[An anatomical case of obstructive jaundice due to the rapid enlargement of hepatic peribiliary cysts in end-stage alcoholic liver cirrhosis].

A man in his 60s had end-stage alcoholic cirrhosis. About six months before his death, hepatic peribiliary cysts (HPBC) rapidly increased, and he developed jaundice and liver failure. The pathological autopsy performed after his death revealed that his intrahepatic bile duct was pressured due to multiple cysts caused by HPBC, which resulted in liver failure. Some cases of HPBC have been associated with alcoholic cirrhosis;however, no other cases of increased HPBC in a short period of time have been reported. Although identifying the cause of increased HPBC in a short time is difficult in this case, it may be have been caused by continuous alcohol drinking after the onset of HPBC. Most patients with HPBC have liver cirrhosis and obstructive jaundice that may promote liver failure as in this case. Therefore, patients with HPBC should not only be instructed for abstinence but also promptly consider effective treatments in the event of obstructive jaundice to prevent liver dysfunction.

Base-excision restriction enzymes: expanding the world of epigenetic immune systems.

The restriction enzymes examined so far are phosphodiesterases, which cleave DNA strands by hydrolysing phosphodiester bonds. Based on the mobility of restriction-modification systems, recent studies have identified a family of restriction enzymes that excise a base in their recognition sequence to generate an abasic (AP) site unless the base is properly methylated. These restriction glycosylases also show intrinsic but uncoupled AP lyase activity at the AP site, generating an atypical strand break. Action of an AP endonuclease at the AP site may generate another atypical break, rejoining/repairing of which is difficult. This PabI family of restriction enzymes contain a novel fold (HALFPIPE) and show unusual properties, such as non-requirement of divalent cations for cleavage. These enzymes are present in Helicobacteraceae/Campylobacteraceae and in few hyperthermophilic archaeal species. In Helicobacter genomes, their recognition sites are strongly avoided, and the encoding genes are often inactivated by mutations or replacement, indicating that their expression is toxic for the cells. The discovery of restriction glycosylases generalizes the concept of restriction-modification systems to epigenetic immune systems, which may use any mode of damage to DNA that are considered 'non-self' based on epigenetic modifications. This concept will add to our understanding of immunity and epigenetics.

Elderly onset age is associated with low efficacy of first anti-tumor necrosis factor treatment in patients with inflammatory bowel disease.

The outcomes of patients with elderly onset (EO) inflammatory bowel disease (IBD) treated with anti-tumor necrosis factor (TNF) remains uncertain. The present study evaluated the efficacy and safety of anti-TNF treatment for bio-naïve EO-IBD. Elderly patients were defined as those 60 years and older, and further divided into those with EO (Elderly-EO) and those with non-elderly onset (Elderly-NEO). A total of 432 bio-naïve patients were enrolled in this multicenter observational study, comprising 55 with Elderly-EO (12.7%), 25 with Elderly-NEO (5.8%), and 352 under age 60 (Non-elderly, 81.5%). After 52 weeks of anti-TNF treatment, clinical and steroid-free remission rates were significantly lower in Elderly-EO than in Non-elderly (37.7% and 60.8%; P = 0.001, and 35.9% and 57.8%; P = 0.003, respectively), and comparable between Elderly-NEO and Non-elderly. Multivariate analysis revealed that elderly onset was a significant factor for both clinical remission (OR, 0.49, 95% CI 0.25-0.96) and steroid-free remission (OR, 0.51, 95% CI 0.26-0.99) after 52 weeks of anti-TNF treatment. The rate of cumulative severe adverse events was significantly higher in Elderly-EO than in Non-elderly (P = 0.007), and comparable between Elderly-NEO and Non-elderly. In conclusion, anti-TNF treatment for bio-naïve EO-IBD may be less effective and raise safety concerns.

Genomic differentiation within East Asian Helicobacter pylori.

The East Asian region, including China, Japan and Korea, accounts for half of gastric cancer deaths. However, different areas have contrasting gastric cancer incidences and the population structure of Helicobacter pylori in this ethnically diverse region is yet unknown. We aimed to investigate genomic differences in H. pylori between these areas to identify sequence polymorphisms associated with increased cancer risk. We analysed 381 H. pylori genomes collected from different areas of the three countries using phylogenetic and population genetic tools to characterize population differentiation. The functional consequences of SNPs with a highest fixation index (Fst) between subpopulations were examined by mapping amino acid changes on 3D protein structure, solved or modelled. Overall, 329/381 genomes belonged to the previously identified hspEAsia population indicating that import of bacteria from other regions of the world has been uncommon. Seven subregional clusters were found within hspEAsia, related to subpopulations with various ethnicities, geographies and gastric cancer risks. Subpopulation-specific amino acid changes were found in multidrug exporters (hefC), transporters (frpB-4), outer membrane proteins (hopI) and several genes involved in host interaction, such as a catalase site, involved in H2O2 entrance, and a flagellin site mimicking host glycosylation. Several of the top hits, including frpB-4, hefC, alpB/hopB and hofC, have been found to be differentiated within the Americas in previous studies, indicating that a handful of genes may be key to local geographic adaptation. H. pylori within East Asia are not homogeneous but have become differentiated geographically at multiple loci that might have facilitated adaptation to local conditions and hosts. This has important implications for further evaluation of these changes in relation to the varying gastric cancer incidence between geographical areas in this region.

