Macroautophagy/autophagy is a conserved catabolic process that maintains cellular homeostasis under basal growth and stress conditions. In cancer, autophagy can either prevent or promote tumor growth, at early or advanced stages, respectively. We screened public databases to identify autophagy-related somatic mutations in cancer, using a computational approach to identify cancer mutational target sites, employing exact statistics. The top significant hit was a missense mutation (Y113C) in the MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) protein, which occurred at a significant frequency in cancer, and was detected in early stages in primary tumors of patients with known tumor lineage. The mutation reduced the formation of GFP-LC3B puncta and attenuated LC3B lipidation during Torin1-induced autophagy. Its effect on the direct physical interaction of LC3B with each of the 4 proteins that control its maturation or lipidation was tested by applying a protein-fragment complementation assay and co-immunoprecipitation experiments. Interactions with ATG4A and ATG4B proteases were reduced, yet without perturbing the cleavage of mutant LC3B. Most importantly, the mutation significantly reduced the interaction with the E1-like enzyme ATG7, but not the direct interaction with the E2-like enzyme ATG3, suggesting a selective perturbation in the binding of LC3B to some of its partner proteins. Structure analysis and molecular dynamics simulations of LC3B protein and its mutant suggest that the mutation changes the conformation of a loop that has several contact sites with ATG4B and the ATG7 homodimer. We suggest that this loss-of-function mutation, which attenuates autophagy, may promote early stages of cancer development.

Patient-provider relationships with primary care and need for hospitalisations are related within the complex networks comprising healthcare. Our objective was to analyse mean days hospitalised, using registration status (active or passive listing) with a provider and number of consultations as proxies of patient-provider relationships with primary care, adjusting for morbidity burden, age and sex while analysing the contribution of psychiatric disorders. The Johns Hopkins Adjusted Clinical Groups Case-Mix System was used to classify morbidity burden into Resource Utilization Band (RUB) 0-5.

Ketamine is receiving increasing attention as a rapid-onset antidepressant in patients suffering from major depressive disorder (MDD) with treatment resistance or severe suicidal ideation. Ketamine modulates several neurotransmitter systems, including norepinephrine via the norepinephrine transporter (NET), both peripherally and centrally. The locus coeruleus (LC), which has high NET concentration, has been attributed to brain networks involved in depression. Thus we investigated the effects of single-dose of racemic ketamine on the LC using resting state functional MRI.

Cyanobacteria of biological soil crusts (BSCs) represent an important part of circumpolar and Alpine ecosystems, serve as indicators for ecological condition and climate change, and function as ecosystem engineers by soil stabilization or carbon and nitrogen input. The characterization of cyanobacteria from both polar regions remains extremely important to understand geographic distribution patterns and community compositions. This study is the first of its kind revealing the efficiency of combining denaturing gradient gel electrophoresis (DGGE), light microscopy and culture-based 16S rRNA gene sequencing, applied to polar and Alpine cyanobacteria dominated BSCs. This study aimed to show the living proportion of cyanobacteria as an extension to previously published meta-transcriptome data of the same study sites. Molecular fingerprints showed a distinct clustering of cyanobacterial communities with a close relationship between Arctic and Alpine populations, which differed from those found in Antarctica. Species richness and diversity supported these results, which were also confirmed by microscopic investigations of living cyanobacteria from the BSCs. Isolate-based sequencing corroborated these trends as cold biome clades were assigned, which included a potentially new Arctic clade of Oculatella. Thus, our results contribute to the debate regarding biogeography of cyanobacteria of cold biomes.

Many people with ALS have been suggested to have a "nice" personality, but most ALS personality studies to date have had limited numbers of participants and have not taken into account personality differences between genders. We used Big Five Inventory data obtained from an online questionnaire looking for risk factors for ALS to investigate personality traits in large numbers of people with ALS and controls.

LZ-106, a newly synthetized analog of quinolone, has been shown to be highly effective in non-small cell lung cancer (NSCLC) in both cultured cells and xenograft mouse model with low toxicity, yet the molecular mechanisms still require exploration. Here, we substantiated the involvement of P53 activation in intracellular reactive oxygen species (ROS) generation upon LZ-106 treatment and related P53 to the ROS-induced viability inhibition and apoptosis, which was exhibited in the previous research. P53 was shown to play an indispensable role in the elevated levels of intracellular ROS in LZ-106-treated NSCLC cells through ROS detection. We further identified the anti-proliferation effect of LZ-106 in NSCLC cells through G1 phase cell cycle arrest by cell cycle analysis, with the expression analysis of the key proteins, and discovered that the cell cycle arrest effect is also mediated by induction of ROS in a P53-dependent manner. In addition, the tumor suppression effect exhibited in vivo was demonstrated to be similar to that in vitro, which requires the participation of P53. Thus, LZ-106 is a potent antitumor drug possessing potent proliferation inhibition and apoptosis induction ability through the P53-dependent ROS modulation both in vitro and in vivo.

