bluuebunny/crossref_metadata_2025_split huggingface dataset:
Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
Authors: Malm Magdalena , Kuo Chih-Chung , Barzadd Mona Moradi , Mebrahtu Aman , Wistbacka Num , Razavi Ronia , Volk Anna-Luisa , Lundqvist Magnus , Kotol David , Edfors Fredrik , Gräslund Torbjörn , Chotteau Veronique , Field Ray , Varley Paul G. , Roth Robert G. , Lewis Nathan E. , Hatton Diane , Rockberg Johan 
Published: August 2021

SummaryBiologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.
Bacterial DNA on the skin surface overrepresents the viable skin microbiome
Authors: Acosta Ellen M , Little Katherine A , Bratton Benjamin P , Lopez Jaime G , Mao Xuming , Payne Aimee , Donia Mohamed S , Devenport Danelle , Gitai Zemer 
Published: August 2021

AbstractThe skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria as compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in-vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation, as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.Significance statementThis study provides a crucial update to the skin microbiome paradigm by showing that viable bacteria of the skin microbiome are primarily localized to hair follicles and other sub-cutaneous structures rather than the skin surface. The native distribution of skin-associated bacteria has not been previously evaluated, and here we show that the skin surface has few intact bacteria while deeper structures are replete with bacterial contents. We used orthogonal approaches to evaluate the source of bacterial DNA and the dynamics of bacterial repopulation on the skin surface to develop an updated model of the skin microbiome. By adjusting the current understanding of the skin microbiome to match this model, we will be able to address outstanding questions in the field.
Molecular signature and target-specificity of inhibitory circuits formed by Martinotti cells in the mouse barrel cortex
Authors: Donato Cristina , Cabezas Carolina , Aguirre Andrea , Lourenço Joana , Potier Marie-Claude , de San Martin Javier Zorrilla , Bacci Alberto 
Published: August 2021

AbstractIn the neocortex, fast synaptic inhibition orchestrates both spontaneous and sensory-evoked activity. GABAergic interneurons (INs) inhibit pyramidal neurons (PNs) directly, modulating their output activity and thus contributing to balance cortical networks. Moreover, several IN subtypes also inhibit other INs, forming specific disinhibitory circuits, which play crucial roles in several cognitive functions. Here, we studied a homogeneous subpopulation of somatostatin (SST)-positive INs, the Martinotti cells (MCs) in layer 2/3 of the mouse barrel cortex (both sexes). MCs are a prominent IN subclass inhibiting the distal portion of PN apical dendrites, thus controlling dendrite electrogenesis and synaptic integration. Yet, it is poorly understood whether MCs inhibit other elements of the cortical circuits, and the connectivity properties with non-PN targets are unknown. We found that MCs have a strong preference for PN dendrites, but they also considerably connect with parvalbumin (PV)-positive, vasoactive intestinal peptide (VIP)-expressing and layer 1 (L1) INs. Remarkably, GABAergic synapses from MCs exhibited clear cell-type-specific short-term plasticity. Moreover, whereas the biophysical properties of MC-PN synapses were consistent with distal dendritic inhibition, MC-IN synapses exhibited characteristics of fast perisomatic inhibition. Finally, MC-PN connections used α5-containing GABAARs, but this subunit was not expressed by the other INs targeted by MCs. We reveal a specialized connectivity blueprint of MCs within different elements of superficial cortical layers. In addition, our results identify α5-GABAARs as the molecular fingerprint of MC-PN dendritic inhibition. This is of critical importance, given the role of α5-GABAARs in cognitive performance and their involvement in several brain diseases.Significance statementMartinotti cells (MCs) are a prominent subclass of SST-expressing GABAergic INs, specialized in controlling distal dendrites of PNs and taking part in several cognitive functions. Here we characterize the connectivity pattern of MCs with other INs in the superficial layers (L1 and L2/3) of the mouse barrel cortex. We found that the connectivity pattern of MCs with PNs as well as PV, VIP and L1 INs exhibit target-specific plasticity and biophysical properties. The stark specificity of α5-GABAARs at MC-PN synapses, and the lack or functional expression of this subunit by other cell types, define the molecular identity of MC-PN connections and the exclusive involvement of this outstanding inhibitory circuits in α5-dependent cognitive tasks.
Prediction of allosteric sites and signalling: insights from benchmarking datasets
Authors: Wu Nan , Strömich Léonie , Yaliraki Sophia N. 
Published: August 2021

