bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia
Authors: Shinde Swapnil Rohidas , Mick David U. , Aoki Erika , Rodrigues Rachel B. , Gygi Steven P. , Nachury Maxence V. 
Published: September 2022

ABSTRACTMany G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as Hedgehog morphogens. Lysine 63-linked ubiquitin (K63Ub) chains mark GPCRs for regulated removal from cilia, but the molecular basis of K63Ub recognition inside cilia remains elusive. Here we show that the BBSome –the trafficking complex in charge of retrieving GPCRs from cilia– engages the ancestral endosomal sorting factor TOM1L2 (Target of Myb1-Like 2) to recognize UbK63 chains within cilia. TOM1L2 directly binds to UbK63 chains and to the BBSome and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin and the GPCRs SSTR3, Smoothened and GPR161 inside cilia. Strikingly, the single cell algaChlamydomonasalso requires its TOM1L2 orthologue to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.
Human olfactory neuronal cells through nasal biopsy: molecular characterization and utility in brain science
Authors: Yang Kun , Ishizuka Koko , Lane Andrew P. , Narita Zui , Hayashida Arisa , Lema Yukiko Y. , Heffron Emma , Loudd Haydn , Schumacher Maeve , Kano Shin-Ichi , Tomoda Toshifumi , Kamiya Atsushi , Ma Minghong , Geman Donald , Younes Laurent , Sawa Akira 
Published: September 2022

ABSTRACTBiopsy is crucial in clinical medicine to obtain tissues and cells that directly reflect the pathological changes of each disease. However, the brain is an exception due to ethical and practical challenges. Nasal biopsy, which captures the olfactory neuronal epithelium, has been considered as an alternative method of obtaining neuronal cells from living patients. Multiple groups have enriched olfactory neuronal cells (ONCs) from biopsied nasal tissue. ONCs can be obtained from repeated biopsies in a longitudinal study, providing mechanistic insight associated with dynamic changes along the disease trajectory and treatment response. Nevertheless, molecular characterization of biopsied nasal cells/tissue has been insufficient. Taking advantage of recent advances in next-generation sequencing technologies at the single-cell resolution and related rich public databases, we aimed to define the neuronal characteristics, homogeneity, and utility of ONCs. We applied single-cell and bulk RNA sequencing for ONCs, analyzing and comparing the data with multiple public datasets. We observed that the molecular signatures of ONCs are similar to those of neurons, distinct from major glial cells. The signatures of ONCs resemble those of developing neurons and share features of excitatory neurons in the prefrontal and cingulate cortex. The high homogeneity of ONCs is advantageous in pharmacological, functional, and protein studies. Accordingly, we provide two proof-of-concept examples for functional and protein studies, solidifying the utility of ONCs in studying objective biomarkers and molecular mechanisms for brain disorders. The ONCs may also be useful in the studies for the olfactory epithelium impairment and the resultant mental dysfunction elicited by SARS-CoV-2.SIGNIFICANCE STATEMENTTo study dynamic changes and underlying mechanisms along disease trajectory and treatment response in neuropsychiatric disorders, olfactory neuronal cells (ONCs) enriched from biopsied nasal tissue may provide a crucial tool. Because ONCs can be obtained from repeated biopsies in a longitudinal study, this tool has been believed to be useful and complementary to postmortem brains and induced pluripotent stem cell-derived neurons. Nevertheless, molecular characterization of biopsied nasal cells/tissue has been insufficient, which hampers a broader use of this resource. Taking advantage of recent advances in next-generation sequencing technologies at the single-cell resolution and related rich public databases, the present study defines ONCs’ neuronal characteristics, homogeneity, and unique utility for the first time.
Molecular characters and phylogenetic analysis of Clostridium perfringens from different regions in China, from 2013 to 2021
Authors: Zhong Jia xin , Zheng Hao ran , Wang Yuan yuan , Bai Lu lu , Du Xiao li , Wu Yuan , Lu Jin xing 
Published: September 2022

