bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-molecule fluorescence microscopy demonstrates fast dynamics of the variant surface glycoprotein coat on living trypanosomes
Authors: Schwebs Marie , Paul Torsten , Glogger Marius , Forster Leonard , Morriswood Brooke , Teßmar Jörg , Groll Jürgen , Engstler Markus , Kollmannsberger Philip , Fenz Susanne 
Published: August 2022

AbstractThe fluidity of Trypanosoma brucei’s dense coat of GPI-anchored variant surface glycoproteins (VSGs) is fundamental for the survival of the parasite. In order to maintain the integrity of the coat, it is recycled on the time scale of a few minutes. This is surprisingly fast as endo- and exocytosis take place in the same small membrane invagination called the flagellar pocket. Here, we present measurements of VSG dynamics on the single-molecule level in living trypanosomes. A large number of short protein trajectories sampling the parasite’s surface were analysed in two distinct scenarios: diffusion and directed motion. To this end, we employed a previously published algorithm and implemented two extensions to consider rim effects as well as localisations errors inherent to single-mole tracking. Neglect of the latter can have a significant distortive effect on the measured diffusion coefficient; in our case resulting in an underestimation by 20 %. We found large heterogeneity in the local diffusion coefficients and velocities with a surprisingly high average value of and , respectively. To decide on the locally dominant motion mode, we present a guideline based on random walk simulations. We find that VSG dynamics is indeed dominated by diffusion. Complementary simulations on long time scales not accessible in the experiment showed that passive VSG randomisation is fast enough to prevent re-endocytosis newly exocytosed VSGs and to accomplish turnover of the full VSG coat within a few minutes.Author summarySingle-molecule tracking in biological systems often suffers from trajectories being too short to obtain statistically robust decisions on the present motion mode. We faced this issue when investigating the dynamics of the protein surface coat of African trypanosomes. To address the question whether diffusion or directed motion governs coat dynamics, we have adopted an algorithm based on temporal decomposition and spatial binning of an ensemble of single-molecule trajectories. We introduced several extensions to the original approach, including the consideration of localisation errors inherent to single-molecule tracking. This improvement alone already prevented the diffusion coefficient from being underestimated by 20 %. We analysed the coat dynamics in two scenarios, diffusion and directed motion, and offer a decision guideline to identify the locally dominating motion mode. Our extended algorithm is available to the scientific community via GitHub. For the trypanosome surface coat we found that the motion is indeed mainly characterised by diffusion with a surprisingly high diffusion coefficient. This finding solves a long-standing question how the parasite maintains its protein coat.
Novel algorithms for improved detection and analysis of fluorescent signal fluctuations
Authors: Stopper Gebhard , Caudal Laura C. , Rieder Phillip , Gobbo Davide , Felix Lisa , Everaerts Katharina , Bai Xianshu , Stopper Laura , Rose Christine R. , Scheller Anja , Kirchhoff Frank 
Published: August 2022

AbstractFluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra-as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and avnalysis of fluorescence signals requires to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation of fluorescent detection of messenger molecules and automated detection of fluorescence fluctuations.Author summaryAnalyzing molecular signalling is crucial in understanding intra- and intercellular communication. These signals are visualized using fluorescent dyes or genetically encoded fluorescence indicators. In the brain, Ca2+ signals of glial cells are essential in deciphering complex regulatory functions in health and disease. Due to signal heterogeneity, detection and analysis are highly challenging. They can be stationary, with low amplitude and localized in cell processes, occur as prominent somatic signals or propagate as waves across cellular networks.We have developed two algorithms to analyze fluorescence transients, each tackling a specific problem. PBasE performs automatic and adaptive background correction, removing basal fluorescence fluctuations. CoRoDe automatically extracts regions of interest, explicitly including temporal information to obtain a precise segmentation, which is essential for accurate transient extraction. Combined, these algorithms are able to detect regions exhibiting low amplitude transients with small spatial extent as well as large, high amplitude signals. Extracted transients are categorized based on their peak amplitude, allowing detailed analyses by comparing changes of specific properties. In order to make these algorithms accessible, an interactive application, called Msparkles, has been designed.
Competitive HIV budding suggests that a self-packaging gRNA:Gag-Pol complex directs HIV assembly and enforces infectivity
Authors: Durden Haley , Saha Ipsita , Preece Benjamin , MacArthur Brian , Petersen Abby , Peppel Wiley , Gallegos Rodrigo , Saffarian Saveez 
Published: August 2022