AbstractAllostery is a pervasive mechanism which regulates the activity of proteins in living systems through binding of a molecule at a distant site from the orthosteric site of the protein. The universality of allosteric regulation complemented by the benefits of highly specific, potentially non-toxic and protein activity modulating allosteric drugs makes uncovering allosteric sites on proteins invaluable for drug discovery. However, there are few computational methods to effectively predict them. Bond-to-bond propensity analysis, a recently developed method, has successfully predicted allosteric sites for a diverse group of proteins with only the knowledge of the orthosteric sites and the corresponding ligands in 19 of 20 cases. The method is based on an energy-weighted atomistic protein graph and allows for computationally highly efficient analysis in atomistic detail. We here extended the analysis onto 432 structures of 146 proteins from two existing benchmarking datasets for allosteric proteins: ASBench and CASBench. We further refined the metrics to account for the cumulative effect of residues with high propensities and the crucial residues in a given site with two additional measures. The allosteric site is recovered for 95/113 proteins (99/118 structures) from ASBench and 32/33 proteins (304/314 structures) from CASBench, with the only a priori knowledge being the orthosteric site residues. Knowing the orthosteric ligands of the protein, the allosteric site is identified for 32/33 proteins (308/314 structures) from CASBench.
Controlling for variable transposition rate with an age-adjusted site frequency spectrum
Authors: Horvath Robert , Menon Mitra , Stitzer Michelle , Ross-Ibarra Jeffrey 
Published: August 2021

AbstractRecognition of the important role of transposable elements (TEs) in eukaryotic genomes quickly led to a burgeoning literature modeling and estimating the effects of selection on TEs. Much of the empirical work on selection has focused on analyzing the site frequency spectrum (SFS) of TEs. But TEs differ from standard evolutionary models in a number of ways that can impact the power and interpretation of the SFS. For example, rather than mutating under a clock-like model, transposition often occurs in bursts which can inflate particular frequency categories compared to expectations under a standard neutral model. If a TE burst has been recent, the excess of low frequency polymorphisms can mimic the effect of purifying selection. Here, we investigate how transposition bursts affect the frequency distribution of TEs and the correlation between age and allele frequency. Using information on the TE age distribution, we propose an age-adjusted site frequency spectrum to compare TEs and neutral polymorphisms to more effectively evaluate whether TEs are under selective constraints. We show that our approach can minimize instances of false inference of selective constraint, but also allows for a correct identification of even weak selection affecting TEs which experienced a transposition burst and is robust to at least simple demographic changes. The results presented here will help researchers working on TEs to more reliably identify the effects of selection on TEs without having to rely on the assumption of a constant transposition rate.
Brain transcriptomic profiling reveals common alterations across neurodegenerative and psychiatric disorders
Authors: Sadeghi Iman , Gispert Juan D. , Palumbo Emilio , Muñoz-Aguirre Manuel , Wucher Valentin , D’Argenio Valeria , Santpere Gabriel , Navarro Arcadi , Guigo Roderic , Vilor-Tejedor Natàlia 
Published: August 2021