ABSTRACTClostridium perfringens (C. perfringens) is a significant foodborne pathogen and a common cause of intestinal diseases in both animals and humans. Altogether, 186 isolates were obtained from humans (n = 147), animals (n = 25), and food (n = 14), comprising 174 type A strains (93.55%), 11 type F strains (5.91%), and one type D strain (0.54%); and were analyzed by multilocus sequence typing (MLST) and antimicrobial susceptibility testing. Additionally, some specific ST complexes were analyzed by cgMLST and cgSNP to investigate genetic relatedness. MLST indicated the most prevalent STs of C. perfringens of human and animal origin were as follows: ST221 (5/147), ST62 (4/147), ST408 (4/147), and ST493 (4/147) were predominant in humans, while ST479 (5/25) was the major type in animals. Within the same ST complex, genetically unrelated relationships or potential clustering/transmission events were further recognized by cgMLST and cgSNP, illustrating that these two methods are valuable in defining outbreaks and transmission events. All tested isolates were susceptible to vancomycin and meropenem. The rates of resistance to metronidazole, penicillin, cefoxitin, moxifloxacin, and clindamycin were low (metronidazole: 1.08%; penicillin: 9.68%; cefoxitin: 0.54%; moxifloxacin: 6.45%; and chloramphenicol: 3.76%). Interestingly, 49.66% of human origin were clindamycin-resistant, and 18.2% were penicillin-insensitive. Importantly, the portion of MDR isolates was significantly lower than in previous reports. The study provides an overview of the epidemiological characteristics of C. perfringens with different origins and hosts in China. C. perfringens demonstrated remarkable genetic diversity and distinct molecular features compared to antibiotic-resistance profiles from other studies.IMPORTANCEC. perfringens is one of the most common bacterial causes of foodborne illness globally, responsible for several food-poisoning outbreaks each year. This study provides an overview of C. perfringens isolates from different hosts and regions in China according to MLST, antibiotic-resistance characters, cgMLST, and cgSNP analyses, showing high genetic diversity and identifying potential clustering and transmission events. The antimicrobial profile in this study was distinct from that of a previous report with a much lower MDR rate, indicating that C. perfringens in China needs further investigation.
Compounds derived from N,N-dimethyldithiocarbamate are effective copper-dependent antimicrobials against Streptococcus pneumoniae
Authors: Menghani Sanjay V. , Sanchez-Rosario Yamil , Pok Chansorena , Liu Renshuai , Gao Feng , O’Brien Henrik , Neubert Miranda J. , Ochoa Klariza , Hellinger Riley D. , Wang Wei , Johnson Michael D. L. 
Published: September 2022

AbstractN,N-dimethyldithiocarbamate (DMDC) is a potent copper-dependent antimicrobial against several pathogens, including Streptococcus pneumoniae. Despite the availability of several vaccines against multiple disease-causing strains of S. pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. We derived novel compounds from DMDC and tested their effectiveness as copper-dependent antimicrobials against S. pneumoniae through in vitro growth and killing curves. Compounds that caused a growth defect and were bactericidal in vitro were tested against other strains of S. pneumoniae and in complex with different transition metals. We found two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein “Compound 3” and “Compound 4”), were effective against TIGR4, D39, and ATCC® 6303™ (a type 3 capsular strain) and further increased the internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. We found that both Compound 3 and Compound 4 were bacteriostatic in combination with zinc. We tested Compound 3 and Compound 4 in vivo against a murine pneumonia model, finding that Compound 3, and not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. We found that the combination of Compound 3 and copper made the pneumococcus more susceptible to activated macrophage mediated killing via an in vitro macrophage killing assay. Collectively, we demonstrate that derivatizing DMDC holds promise as potent bactericidal antibiotics against S. pneumoniae.
Reduced ribosome activity influences the non-uniform evolution of 16S rRNA hypervariable regions
Authors: Bose Nikhil , Moore Sean D. 
Published: September 2022

Abstract16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain hypervariable regions that can help distinguish different genera. However, intra-genus distinction is often difficult due to high sequence identities among closely related species. Although common tools for 16S sequence taxonomic classification weight residue variations equally during comparisons, specific residues within hypervariable regions have not drifted evenly through evolution, suggesting that portions of them may be biologically important. We developed an in vivo test system where 16S variants coexisted among natural ribosome populations which allowed their fitness to be evaluated. We found that versions with evolutionarily disparate hypervariable regions were underpopulated in ribosomes and active translation pools, even for a single nucleotide polymorphism (SNP), which indicates functional constraints to the free evolutionary drift of hypervariable regions. Using an in silico method (positional relative entropy), we analyzed over 12,000 16S V3-V4 sequences across Escherichia and Shigella strains and identified species that can be distinguished by position-specific SNPs present in multiple 16S alleles in a genome. When we evaluated these informative SNPs with our in vivo system, we discovered that ribosomes harboring them were compromised, suggesting that their evolution is indeed biologically constrained. Overall, this study demonstrates that SNPs within hypervariable regions are not necessarily inconsequential and that common computational approaches for taxonomic 16S rRNA sequence classification should not assume an even probability of residues at each position.ImportanceHypervariable regions within 16S rRNA genes are commonly analyzed to determine microbial diversity. However, because sequences within a genus are highly similar, strain- or species-specific identification is often uncertain. Because there are no established functions of hypervariable regions, residue variations within them are often evenly weighted when making taxonomic comparisons. This study established that 16S rRNAs with naturally occurring variations in hypervariable regions can affect ribosome quality, indicating that their residues should not be weighted equally during taxonomic sequence classifications.
Continuous directed evolution of a feedback-resistant Arabidopsis arogenate dehydratase in plantized E. coli
Authors: Leong Bryan J. , Hanson Andrew D. 
Published: September 2022