Genome-wide association study of gastric cancer- and duodenal ulcer-derived Helicobacter pylori strains reveals discriminatory genetic variations and novel oncoprotein candidates.

Genome-wide association studies (GWASs) can reveal genetic variations associated with a phenotype in the absence of any hypothesis of candidate genes. The problem of false-positive sites linked with the responsible site might be bypassed in bacteria with a high homologous recombination rate, such as Helicobacter pylori, which causes gastric cancer. We conducted a small-sample GWAS (125 gastric cancer cases and 115 controls) followed by prediction of gastric cancer and control (duodenal ulcer) H. pylori strains. We identified 11 single nucleotide polymorphisms (eight amino acid changes) and three DNA motifs that, combined, allowed effective disease discrimination. They were often informative of the underlying molecular mechanisms, such as electric charge alteration at the ligand-binding pocket, alteration in subunit interaction, and mode-switching of DNA methylation. We also identified three novel virulence factors/oncoprotein candidates. These results provide both defined targets for further informatic and experimental analyses to gain insights into gastric cancer pathogenesis and a basis for identifying a set of biomarkers for distinguishing these H. pylori-related diseases.

Corrigendum: Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

[This corrects the article DOI: 10.3389/fmicb.2020.01628.].

Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

Bridge to surgery using a self-expandable metallic stent for stages II-III obstructive colorectal cancer.

BACKGROUND: Bridge to surgery (BTS) using a self-expandable metallic stent (SEMS) for the treatment of obstructive colorectal cancer improves the patient's quality of life. This study aimed to examine prognostic factors of obstructive colorectal cancer. METHODS: We analyzed stage II-III resectable colon cancer cases (Cur A) retrospectively registered between January 2005 and December 2017. Overall, 117 patients with Cur A obstructive colorectal cancer were evaluated: 67 of them underwent emergency surgery (ES Group) and 50 of them after BTS with SEMS placement (BTS group). We compared surgical results and prognoses between the two groups. RESULTS: A total of 50 patients underwent endoscopic SEMS placement, which technical success of 96% and morbidity rate of 18%. Primary anastomosis rates were 77.6% in ES and 95.7% in BTS (p <  0.001); postoperative complication, 46.3% in ES and 10.5% in BTS (p <  0.001); pathological findings of lymphatic invasion, 66.7% in ES and 100% in BTS (p <  0.001); venous invasion were 66.8% in ES and 92% in BTS (p = 0.04); and recurrence of 25.4% in ES and 39.1% in BTS. The 3-year overall survival was significantly different between two groups (ES, 86.8%:BTS, 58.8%), BTS is worse than ES (log-rank test; p <  0.001). Venous invasion independently predicted worsened recurrence-free and overall survival. CONCLUSIONS: The vascular invasiveness was correlated with tumor progression after SEMS placement, and the survival rate was lower in BTS. SEMS potentially worsens prognostic outcomes in stage II-III obstructive colorectal cancer.

Detection of DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis of Circular Bacterial Chromosomes.

Double-strand breakage of DNA is a process central to life and death in DNA-coded organisms. Its sensitive and quantitative detection is realized by pulsed-field gel electrophoresis of a huge (Mb) circular chromosome. A single double-strand break at one of its millions of potential sites will make it linear and release it from branches of an agarose jungle. Then the huge fragments will move according to their size. We developed this method to analyze formation of DNA double-strand breaks and their processing in E. coli. Here we detail our protocol taking the example of chromosome breaks caused by action of a restriction enzyme in vivo. It is important to prevent formation of irrelevant double-strand breaks.

A prospective multicenter observational study evaluating the risk of periendoscopic events in patients using anticoagulants: the Osaka GIANT Study.