ABSTRACTNeurodegenerative and neuropsychiatric disorders (ND-NPs) are multifactorial, polygenic and complex behavioral phenotypes caused by brain abnormalities. Large-scale collaborative efforts have tried to identify the genetic architecture of these conditions. However, specific and shared underlying molecular pathobiology of brain illnesses is not clear. Here, we examine transcriptome-wide characterization of eight conditions, using a total of 2,633 post-mortem brain samples from patients with Alzheimer’s disease (AD), Parkinson’s disease (PD), Progressive Supranuclear Palsy (PSP), Pathological Aging (PA), Autism Spectrum Disorder (ASD), Schizophrenia (Scz), Major Depressive Disorder (MDD), and Bipolar Disorder (BP)–in comparison with 2,078 brain samples from matched control subjects.Similar transcriptome alterations were observed between NDs and NPs with the top correlations obtained between Scz-BP, ASD-PD, AD-PD, and Scz-ASD. Region-specific comparisons also revealed shared transcriptome alterations in frontal and temporal lobes across NPs and NDs. Co-expression network analysis identified coordinated dysregulations of cell-type-specific modules across NDs and NPs. This study provides a transcriptomic framework to understand the molecular alterations of NPs and NDs through their shared- and specific gene expression in the brain.
Structural basis for feedforward control in the PINK1/parkin pathway
Authors: Sauvé Véronique , Sung George , MacDougall Emma , Kozlov Guennadi , Saran Anshu , Fakih Rayan , Fon Edward A. , Gehring Kalle 
Published: August 2021

ABSTRACTPINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
Refutation of traumatic insemination in the Drosophila bipectinata species complex: Hypothesis fails critical tests
Authors: Polak Michal , McEvey Shane F. 
Published: August 2021

AbstractTraumatic insemination (TI) is a rare reproductive behaviour characterized by the transfer of sperm to the female via puncture wounds inflicted across her body wall. Here, we challenge the claim made by Kamimura (2007) that males of species of the Drosophila bipectinata complex utilize a pair of claw-like processes (“claws”) to traumatically inseminate females: the claws are purported to puncture the female body wall and genital tract, and to inject sperm through the wounds into the genital tract, bypassing the vaginal opening, the route of sperm transfer occurring in other Drosophila. This supposed case of TI is widely cited and featured in prominent subject reviews. We examined high-resolution scanning electron micrographs of the claws and failed to discover any obvious “groove” for sperm transport. We demonstrated that sperm occurred in the female reproductive tract as a single integrated unit when mating flies were experimentally separated, inconsistent with the claim that sperm are injected via paired processes. The aedeagus in the bipectinata complex was imaged, and shown to deliver sperm through the vaginal opening. Laser ablation of the sharp terminal ends of the claws failed to inhibit insemination. The results refute the claim of TI in the Drosophila bipectinata species complex.
Sex-dependent resource defense in a nectar-feeding bat
Authors: Wintergerst Sabine , Winter York , Nachev Vladislav 
Published: August 2021

AbstractAggressive resource defense spans from the transient monopolization of a resource up to the long-term maintenance of a territory. While such interference competition is common in nectar-feeding birds, reports in nectar-feeding bats are rare.Glossophagabats have been observed to temporarily defend flowers but the extent of this monopolization, its effects on nectar intake, and underlying sexual differences have remained unknown. We investigated resource defense behavior ofGlossophaga muticain the laboratory. We presented bats with two patches of computer-controlled artificial flowers and tracked individual nectar intake. Furthermore, we established an automated method for detecting aggressive interactions at the artificial flowers. Theoretical models of interference competition predict more aggressive interactions when resources are spatially more clumped. To test this, we varied resource distribution across two patches from clumped to dispersed and monitored bats’ interactions in one male, one female, and four mixed-sex groups. Males engaged in aggressive interactions more often than females and in each group some individuals defended clumped artificial flowers against others. Subordinate males experienced a substantial decrease in nectar intake, while females were only marginally affected by male aggression. These results suggest that aggressive interactions and their effect on nectar intake are sex-dependent inG. mutica. Furthermore, aggressive interactions were more frequent and resource defense was only successful when resources were clumped. Our experimental set-up allowed us to perform an automated test of models of interference competition with a mammal under controlled laboratory conditions. This approach may pave the way for similar studies with other animals.Lay summaryMales bully other males to get more food, but only when food is easy to defend. When flowers are spread out nectar-feeding bats rarely engage in fights. However, when there are rich flowers in one spot and no flowers elsewhere, some males start attacking others, denying them access to the nectar. Females do not seem bothered by such male bullies, but when there are no males around, some females become bullies themselves.
Seizures, behavioral deficits and adverse drug responses in two new genetic mouse models of HCN1 epileptic encephalopathy
Authors: Merseburg Andrea , Kasemir Jacquelin , Buss Eric W. , Leroy Felix , Bock Tobias , Porro Alessandro , Barnett Anastasia , Tröder Simon E. , Engeland Birgit , Stockebrand Malte , Moroni Anna , Siegelbaum Steven A , Isbrandt Dirk , Santoro Bina 
Published: August 2021

