same epochs and similar age–metallicity relationship. However, clear indications of a spatial gradient were also found, with mean age increasing with radius and star formation episodes being more prolonged in the centre. While some galaxy evolution models predict bursty SFHs in dwarf galaxies and thus a secular origin of the observed SFH cannot be excluded in Fornax, other evidence points to possible mergers or interactions as the cause of its bursty SFH. In particular, we calculated the Fornax orbit relative to the closest dwarfs and the Milky Way and observed a correspondence between the main intermediate-age and young events and peri-passages of Fornax around the Milky Way, possibly indicating tidally induced star formation.
Periods and classifications of RR Lyrae stars in the globular cluster M15
Authors: Hoffman Andrew M , Murakami Yukei S , Zheng WeiKang , Stahl Benjamin E , Filippenko Alexei V
Published: January 2021

We present measurements of the periods, amplitudes, and types of 74 RR Lyrae stars in the globular cluster M15 derived from Nickel 1 m telescope observations conducted at Lick Observatory in 2019 and 2020. Of these RR Lyrae stars, two were previously reported but without a determination of the period. In addition, we identify five Type II Cepheid variable stars for which we report three novel period determinations, and a further 34 stars with uncertain classifications and periods. We discuss the development and subsequent application to our data of a new python package, Period-determination and Identification Pipeline Suite (pips), based on a new adaptive free-form fitting technique to detect the periods of variable stars with a clear treatment of uncertainties.
Statistical microlensing towards magnified high-redshift star clusters
Authors: Dai Liang
Published: January 2021

We study light variability of gravitationally magnified high-redshift star clusters induced by a foreground population of microlenses. This arises as the incoherent superposition of light variations from many source stars traversing the random magnification pattern on the source plane. The light curve resembles a scale-invariant, Gaussian process on time-scales of years to decades, while exhibits rapid and frequent micro-caustic crossing flares of larger amplitudes on time-scales of days to months. For a concrete example, we study a young Lyman-continuum-leaking star cluster in the Sunburst Arc at z = 2.37. We show that one magnified image happens to be intervened by a foreground galaxy, and hence should exhibit a variable flux at the 1–$2{{\ \rm per\ cent}}$ level, which is measurable in space with ∼1–$3\,$ ks exposures on the Hubble Space Telescope and more easily with the James Webb Space Telescope, or even from the ground using a ∼4-m telescope without adaptive optics. Detailed measurement of this variability can help determine the absolute macro magnification and hence the intrinsic mass and length scales of the star cluster, test synthetic stellar population models, and probe multiplicity of massive stars. Furthermore, monitoring the other lensed images of the star cluster, which are free from significant intervention by foreground microlenses, can allow us to probe planetary to stellar mass compact objects constituting as little as a few per cent of the dark matter. Given the typical surface density of intracluster stars, we expect this phenomenon to be relevant for other extragalactic star clusters lensed by galaxy clusters.
Simulating dust grain-radiation coupling on a moving mesh
Authors: McKinnon Ryan , Kannan Rahul , Vogelsberger Mark , O’Neil Stephanie , Torrey Paul , Li Hui
Published: January 2021

We present a model for the interaction between dust and radiation fields in the radiation hydrodynamic code arepo-rt, which solves the moment-based radiative transfer equations on an unstructured moving mesh. Dust is directly treated using live simulation particles, each of which represent a population of grains that are coupled to hydrodynamic motion through a drag force. We introduce methods to calculate radiation pressure on and photon absorption by dust grains. By including a direct treatment of dust, we are able to calculate dust opacities and update radiation fields self-consistently based on the local dust distribution. This hybrid scheme coupling dust particles to an unstructured mesh for radiation is validated using several test problems with known analytic solutions, including dust driven via spherically symmetric flux from a constant luminosity source and photon absorption from radiation incident on a thin layer of dust. Our methods are compatible with the multifrequency scheme in arepo-rt, which treats UV, optical photons as single scattered and IR photons as multi scattered. At IR wavelengths, we model heating of and thermal emission from dust. Dust and gas are not assumed to be in local thermodynamic equilibrium but transfer energy through collisional exchange. We estimate dust temperatures by balancing these dust-radiation and dust-gas energy exchange rates. This framework for coupling dust and radiation can be applied in future radiation hydrodynamic simulations of galaxy formation.
Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing
Authors: Athapattu Uditha S , Amarasekara Charuni A , Immel Jacob R , Bloom Steven , Barany Francis , Nagel Aaron C , Soper Steven A
Published: January 2021

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s−1 and a processivity of >10.5 kb at 25°C.
Propofol and sevoflurane affect intra-operative memory formation of words differently
Authors: Orena Eleonora F. , Landucci Francesco , Ayadi Roberta , Caldiroli Dario , Papagno Costanza
Published: January 2021

BACKGROUND

Memory priming seems possible even during apparently adequate anaesthesia. However, the effects of different anaesthetics and type of stimuli, by virtue of their specific neural underpinnings, have not been considered.