SummaryTo resolve the assembly mechanism of infectious HIV virions, we tested the ability of HIV to assemble infectious virions in the presence of a titrated mix of infectious/ non-infectious proviral genomes. The analysis of our assembly competitions shows that during translation, 15 ± 5 Gag-Pols bind back to their parental gRNA creating a gRNA1: Gag-Pol15 complex. This complex initiates the infectious virion assembly through interactions mediated by cis packaged Gag/Gag-pols and the plasma membrane. Our analysis also shows the number of Gag-Pol and Env proteins packaged in an infectious HIV virion and the minimum functional units of these proteins required for viral infectivity. We suggest that aside from orchestrating the infectious virion assembly the gRNA1: Gag-Pol15 complex plays a major role in HIV evolution and likely hampers effectiveness of antiviral therapies.One-Sentence SummaryCompetitive HIV budding reveals gRNA1: Gag-Pol15 complex orchestrating viral assembly.
CaMKII activity spreads by inter-holoenzyme phosphorylation
Authors: Lučić Iva , Héluin Léonie , Jiang Pin-Lian , Castro Scalise Alejandro G. , Wang Cong , Franz Andreas , Wahl Markus , Liu Fan , Plested Andrew J.R. 
Published: August 2022

AbstractThe dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naïve holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.
The cost of sympatry: spatio-temporal patterns in leopard dietary and physiological responses to tiger competition in Rajaji Tiger Reserve, India
Authors: Patel Shiv Kumari , Ruhela Sourabh , Biswas Suvankar , Bhatt Supriya , Pandav Bivash , Mondol Samrat 
Published: August 2022

1.AbstractApex predators have critical roles in maintaining the structure of ecosystem functioning by controlling intraguild subordinate populations. Such dominant-subordinate interactions involve agonistic interactions including direct (death/displacement) or indirect (physiological and/or health implications) impacts on the subordinates. As these indirect effects are often mediated through physiological processes, it is important to quantify such responses for better understanding of population parameters.We used a well-known sympatric large carnivore intraguild system involving tiger (Panthera tigris) and leopard (Panthera pardus) to understand the dietary and physiological responses under a spatio-temporal gradient of tiger competition pressures in Rajaji Tiger Reserve (RTR), a major source tiger population of the western Terai-Arc Landscape, India between 2015-2020. The park provided a natural experimental set-up for tiger competition with the eastern part (ERTR) having high tiger density and the western (WRTR) part with functionally no competition from tigers.We conducted systematic faecal sampling in the winters of 2015 and 2020 from ERTR and WRTR to assess diet and physiological measures. Analyses of leopard-confirmed faeces suggest a dietary-niche separation as a consequence of tiger competition. In 2020, we found increased occurrence of large-bodied prey species without tiger competition in WRTR. Physiological measures followed the dietary responses where leopards with large-sized prey in diet showed higher fT3M and lower fGCM measures in WRTR. In contrast, ERTR leopards showed lower levels of fT3M as well as fGCM in 2020, possibly due to intense competition from tigers. Overall, these pattens strongly indicate a physiological cost of sympatry where competition with dominant tigers resulted in elevated nutritional stress.The combination of the natural habitat providing unique experimental setup, spatio-temporal sampling strategy and multidisciplinary approaches provide critical conservation perspectives for leopards, particularly in the context of recent increase in tiger numbers across India. We recommend expansion of leopard monitoring and population estimation efforts to buffers, developing appropriate plans for human-leopard conflict mitigation and intensive efforts to understand leopard population dynamics patterns to ensure their persistence during the ongoing Anthropocene.
Faster detection of “darks” than “brights” by monkey superior colliculus neurons
Authors: Malevich Tatiana , Zhang Tong , Baumann Matthias P. , Bogadhi Amarender R. , Hafed Ziad M. 
Published: August 2022