AbstractContinuous directed evolution (CDE) is a powerful tool for enzyme engineering due to the depth and scale of evolutionary search that it enables. If suitably controlled and calibrated, CDE could be widely applied in plant breeding and biotechnology to improve plant enzymes ex planta. We tested this concept by evolving Arabidopsis arogenate dehydratase (AtADT2) for resistance to feedback inhibition. We used an Escherichia coli platform with a phenylalanine biosynthesis pathway reconfigured (‘plantized’) to mimic the plant pathway, a T7RNA polymerase-base deaminase hypermutation system (eMutaT7), and 4-fluorophenylalanine as selective agent. Selection schemes were pre-validated using a known feedback-resistant AtADT2 variant. We obtained variants that had 4-fluorophenylalanine resistance at least matching the known variant and that carried mutations in the ACT domain responsible for feedback inhibition. We conclude that ex planta CDE of plant enzymes in a microbial platform is a viable way to tailor characteristics that involve interaction with small molecules.
Global mosquito virome profiling and mosquito spatial diffusion pathways revealed by marker-viruses
Authors: Zhao Lu , Yu Ping , Shi Chenyan , Jia Lijia , Evans Atoni , Wang Xiaoyu , Wu Qun , Xiong Guodian , Ming Zhaoyan , Salazar Ferdinand , Agwanda Bernard , Bente Dennis , Wang Fei , Liu Di , Yuan Zhiming , Xia Han 
Published: September 2022

AbstractMosquitoes are vectors of numerous emergence and reemergence of mosquito-borne diseases, leading to an overwhelming global challenge. The boom in metagenomic studies promoted the increase of mosquito viruses being described, and studies from different regions of the world showed that mosquitoes harbor abundant and diverse viromes. However, there is still a lack of large-scale systematic comparison of viromes among various ecological factors such as the mosquito species/genera, location, etc, and consistent patterns associated with these factors are not clear. Here, we provide an overview of virome profiling that integrated the perspective of mosquito genus, locations, and climates based on the global scale, and redefined the ‘core-virome’. Our results also further implicate some mosquito-associated viruses strongly associated with the ecological factors and highlighted the evidence of mosquitoes’ cross-regional movements by the candidate marker viruses. The study may be helpful in gaining new insights into strategies to prevent arbovirus epidemics.
Ribosomal RNA 2’-O-methylation dynamics impact cell fate decisions
Authors: Häfner SJ , Jansson MD , Altinel K , Kraushar ML , Andersen KL , Abay-Nørgaard Z Caldwell , Fontenas M , Sørensen DM , Gay DM , Arendrup FS , Tehler D , Krogh N , Nielsen H , Kirkeby A , Lund AH 
Published: September 2022

AbstractTranslational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome itself exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2’-O-methylation of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here we explore changes in rRNA 2’-O-methylation during mouse brain development and during tri-lineage differentiation of human embryonic stem cells. We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site which impacts neuronal differentiation. Modulation of its methylation levels affects ribosome association of the Fragile X Mental Retardation Protein and translation of WNT pathway-related mRNAs. Together, the data reveals ribosome heterogeneity through rRNA 2’-O-methylation during early development and differentiation and suggests a direct role for ribosomes in regulating translation during cell fate acquisition.
The role of the γ subunit in the photosystem of the lowest-energy phototrophs
Authors: Namoon Dowrung , Rudling Nicola M , Canniffe Daniel P 
Published: September 2022

ABSTRACTPurple phototrophic bacteria use a core ‘photosystem’ consisting of light harvesting antenna complex 1 (LH1) surrounding the reaction centre (RC), which primarily absorbs far-red–near-infrared light and converts it to chemical energy. Species in the Blastochloris genus, which are able to use light >1000nm for photosynthesis, use bacteriochlorophyll (BChl) b rather than the more common BChl a as their major photopigment, and also uniquely assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple open reading frames in their genomes. In order to assign a role to this subunit, we deleted the four LH1γ-encoding genes in the model Blastochloris viridis. Interestingly, growth under halogen bulbs routinely used for cultivation of anoxygenic phototrophs yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC complex without LH1, but growth under white light from fluorescent bulbs yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose density gradient fractionation demonstrate that the mutant grown in white light assembles RC–LH1, albeit with an absorption maximum blue-shifted by 46 nm relative to the WT complex. Wavelengths between 900–1000 nm transmit poorly through the atmosphere due to strong absorption by water, thus our results provide an evolutionary rationale for the incorporation of the γ subunit into the LH1 ring; this polypeptide red-shifts the absorption maximum of the complex to a range of the spectrum where the photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids carrying genes encoding natural LH1γ variants and demonstrate that the polypeptide found in the WT complex red-shifts absorption back to 1018 nm, but incorporation of a distantly-related variant results in only a moderate red-shift. This result suggests that tuning the absorption maximum of this organism is possible, and may permit light capture past the current low-energy limit of natural photosynthesis.
An approach towards identifying the site of interaction between the Streptomyces phage ØC31 protein Gp3 and the coiled-coil domain of its serine integrase
Authors: Halablab Monalissa , Colloms Sean , Kane Steven 
Published: September 2022