Efficient extraction of knowledge from biological data requires the development of structured vocabularies to unambiguously define biological terms. This paper proposes descriptions and definitions to disambiguate the term 'single-exon gene'. Eukaryotic Single-Exon Genes (SEGs) have been defined as genes that do not have introns in their protein coding sequences. They have been studied not only to determine their origin and evolution but also because their expression has been linked to several types of human cancer and neurological/developmental disorders and many exhibit tissue-specific transcription. Unfortunately, the term 'SEGs' is rife with ambiguity, leading to biological misinterpretations. In the classic definition, no distinction is made between SEGs that harbor introns in their untranslated regions (UTRs) versus those without. This distinction is important to make because the presence of introns in UTRs affects transcriptional regulation and post-transcriptional processing of the mRNA. In addition, recent whole-transcriptome shotgun sequencing has led to the discovery of many examples of single-exon mRNAs that arise from alternative splicing of multi-exon genes, these single-exon isoforms are being confused with SEGs despite their clearly different origin. The increasing expansion of RNA-seq datasets makes it imperative to distinguish the different SEG types before annotation errors become indelibly propagated in biological databases. This paper develops a structured vocabulary for their disambiguation, allowing a major reassessment of their evolutionary trajectories, regulation, RNA processing and transport, and provides the opportunity to improve the detection of gene associations with disorders including cancers, neurological and developmental diseases.

New methodology must be developed to improve the ability to characterize the growing number of amino acid sequences, which vastly exceeds the number of experimentally determined protein structures. Homologous proteins can be used as structural templates for modeling proteins that do not have experimentally determined structures. However, in many cases, there are no homologous proteins (typically <30% sequence identity) with determined structures from which a query sequence can be reliably modeled. The aim of protein threading is to use features, such as secondary structure, solvent accessibility and torsional angles, in addition to sequence patterns to identify structural templates from the protein databank to assist for full-length atomic-level structural modeling. However, there are still numerous protein sequences for which correct templates cannot be recognized. This raises the question as to what attributes allow query sequences to be matched to the correct but distantly homologous templates. To aid the investigation into this question and to provide genome-score protein structure for the biological community, a database called THE-DB (threading hard and easy protein database) has been developed in which it becomes possible to analyze over 15 000 query sequences from the Escherichia coli (E. coli) K12 and human proteomes, as well as to find their three-dimensional templates derived from the state-of-the-art threading algorithms which is not feasible with existing protein template databases. The E. coli K12 and human data can be downloaded in bulk from the THE-DB page.

Understanding the molecular evolutionary basis of social behavior is a major challenge in evolutionary biology. Social insects evolved a complex language of chemical signals to coordinate thousands of individuals. In the fire ant Solenopsis invicta, chemical signals are involved in the determination of a polymorphic social organization. Single-queen (monogyne) or multiqueen (polygyne) social structure is determined by the "social chromosome," a nonrecombining region containing ∼504 genes with two distinct haplotypes, SB and Sb. Monogyne queens are always SBB, while polygyne queens are always SBb. Workers discriminate monogyne from polygyne queens based on olfactory cues. Here, we took an evolutionary genomics approach to search for candidate genes in the social chromosome that could be responsible for this discrimination. We compared the SB and Sb haplotypes and analyzed the evolutionary rates since their divergence. Notably, we identified a cluster of 23 odorant receptors in the nonrecombining region of the social chromosome that stands out in terms of nonsynonymous changes in both haplotypes. The cluster includes twelve genes formed by recent Solenopsis-specific duplications. We found evidence for positive selection on several tree branches and significant differences between the SB and Sb haplotypes of these genes. The most dramatic difference is the complete deletion of two of these genes in Sb. These results suggest that the evolution of polygyne social organization involved adaptations in olfactory genes and opens the way for functional studies of the molecular mechanisms underlying social behavior.

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.