Background and study aims An increasing number of patients have been using anticoagulants including anti-vitamin K antagonists (VKAs) and direct oral anticoagulants (DOACs); however, in patients using anticoagulants, limited data are available with regard to the risks of gastrointestinal bleeding and thromboembolic events during the peri-endoscopic period. We aimed to evaluate the peri-endoscopic bleeding and thrombotic risks in patients administered VKAs or DOACs. Patients and methods Consecutive patients using anticoagulants who underwent endoscopic biopsy, mucosal resection, or submucosal dissection were prospectively enrolled across 11 hospitals. The primary outcome assessed was difference in incidence of post-procedural gastrointestinal bleeding in patients using VKAs and DOACs. Duration of hospitalization and peri-procedural thromboembolic events were also compared. Results We enrolled 174 patients using VKAs and 37 using DOACs. In total, 16 patients using VKA were excluded from the analysis because of cancellation of endoscopic procedures and contraindications to the use of DOACs; 128 (81 %) patients using VKAs and 17 (46 %) using DOACs received heparin-bridging therapy (HB). The rate of post-procedural gastrointestinal bleeding in DOAC users was similar to that in VKA users (16.2 % vs. 16.4 %, P  = 1.000). Duration of hospitalization was significantly longer in patients using VKAs than in those using DOACs (median 15 vs. 7 days, P  < 0.0001). Myocardial infarction occurred during pre-endoscopic HB in one patient using VKAs. Conclusion DOAC administration showed similar post-procedural gastrointestinal bleeding risk to VKA administration in patients undergoing endoscopic procedures, but it shortened the hospital stay.

Clinical use of molecular targeted agents for primary small bowel adenocarcinoma: A multicenter retrospective cohort study by the Osaka Gut Forum.

Primary small bowel adenocarcinoma (SBA) is a rare cancer for which effective treatment strategies have not yet been established. The results of previous retrospective studies suggest that chemotherapy contributes to a longer survival time in patients with SBA. However, there are few case reports about the efficacy of molecular targeted agent-containing chemotherapy for SBA. In the present study, the treatment and follow-up data of patients with SBA who received chemotherapy with or without molecular targeted agents were retrospectively analyzed. Each patient was treated in one of ten hospitals participating in the Osaka Gut Forum between April 2006 and March 2014. The following factors were evaluated: Age, sex, Eastern Cooperative Oncology Group performance status (PS), tumor location, tumor differentiation, chemotherapy regimen, resection of primary tumor, tumor biomarker expression, distant metastasis, best response under chemotherapy, time to disease progression, subsequent treatments, survival status and treatment toxicity. A total of 27 patients (17 males and 10 females; mean age, 63.4 years old; range, 36-83 years old) received chemotherapy due to non-curative tumor resection, unresectable tumor or post-operative recurrence. The median overall survival time was 14.8 months (range, 2-58 months). A univariate analysis revealed a PS of 0 (P=0.0228) and treatment with platinum-based chemotherapy (P=0.0048) were significant factors for an improved prognosis. An age-adjusted multivariate analysis also revealed that a platinum-based regimen was a significant positive prognostic factor (P=0.0373). Molecular targeted agents were administered to 8 patients, for whom it was their first- or second-line therapy. Among the 17 patients who received oxaliplatin-based chemotherapy as a first-line chemotherapy, a PS of 0 (P=0.0255) and treatment with bevacizumab (P=0.0121) were significant positive prognostic factors. Toxicities higher than Grade 3 occurred in 8/27 patients with SBA; however, serious side effects due to the molecular targeted agents were not experienced. The results of the present study indicate that chemotherapy containing molecular targeted agents is a well-tolerated and effective treatment option for SBA.

Restriction glycosylases: involvement of endonuclease activities in the restriction process.

All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction­modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Prognostic Factors of Oral Intake After Endoscopic Gastroduodenal Stent Placement for Advanced Gastric Cancer.

Gastroduodenal stents are effective for gastric outlet obstruction (GOO) due to gastric cancer. However, some patients are unable to eat again soon. We retrospectively analyzed the cause of short-feeding periods. Between 2011 and 2015, we performed stent placement in 22 patients who could not eat solids. The effects of clinical characteristics on duration of oral intake were analyzed using Cox proportional hazards models. Univariate analyses revealed that the degree of GOO [no oral intake/liquids only; hazard ratio (HR), 10.9; 95% confidence interval (CI), 2.5-48.1; P=0.003], performance status score (2 or 3/0 or 1; HR, 5.7; 95% CI, 1.8-16.9; P=0.004), and poststenting chemotherapy (no/yes; HR, 5.7; 95% CI, 1.9-18.9; P=0.002) were significant factors for cessation of oral intake. Multivariate analysis showed that GOO and chemotherapy were significant factors. Gastroduodenal stents were less effective for patients with severe stenosis or without poststenting chemotherapy.

Population Evolution of Helicobacter pylori through Diversification in DNA Methylation and Interstrain Sequence Homogenization.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

A Novel Approach to Helicobacter pylori Pan-Genome Analysis for Identification of Genomic Islands.

