bluuebunny/crossref_metadata_2025 huggingface dataset:
Pan-cancer distribution of cleaved cell-surface Amphiregulin, the target of the GMF-1A3 antibody drug conjugate
Authors: Lofgren Kristopher A. , Reker Nicolette C. , Sreekumar Sreeja , Kenny Paraic A.
Published: March 2022

Amphiregulin (AREG) is a transmembrane protein which, following TACE/ADAM17-dependent cleavage, releases a soluble Epidermal Growth Factor Receptor ligand domain that promotes proliferation of normal and malignant cells. Expression of Amphiregulin has been described by immunohistochemistry in several tumor types, including lung, prostate, head and neck, gastric, pancreatic and breast cancers but evidence for a functional requirement for Amphiregulin in these malignancies is more limited. We have previously described the development of a monoclonal antibody, GMF-1A3, that selectively recognizes the Amphiregulin epitope that is revealed following cleavage by TACE/ADAM17 and demonstrated that drug conjugates of this antibody have anti-tumor activity in mouse models. By directly evaluating Amphiregulin cleavage, immunohistochemistry on tissue specimens using this antibody can be used to evaluate the extent to which Amphiregulin is being proteolytically processed in cancer, which is a more direct measure of Amphiregulin activity. As a potential companion diagnostic for this antibody-drug conjugate, this immunohistochemistry assay allows identification of tumors with high levels of the cleaved Amphiregulin target. Here we evaluate levels of cleaved Amphiregulin in 370 specimens from 10 tumor types and demonstrate that it is widely expressed in solid tumors and is especially common (more than 50% of cases) in breast, prostate, liver and lung cancer.
Characterization of Morreton Virus (MORV) as a Novel Oncolytic Virotherapy Platform for Liver Cancers
Authors: Nagalo Bolni Marius , Zhou Yumei , Loeuillard Emilien J. , Dumbauld Chelsae , Barro Oumar , Elliott Natalie M. , Baker Alexander T. , Arora Mansi , Bogenberger James M. , Meurice Nathalie , Petit Joachim , Uson Junior Pedro Luiz Serrano , Aslam Faaiq , Chamcheu Jean Christopher , Simoes Camila C. , Cannon Martin J. , Basnakian Alexei , Post Steven R. , Buetow Kenneth , Barrett Michael T. , Duda Dan G. , Jacobs Bertram , Vile Richard , Barry Michael A. , Roberts Lewis R. , Ilyas Sumera , Borad Mitesh J.
Published: March 2022

Morreton virus (MORV) is a novel oncolyticVesiculovirus, genetically distinct from vesicular stomatitis virus (VSV). we report that MORV induced potent cytopathic effects in a panel of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines. In addition, high intranasal doses of MORV were not associated with significant adverse effects and were well tolerated in mice bearing liver tumor xenografts and syngeneic liver cancers. Furthermore, single intratumoral treatments with MORV (1 x 107TCID50) triggered a robust antitumor immune response leading to substantial tumor regression and disease control in a syngeneic CCA model, using 10-fold lower dose compared to VSV (1 x 108TCID50). In addition, MORV and VSV both induced prominent tumor growth delay in immunodeficient mice bearing Hep3B hepatocellular carcinoma (HCC) but not in mice bearing HuCCT-1 CCA xenografts. Our findings indicate that wild-type MORV is safe and can induce potent tumor regression in HCC and CCA animal models without adverse events via immune-mediated and immune-independent mechanisms. Further development and clinical translation of MORV as virotherapy for liver cancers are warranted.
Organization of thalamocortical structural covariance and a corresponding 3D atlas of the mouse thalamus
Authors: Yee Yohan , Ellegood Jacob , French Leon , Lerch Jason P.
Published: March 2022

For information from sensory organs to be processed by the brain, it is usually passed to appropriate areas of the cerebral cortex. Almost all of this information passes through the thalamus, a relay structure that reciprocally connects to the vast majority of the cortex. The thalamus facilitates this information transfer through a set of thalamocortical connections that vary in cellular structure, molecular profiles, innervation patterns, and firing rates. Additionally, corticothalamic connections allow for intracortical information transfer through the thalamus. These efferent and afferent connections between the thalamus and cortex have been the focus of many studies, and the importance of cortical connectivity in defining thalamus anatomy is demonstrated by multiple studies that parcellate the thalamus based on cortical connectivity profiles.