AbstractDe novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo mutations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Hcn1G380D/+ animals had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from HCN1 patients, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the need to tailor effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective.
Adolescent development of multiscale structural wiring and functional interactions in the human connectome
Authors: Park Bo-yong , Paquola Casey , Bethlehem Richard A. I. , Benkarim Oualid ,   Neuroscience in Psychiatry Network (NSPN) Consortium, Mišić Bratislav , Smallwood Jonathan , Bullmore Edward T. , Bernhardt Boris C. 
Published: August 2021

AbstractAdolescence is a time of profound changes in the physical wiring and function of the brain. Here, we analyzed structural and functional brain network development in an accelerated longitudinal cohort spanning 14–25 years (n = 199). Core to our work was an advanced in vivo model of cortical wiring incorporating MRI features of cortico-cortical proximity, microstructural similarity, and white matter tractography. Longitudinal analyses assessing age-related changes in cortical wiring identified a continued differentiation of multiple cortico-cortical structural networks in youth. Studying resting-state functional MRI measures in the same participants at baseline, we found that regions with more similar structural wiring were more likely to be functionally coupled. Moreover, longitudinal structural wiring changes, particularly between sensory/unimodal and default mode networks, were reflected in tendencies for increased differentiation in brain function. These longitudinal findings provide new insights into adolescent development of human brain structure and function, illustrating how structural wiring interacts with the maturation of macroscale functional hierarchies.
Natural selection does not affect the estimates of effective population size based on linkage disequilibrium
Authors: Novo Irene , Santiago Enrique , Caballero Armando 
Published: August 2021

AbstractThe effective population size (Ne) is a key parameter to quantify the magnitude of genetic drift and inbreeding, with important implications in human evolution. The increasing availability of high-density genetic markers allows the estimation of historical changes in Ne across time using measures of genome diversity or linkage disequilibrium between markers. Selection is expected to reduce diversity and Ne, and this reduction is modulated by the heterogeneity of the genome in terms of recombination rate. Here we investigate by computer simulations the consequences of selection (both positive and negative) and of recombination rate heterogeneity in the estimation of historical Ne. We also investigate the relationship between diversity parameters and Ne across the different regions of the genome using human marker data. We show that the estimates of historical Ne obtained from linkage disequilibrium between markers (NeLD) are virtually unaffected by selection. In contrast, those estimates obtained by coalescence mutation-recombination-based methods can be strongly affected by it, what could have important consequences for the estimation of human demography. The simulation results are supported by the analysis of human data. The estimates of NeLD obtained for particular genomic regions do not correlate with recombination rate, nucleotide diversity, polymorphism, background selection statistic, minor allele frequency of SNPs, loss of function and missense variants and gene density. This suggests that NeLD measures are merely indicative of demographic changes in population size across generations.Author summaryThe inference of the demographic history of populations is of great relevance in evolutionary biology. This inference can be made from genomic data using coalescence methods or linkage disequilibrium methods. However, the assessment of these methods is usually made assuming neutrality (absence of selection). Here we show by computer simulations and analyses of human data that the estimates of historical effective population size obtained from linkage disequilibrium between markers are unaffected by natural selection, either positive or negative. In contrast, estimates obtained by coalescence mutation-recombination-based methods can be strongly affected by it, which could have important consequences for recent estimations of human demography. Thus, only linkage disequilibrium methods appear to provide unbiased estimates of the population census size.