Insights into the Molecular Evolution of the PDZ/LIM Family and Identification of a Novel Conserved Protein Motif

The PDZ and LIM domain-containing protein family is encoded by a diverse group of genes whose phylogeny has currently not been analyzed. In mammals, ten genes are found that encode both a PDZ- and one or several LIM-domains. These genes are: ALP, RIL, Elfin (CLP36), Mystique, Enigma (LMP-1), Enigma homologue (ENH), ZASP (Cypher, Oracle), LMO7 and the two LIM domain kinases (LIMK1 and LIMK2). As conventional alignment and phylogenetic procedures of full-length sequences fell short of elucidating the evolutionary history of these genes, we started to analyze the PDZ and LIM domain sequences themselves. Using information from most sequenced eukaryotic lineages, our phylogenetic analysis is based on full-length cDNA-, EST-derived- and genomic- PDZ and LIM domain sequences of over 25 species, ranging from yeast to humans. Plant and protozoan homologs were not found. Our phylogenetic analysis identifies a number of domain duplication and rearrangement events, and shows a single convergent event during evolution of the PDZ/LIM family. Further, we describe the separation of the ALP and Enigma subfamilies in lower vertebrates and identify a novel consensus motif, which we call ‘ALP-like motif’ (AM). This motif is highly-conserved between ALP subfamily proteins of diverse organisms. We used here a combinatorial approach to define the relation of the PDZ and LIM domain encoding genes and to reconstruct their phylogeny. This analysis allowed us to classify the PDZ/LIM family and to suggest a meaningful model for the molecular evolution of the diverse gene architectures found in this multi-domain family

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

'What you suggest is not what I expected': How pre-consultation expectations affect shared decision-making in patients with low back pain

Objective: Existing studies on shared decision-making (SDM) have hardly taken into consideration that patients could have independently developed expectations prior to their consultation with a healthcare provider, nor have studies explored how such expectations affect SDM. Therefore, we explore how pre-consultation expectations affect SDM in patients with low back pain. Methods: We performed a qualitative study through telephone interviews with 10 patients and seven care professionals (physicians, nurse, physician assistants) and 63 in-person observations of patient-physician consultations in an outpatient clinic in the Netherlands. Transcripts were analyzed through an open coding process. Results: A discrepancy existed between what patients expected and what care professionals could offer. Professionals perceived they had to undertake additional efforts to address patients' ‘unrealistic’ expectations while attempting SDM. Patients, in turn, were often dissatisfied with the outcomes of the SDM encounter, as they believed their own expectations were not reflected in the final decision. Conclusion: Unaddressed pre-consultation expectations form a barrier to constructive SDM encounters. Practical implications: Patients’ pre-consultation expectations need to be explored during the SDM encounter. To achieve decisions that are truly shared by care professionals and patients, patients’ pre-consultation expectations should be better incorporated into SDM models and education

'What you suggest is not what I expected': How pre-consultation expectations affect shared decision-making in patients with low back pain

OBJECTIVE: Existing studies on shared decision-making (SDM) have hardly taken into consideration that patients could have independently developed expectations prior to their consultation with a healthcare provider, nor have studies explored how such expectations affect SDM. Therefore, we explore how pre-consultation expectations affect SDM in patients with low back pain. METHODS: We performed a qualitative study through telephone interviews with 10 patients and seven care professionals (physicians, nurse, physician assistants) and 63 in-person observations of patient-physician consultations in an outpatient clinic in the Netherlands. Transcripts were analyzed through an open coding process. RESULTS: A discrepancy existed between what patients expected and what care professionals could offer. Professionals perceived they had to undertake additional efforts to address patients' 'unrealistic' expectations while attempting SDM. Patients, in turn, were often dissatisfied with the outcomes of the SDM encounter, as they believed their own expectations were not reflected in the final decision. CONCLUSION: Unaddressed pre-consultation expectations form a barrier to constructive SDM encounters. PRACTICAL IMPLICATIONS: Patients' pre-consultation expectations need to be explored during the SDM encounter. To achieve decisions that are truly shared by care professionals and patients, patients' pre-consultation expectations should be better incorporated into SDM models and education

