bluuebunny/crossref_metadata_2025_split huggingface dataset:
Orthogonal Gene Engineering Enables CD8+ T Cells to Control Tumors through a Novel PD-1+TOX-indifferent Synthetic Effector State
Authors: Corria-Osorio Jesus , Carmona Santiago J. , Stefanidis Evangelos , Andreatta Massimo , Muller Tania , Ortiz-Miranda Yaquelin , Seijo Bili , Castro Wilson , Jimenez-Luna Cristina , Scarpellino Leonardo , Ronet Catherine , Spill Aodrenn , Lanitis Evripidis , Luther Sanjiv A. , Romero Pedro , Irving Melita , Coukos George 
Published: February 2022

SummaryAdoptive immunotherapy offers opportunities to reprogram T cells and the tumor microenvironment. Orthogonal engineering of adoptively transferred T cells with an IL-2Rβγ-binding IL-2 variant, PD1-decoy and IL-33 led to cell-autonomous T-cell expansion, T-cell engraftment and tumor control in immunocompetent hosts through reprogramming of both transferred and endogenous CD8+ cells. Tumor-infiltrating lymphocytes adopted a novel effector state characterized by TOX suppression and specific expression of multiple effector molecules, most prominently granzyme C. While the IL-2 variant promoted CD8+ T-cell stemness and persistence, and was associated with downregulation of TOX, the combination with IL-33 was necessary to trigger the novel polyfunctional effector state. Rational T-cell engineering without host lymphodepletion enables optimal reprogramming of adoptively transferred T cells as well as mobilization of endogenous immunity into new functional CD8+ states mediating tumor control.
DJ-1 glyoxalase activity makes a modest contribution to cellular defense against methylglyoxal damage in neurons
Authors: Mazza Melissa Conti , Shuck Sarah , Lin Jiusheng , Moxley Michael A. , Termini John , Cookson Mark R. , Wilson Mark A. 
Published: February 2022

AbstractHuman DJ-1 is a cytoprotective protein whose absence causes Parkinson’s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.
Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome
Authors: Evans Sasha L. , Al-Hazeem Monsour M. J. , Mann Daniel , Smetacek Nicolas , Beavil Andrew J. , Sun Yaqi , Chen Taiyu , Dykes Gregory F. , Liu Lu-Ning , Bergeron Julien R. C. 
Published: February 2022

AbstractCarboxysomes are proteaceous bacterial microcompartments (BMCs) that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating carbonic anhydrase and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyses the dehydration of bicarbonate into CO2, the first step of the Calvin–Benson–Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural characterization of native carboxysomes, including the shell and the internal organization, is currently limited to low-resolution tomography studies. Notably, the degree of heterogeneity of the shell, and the internal arrangement of enzymes, remain poorly understood. Here, we report the structural characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryo-EM. We determine the structure of RuBisCO enzyme at 2.9 Å resolution. In addition, we obtain low-resolution maps of the icosahedral protein shell and the concentric interior organisation. In combination with artificial intelligence (AI)-driven modelling approaches, we exploited these maps to propose a complete atomic model of an intact carboxysome. This study provides insight into carboxysome structure and protein-protein interactions involved in carboxysome assembly. Advanced knowledge about carboxysome architecture and structural plasticity is critical for not only a better understanding of biological carbon fixation mechanism but also repurposing carboxysomes in synthetic biology for biotechnological applications.
Structural insights into Charcot-Marie-Tooth disease-linked mutations in human GDAP1
Authors: Sutinen Aleksi , Nguyen Giang Thi Tuyet , Raasakka Arne , Muruganandam Gopinath , Loris Remy , Ylikallio Emil , Tyynismaa Henna , Bartesaghi Luca , Ruskamo Salla , Kursula Petri 
Published: February 2022

ABSTRACTCharcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R as well as CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behaviour, but both present a large decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side chain interaction network between helices α3, α6, and α7, which is affected by CMT mutations, as well as a hinge in the long helix α6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra- and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and eventually, insufficient motor and sensory neuron function.
Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2
Authors: Alderson T. Reid , Pritišanac Iva , Kolarić Đesika , Moses Alan M. , Forman-Kay Julie D. 
Published: February 2022

AbstractThe AlphaFold Protein Structure Database contains predicted structures for millions of proteins. For the majority of human proteins that contain intrinsically disordered regions (IDRs), which do not adopt a stable structure, it is generally assumed these regions have low AlphaFold2 confidence scores that reflect low-confidence structural predictions. Here, we show that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. By comparison to experimental NMR data for a subset of IDRs that are known to conditionally fold (i.e., upon binding or under other specific conditions), we find that AlphaFold2 often predicts the structure of the conditionally folded state. Based on databases of IDRs that are known to conditionally fold, we estimate that AlphaFold2 can identify conditionally folding IDRs at a precision as high as 88% at a 10% false positive rate, which is remarkable considering that conditionally folded IDR structures were minimally represented in its training data. We find that human disease mutations are nearly 5-fold enriched in conditionally folded IDRs over IDRs in general, and that up to 80% of IDRs in prokaryotes are predicted to conditionally fold, compared to less than 20% of eukaryotic IDRs. These results indicate that a large majority of IDRs in the proteomes of human and other eukaryotes function in the absence of conditional folding, but the regions that do acquire folds are more sensitive to mutations. We emphasize that the AlphaFold2 predictions do not reveal functionally relevant structural plasticity within IDRs and cannot offer realistic ensemble representations of conditionally folded IDRs.Significance StatementAlphaFold2 and other machine learning-based methods can accurately predict the structures of most proteins. However, nearly two-thirds of human proteins contain segments that are highly flexible and do not autonomously fold, otherwise known as intrinsically disordered regions (IDRs). In general, IDRs interconvert rapidly between a large number of different conformations, posing a significant problem for protein structure prediction methods that define one or a small number of stable conformations. Here, we found that AlphaFold2 can readily identify structures for a subset of IDRs that fold under certain conditions (conditional folding). We leverage AlphaFold2’s predictions of conditionally folded IDRs to quantify the extent of conditional folding across the tree of life, and to rationalize disease-causing mutations in IDRs.Classifications: Biological Sciences; Biophysics and Computational Biology
Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces
Authors: Francis Emmet , Xiao Hugh , Teng Lay Heng , Heinrich Volkmar 
Published: February 2022

AbstractComplex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy (RICM) and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.SummaryThe detailed analysis of immune-cell spreading on antibody-coated surfaces establishes that active cytoskeletal protrusion rather than passive substrate adhesion drives phagocytic spreading.
Synthetic lethality between toxic amino acids, retrograde target genes and chaperones in Saccharomyces cerevisiae
Authors: Druseikis Marina E. , Covo Shay 
Published: February 2022