Here, we examine correlated morphological variation between the thalamus and cortex, or thalamocortical structural covariance. For each voxel in the thalamus as a seed, we construct a cortical structural covariance map that represents correlated cortical volume variation, and examine whether high structural covariance is observed in cortical areas that are functionally relevant to the seed. Then, using these cortical structural covariance maps as features, we subdivide the thalamus into six non-overlapping regions (clusters of voxels), and assess whether cortical structural covariance is associated with cortical connectivity that specifically originates from these regions.

We show that cortical structural covariance is high in areas of the cortex that are functionally related to the seed voxel, cortical structural covariance varies along cortical depth, and sharp transitions in cortical structural covariance profiles are observed when varying seed locations in the thalamus. Subdividing the thalamus based on structural covariance, we additionally demonstrate that the six thalamic clusters of voxels stratify cortical structural covariance along the dorsal-ventral, medial-lateral, and anterior-posterior axes. These cluster-associated structural covariance patterns are prominently detected in cortical regions innervated by fibers projecting out of their related thalamic subdivisions.

Together, these results advance our understanding of how the thalamus and the cortex couple in their volumes. Our results indicate that these volume correlations reflect functional organization and structural connectivity, and further provides a novel segmentation of the mouse thalamus that can be used to examine thalamic structural variation and thalamocortical structural covariation in disease models.
Calcium-induced environmental adaptability of the blood protein vitronectin
Authors: Tian Ye , Shin Kyungsoo , Aleshin Alexander E. , Im Wonpil , Marassi Francesca M.
Published: March 2022

The adaptability of proteins to their work environments is fundamental for cellular life. Here we describe how the hemopexin-like (HX) domain of the multifunctional blood glycoprotein vitronectin (Vn) binds Ca2+ to adapt to excursions of temperature and shear stress. Using X-ray crystallography and molecular dynamics (MD) simulations, nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSF), we describe how Ca2+ and its flexible hydration shell enable the protein to perform conformational changes, that relay beyond the Ca2+ binding site to alter the number of polar contacts and confer conformational stability. By means of mutagenesis, we identify key residues that cooperate with Ca2+ to promote protein stability, and we show that Ca2+ association confers protection against shear stress, a property that provides conformational advantage for proteins that circulate in the vasculature like Vn. The data reveal a mechanism of adaptation.

Significance Statement

The protein vitronectin (Vn) plays important roles in cell adhesion and migration, bone remodeling and immunity. It circulates in blood, but it is also found in the extracellular matrix, and it accumulates with plaques associated with age-related macular degeneration, Alzheimer’s disease, atherosclerosis and other degenerative disorders. Vn is a calcium-binding protein, and here, we show that calcium helps Vn alter its structure in response to diverse environmental conditions. The results shed light on the way in which Vn adapts to its surroundings. This structural knowledge is important for the development of diagnostic, preventive or therapeutic approaches.
Thermodynamic stabilization of the von Willebrand Factor A1 domain due to loss-of-function disease-related mutations
Authors: Sandoval-Pérez Angélica , Mejía-Restrepo Valeria , Aponte-Santamaría Camilo
Published: March 2022

The von Willebrand disease (vWD) is the most common hereditary bleeding disorder, caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets at sites of vascular lesion. vWF carries out this essential homeostatic task, via the specific protein-protein interaction between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIBα). Upon the vWF activation triggered by the shear of the flowing blood. The two naturally occurring mutations G1324A and G1324S at the A1 domain, near the GPIBα binding site, result in a dramatic decrease of platelets adhesion, a bleeding disorder classified as type 2M vWD. However, it remained unclear how these two supposedly minor modifications lead to this drastic phenotypic response. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and non-equilibrium MD-based free energy simulations. Our data confirm that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. These mutations locally diminished the flexibility of the binding site to GPIBα and induced a conformational change that affected the nearby secondary structure elements. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously-reported mutation-augmented melting temperatures. Overall, our results support the idea of thermodynamic conformational restriction of A1— before the binding to GPIBα—as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.
Prokaryotic genome design is ruled by the mechanical properties and complexity of the DNA sequence
Authors: Tatsuke Tsuneyuki
Published: March 2022