AbstractVisual processing is segregated into ON and OFF channels as early as in the retina, and the superficial (output) layers of the primary visual cortex are dominated by neurons preferring dark stimuli. However, it is not clear how the timing of neural processing differs between “darks” and “brights” in general, especially in light of psychophysical evidence; it is also equally not clear how subcortical visual pathways that are critical for active orienting represent stimuli of positive (luminance increments) and negative (luminance decrements) contrast polarity. Here, we recorded from all visually-responsive neuron types in the superior colliculus (SC) of two male rhesus macaque monkeys. We presented a disc (0.51 deg radius) within the response fields (RF’s) of neurons, and we varied, across trials, stimulus Weber contrast relative to a gray background. We also varied contrast polarity. There was a large diversity of preferences for darks and brights across the population. However, regardless of individual neural sensitivity, most neurons responded significantly earlier to dark than bright stimuli. This resulted in a dissociation between neural preference and visual response onset latency: a neuron could exhibit a weaker response to a dark stimulus than to a bright stimulus of the same contrast, but it would still have an earlier response to the dark stimulus. Our results highlight an additional candidate visual neural pathway for explaining behavioral differences between the processing of darks and brights, and they demonstrate the importance of temporal aspects in the visual neural code for orienting eye movements.Significance statementObjects in our environment, such as birds flying across a bright sky, often project shadows (or images darker than the surround) on our retina. We studied how primate superior colliculus (SC) neurons visually process such dark stimuli. We found that the overall population of SC neurons represented both dark and bright stimuli equally well, as evidenced by a relatively equal distribution of neurons that were either more or less sensitive to darks. However, independent of sensitivity, the great majority of neurons detected dark stimuli earlier than bright stimuli, evidenced by a smaller response latency for the dark stimuli. Thus, SC neural response latency can be dissociated from response sensitivity, and it favors the faster detection of dark image contrasts.
Sequencing the orthologs of human autosomal forensic short tandem repeats provides individual- and species-level identification in African great apes
Authors: Fedele Ettore , Wetton Jon H. , Jobling Mark A. 
Published: August 2022

AbstractBackgroundGreat apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). Here we assess in African great ape DNAs the human-based ForenSeq DNA Sequencing Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification.ResultsThe Forenseq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges, but variation in repeat array structures and little relationship with the known phylogeny, suggesting stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity, indicating a possible mutational difference to humans.ConclusionsDespite some variability in amplification success, orthologs of most human aSTRs in the ForenSeq DNA Sequencing Prep Kit can be analysed in African great apes. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci.
Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins
Authors: Agam Ganesh , Gebhardt Christian , Popara Milana , Mächtel Rebecca , Folz Julian , Ambrose Benjamin , Chamachi Neharika , Chung Sang Yoon , Craggs Timothy D. , de Boer Marijn , Grohmann Dina , Ha Taekjip , Hartmann Andreas , Hendrix Jelle , Hirschfeld Verena , Hübner Christian G. , Hugel Thorsten , Kammerer Dominik , Kang Hyun-Seo , Kapanidis Achillefs N. , Krainer Georg , Kramm Kevin , Lemke Edward , Lerner Eitan , Margeat Emmanuel , Martens Kristen , Michaelis Jens , Mitra Jaba , Moya Muñoz Gustavo G. , Quast Robert , Robb Nicole B. , Sattler Michael , Schlierf Michael , Schneider Jonathan , Schröder Tim , Sefer Anna , Tan Piau Siong , Thurn Johann , Tinnefeld Philip , van Noort John , Weiss Shimon , Wendler Nicolas , Zijlstra Niels , Barth Anders , Seidel Claus A. M. , Lamb Don C. , Cordes Thorben 
Published: August 2022

