This work reviews biofilm investigation techniques and highlights the benefits and drawbacks of each approach focusing especially on Pseudomonas syringae and may serve as a comprehensive guide for ...any early-career researchers starting with the topic of biofilm. Each approach with applications of individual microscopy and spectroscopy techniques is summarized together with characterization of Pseudomonas syringae and its role in pathogenesis.
Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic ...variation in 1054 unsolved STGD and STGD-like probands.
Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays.
In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband.
Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently ...required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.
Keratoconus is a relatively frequent disease leading to severe visual impairment. Existing therapies are imperfect and clinical management may benefit from improved understanding of mechanisms ...leading to this disease. We aim to investigate the replication of 11 single nucleotide polymorphisms (SNPs) with keratoconus.
SNPs from loci previously found in association with keratoconus were genotyped in 165 keratoconus cases of Caucasian Czech origin (108 males and 57 females) and 193 population and gender-matched controls. They included rs1536482 (COL5A1), rs4839200 (KCND3), rs757219 and rs214884 (IMMP2L), rs1328083 and rs1328089 (DAOA), rs2721051 (FOXO1), rs4894535 (FNDC3B), rs4954218 (MAP3K19, RAB3GAP1), rs9938149 (ZNF469) and rs1324183 (MPDZ). A case-control association analysis was assessed using Fisher's exact tests.
The strongest association was found for rs1324183 (allelic test OR = 1.58; 95% CI, 1.10-2.24, p = 0.01). Statistically significant values were also obtained for rs2721051 (allelic test OR = 1.72; 95% CI, 1.07-2.77, p = 0.025) and rs4954218 (allelic test OR = 1.53; 95% CI, 1.01-2.34; p = 0.047) which showed an opposite effect direction compared to previously reported one.
Independent replication of association between two SNPs and keratoconus supports the association of these loci with the risks for the disease development, while the effect of rs4954218 warrants further investigation. Understanding the role of the genetic factors involved in keratoconus etiopathogenesis may facilitate development of novel therapies and an early detection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Crohn’s disease (CD) can be associated with a wide range of extraintestinal manifestations (EIMs), including neurological ones. Published studies differ in their conclusions about the ...epidemiology and etiopathogenesis of neurological EIMs. The aims of this study were to demonstrate the presence and find risk factors of peripheral (somatic and autonomic) neuropathy patients with severe CD on anti-TNFα biological therapy.
Material and methods
A clinical examination focusing on detection of peripheral sensor-motor nervous dysfunction (including Sudoscan) and examination of autonomic nervous system dysfunction (using Ewing´s battery tests and spectral analysis) together with laboratory tests and collection of demographic data followed by administration of questionnaires were performed on a total of 30 neurologically asymptomatic outpatients with severe CD on anti-TNFα biological therapy.
Results
Peripheral sensor-motor nervous function via clinical neurological examination was pathological in 36.7% and Sudoscan in 33.3% of cases. Statistically significant associations between vibration perception test and age, CD and biological therapy duration, body mass index and Crohn’s Disease Activity Index were proved while statistically significant associations between temperature perception test and age and BMI were proved as well. Additionally, a decrease of total protein in a patient´s serum below the physiological cut-off in the 6 months prior to measurement was associated with a pathological result of a Sudoscan. Cardiovascular autonomic neuropathy based on Ewing´s battery tests was present in 56.7% of patients, no statistically significant risk factors were found. Our peripheral neuropathy questionnaire correlated with the results of the Sudoscan test and some tests of the clinical examination of peripheral sensor-motor nervous function (discriminatory contact perception test, temperature perception test).
Conclusions
This study demonstrated a relatively high prevalence of peripheral (especially autonomic) neuropathy and verified some risk factors for the development of peripheral somatic neuropathy in asymptomatic patients with severe form of CD on anti-TNFα biological therapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We aim to report the ocular phenotype and molecular genetic findings in two Czech families with Sorsby fundus dystrophy and to review all the reported
pathogenic variants. Two probands with Sorsby ...fundus dystrophy and three first-degree relatives underwent ocular examination and retinal imaging, including optical coherence tomography angiography. The DNA of the first proband was screened using a targeted ocular gene panel, while, in the second proband, direct sequencing of the
coding region was performed. Sanger sequencing was also used for segregation analysis within the families. All the previously reported
variants were reviewed using the American College of Medical Genetics and the Association for Molecular Pathology interpretation framework. A novel heterozygous variant, c.455A>G p.(Tyr152Cys), in
was identified in both families and potentially de novo in one. Optical coherence tomography angiography documented in one patient the development of a choroidal neovascular membrane at 54 years. Including this study, 23 heterozygous variants in
have been reported as disease-causing. Application of gene-specific criteria denoted eleven variants as pathogenic, eleven as likely pathogenic, and one as a variant of unknown significance. Our study expands the spectrum of
pathogenic variants and highlights the importance of optical coherence tomography angiography for early detection of choroidal neovascular membranes.
In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3–q24.12. Whole-genome ...sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal “endothelium” in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.
In the past few decades, society has faced rapid development and spreading of antimicrobial resistance due to antibiotic misuse and overuse and the immense adaptability of bacteria. Difficulties in ...obtaining effective antimicrobial molecules from natural sources challenged scientists to develop synthetic molecules with antimicrobial effect. We developed modular molecules named LEGO-Lipophosphonoxins (LEGO-LPPO) capable of inducing cytoplasmic membrane perforation. In this structure-activity relationship study we focused on the role of the LEGO-LPPO hydrophobic module directing the molecule insertion into the cytoplasmic membrane. We selected three LEGO-LPPO molecules named C9, C8 and C7 differing in the length of their hydrophobic chain and consisting of an alkenyl group containing one double bond. The molecule with the long hydrophobic chain (C9) was shown to be the most effective with the lowest MIC and highest perforation rate both
in vivo
and
in vitro
. We observed high antimicrobial activity against both G
+
and G
−
bacteria with significant differences in LEGO-LPPOs mechanism of action on these two cell types. We observed a highly cooperative mechanism of LEGO-LPPO action on G
−
bacteria as well as on liposomes resembling G
−
bacteria. LEGO-LPPO action on G
−
bacteria was significantly slower compared to G
+
bacteria suggesting the role of the outer membrane in affecting the LEGO-LPPOs perforation rate. This notion was supported by the higher sensitivity of the
E. coli
strain with a compromised outer membrane. Finally, we noted that the composition of the cytoplasmic membrane affects the activity of LEGO-LPPOs since the presence of phosphatidylethanolamine increases their membrane disrupting activity.
We developed modular antimicrobial compounds capable of inducing cytoplasmic membrane perforation. This structure-activity relationship study focuses on the role of the length of their alkenyl hydrophobic module.
Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD ...cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped ...to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.−339_361dup for CHED1 and c.−370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.−274T>G and c.−307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.
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