Genet Sel Evol. 2023 Apr 11;55(1):25. doi: 10.1186/s12711-023-00800-7.

NO ABSTRACT

PMID:37041461 | DOI:10.1186/s12711-023-00800-7

Methods Mol Biol. 2023;2621:73-89. doi: 10.1007/978-1-0716-2950-5_6.

ABSTRACT

Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissues stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA's integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation-sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we describe Capture MRE-seq, a new MRE-seq protocol tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).

PMID:37041441 | DOI:10.1007/978-1-0716-2950-5_6

ISME J. 2023 Apr 11. doi: 10.1038/s41396-023-01405-0. Online ahead of print.

ABSTRACT

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

PMID:37041326 | DOI:10.1038/s41396-023-01405-0

Neurology. 2023 Apr 11:10.1212/WNL.0000000000207241. doi: 10.1212/WNL.0000000000207241. Online ahead of print.

ABSTRACT

OBJECTIVE: Birk-Landau-Perez syndrome is a genetic disorder caused by biallelic pathogenic variants in SLC30A9 presenting with a complex movement disorder, developmental regression, oculomotor abnormalities and renal impairment. So far it has been reported in only two families. We describe the clinical phenotype of eight further individuals from four unrelated families with SLC30A9-related disease.

METHOD: Following detailed clinical phenotyping, one family underwent research whole-genome sequencing (WGS), one research whole-exome sequencing (WES) and two diagnostic WGS. Variants of interest were assessed for pathogenicity using in silico prediction tools, homology modelling and where relevant, sequencing of cDNA for splicing impact.

RESULTS: In two unrelated families of Pakistani origin (one consanguineous, one not), the same homozygous missense variant in SLC30A9 (c.1253G>T, p.Gly418Val) was identified. Family 1 included two affected brothers, and Family 2 one affected boy. In Family 3, also consanguineous, there were four affected siblings homozygous for the variant c.1049delCAG, pAla350del. The fourth family was non-consanguineous: the one affected individual was compound heterozygous for c.1083dup, p.Val362Cysfs*5 and c.1413A>G, p.Ser471=. Despite phenotypic variability between the four families, all affected patients manifested with a progressive hyperkinetic movement disorder, associated with oculomotor apraxia and ptosis. None had evidence of severe renal impairment. For the novel missense variant, the conformation of the loop domain and packing of transmembrane helices are likely to be disrupted based on structure modelling. Its presence in two unrelated Pakistani families suggests a possible founder variant. For the synonymous variant p.Ser471=, an impact on splicing was confirmed through cDNA analysis.

DISCUSSION: Pathogenic variants in SLC30A9 cause a progressive autosomal recessive neurological syndrome associated with a complex hyperkinetic movement disorder. Our report highlights the expanding disease phenotype, which can present with a wider spectrum of severity than has previously been recognised.

PMID:37041080 | DOI:10.1212/WNL.0000000000207241

J Control Release. 2023 Apr 9:S0168-3659(23)00258-4. doi: 10.1016/j.jconrel.2023.04.009. Online ahead of print.

ABSTRACT

Many diseases affecting the central nervous system (CNS) are deadly but less understood, leading to impaired mental and motor capabilities and poor patient prospects. Gene therapy is a promising therapeutic modality for correcting many genetic disorders, expanding in breadth and scope with further advances. This review summarizes the candidate CNS disorders for gene therapy, mechanisms of gene therapy, and recent clinical advances and limitations of gene therapy in CNS disorders. We highlight that improving delivery across CNS barriers, safety, monitoring techniques, and multiplexing therapies are predominant factors in advancing long-term outcomes from gene therapy.

PMID:37040842 | DOI:10.1016/j.jconrel.2023.04.009

Cell Metab. 2023 Apr 4:S1550-4131(23)00091-8. doi: 10.1016/j.cmet.2023.03.013. Online ahead of print.