AbstractSingle-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than ± 0.06, corresponding to an interdye distance precision of ≤ 0.2 nm and accuracy of ≤ 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to ≲ 10% of the Förster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.
Cassette recombination dynamics within chromosomal integrons are regulated by toxin–antitoxin systems
Authors: Richard Egill , Darracq Baptiste , Littner Eloi , Vit Claire , Whiteway Clémence , Bos Julia , Fournes Florian , Garriss Geneviève , Conte Valentin , Lapaillerie Delphine , Parissi Vincent , Rousset François , Skovgaard Ole , Bikard David , Rocha Eduardo P. C. , Mazel Didier , Loot Céline 
Published: August 2022

AbstractIntegrons are adaptive bacterial devices that rearrange promoter less gene cassettes into variable ordered arrays under stress conditions, to sample combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here we show a key role of promoter-containing toxin–antitoxin (TA) cassettes as abortive systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.TeaserThe accumulation of cassette functions in integrons is ensured by toxin–antitoxin systems which kill the cell when the cassette excision rate is too high.
Genome-wide single cell annotation of the human protein-coding genes
Authors: Karlsson Max , Álvez María Bueno , Shi Mengnan , Méar Loren , Schutten Rutger , Hikmet Feria , Digre Andreas , Katona Borbala , Vuu Jimmy , Bosic Martina , Sjöstedt Evelina , Edfors Fredrik , Oksvold Per , von Feilitzen Kalle , Zwahlen Martin , Forsberg Mattias , Johansson Fredric , Mulder Jan , Hökfelt Tomas , Luo Younglun , Butler Lynn , Zhong Wen , Mardinoglu Adil , Sivertsson Åsa , Ponten Fredrik , Zhang Cheng , Lindskog Cecilia , Fagerberg Linn , Uhlén Mathias 
Published: August 2022

AbstractAn important quest for the life science community is to deliver a complete annotation of the human building-blocks of life, the genes and the proteins. Here, we report on a genome-wide effort to annotate all protein-coding genes based on single cell transcriptomics data representing all major tissues and organs in the human body, integrated with data from bulk transcriptomics and antibody-based tissue profiling. Altogether, 25 tissues have been analyzed with single cell transcriptomics resulting in genome-wide expression in 444 single cell types using a strategy involving pooling data from individual cells to obtain genome-wide expression profiles of individual cell type. We introduce a new genome-wide classification tool based on clustering of similar expression profiles across single cell types, which can be visualized using dimensional reduction maps (UMAP). The clustering classification is integrated with a new “tau” score classification for all protein-coding genes, resulting in a measure of single cell specificity across all cell types for all individual genes. The analysis has allowed us to annotate all human protein-coding genes with regards to function and spatial distribution across individual cell types across all major tissues and organs in the human body. A new version of the open access Human Protein Atlas (www.proteinatlas.org) has been launched to enable researchers to explore the new genome-wide annotation on an individual gene level.
Genome-wide analysis of lncRNAs points to their roles in the modulation of developmental regulator expression during plant male germline development
Authors: Lohani Neeta , Golicz Agnieszka A. , Allu Annapurna D. , Bhalla Prem L. , Singh Mohan B. 
Published: August 2022

AbstractLncRNAs can function in regulating of gene expression, but their roles as essential regulators of developmental processes and organismal phenotypes remain largely unclear. Especially the roles of lncRNAs in plants are largely unexplored. However, it has been proposed that plant lncRNAs act as regulators of protein-coding genes during development and that the similar roles of animal and plant lncRNAs result from convergent evolution. Since pollen development follows an established program with well-defined and characterized stages, we have used it as a model for studying plant lncRNAs and their roles in reproductive development. We investigated of lncRNA expression and function during pollen formation in field mustard (Brassica rapa). Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4,347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.