Although we represent the genetic code using four letters, namely A, T, G, and C, it is a chemical entity with sequence-dependent physical properties and a spatial extent. In other words, it can be said that genetic information is compressed and recorded in the genomic DNA as a storage medium with a spatial spread. This study analyzed prokaryotic genomes in terms of their physical properties and complexity. The findings clearly indicated that a mathematic equation can be used to express the relationship between the length of a genome and the upper limit of its rigidity. Furthermore, many prokaryotes have been shown to maximize DNA complexity under various physical constraints. Here, this relationship between rigidity and complexity was found to be shaped like an entropy function. It was also clarified that codon usage tendencies are affected by the physical properties and complexity of the DNA sequence. Moreover, the analysis of codons encoding two consecutive synonymous amino acids suggests that many prokaryotic codon combinations tend to increase genomic DNA complexity. This study revealed the macro-level rules for packing genomic DNA into prokaryotic cells.
Programming bacteria for multiplexed DNA detection
Authors: Cheng Yu-Yu , Chen Zhengyi , Cao Xinyun , Ross Tyler D. , Falbel Tanya G. , Burton Briana M. , Venturelli Ophelia S.
Published: March 2022

DNA is a universal and programmable signal of living organisms. Here we developed cell-based DNA sensors by engineering the naturally competent bacteriumBacillus subtilis(B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity and sensitivity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA sensing mechanism and simple detection procedure could enable programmable DNA sensing for broad applications.
Quantitative Systems Pharmacology Modeling Framework of Autophagy in Tuberculosis: Application to Adjunctive Metformin Host-Directed Therapy
Authors: Mehta Krina , Guo Tingjie , Wallis Robert , van der Graaf Piet H. , van Hasselt J.G. Coen
Published: March 2022

Background

Quantitative systems pharmacology (QSP) modeling of the host-immune response against Mtb can inform rational design of host-directed therapies (HDTs). We aimed to develop a QSP framework to evaluate the effects of metformin-associated autophagy-induction in combination with antibiotics.

Methods

A QSP framework for autophagy was developed by extending a model for host-immune response to include AMPK-mTOR-autophagy signalling. This model was combined with pharmacokinetic-pharmacodynamic models for metformin and antibiotics against Mtb. We compared the model predictions to mice infection experiments, and derived predictions for pathogen and host-associated dynamics in humans treated with metformin in combination with antibiotics.

Results

The model adequately captured the observed bacterial load dynamics in mice Mtb infection models treated with metformin. Simulations for adjunctive metformin therapy in newly diagnosed patients suggested a limited yet dose-dependent effect of metformin on reducing the intracellular bacterial load and selected pro-inflammatory cytokines. Our predictions suggest that metformin may provide beneficiary effects when overall bacterial load, or extracellular-to-intracellular bacterial ratio is low, either early after infection or late during antibiotic treatment.

Conclusions

We present the first QSP framework for HDTs against Mtb, linking cellular-level autophagy effects to disease progression. This framework may be extended to guide design of HDTs against Mtb.
Bartonella taylorii: A model organism for studying Bartonella infection in vitro and in vivo
Authors: Fromm Katja , Boegli Alexandra , Ortelli Monica , Wagner Alexander , Bohn Erwin , Malmsheimer Silke , Wagner Samuel , Dehio Christoph
Published: March 2022

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species has hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain B. taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro could be correlated to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.
Cellular stress in brain organoids is limited to a distinct and bioinformatically removable subpopulation
Authors: Vértesy Ábel , Eichmueller Oliver L. , Naas Julia , Novatchkova Maria , Esk Christopher , Balmaña Meritxell , Ladstaetter Sabrina , Bock Christoph , Haeseler Arndt von , Knoblich Juergen A.
Published: March 2022

Organoids enable disease modeling in complex and structured human tissue,in vitro. Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.
Flotillin-2 regulates EGFR activation, degradation, and cancer growth
Authors: Wisniewski David J. , Liyasova Mariya S. , Korrapati Soumya , Zhang Xu , Ratnayake Shashikala , Chen Qingrong , Gilbert Samuel F. , Catalano Alexis , Voeller Donna , Meerzaman Daoud , Guha Udayan , Porat-Shliom Natalie , Annunziata Christina M. , Lipkowitz Stanley
Published: March 2022

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 decreased EGFR sjgnaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferationin vitroandin vivo.
A WAX INDUCER1/SHINE transcription factor controls cuticular wax in barley
Authors: McAllister Trisha , Campoli Chiara , Eskan Mhmoud , Liu Linsan , McKim Sarah M.
Published: March 2022