ABSTRACT

Nonalcoholic steatohepatitis (NASH) prevalence is rising with no pharmacotherapy approved. A major hurdle in NASH drug development is the poor translatability of preclinical studies to safe/effective clinical outcomes, and recent failures highlight a need to identify new targetable pathways. Dysregulated glycine metabolism has emerged as a causative factor and therapeutic target in NASH. Here, we report that the tripeptide DT-109 (Gly-Gly-Leu) dose-dependently attenuates steatohepatitis and fibrosis in mice. To enhance the probability of successful translation, we developed a nonhuman primate model that histologically and transcriptionally mimics human NASH. Applying a multiomics approach combining transcriptomics, proteomics, metabolomics, and metagenomics, we found that DT-109 reverses hepatic steatosis and prevents fibrosis progression in nonhuman primates, not only by stimulating fatty acid degradation and glutathione formation, as found in mice, but also by modulating microbial bile acid metabolism. Our studies describe a highly translatable NASH model and highlight the need for clinical evaluation of DT-109.

PMID:37040763 | DOI:10.1016/j.cmet.2023.03.013

Immunity. 2023 Apr 4:S1074-7613(23)00126-7. doi: 10.1016/j.immuni.2023.03.006. Online ahead of print.

ABSTRACT

Interferon-γ (IFN-γ) is a key cytokine in response to viral or intracellular bacterial infection in mammals. While a number of enhancers are described to promote IFN-γ responses, to the best of our knowledge, no silencers for the Ifng gene have been identified. By examining H3K4me1 histone modification in naive CD4+ T cells within Ifng locus, we identified a silencer (CNS-28) that restrains Ifng expression. Mechanistically, CNS-28 maintains Ifng silence by diminishing enhancer-promoter interactions within Ifng locus in a GATA3-dependent but T-bet-independent manner. Functionally, CNS-28 restrains Ifng transcription in NK cells, CD4+ cells, and CD8+ T cells during both innate and adaptive immune responses. Moreover, CNS-28 deficiency resulted in repressed type 2 responses due to elevated IFN-γ expression, shifting Th1 and Th2 paradigm. Thus, CNS-28 activity ensures immune cell quiescence by cooperating with other regulatory cis elements within the Ifng gene locus to minimize autoimmunity.

PMID:37040761 | DOI:10.1016/j.immuni.2023.03.006

Research (Wash D C). 2023;6:0041. doi: 10.34133/research.0041. Epub 2023 Jan 30.

ABSTRACT

Tumor metastasis is a hallmark of colorectal cancer (CRC), in which exosome plays a crucial role with its function in intercellular communication. Plasma exosomes were collected from healthy control (HC) donors, localized primary CRC and liver-metastatic CRC patients. We performed proximity barcoding assay (PBA) for single-exosome analysis, which enabled us to identify the alteration in exosome subpopulations associated with CRC progression. By in vitro and in vivo experiments, the biological impact of these subpopulations on cancer proliferation, migration, invasion, and metastasis was investigated. The potential application of exosomes as diagnostic biomarkers was evaluated in 2 independent validation cohorts by PBA. Twelve distinct exosome subpopulations were determined. We found 2 distinctly abundant subpopulations: one ITGB3-positive and the other ITGAM-positive. The ITGB3-positive cluster is rich in liver-metastatic CRC, compared to both HC group and primary CRC group. On the contrary, ITGAM-positive exosomes show a large-scale increase in plasma of HC group, compared to both primary CRC and metastatic CRC groups. Notably, both discovery cohort and validation cohort verified ITGB3+ exosomes as potential diagnostic biomarker. ITGB3+ exosomes promote proliferation, migration, and invasion capability of CRC. In contrast, ITGAM+ exosomes suppress CRC development. Moreover, we also provide evidence that one of the sources of ITGAM+ exosomes is macrophage. ITGB3+ exosomes and ITGAM+ exosomes are proven 2 potential diagnostic, prognostic, and therapeutic biomarkers for management of CRC.