ABSTRACTStreptomyces phage ØC31 encodes a serine integrase which recombines the host (attB) and phage (attP) attachment sites to form new attL and attR sites. For attL and attR to be recombined by the integrase, the presence of its cognate recombination directionality factor (RDF) is required. It is hypothesized that the RDF binds the coiled-coil domain of the integrase to accomplish this switch in recombination directionality. Yet, nothing is known about the site of interaction between the RDF and integrase. Here, we tried to identify the region on the coiled-coil domain of ØC31 integrase to which its cognate RDF (gp3) might bind. Mutant integrases fused to their cognate RDF (gp3) were created and selected for inactivity in attL x attR recombination. It was not possible to characterize the mutants and identify the Int-RDF binding region, due to unforeseen errors that occurred during the construction of the mutant library, but we demonstrated that simple experimental approaches could be used to identify this region. Despite this, an integrase mutant (P398L D595N) fused to gp3 was characterized. This mutant was catalyzing a more unidirectional attL x attR recombination reaction with reduced attP x attB recombination compared to the wild-type integrase. This mutant was selected since it could be used within the field of molecular biology to construct inversion switches which are a key element by which cells can be computerized.By the time this work was uploaded on bioRxiv, there is now a publication that investigated the Int-RDF interaction interface and several residues at the base of the coiled-coil that affected both the interaction and recombinase activity were identified (Paul C M Fogg, Ellen Younger, Booshini D Fernando, Thanafez Khaleel, W Marshall Stark, Margaret C M Smith, Recombination directionality factor gp3 binds PhiC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif, Nucleic Acids Research, Volume 46, Issue 3, 16 February 2018, Pages 1308-1320, https://doi.org/10.1093/nar/gkx1233).
GTP as an enzymatic synchronizer
Authors: Guo Chen-Jun , Zhou Xian , Liu Ji-Long 
Published: September 2022

AbstractSynchronizer is the key component of modern automobile gearbox, which coordinates the deceleration and acceleration of two groups of gears. In industry, synchronizer is composed of several components with fine structure. Here, we show that guanosine triphosphate (GTP) has the property of enzyme synchronizer. Through cryo-electron microscopy (cryo-EM) and 3D variability analysis, we obtain the full spectrum conformation of CTP synthase at near atomic resolution. In the catalytic process of CTP synthase, we find that GTP synchronizes the reaction of two independent domains, namely, glutamine-dependent aminotransferase (GAT) domain and amidoligase (AL) domain. The wing structure acts as a striker, and then stimulates the intermediate ammonia to complete the catalytic cycle through the internal gas channel. Different from being degraded to regulate tubulins or G-proteins, GTP acts as an enzymatic synchronizer to coordinate the reactions catalyzed by the two independent domains of CTP synthase.
Novel Biosensor Identifies Ruxolitinib as a Potent and Cardioprotective CaMKII Inhibitor
Authors: Gaido Oscar E. Reyes , Granger Jonathan M. , Nkashama Lubika J. , Lin Brian L. , Long Alan , Mesubi Olurotimi O. , Schole Kate L. , Terrilion Chantelle E. , Liu Jun O. , Luczak Elizabeth D. , Anderson Mark E. 
Published: September 2022

AbstractCa2+/Calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes heart injury and arrhythmias—two major sources of mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked if any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered a novel fluorescent biosensor, CaMKAR (CaMKII Activity Reporter), which features superior sensitivity, kinetics, and tractability for high throughput screening. Using this tool, we carried a drug repurposing screen (4,475 compounds in clinical use) in human cells expressing autonomously active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. Standout among these, ruxolitinib, an orally bioavailable and U.S Food and Drug Administration (FDA)-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice at concentrations equivalent to human doses. 10-minute treatment in mice was sufficient to prevent atrial fibrillation— the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice behaved normally in established cognitive assays. Our results suggest that human CaMKII inhibition is feasible and safe, and support prompt clinical investigation of ruxolitinib for cardiac indications.One Sentence SummaryWe developed a CaMKII biosensor suitable for high throughput screening and identified ruxolitinib as a CaMKII inhibitor capable of rescuing cardiac arrhythmia.