All land plants seal their above ground body parts with a lipid-rich hydrophobic barrier called the cuticle that protects tissues from dehydration and other terrestrial threats. Mutational studies in several model species, including barley, have resolved multiple loci regulating cuticular metabolism and development. Of particular importance are the eceriferum (cer) mutants characterized by visual alterations in cuticular wax. In barley, some cer mutants, such as cer-x lines, show defects in the distinctive β-diketone-enriched wax bloom on reproductive stage leaf sheaths, stems and spikes. In our study we exploited extensive allelic populations, near-isogenic lines and powerful genotyping platforms to identify variation in the HvWAX INDUCER1 (HvWIN1) gene as causal for cer-x mutants. We show that HvWIN1 function reduces cuticular permeability, promotes the accumulation of β-diketones, and regulates cuticular metabolic gene expression. Analyses across the barley pangenome and hundreds of exome-capture datasets revealed high sequence conservation of HvWIN1 but also two non-synonymous variants exclusive to cultivated germplasm. Taken together, we suggest that variation in HvWIN1 controls multiple cuticular features in barley by controlling the expression of genes involved in cuticle development.
Robust high-throughput phenotyping with deep segmentation enabled by a web-based annotator
Authors: Yuan Jialin , Kaur Damanpreet , Zhou Zheng , Nagle Michael , Kiddle Nicholas George , Doshi Nihar A. , Behnoudfar Ali , Peremyslova Ekaterina , Ma Cathleen , Strauss Steven H. , Fuxin Li
Published: March 2022

The abilities of plant biologists and breeders to characterize the genetic basis of physio-logical traits is limited by their abilities to obtain quantitative data representing precise details of trait variation, and particularly to collect this data at a high-throughput scale at low cost. Although deep learning methods have demonstrated unprecedented potential to automate plant phenotyping, these methods commonly rely on large training sets that can be time-consuming to generate. Intelligent algorithms have therefore been proposed to enhance the productivity of these annotations and reduce human efforts. We propose a high-throughput phenotyping system which features a Graphical User Interface (GUI) and a novel interactive segmentation algorithm: Semantic-Guided Interactive Object Segmentation (SGIOS). By providing a user-friendly interface and intelligent assistance with annotation, this system offers potential to streamline and accelerate the generation of training sets, reducing the effort required by the user. Our evaluation shows that our proposed SGIOS model requires fewer user inputs compared to the state-of-art models for interactive segmentation. As a case study in the use of the GUI applied for genetic discovery in plants, we present an example of results from a preliminary genome-wide association study (GWAS) ofin plantaregeneration inPopulus trichocarpa(poplar). We further demonstrate that the inclusion of semantic prior map with SGIOS can accelerate the training process for future GWAS, using a sample of a dataset extracted from a poplar GWAS ofin vitroregeneration. The capabilities of our phenotyping system surpass those of humans unassisted to rapidly and precisely phenotype our traits of interest. The scalability of this system enables large-scale phenomic screens that would otherwise be time-prohibitive, thereby providing increased power for GWAS, mutant screens, and other studies relying on large sample sizes to characterize the genetic basis of trait variation. Our user-friendly system can be used by researchers lacking a computational background, thus helping to democratize the use of deep segmentation as a tool for plant phenotyping.
Oligomer-to-Monomer Transition Underlies the Chaperone Function of AAGAB in AP1/AP2 Assembly
Authors: Tian Yuan , Datta Ishara , Yang Rui , Wan Chun , Wang Bing , He Huan , Li Suzhao , Shen Jingshi , Yin Qian
Published: March 2022

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homotetramer. AAGAB tetramerization is mediated by its C-terminal domain, which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the tetramerization domain (TD), revealing a dimer of dimer assembly. Interestingly, AAGAB uses the same TD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of TD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.
In vitro susceptibility to β-lactam antibiotics and viability of Neisseria gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum β-lactamases
Authors: Kandinov Ilya , Gryadunov Dmitry , Vinokurova Alexandra , Antonova Olga , Kubanov Alexey , Solomka Victoria , Shagabieva Julia , Deryabin Dmitry , Shaskolskiy Boris
Published: March 2022

N. gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum β-lactamases (ESBLs) have been obtained in vitro and studied for their viability and susceptibility to β-lactam antibiotics. The artificial pblaTEM-1 and pblaTEM-20 plasmids were constructed by site-directed mutagenesis from a pblaTEM-135 plasmid of the Toronto/Rio type, which was extracted from the clinical isolate. MIC values were determined for a series of β-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem. The N. gonorrhoeae strain carrying the pblaTEM-20 plasmid exhibited a high level of resistance to penicillins and II-IV generation cephalosporins (MIC ≥ 2 mg/L) but not to carbapenems (MIC ≤ 0.008 mg/L). However, this strain stopped growing after 6 hours of cultivation. The reduced viability was not associated with the plasmid loss but can be explained by the following factors: the presence of the plasmid itself, which requires additional costs for its reproduction, and the expression of ESBL, which can affect the structure of the peptidoglycan layer in the cell membrane. The cell growth was mathematically modeled using the generalized Verhulst equation. The parameter, characterizing the maximum possible number of cells grown under given conditions, decreased within the wild type (without plasmids) - pblaTEM-135 - pblaTEM-1 - pblaTEM-20 series, i.e., the plasmid-bearing strains had reduced viability compared to that of the wild-type strain. The kinetics for the cell death of N. gonorrhoeae strains without the pblaTEM-20 plasmid in the presence of ceftriaxone can be described by a modified Chick-Watson law. For the N. gonorrhoeae strain that harbors the pblaTEM-20 plasmid, the kinetics for cell number changes with time reflected several processes: the hydrolysis of ceftriaxone by TEM-20 β-lactamase, growth and gradual death of cells. The demonstrated reduced viability of N. gonorrhoeae strains with the pblaTEM-20 plasmid probably explains the absence of clinical isolates of N. gonorrhoeae that produce ESBL.
Structure and function of diadenylate cyclase DacM fromMycoplasma ovipneumoniae
Authors: Hao Xiujing , Zhou Xinhui , Zhang Ying , Han Yang , Xu Zhaokun , Ma Chunji , Luo Haixia , Qi Kemin , Fan Shilong , Li Min
Published: March 2022

Cyclic diadenosine monophosphate (c-di-AMP) is a second-messenger nucleotide that is produced by many bacteria. C-di-AMP can not only regulate bacterial growth, cell-wall homeostasis, ion transport and gene transcription, but can also be recognized by multiple sensor / receptor proteins in infected host cells to trigger an innate immune response.Mycoplasma ovipneumoniaecauses non-progressive pneumonia in both sheep and goats. Here, we analyzed c-di-AMP signaling inM. ovipneumoniae, which is a genome-reduced obligately pathogenic bacterium. Our results demonstrate that these bacteria can produce c-di-AMP, and we could identify the diadenylate cyclase, which was named DacM. The enzyme was found to utilize both ATP and ADP to synthesize c-di-AMP, resembling CdaM from a novel family of diadenylate cyclases first found inMycoplasma pneumoniae. Furthermore, we present the crystal structures of DacM in the apo state and substrate-bound state at 3 Å and 1.9 Å resolution, respectively. Mutation of residues Asp112, Gly113, Tyr128, Phe129, and Arg143 surrounding the active sites to Ala were lethal to DacM enzymatic activity. These structures provide valuable insights into the biochemistry of c-di-AMP, and offer a basis for the structure-based design of new drugs for animal husbandry.
Alpha-1-antitrypsin binds to the glucocorticoid receptor with biological significance in macrophages
Authors: Bai Xiyuan , Bai An , Tomasicchio Michele , Hagman James R. , Buckle Ashley M. , Gupta Arnav , Kadiyala Vineela , Bevers Shaun , Serban Karina A. , Kim Kevin , Feng Zhihong , Spendier Kathrin , Hagen Guy , Fornis Lorelenn , Griffith David E. , Dzieciatkowska Monika , Sandhaus Robert A. , Gerber Anthony N. , Chan Edward D.
Published: March 2022

Alpha-1-antitrypsin (AAT), a serine protease inhibitor produced mainly by the liver, is the third most abundant protein in plasma. While a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Since AAT has significant anti-inflammatory properties affecting many cell types including macrophages, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in macrophages. We report the novel finding that AAT binds to GR in macrophages using several approaches, including co-immunoprecipitation, mass spectrometry, microscale thermophoresis, and molecular modeling. The mass spectrometry data are availableviaProteomeXchange with identifier PXD030989. We further demonstrate that AAT induction of angiopoietin-like 4 protein and AAT inhibition of lipopolysaccharide-induced nuclear factor-kappa B activation and interleukin-8 production are mediated, in part, through AAT–GR interaction. Furthermore, this interaction contributes to a host-protective role against mycobacteria in macrophages. The interaction of AAT and GR described in this study identifies a mechanism for the antiinflammatory and host-defense properties of AAT.
The role of Cycloastragenol at the intersection of Nrf-2/ARE, telomerase, and proteasome activity
Authors: Yılmaz Sinem , Bedir Erdal , Kırmızıbayrak Petek Ballar
Published: March 2022