OBJECTIVE

To determine if intra-operative implicit memory is affected by the type of anaesthesia (propofol or sevoflurane) or by the type of stimuli (abstract or concrete words).

DESIGN

Two consecutive, randomised controlled experiments.

SETTING

Neurological institute in Milan, Italy.

PATIENTS

Forty-three patients undergoing anaesthesia with propofol (experiment 1) and 32 patients undergoing anaesthesia with sevoflurane (experiment 2). Patients were ASA I or II, age 18 to 65 years, native Italian speakers, right-handed and without any condition affecting memory or hearing.

INTERVENTION

During anaesthesia, the patients heard a list of either concrete or abstract words or no words at all (controls). Explicit memory was tested with an explicit recall task and the Brice Interview; implicit memory was assessed through a word stem completion test.

OUTCOME MEASURES

The number of explicitly recalled words, positivity to the Brice Interview, the proportion of target and nontarget hits, and a derived implicit memory score.

RESULTS

With propofol, the proportion of target hits was significantly greater than the proportion of nontarget hits for the concrete word experimental group (P = 0.018). The implicit memory score of the concrete word experimental group was significantly higher than the score of both the abstract word experimental group (P 
= 0.000) and the concrete word control group (P = 0.023). With sevoflurane, the proportion of target hits was significantly higher than the proportion of nontarget hits for the abstract word experimental group only (P = 0.027). No patients had a BIS above 60 and no one could recall intra-operative events or words.

CONCLUSION

Intra-operative memory for words can form during apparently adequate BIS-guided anaesthesia but is modified by propofol or sevoflurane acting on different brain targets. Further studies on larger samples and using neuroimaging techniques are needed.

TRIAL REGISTRATION

Clinicaltrials.gov identifier: NCT03727464.
Genetic depletion studies define receptor usage by virulent hantaviruses in human endothelial cells
Authors: Dieterle M. Eugenia , Solà-Riera Carles , Jangra Rohit K. , Ye Chunyan , Goodfellow Samuel M. , Mittler Eva , Kasikci Ezgi , Bradfute Steven B. , Klingström Jonas , Chandran Kartik
Published: January 2021

Hantaviruses are a large group of RNA viruses that include known epidemic threats and other agents poised for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures alike are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Previous work has implicated several cell-surface molecules, most notably β3- and β1-containing integrin heterodimers, decay-accelerating factor (DAF), and the cadherin superfamily protein protocadherin-1 (PCDH1), in hantavirus entry in endothelial cells, the major targets of viral infection in humans. Despite the fact that β3/β1 integrins have been presumed to be the major hantavirus entry receptors for over two decades, rigorous genetic evidence supporting their requirement, and that of DAF as an entry cofactor, is lacking. Here, we used CRISPR/Cas9 engineering to knock out four candidate hantaviral receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells. PCDH1 loss substantially reduced entry and infection by a subset of hantaviruses endemic to the Americas. In contrast, the loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by any of a large panel of hantaviruses tested. We conclude that the major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.
Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies
Authors: Greaney Allison J. , Loes Andrea N. , Crawford Katharine H.D. , Starr Tyler N. , Malone Keara D. , Chu Helen Y. , Bloom Jesse D.
Published: January 2021

The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent serum antibodies are impacted by all mutations to the spike’s receptor-binding domain (RBD), the main target of serum neutralizing activity. Binding by polyclonal serum antibodies is affected by mutations in three main epitopes in the RBD, but there is substantial variation in the impact of mutations both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD’s receptor binding motif. The most important site is E484, where neutralization by some sera is reduced >10-fold by several mutations, including one in emerging viral lineages in South Africa and Brazil. Going forward, these serum escape maps can inform surveillance of SARS-CoV-2 evolution.
Glycan reactive anti-HIV-1 antibodies bind the SARS-CoV-2 spike protein but do not block viral entry
Authors: Mannar Dhiraj , Leopold Karoline , Subramaniam Sriram
Published: January 2021

The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via FcγRII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.
Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations
Authors: Rovšnik Urška , Zhuang Yuxuan , Forsberg Björn O , Carroni Marta , Yvonnesdotter Linnea , Howard Rebecca J , Lindahl Erik
Published: January 2021

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here we report cryo-electron microscopy (cryo-EM) structures of the proton-activatedGloeobacter violaceusligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and sidechain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones, and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results support a dissection of protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.
Phosphoproteomic Landscape of AML Cells Treated with the ATP-Competitive CK2 Inhibitor CX-4945
Authors: Rosales Mauro , Rodríguez-Ulloa Arielis , Besada Vladimir , Ramón Ailyn C. , Pérez George V. , Ramos Yassel , Guirola Osmany , González Luis J. , Zettl Katharina , Wiśniewski Jacek R. , Perera Yasser , Perea Silvio E.
Published: January 2021