Whole-genome duplication (WGD) has been a major evolutionary driver of increased genomic complexity in vertebrates. One such event occurred in the salmonid family ∼80 Ma (Ss4R) giving rise to a plethora of structural and regulatory duplicate-driven divergence, making salmonids an exemplary system to investigate the evolutionary consequences of WGD. Here, we present a draft genome assembly of European grayling (Thymallus thymallus) and use this in a comparative framework to study evolution of gene regulation following WGD. Among the Ss4R duplicates identified in European grayling and Atlantic salmon (Salmo salar), one-third reflect nonneutral tissue expression evolution, with strong purifying selection, maintained over ∼50 Myr. Of these, the majority reflect conserved tissue regulation under strong selective constraints related to brain and neural-related functions, as well as higher-order protein-protein interactions. A small subset of the duplicates have evolved tissue regulatory expression divergence in a common ancestor, which have been subsequently conserved in both lineages, suggestive of adaptive divergence following WGD. These candidates for adaptive tissue expression divergence have elevated rates of protein coding- and promoter-sequence evolution and are enriched for immune- and lipid metabolism ontology terms. Lastly, lineage-specific duplicate divergence points toward underlying differences in adaptive pressures on expression regulation in the nonanadromous grayling versus the anadromous Atlantic salmon. Our findings enhance our understanding of the role of WGD in genome evolution and highlight cases of regulatory divergence of Ss4R duplicates, possibly related to a niche shift in early salmonid evolution.

How is endometriosis associated with adverse maternal, fetal and neonatal outcomes of pregnancy?

The development of tissue fibrosis is complex and at the present time, not fully understood. Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA disease) have been described in patients with mutations in NHL repeat-containing protein 2 (NHLRC2). However, the molecular functions of NHLRC2 are uncharacterized. Herein, we identified putative interacting partners for NHLRC2 using proximity-labeling mass spectrometry. We also investigated the function of NHLRC2 using immortalized cells cultured from skin biopsies of FINCA patients and normal fibroblasts with NHLRC2 knock-down and NHLRC2 overexpressing gene modifications. Transmission electron microscopy analysis of immortalized cell cultures from three FINCA patients demonstrated multilamellar bodies and distinctly organized vimentin filaments. Additionally, two of three cultures derived from patient skin biopsies contained cells that exhibited features characteristic of myofibroblasts. Altogether, the data presented in this study show for the first time that NHLRC2 is involved in cellular organization through regulation of the cytoskeleton and vesicle transport. We conclude that compound heterozygous p.Asp148Tyr and p.Arg201GlyfsTer6 mutations in NHLRC2 lead to severe tissue fibrosis in humans by enhancing the differentiation of fibroblasts to myofibroblasts.

Techniques for dose reduction in computed tomography (CT) are receiving increasing attention. Lowering the tube current in front of the patient, known as risk-organ-based tube current modulation (RTM), represents a new approach. Physical dose parameters can determine the exposure but are not able to assess the biological-X-ray interactions. The purpose of this study was to establish a biological phantom model to evaluate the effect of RTM on X-ray-induced DNA double-strand breaks (DSBs). In breast phantoms and in the location of the spine in an Alderson phantom, isolated human blood lymphocytes were irradiated using a 128-slice CT scanner. A standard thoracic CT protocol (120 kV, 110 ref. mAs, anatomy-based tube current modulation, pitch 0.6, scan length 30 cm) with and without RTM was used. X-ray-induced DSBs were quantified in isolated blood lymphocytes using immunofluorescence microscopy after staining for the phosphorylated histone variant γ-H2AX. Using RTM, the resulting DNA damage reduction was 41% in superficial breast locations (P = 0.0001), 28% in middle breast locations (P = 0.0003) and 29% in lower breast locations (P = 0.0001), but we found a DNA damage increase of 36% in superficial spine locations (P = 0.0001) and of 26% in deep spine locations (P = 0.0001). In summary, we established a biological phantom model that is suitable for detecting DNA damage in distinct organs. In addition, we were able to show that, using RTM, X-ray-induced DNA damage in the breast can be significantly reduced; however, there is a significant increase in DSBs in the location of the spine.

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) that is characterized by intractable nasal polyp formation. Eosinophil-derived neurotoxin (EDN) is an eosinophil granule protein that is closely related to allergic inflammation, but the pathological implications of EDN in ECRS remain unknown. In this study, we evaluated the function of EDN in ECRS pathogenesis and assessed its potential as a disease activity marker. Serum EDN levels were significantly higher in patients with ECRS than in those with other nasal and paranasal diseases, and were positively correlated with clinical disease activity. Production of EDN from isolated human eosinophils was induced by stimulation with IL-5 in vitro. Human nasal epithelial cells were stimulated with EDN, and the resultant changes in gene expression were detected by RNA sequencing. Pathway analysis revealed that the major canonical pathway affected by EDN stimulation was 'regulation of the epithelial-mesenchymal transition pathway'; the only gene in this pathway to be up-regulated was matrix metalloproteinase 9 (MMP-9). Consistent with this, immunostaining analysis revealed intense staining of both EDN and MMP-9 in nasal polyps from patients with ECRS. In conclusion, our data demonstrate that serum EDN level is a useful marker for the evaluation of ECRS severity. Furthermore, EDN induces production of MMP-9 from the nasal epithelium, which may be involved in the pathogenesis of ECRS.