Aging is well-characterized by the gradual decline of cellular functionality. As redox balance, proteostasis, and telomerase systems have been found to be associated with aging and age-related diseases, targeting these systems by small compounds has been considered as a promising therapeutic approach. Cycloastragenol (CA), a small molecule telomerase activator obtained fromAstragalusspecies, has been reported to have positive effects on several age-related pathophysiologies, including ischemia, glucose intolerance, diabetes, and neurodegenerative diseases. Although CA has received intense attention as a promising compound for aging and age-related diseases, the mechanisms underlying CA activity related to redox balance, proteasome, and telomerase has not yet been reported. Here, we presented that CA increased Nrf-2 activity leading to upregulation of cytoprotective enzymes and attenuation of oxidative stress-induced ROS levels. Furthermore, CA enhanced telomerase activity by increasing not only the expression of hTERT but also its nuclear localization via the Hsp90-chaperon complex. CA alleviated the oxidative stress-induced mitochondrial hTERT levels while increasing the nuclear hTERT levels. Additionally, the proteasome activity and assembly were increased by CA. Strikingly, our data revealed that CA-mediated neuroprotection requires both Nrf-2 and hTERT induction. In conclusion, this study is the first report describing the effect of CA on these aging-related three major cellular pathways and their interrelationships. As the proteasome activator effect of CA is dependent on induction of telomerase activity, which is mediated by Nrf-2 system, CA has a great potential for healthier aging and prevention or treatment of age-related diseases by positively affecting these three cellular pathways.
Double NPY motifs at the N-terminus of Sso2 synergistically bind Sec3 to promote membrane fusion
Authors: Peer Maximilian , Yuan Hua , Zhang Yubo , Korbula Katharina , Novick Peter J. , Dong Gang
Published: March 2022

Exocytosis is an active vesicle trafficking process by which eukaryotes secrete materials to the extracellular environment and insert membrane proteins into the plasma membrane. The final step of exocytosis in yeast involves the assembly of two t-SNAREs, Sso1/2 and Sec9, with the v-SNARE, Snc1/2, on secretory vesicles. The rate-limiting step in this process is the formation of a binary complex of the two t-SNAREs. Despite a previous report of acceleration of binary complex assembly by Sec3, it remains unknown how Sso2 is efficiently recruited to the vesicle-docking site marked by Sec3. Here we report a crystal structure of the pleckstrin homology (PH) domain of Sec3 in complex with a nearly full-length version of Sso2 lacking only its C-terminal transmembrane helix. The structure shows a previously uncharacterized binding site for Sec3 at the N-terminus of Sso2, consisting of two highly conserved triple residue motifs (NPY: Asn-Pro-Tyr). We further reveal that the two NPY motifs bind Sec3 synergistically, which together with the previously reported binding interface constitute dual-site interactions between Sso2 and Sec3 to drive the fusion of secretory vesicles at target sites on the plasma membrane.

Significance

SNARE assembly, which involves one v-SNARE with two t-SNARE proteins, drives the fusion of vesicles to target compartments. The rate-limiting step in SNARE assembly is the assembly of the two t-SNARE proteins on the target membrane. Previous studies in yeast showed that Sec3, a component of the exocyst vesicle tethering complex, directly interacts with the t-SNARE protein Sso2 to promote fast assembly of an Sso2-Sec9 binary t-SNARE complex. This paper presents a new crystal structure of the Sec3 PH domain in complex with a nearly full-length version of Sso2, which reveals a previously unknown binding site for Sec3 at the N-terminus of Sso2. Our work demonstrates that the dual-site interactions between Sso2 and Sec3 plays an essential role in promoting the fusion of secretory vesicles at target sites on the plasma membrane.
Blocking abundant RNA transcripts by high-affinity oligonucleotides during transcriptome library preparation
Authors: Everaert Celine , Verwilt Jasper , Verniers Kimberly , Vandamme Niels , Rubio Alvaro Marcos , Vandesompele Jo , Mestdagh Pieter
Published: March 2022

RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3’ end sequencing and long-read sequencing, and MALAT1 in single-cell 3’ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Furthermore, our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
Phytochemical-induced mucin accumulation in the gastrointestinal lumen is independent of the microbiota
Authors: Forgie Andrew J. , Ju Tingting , Tollenaar Stephanie L. , Willing Benjamin P.
Published: March 2022