Genomes of a given bacterial species can show great variation in gene content and thus systematic analysis of the entire gene repertoire, termed the pan-genome, is important for understanding bacterial intra-species diversity, population genetics, and evolution. Here, we analyzed the pan-genome from 30 completely sequenced strains of the human gastric pathogen Helicobacter pylori belonging to various phylogeographic groups, focusing on 991 accessory (not fully conserved) orthologous groups (OGs). We developed a method to evaluate the mobility of genes within a genome, using the gene order in the syntenically conserved regions as a reference, and classified the 991 accessory OGs into five classes: Core, Stable, Intermediate, Mobile, and Unique. Phylogenetic networks based on the gene content of Core and Stable classes are highly congruent with that created from the concatenated alignment of fully conserved core genes, in contrast to those of Intermediate and Mobile classes, which show quite different topologies. By clustering the accessory OGs on the basis of phylogenetic pattern similarity and chromosomal proximity, we identified 60 co-occurring gene clusters (CGCs). In addition to known genomic islands, including cag pathogenicity island, bacteriophages, and integrating conjugative elements, we identified some novel ones. One island encodes TerY-phosphorylation triad, which includes the eukaryote-type protein kinase/phosphatase gene pair, and components of type VII secretion system. Another one contains a reverse-transcriptase homolog, which may be involved in the defense against phage infection through altruistic suicide. Many of the CGCs contained restriction-modification (RM) genes. Different RM systems sometimes occupied the same (orthologous) locus in the strains. We anticipate that our method will facilitate pan-genome studies in general and help identify novel genomic islands in various bacterial species.

Genome-wide survey of codons under diversifying selection in a highly recombining bacterial species, Helicobacter pylori.

Selection has been a central issue in biology in eukaryotes as well as prokaryotes. Inference of selection in recombining bacterial species, compared with clonal ones, has been a challenge. It is not known how codons under diversifying selection are distributed along the chromosome or among functional categories or how frequently such codons are subject to mutual homologous recombination. Here, we explored these questions by analysing genes present in >90% among 29 genomes of Helicobacter pylori, one of the bacterial species with the highest mutation and recombination rates. By a method for recombining sequences, we identified codons under diversifying selection (dN/dS> 1), which were widely distributed and accounted for ∼0.2% of all the codons of the genome. The codons were enriched in genes of host interaction/cell surface and genome maintenance (DNA replication,recombination, repair, and restriction modification system). The encoded amino acid residues were sometimes found adjacent to critical catalytic/binding residues in protein structures.Furthermore, by estimating the intensity of homologous recombination at a single nucleotide level, we found that these codons appear to be more frequently subject to recombination.We expect that the present study provides a new approach to population genomics of selection in recombining prokaryotes.

The Landscape of Realized Homologous Recombination in Pathogenic Bacteria.

Recombination enhances the adaptive potential of organisms by allowing genetic variants to be tested on multiple genomic backgrounds. Its distribution in the genome can provide insight into the evolutionary forces that underlie traits, such as the emergence of pathogenicity. Here, we examined landscapes of realized homologous recombination of 500 genomes from ten bacterial species and found all species have "hot" regions with elevated rates relative to the genome average. We examined the size, gene content, and chromosomal features associated with these regions and the correlations between closely related species. The recombination landscape is variable and evolves rapidly. For example in Salmonella, only short regions of around 1 kb in length are hot whereas in the closely related species Escherichia coli, some hot regions exceed 100 kb, spanning many genes. Only Streptococcus pyogenes shows evidence for the positive correlation between GC content and recombination that has been reported for several eukaryotes. Genes with function related to the cell surface/membrane are often found in recombination hot regions but E. coli is the only species where genes annotated as "virulence associated" are consistently hotter. There is also evidence that some genes with "housekeeping" functions tend to be overrepresented in cold regions. For example, ribosomal proteins showed low recombination in all of the species. Among specific genes, transferrin-binding proteins are recombination hot in all three of the species in which they were found, and are subject to interspecies recombination.

Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance.

BACKGROUND: R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. RESULTS: Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East Asia and the Americas. In Malaysia, hrgC was horizontally transferred from hspEAsia to hpAsia2 strains. CONCLUSIONS: The PabI family of RM system behaves as a mobile, selfish genetic element, similar to the other families of Type II RM systems. Our analysis additionally revealed some cases of long-term inheritance. The distribution of the hrgC gene replacing the PabI family in the subpopulations of H. pylori, hspAmerind, hspEAsia and hpAsia2, corresponds to the two human migration events, one from East Asia to Americas and the other from China to Malaysia.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.