Skeletal muscle regeneration in adults is attributed to the presence of satellite stem cells that proliferate, differentiate, and eventually fuse with injured myofibers. However, the signaling mechanisms that regulate satellite cell homeostasis and function remain less understood. While IKKβ-mediated canonical NF-κB signaling has been implicated in the regulation of myogenesis and skeletal muscle mass, its role in the regulation of satellite cell function during muscle regeneration has not been fully elucidated. Here, we report that canonical NF-κB signaling is induced in skeletal muscle upon injury. Satellite cell-specific inducible ablation of IKKβ attenuates skeletal muscle regeneration in adult mice. Targeted ablation of IKKβ also reduces the number of satellite cells in injured skeletal muscle of adult mice, potentially through inhibiting their proliferation and survival. We also demonstrate that the inhibition of specific components of the canonical NF-κB pathway causes precocious differentiation of cultured satellite cells both ex vivo and in vitro. Finally, our results highlight that the constitutive activation of canonical NF-κB signaling in satellite cells also attenuates skeletal muscle regeneration following injury in adult mice. Collectively, our study demonstrates that the proper regulation of canonical NF-κB signaling is important for the regeneration of adult skeletal muscle.

Post-transcriptional regulation of RNAs is critical to the diverse range of cellular processes. The volume of functional genomic data focusing on post-transcriptional regulation logics continues to grow in recent years. In the current database version, POSTAR2 (http://lulab.life.tsinghua.edu.cn/postar), we included the following new features and data: updated ∼500 CLIP-seq datasets (∼1200 CLIP-seq datasets in total) from six species, including human, mouse, fly, worm, Arabidopsis and yeast; added a new module 'Translatome', which is derived from Ribo-seq datasets and contains ∼36 million open reading frames (ORFs) in the genomes from the six species; updated and unified post-transcriptional regulation and variation data. Finally, we improved web interfaces for searching and visualizing protein-RNA interactions with multi-layer information. Meanwhile, we also merged our CLIPdb database into POSTAR2. POSTAR2 will help researchers investigate the post-transcriptional regulatory logics coordinated by RNA-binding proteins and translational landscape of cellular RNAs.

Heat-stable nucleoid structuring protein (H-NS) plays a crucial role in gene silencing within prokaryotic cells and is important in pathogenesis. It was reported that H-NS silences nearly 5% of the genome, yet the molecular mechanism of silencing is not well understood. Here, we employed a highly-sensitive single-molecule counting approach, and measured the dissociation constant (KD) of H-NS binding to single DNA binding sites. Charged residues in the linker domain of H-NS provided the most significant contribution to DNA binding affinity. Although H-NS was reported to prefer A/T-rich DNA (a feature of pathogenicity islands) over G/C-rich DNA, the dissociation constants obtained from such sites were nearly identical. Using a hairpin unzipping assay, we were able to uncouple non-specific DNA binding steps from nucleation site binding and subsequent polymerization. We propose a model in which H-NS initially engages with non-specific DNA via reasonably high affinity (∼60 nM KD) electrostatic interactions with basic residues in the linker domain. This initial contact enables H-NS to search along the DNA for specific nucleation sites that drive subsequent polymerization and gene silencing.

Typically not assisted by proofreading, the RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses may need to independently control its fidelity to fulfill virus viability and fitness. However, the precise mechanism by which the RdRP maintains its optimal fidelity level remains largely elusive. By solving 2.1-2.5 Å resolution crystal structures of the classical swine fever virus (CSFV) NS5B, an RdRP with a unique naturally fused N-terminal domain (NTD), we identified high-resolution intra-molecular interactions between the NTD and the RdRP palm domain. In order to dissect possible regulatory functions of NTD, we designed mutations at residues Y471 and E472 to perturb key interactions at the NTD-RdRP interface. When crystallized, some of these NS5B interface mutants maintained the interface, while the others adopted an 'open' conformation that no longer retained the intra-molecular interactions. Data from multiple in vitro RdRP assays indicated that the perturbation of the NTD-RdRP interactions clearly reduced the fidelity level of the RNA synthesis, while the processivity of the NS5B elongation complex was not affected. Collectively, our work demonstrates an explicit and unique mode of polymerase fidelity modulation and provides a vivid example of co-evolution in multi-domain enzymes.

Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally-resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 International Society for Advancement of Cytometry.