AbstractThe toxicity of non-proteinogenic amino acids has been known for decades. Numerous reports describe their antimicrobial/anticancer potential. However, these molecules are often toxic to the host as well, therefore a synthetic lethality approach can be beneficial. Here we investigated the potential synthetic lethality between toxic amino acids, the retrograde pathway, and molecular chaperones. In Saccharomyces cerevisiae, mitochondrial retrograde (RTG) pathway activation induces transcription of RTG-target genes, which replenishes alpha- ketoglutarate and glutamate; both metabolites are required for arginine and lysine biosynthesis. We previously reported that tolerance of canavanine, a toxic arginine derivative, requires an intact RTG pathway, and low-dose canavanine exposure reduces the expression of RTG-target genes. At higher concentrations, canavanine causes protein misfolding. Here we show that in WT, low-dose thialysine exposure, a toxic lysine analog, has a similar effect as canavanine on RTG-target gene expression. To study if single amino acid deficiency and mild protein misfolding stress elicit a similar effect on RTG-target genes, we compared expression of heat shock protein (HSP) mutants grown without arginine or lysine to WT. Arginine deprivation induces RTG-target gene expression in sse2Δ, hsp78Δ, and mdj1Δ, but CIT2 and DLD3 are reduced in cpr7Δ. Lysine deprivation has the opposite effect on RTG-target gene expression in HSP mutants. Interestingly, exposure of HSP mutants to canavanine and thialysine reversed the trend of RTG-target gene expression in several cases. The RTG-target expression pattern in HSP mutants had a predictive value in canavanine sensitivity - the mutant with the lowest expression was the most sensitive - but this was not the case for thialysine. Some, but not all, mutants in RTG-target genes are sensitive to canavanine and thialysine; additional mutation in a certain HSP can exacerbate this sensitivity. Overall, we show that inhibiting molecular chaperones, RTG-target genes, or both can sensitize cells to low doses of toxic amino acids.
Detection of specific uncultured bacteriophages by fluorescence in situ hybridisation in pig microbiome
Authors: Ostenfeld Line Jensen , Munk Patrick , Aarestrup Frank M. , Otani Saria 
Published: February 2022

AbstractMicrobial communities have huge impacts on their ecosystems and local environments spanning from marine and soil communities to the mammalian gut. Bacteriophages (phages) are important drivers of population control and diversity in the community, but our understanding of complex microbial communities is halted by biased detection techniques. Metagenomics have provided a method of novel phage discovery independent of in vitro culturing techniques and have revealed a large proportion of understudied phages. Here, five large phage genomes, that were previously assembled in silico from pig faecal metagenomes, are detected and observed directly in their natural environment using a modified phageFISH approach, and combined with methods to decrease bias against large phages. These phages are uncultured with unknown hosts. The specific phages were detected by PCR and fluorescent in situ hybridisation in their original faecal samples as well as across other faecal samples. Co-localisation of bacterial signals and phage signals allowed detection of the different stages of phage life cycle. All phages displayed examples of early infection, advanced infection, burst, and free phages. To our knowledge, this is the first detection of jumbophages in faeces, which were investigated independently of culture, host identification, and size, and based solely on the genome sequence. This approach opens up opportunities for characterisation of novel in silico phages in vivo from a broad range of gut microbiomes.
With a pinch of salt: metagenomic insights into Namib Desert salt pan microbial mats and halites reveal functionally adapted and competitive communities
Authors: Martínez-Alvarez Laura , Ramond Jean-Baptiste , Vikram Surendra , León-Sobrino Carlos , Maggs-Kölling Gillian , Cowan Don A. 
Published: February 2022

AbstractSalt pans or playas, which are saline-rich springs surrounded by halite evaporates in arid environments, have played an essential role in landscape erosion during the formation of the Namib Desert and are numerous in its central region. In this study, we used shotgun metagenomics to investigate the phylogenetic and functional capacities of the microbial communities from two salt pans (namely, Eisefeld and Hosabes) located in the Central Namib Desert, located in Southwest Africa. We studied the source and sink sediment mat communities of the saline streams, as well as those from two halites (crystallized structures on the stream margins). The microbial assemblages and potential functions were distinct in both niches. Independently from their localization (Eisfeld vs Hosabes and source vs sink), the sediment mat communities were dominated by members of theAlpha-andGamma-proteobacteriaclasses, while halites were Archaea-dominated and also contained high abundances of the extremely halophilic bacteriumSalinibactersp. (phylumBacteroidota). Photoheterotrophy and chemoheterotrophy were the principal lifestyles in both niches, with halite communities having a reduced diversity of metabolic pathways. Intense microbial-virus interactions in both niches were implied by the widespread detection of CRISPR-Cas defense systems. We identified a putatively novel clade of type II CRISPR-Cas systems, as well as novel candidate viral lineages of the class Caudoviricetes and of Halobacteriales-infecting haloviruses. Putative gene transfer agent-like sequences within theAlphaproteobacteriawere identified in the sediment mat communities. These horizontal gene transfer elements have the potential to drive genome plasticity and evolution of theAlphaproteobacteriain the Namib Desert salt pan microbiomes.ImportanceThe hyperarid Namib Desert is one of the oldest deserts on Earth. It contains multiple clusters of playas which are saline-rich springs surrounded by halite evaporites. Playas are of great ecological importance and their indigenous (poly)extremophilic microorganisms are potentially involved in the precipitation of minerals such as carbonates and sulfates and have been of great biotechnological importance. While there has been a considerable amount of microbial ecology research preformed on various Namib Desert edaphic microbiomes, little is known about the microbial communities inhabiting its multiple playas. In this work, we therefore provide a comprehensive taxonomic and functional potential characterization of the microbial, including viral, communities of sediment mats and halites from two distant Namib Desert, contributing towards a better understanding of the ecology of this biome.
Reproducibility metrics for CRISPR screens
Authors: Billmann Maximilian , Ward Henry N. , Aregger Michael , Costanzo Michael , Andrews Brenda J. , Boone Charles , Moffat Jason , Myers Chad L. 
Published: February 2022

ABSTRACTCRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal.
Cohesin mutation sensitizes cancer cells to anti-PD-1 therapy through endogenous retrovirus-mediated PD-L1 upregulation
Authors: Han Yumin , Peng Fangfei , Chang Yunqi , Liu Tingting , Shen Jiayan , Chen Zizhuo , Dong Qian , Zhou Ping , Jiang Feng , Xiang Honggang , Zhu Hong , Qing Chen , Kong Xiangyin , Ding Jian , Lang Jing-Yu 
Published: February 2022

AbstractImmune checkpoint therapy shows impressive and durable clinical responses in cancer patients, but the genetic determinants that enable cancer cells to respond to anti-PD-1 therapy are still elusive. Herein, we identified thatNIPBLdeficiency promotes endogenous retrovirus (ERV) expression in tumour cells, which in turn inactivates CD8+ tumour-infiltrating lymphocytes (TILs) via the PD-L1/PD-1 inhibitory checkpoint pathway. Mechanistically,NIPBLdeficiency impairs DNMT1 transcription, preventing DNMT1 from suppressing ERV expression in tumour cells; ERVs stimulate PD-L1 expression by inducing the STAT2-IRF9 complex, a downstream event of double-stranded RNA (dsRNA)-MAVS-IRF3 signalling, and thereby suppress CD8 TIL-mediated immunity. An anti-PD-1 monoclonal antibody achieved remarkable therapeutic effects inNipbl-deficient syngeneic tumour models and improved host survival by eliciting an antitumour memory immune response. Cancer patients harbouring mutations of cohesin subunits and regulators plus DNMT1 had significantly better responses to anti-PD-1 therapy than their non-mutated counterparts did. Our study reveals a novel mechanism by which cohesin complex deregulation stimulates ERV expression by impairing DNMT1 expression and fosters an immunosuppressive tumour microenvironment by activating the PD-L1/PD-1 inhibitory checkpoint.
Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila Melanogaster (Pupal Retina)
Authors: Naz Rubina , Iqbal Anwar , Saeed Asma , Khan Alamzeb , Shutaywi Meshal , Cioca Adriana Lavinia , Vrinceanu Narcisa 
Published: February 2022

AbstractA novel pair of protein Tyrosine Phosphatases in Drosophila Melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized having fundamental role in cellular processes including effects on metabolism, cell proliferation and differentiation. This study presents comparative homology modeling of low molecular weight phosphotyrosine protein phosphatase (PTPs) from Drosophila melanogaster (Dr-PTPs) and their complexation with potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homology is approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel β sheet with flanking α helices on both sides, showing two right-handed β-α-β motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs activation. The structure guided studies of both proteins clearly reveal a common mechanism of action, inhibitor binding at the active site and will expected to contribute towards the basic understanding of functional association of this enzyme with other molecules.