Figure 1

<p>Exon structure, domain composition and the six basic forms of PDZ/LIM genes. (A) The exon composition of the human PDZ-LIM domain encoding genes in alphabetical order. Indicated are <i>ALP</i> (ENSG00000154553), <i>ZASP</i> (ENSG00000122367, Elfin (ENSG00000107438), ENIGMA (ENSG00000196923), <i>Enigma-Homolog</i> (ENH) (ENSG00000163110) <i>LIMK1</i> (ENSG00000106683, OTTHUMG00000023448), <i>LIMK2</i> (ENSG00000182541), <i>LMO7</i> (ENSG00000136153), <i>Mystique</i> (ENSG00000120913) and RIL (ENSG00000131435). Domains are color coded on the exons: LIM yellow, PDZ blue, CH red and ZM motif green, while transcription start sites are indicated after non coding regions (colored white) with a small arrow on top. (B) Presence of domain architectures for PDZ and LIM genes and their species distribution. Six basic gene structures can be found amongst the different taxons. The <i>tungus gene</i>, found in the two <i>arthropod species investigated and the nematode homolog Eat-1 both</i> encode one N-terminal PDZ and four C-terminal LIM domains. Eat-1 has been described earlier as the <i>Caenorhabditis elegans</i> ALP/Enigma gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000189#pone.0000189-McKeown1" target="_blank">[50]</a>. Only a single <i>LIMK</i> gene was found per invertebrate species examined, and the <i>LMO7</i> homolog lacks the CH domain (<i>CG31534</i>). The LMO7 gene of <i>Drosophila</i> melanogaster lacks not only the CH domain but also the PDZ domain (not shown, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000189#pone.0000189.s002" target="_blank">Supplemental table S1</a>). As not all ALP and Enigma subfamily members share the ZM domain (ZASP and ALP contain 2 and Elfin one ZM motif) we have excluded the ZM motif from these groups and show only a ZM motif for eat-1/tungus in this figure.</p

Figure 7

<p>Chromosomal locations of ALP and Enigma subfamily genes. Shown are four species were PDZ/LIM genes are co-localized on the same chromosome. Numbers indicated are distances in mega basepairs (bp). If genes were found on individual chromosomes they are not shown here. Further not shown are the results for <i>Pan troglodytes</i> and <i>Macaca mulatta</i> which show exactly the same chromosomal distribution as observed for the humans. Not shown is also the data for <i>Rattus norwegicus</i>, where LIMK1 was co localized with LMO7, but no other combination was found. No combinations whatsoever were found in Mus musculus and in Canis familaris. It must be noted that for some of the species investigated the genomes are not completely sequenced and/or fully assembled.</p

Figure 6

<p>Evolutionary model for the PDZ and LIM encoding genes. The most parsimonious model derived from our phylogenetic analysis shows that the PDZ domain of all 10 different PDZ/LIM encoding genes share a common ancestor, with closest homology to LMO7. The combination of this PDZ domain with a LIM domain formed the common ancestor for both the LMO7 and the ALP/Enigma lineages. The single LIM domain in the ALP/Enigma lineage (closest to LIM2 in <i>eat-1/tungus)</i> then duplicated and gave rise to a PDZ two LIM domain structure. Subsequently the duplicated LIM (closest to LIM3 in <i>eat-1/tungus)</i>, duplicated twice and generated a PDZ four LIM structure similar to <i>eat-1/tungus</i>. From this gene structure, through gene duplication and subsequent domain loss (loosing either three LIM domains (LIM2–4) or only 1 LIM domain (LIM1) for the ALP and Enigma subfamilies, respectively), the ALP/Enigma subfamilies evolved. The color code used for domains is PDZ (blue), LIM (yellow and green), CH (red), Kinase domain (black) and AM-motif (pink).</p