PMID:37040507 | PMC:PMC10076010 | DOI:10.34133/research.0041

Proc Natl Acad Sci U S A. 2023 Apr 18;120(16):e2120826120. doi: 10.1073/pnas.2120826120. Epub 2023 Apr 11.

ABSTRACT

In newborn humans, and up to approximately 2 y of age, calvarial bone defects can naturally regenerate. This remarkable regeneration potential is also found in newborn mice and is absent in adult mice. Since previous studies showed that the mouse calvarial sutures are reservoirs of calvarial skeletal stem cells (cSSCs), which are the cells responsible for calvarial bone regeneration, here we hypothesized that the regenerative potential of the newborn mouse calvaria is due to a significant amount of cSSCs present in the newborn expanding sutures. Thus, we tested whether such regenerative potential can be reverse engineered in adult mice by artificially inducing an increase of the cSSCs resident within the adult calvarial sutures. First, we analyzed the cellular composition of the calvarial sutures in newborn and in older mice, up to 14-mo-old mice, showing that the sutures of the younger mice are enriched in cSSCs. Then, we demonstrated that a controlled mechanical expansion of the functionally closed sagittal sutures of adult mice induces a significant increase of the cSSCs. Finally, we showed that if a calvarial critical size bone defect is created simultaneously to the mechanical expansion of the sagittal suture, it fully regenerates without the need for additional therapeutic aids. Using a genetic blockade system, we further demonstrate that this endogenous regeneration is mediated by the canonical Wnt signaling. This study shows that controlled mechanical forces can harness the cSSCs and induce calvarial bone regeneration. Similar harnessing strategies may be used to develop novel and more effective bone regeneration autotherapies.

PMID:37040407 | DOI:10.1073/pnas.2120826120

Microbiology (Reading). 2023 Apr;169(4). doi: 10.1099/mic.0.001319.

ABSTRACT

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

PMID:37040165 | DOI:10.1099/mic.0.001319

J Natl Cancer Inst. 2023 Apr 11:djad062. doi: 10.1093/jnci/djad062. Online ahead of print.

ABSTRACT

BACKGROUND: Adenoid Cystic Carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the co-existence within tumor tissues of two distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these two cell-types, and their differential vulnerability to anti-tumor treatments, remain unknown.

METHODS: Using single-cell RNA-sequencing (scRNA-seq), we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDX) of human ACCs. Using prospective xeno-transplantation experiments, we compared the tumor-initiating capacity of the two cell-types, and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the two cell-types and tested their role as lineage-specific therapeutic targets.

RESULTS: Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (ATRA, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells, and in vivo anti-tumor activity against PDX models of ACC.

CONCLUSIONS: In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

PMID:37040084 | DOI:10.1093/jnci/djad062

Bioinformatics. 2023 Apr 11:btad186. doi: 10.1093/bioinformatics/btad186. Online ahead of print.

ABSTRACT

MOTIVATION: The accurate prediction of complex phenotypes such as metabolic fluxes in living systems is a grand challenge for systems biology and central to efficiently identifying biotechnological interventions that can address pressing industrial needs. The application of gene expression data to improve the accuracy of metabolic flux predictions using mechanistic modeling methods such as Flux Balance Analysis (FBA) has not been previously demonstrated in multi-tissue systems, despite their biotechnological importance. We hypothesized that a method for generating metabolic flux predictions informed by relative expression levels between tissues would improve prediction accuracy.

RESULTS: Relative gene expression levels derived from multiple transcriptomic and proteomic datasets were integrated into Flux Balance Analysis predictions of a multi-tissue, diel model of Arabidopsis thaliana's central metabolism. This integration dramatically improved the agreement of flux predictions with experimentally based flux maps from 13C Metabolic Flux Analysis (13C-MFA) compared with a standard parsimonious FBA approach. Disagreement between FBA predictions and MFA flux maps, as measured by weighted averaged percent error values, dropped from between 169-180% and 94-103% in high light and low light conditions, respectively, to between 10-13% and 9-11%, depending on the gene expression dataset used. The incorporation of gene expression data into the modeling process also substantially altered the predicted carbon and energy economy of the plant.