The mucus layer is critical to gastrointestinal health and ecology. Dietary phytochemicals are well documented to stimulate mucus production and secretion, but the underlying mechanism and effects on gut health are poorly understood. We fed germ-free and conventional mice diets containing approximately 0.4% of polyphenols per gram to determine if the phytochemical-induced accumulation of mucin in the gastrointestinal lumen is dependent on the microbiota. In addition, we assess how increased mucin shapes microbial communities in conventional mice. Germ-free mice receiving a pea (Pisum sativuum) seed coat proanthocyanidin-containing diet (PA) had greater levels of fecal mucin compared to the non-proanthocyanidin-containing (NPA) pea seed coat diet control (P < 0.05), confirming that fecal mucin accumulation is independent of the gut microbiota. Conventional mice fed the PA diet and a red osier dogwood (ROD; Cornus sericea) extract diet (DW) had higher mucin levels compared to a control diet without phytochemicals (P < 0.01 and P < 0.05, respectively). The increase in luminal mucin was associated with consistent increases in bacterial taxa belonging to Lachnospiraceae and [Clostridium] leptum species and a decrease in Romboutsia species. We conclude that phytochemicals have the ability to alter gut microbial ecology by increasing the amount of mucin in the gastrointestinal lumen.
A core transcriptional response for biofilm formation by Y. pseudotuberculosis
Authors: Mahmud A.K.M.F. , Nilsson K. , Soni D.K. , Choudhury R. , Navais R. , Tuck Simon , Avican K. , Fällman M.
Published: March 2022

Previous transcriptional profiling of the enteropathogen Yersinia pseudotuberculosis during persistent stages of colonisation of mouse cecal lymphoid follicles indicated the possible involvement of biofilm in infection maintenance. Not much is known about the mechanisms responsible for biofilm formation by this pathogen, and most current knowledge is based on results of experiments conducted using the related Y. pestis pathogen that forms biofilm in the flea gut. In this study, we performed transcriptional profiling of Y. pseudotuberculosis in biofilms from different biofilm-inducing conditions, bile exposure, amino acid deprivation and in vivo mimicking conditions with and without oxygen. The comparison of differential expression of genes in biofilm versus planktonic bacteria showed a set of 54 core genes that were similarly regulated, independent of inducing condition. This set included many genes that were previously shown to be associated with biofilms, such as hutG, hsmF, hmsT and cpxP that were upreg-ulated and other genes such as hmsP and rfaH that were downregulated. There were also novel biofilm-associated genes, including genes encoding hypothetical proteins. To identify the genes involved in inducing biofilm formation, the gene expression of bacteria during an early initial phase when biofilm starts to form after induction by bile or amino acid depletion was determined. Comparisons of the resulting gene expression profiles with the profiles of non-induced bacteria incubated for the same period of time showed a set of core genes associated with early biofilm formation. This set included genes involved in quorum sensing, pili biogenesis and genes indicative of a potential metabolic shift involving nitrogen utilisation. Genes encoding components of sugar phosphotransferase systems were also up-regulated during biofilm induction. Assays of biofilm formation by bacteria deleted of some of these core genes showed that strains lacking hpr and luxS, which are known to be important for functional sugar phosphotransferase systems and quorum sensing, as well as glnL encoding a sensory histidine kinase were most negatively affected. Most of the deletion mutant strains tested were affected, but the effect was less severe, suggesting high levels of redundancy in the pathways involved in biofilm formation by this pathogen.
CRISPRcleanRWebApp: an interactive web application for processing genome-wide pooled CRISPR-Cas9 viability screens
Authors: Vinceti Alessandro , de Lucia Riccardo Roberto , Cremaschi Paolo , Perron Umberto , Karacok Emre , Mauri Luca , Fernandez Carlos , Kluczynski Krzysztof Henryk , Anderson Daniel Stephen , Iorio Francesco
Published: March 2022

A limitation of pooled CRISPR-Cas9 viability screens is the high false-positive rate in detectingessential genesarising from copy number-amplified (CNA) regions of the genome. To solve this issue, we developedCRISPRcleanR: a computational method implemented as R/python package and in a dockerized version. CRISPRcleanR detects and corrects biased responses to CRISPR-Cas9 targeting in an unsupervised fashion, accurately reducing false-positive signals, while maintaining sensitivity in identifying relevant genetic dependencies. Here, we presentCRISPRcleanRWebApp, a web-based application enabling access to CRISPRcleanR through an intuitive graphical web-interface. CRISPRcleanRWebAppremoves the complexity of low-level R/python-language user interactions; it provides a user-friendly access to a complete analytical pipeline, not requiring any data pre-processing, and generating gene-level summaries of essentiality with associated statistical scores; it offers a range of interactively explorable plots, while supporting a wider range of CRISPR guide RNAs’ libraries with respect to the original package. CRISPRcleanRWebAppis freely available at:https://crisprcleanr-webapp.fht.org/.