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.
Structural Characteristics and Proton Conductivity of the Gel Within the Electrosensory Organs of Cartilaginous Fishes
Authors: Phillips Molly , Wheeler Alauna , Robinson Matthew J. , Leppert Valerie , Jia Manping , Rolandi Marco , Hirst Linda S. , Amemiya Chris T.
Published: January 2021

The Ampullae of Lorenzini (AoL) of cartilaginous fishes are sensory organs used to detect environmental electric fields. The proximal ends of the organs are externally visible as pores in the skin that lead into gel-filled tubular canals which terminate in rounded chambers filled with highly specialized electrosensory cells. The viscoelastic gel that fills the organs is composed of proteins and polysaccharides that are not yet completely characterized but are thought to play a critical role in the electrosensing mechanism. Although recent studies have identified various components of AoL gel, it has remained unclear how the component molecules are structurally arranged and how their structure influences the overall function of the AoL. Here we present the first microscopic descriptions and x-ray scattering data from AoL gel extracted from spotted ratfish (Hydrolagus colliei). Our results suggest that AoL gel is colloidal in nature and composed of spherical globules that are approximately 10-100 nm in size. We investigated the structural influence of the protein components of the gel specifically by analyzing gel that had been digestedin situvia enzymatic proteolysis. By comparing gel before and after digestion using microscopy, x-ray scattering analyses, and proton conductivity measurements, we directly observed the structural and functional influence of proteins in AoL gel. The findings described here represent the first detailed structural analysis of AoL gel and lay the groundwork for more detailed studies into the specific interactions of molecules inside AoL gel at the nanoscale, with particular reference to their mechanistic role in electrosensing.
Optogenetic stimulation of VIPergic SCN neurons induces photoperiodic changes in the mammalian circadian clock
Authors: Tackenberg Michael C. , Hughey Jacob J. , McMahon Douglas G.
Published: January 2021

Circadian clocks play key roles in how organisms respond to and even anticipate seasonal change in day length, or photoperiod. In mammals, photoperiod is encoded by the central circadian pacemaker in the brain, the suprachiasmatic nucleus (SCN). The subpopulation of SCN neurons that secrete the neuropeptide VIP mediate the transmission of light information within the SCN neural network, suggesting a role for these neurons in circadian plasticity in response to light information that has yet to be directly tested. Here, we usedin vivooptogenetic stimulation of VIPergic SCN neurons followed byex vivoPERIOD 2::LUCIFERASE (PER2::LUC) bioluminescent imaging to test whether activation of this SCN neuron sub-population can induce SCN network changes that are hallmarks of photoperiodic encoding. We found that optogenetic stimulation designed to mimic a long photoperiod indeed altered subsequent SCN entrained phase, increased the phase dispersal of PER2 rhythms within the SCN network, and shortened SCN free-running period – similar to the effects of a true extension of photoperiod. Optogenetic stimulation also induced analogous changes on related aspects of locomotor behaviorin vivo. Thus, selective activation of VIPergic SCN neurons induces photoperiodic network plasticity in the SCN which underpins photoperiodic entrainment of behavior.
SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+
Authors: Lai Alex L. , Freed Jack H.
Published: January 2021

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2’ cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue difference, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.
Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy
Authors: Schmitt Rebecca E , Smith Douglas Y , Cho Dong Seong , Kirkeby Lindsey A , Resch Zachary T , Liewluck Teerin , Niu Zhiyv , Milone Margherita , Doles Jason D
Published: January 2021

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive distal myopathy with rimmed vacuoles that typically manifests in late adolescence/early adulthood. GNE encodes an enzyme that is the rate-limiting step in sialic acid biosynthesis which is necessary for proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study we aimed to address this knowledge gap by querying underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem cell (iPSC) model. Muscle and skin biopsies were acquired from two patients with GNE myopathy that presented with distinct histopathological features. Control and patient-specific iPSCs were derived from skin fibroblasts and differentiated down a skeletal muscle lineage in a three-stage process analogous to muscle development and muscle regeneration. Initial studies revealed: 1) the ability of patient-derived GNE iPSC clones to recapitulate key characteristics of the human pathology including TDP-43 accumulation and evidence of dysregulated autophagy, and 2) a striking defect in myogenic progression of the more severe GNE iPSC clone. Single-cell RNA sequencing time course studies were then performed to more rigorously explore myogenesis defects. Cluster-based bioinformatics analyses revealed clear differences between control and GNE iPSC-derived muscle precursor cells (iMPCs). On a transcriptional level, late stage GNE iMPCs resembled that of early stage control iMPCs, confirming stalled myogenic progression on a molecular level. Comparative expression and pathway studies revealed EIF2 signaling as a top signaling pathway altered in GNE iMPCs. In summary, we report a novelin vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a likely novel contributing mechanism to the etiology of GNE myopathy.

SUMMARY STATEMENT

Development of a novel cell-based model of GNE myopathy, utilizing GNE patient-derived samples, which recapitulates human disease characteristics, uncovered myogenic differentiation defects, and can elucidate possible mechanistic contributors to the disease.