AVAILABILITY: Code is available from https://github.com/Gibberella/ArabidopsisGeneExpressionWeights.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID:37040081 | DOI:10.1093/bioinformatics/btad186

Curr Protoc. 2023 Apr;3(4):e716. doi: 10.1002/cpz1.716.

ABSTRACT

Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This article describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation of mammalian reoviruses in cell culture from virus stocks Alternate Protocol 1: Large-scale propagation (and purification) of mammalian reoviruses in cell culture from virus stocks Basic Protocol 2: Quantification of mammalian reoviruses by plaque assay with neutral red staining Alternate Protocol 2: Quantification of mammalian reoviruses by plaque assay with crystal violet staining Basic Protocol 3: Storage of mammalian reoviruses Support Protocol 1: Growth and maintenance of mouse L929 cells Support Protocol 2: Plating L929 cells.

PMID:37039704 | DOI:10.1002/cpz1.716

Elife. 2023 Apr 11;12:e85439. doi: 10.7554/eLife.85439. Online ahead of print.

ABSTRACT

We develop a method that allows one to test a large number of drug combinations in a single cell culture sample. We rely on the randomness of drug uptake in individual cells as a tool to create and encode drug treatment regimens. A single sample containing thousands of cells is treated with a combination of fluorescently barcoded drugs. We create independent transient drug gradients across the cell culture sample to produce heterogeneous local drug combinations. After the incubation period, the ensuing phenotype and corresponding drug barcodes for each cell are recorded. We use these data for statistical prediction of the treatment response to the drugs in a macroscopic population of cells. To further application of this technology, we developed a fluorescent barcodingmethod that does not require any chemical drug(s) modifications. We also developed segmentation-free image analysis capable of handling large optical fields containing thousands of cells in the sample, even in confluent growth condition. The technology necessary to execute our method is readily available in most biological laboratories, does not require robotic or microfluidic devices, and dramatically reduces resource needs and resulting costs of the traditional high-throughput studies.

PMID:37039628 | DOI:10.7554/eLife.85439

Proteomics. 2023 Apr;23(7-8):e2200011. doi: 10.1002/pmic.202200011.

NO ABSTRACT

PMID:37039310 | DOI:10.1002/pmic.202200011

Gigascience. 2022 Dec 28;12:giad023. doi: 10.1093/gigascience/giad023.

ABSTRACT

BACKGROUND: Leeches have been used in traditional Chinese medicine since prehistoric times to treat a spectrum of ailments, but very little is known about their physiological, genetic, and evolutionary characteristics.

FINDINGS: We sequenced and assembled chromosome-level genomes of 3 leech species (bloodsucking Hirudo nipponia and Hirudinaria manillensis and nonbloodsucking Whitmania pigra). The dynamic population histories and genome-wide expression patterns of the 2 bloodsucking leech species were found to be similar. A combined analysis of the genomic and transcriptional data revealed that the bloodsucking leeches have a presumably enhanced auditory sense for prey location in relatively deep fresh water. The copy number of genes related to anticoagulation, analgesia, and anti-inflammation increased in the bloodsucking leeches, and their gene expressions responded dynamically to the bloodsucking process. Furthermore, the expanded FBN1 gene family may help in rapid body swelling of leeches after bloodsucking, and the expanded GLB3 gene family may be associated with long-term storage of prey blood in a leech's body.

CONCLUSIONS: The high-quality reference genomes and comprehensive datasets obtained in this study may facilitate innovations in the artificial culture and strain optimization of leeches.

PMID:37039117 | DOI:10.1093/gigascience/giad023

Soft Matter. 2023 Apr 11. doi: 10.1039/d2sm01580c. Online ahead of print.