Highlights

CRISPR-Cas9 screens are widely used for the identification of cancer dependencies

In such screens, false-positives arise from targeting copy number amplified genes

CRISPRcleanR corrects this bias in an unsupervised fashion

CRISPRcleanRWebAppis a web user-friendly front-end for CRISPRcleanR

Abstract Figure
Development of a tractable model system to mimic wood-ageing of beer on a lab scale
Authors: Bossaert Sofie , Kocijan Tin , Winne Valérie , Van Opstaele Filip , Schlich Johanna , Herrera-Malaver Beatriz , Verstrepen Kevin J. , De Rouck Gert , Lievens Bart , Crauwels Sam
Published: March 2022

Wood-ageing of conventionally fermented beers is gaining increased attention in the production of sour beers with a noteworthy balance between sourness, wood aroma and flavour complexity. Besides the extraction of wood-derived compounds into the beer, wood-aged sours owe their layered flavour profile to the activity of a variety of ‘wild’ microorganisms that reside in the barrels or that emerge from the brewing or maturation environment. However, until now wood-ageing of craft beers largely remains a process of trial and error that often generates unexpected or undesirable results. Therefore, to better understand the process and develop control strategies to improve the consistency, predictability and overall quality of the resulting beer, more insight is needed into the interactions between the wood, the microorganisms and the maturing beer. Nevertheless, as studying these interactions on an industrial scale is highly challenging, the objective of this study was to develop a reproducible and easy-to-manipulate experimentally tractable system that can be used to study wood-ageing of beer on a lab scale. Barrel-ageing was mimicked in a 0.5 liter glass jar filled with beer and closed off by a wooden disk. Furthermore, the system was equipped with a synthetic community composed of four bacterial species (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevisandPediococcus damnosus) and four fungal species (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciensandSaccharomyces cerevisiae) that represented key microbes previously identified in wood-ageing experiments with 225-liter barrels. In order to test the hypothesis that the barrel-ageing process of beer can be replicated in the simplifiedin-vitrosystem, the system was subjected to 60 days of ageing and microbial community dynamics and beer chemistry were compared with a 38-week industrial barrel-ageing experiment using the same beer. Beer samples were collected at regular time points and subjected to both qPCR assays targeting the eight selected species and chemical analysis. Results revealed thatin vitroageing showed similar trends in the temporal dynamics of the microbial populations and beer chemistry as those observed during 38-weeks of barrel-ageing in 225-liter barrels. Furthermore, results were found to be highly reproducible. Altogether, thein-vitrosystem was found to be a robust and reproducible system that has great potential to perform more in-depth research about the intricate interactions between microbes, wood and maturing beer and to develop control strategies to improve the consistency, predictability and overall quality of the resulting beer.
Increased receptor affinity and reduced recognition by specific antibodies contribute to immune escape of SARS-CoV-2 variant Omicron
Authors: Vogt Anne-Cathrine , Augusto Gilles , Martina Byron , Chang Xinyue , Nasrallah Gheyath , Speiser Daniel E. , Vogel Monique , Bachmann Martin F. , Mohsen Mona O.
Published: March 2022

In this report, we mechanistically reveal how the Variant of Concern (VOC) SARS-CoV-2 Omicron (B.1.1.529) escapes neutralizing antibody responses, by characterization of this variant, and wildtype Wuhan and Delta variant (B.1.617.2). Convalescent sera as well as sera obtained from participants who received two or three doses of mRNA vaccines (Moderna-mRNA-1273® or Pfizer-BNT162b2®) were used for comparison in this study. Our data demonstrate that both the Delta as well as Omicron variants exhibit higher affinity for the receptor ACE2, facilitating infection and causing antibody escape by receptor affinity (affinity escape), due to reduced ability of antibodies to compete with RBD-receptor interaction and virus neutralization. In contrast, only Omicron but not Delta variant escaped antibody recognition, most likely because only Omicron exhibit the mutation at position E484 associated with reduced recognition, resulting in further reduced neutralization (specificity escape). Nevertheless, the immunizations with RNA based vaccines resulted in marked viral neutralization in vitro for all strains, compatible with the fact that Omicron is still largely susceptible to vaccination-induced antibodies, despite affinity- and specificity escape.