ABSTRACT

The vertex model of epithelia describes the apical surface of a tissue as a tiling of polygonal cells, with a mechanical energy governed by deviations in cell shape from preferred, or target, area, A0, and perimeter, P0. The model exhibits a rigidity transition driven by geometric incompatibility as tuned by the target shape index, . For with p*(6) the perimeter of a regular hexagon of unit area, a cell can simultaneously attain both the preferred area and preferred perimeter. As a result, the tissue is in a mechanically soft compatible state, with zero shear and Young's moduli. For p0 < p*(6), it is geometrically impossible for any cell to realize the preferred area and perimeter simultaneously, and the tissue is in an incompatible rigid solid state. Using a mean-field approach, we present a complete analytical calculation of the linear elastic moduli of an ordered vertex model. We analyze a relaxation step that includes non-affine deformations, leading to a softer response than previously reported. The origin of the vanishing shear and Young's moduli in the compatible state is the presence of zero-energy deformations of cell shape. The bulk modulus exhibits a jump discontinuity at the transition and can be lower in the rigid state than in the fluid-like state. The Poisson's ratio can become negative which lowers the bulk and Young's moduli. Our work provides a unified treatment of linear elasticity for the vertex model and demonstrates that this linear response is protocol-dependent.

PMID:37039037 | DOI:10.1039/d2sm01580c

Future Microbiol. 2023 Apr 11. doi: 10.2217/fmb-2022-0261. Online ahead of print.

NO ABSTRACT

PMID:37039024 | DOI:10.2217/fmb-2022-0261

Eur J Neurol. 2023 Apr 10. doi: 10.1111/ene.15819. Online ahead of print.

ABSTRACT

While the incidence of neurological disease is increasing worldwide, treatment remains mostly limited to symptom management. The gut-brain axis, which encompasses the communication routes between microbiota, gut, and brain, has emerged as a crucial area of investigation for identifying new preventive and therapeutic targets in neurological disease. Due to the inter-organ, systemic nature of the gut-brain axis, together with the multitude of biomolecules and microbial species involved, molecular systems biology approaches are required to accurately investigate the mechanisms of gut-brain communication. High-throughput omics profiling, together with computational methodologies such as dimensionality reduction or clustering, machine learning, network inference and genome-scale metabolic models, allow to discover novel biomarkers and elucidate mechanistic insights. In this review, we introduce the general concepts of experimental and computational methodologies for gut-brain axis research and discuss their applications, mainly in human cohorts. We further highlight important aspects concerning rational study design, sampling procedures and data modalities relevant for gut-brain communication, strengths and limitations of methodological approaches and some future perspectives. In conclusion, we review how multi-omics analysis, together with advanced data mining, are essential to functionally characterize the gut-brain axis in neurological disease and finally put forward novel preventive or therapeutic strategies.

PMID:37038632 | DOI:10.1111/ene.15819

Adv Sci (Weinh). 2023 Apr 10:e2207454. doi: 10.1002/advs.202207454. Online ahead of print.

ABSTRACT

Pulmonary fibrosis (PF) is a heterogeneous disease with a poor prognosis. Therefore, identifying additional therapeutic modalities is required to improve outcome. However, the lack of biomarkers of disease progression hampers the preclinical to clinical translational process. Here, this work assesses and identifies progressive alterations in pulmonary function, transcriptomics, and metabolomics in the mouse lung at 7, 14, 21, and 28 days after a single dose of oropharyngeal bleomycin. By integrating multi-omics data, this work identifies two central gene subnetworks associated with multiple critical pathological changes in transcriptomics and metabolomics as well as pulmonary function. This work presents a multi-omics-based framework to establish a translational link between the bleomycin-induced PF model in mice and human idiopathic pulmonary fibrosis to identify druggable targets and test therapeutic candidates. This work also indicates peripheral cannabinoid receptor 1 (CB1 R) antagonism as a rational therapeutic target for clinical translation in PF. Mouse Lung Fibrosis Atlas can be accessed freely at https://niaaa.nih.gov/mouselungfibrosisatlas.

PMID:37038090 | DOI:10.1002/advs.202207454