Intravitreal Dexamethasone Implant in Anti-Vascular Endothelial Growth Factor Pretreated Diabetic Macular Edema-A Swiss Cohort Study.

BACKGROUND/OBJECTIVES: Diabetic macular edema (DME) is a significant cause of visual impairment, often treated with anti-vascular endothelial growth factor (anti-VEGF) agents. However, some patients do not respond adequately to this treatment. This study aims to evaluate the contribution of the intravitreal dexamethasone (DEX) implant as a second-line treatment in DME patients with insufficient response to anti-VEGF therapy or with high treatment burden. METHODS: This retrospective multicenter cohort study was conducted across seven clinical sites in Switzerland. The study included eyes with active DME that had been pretreated with anti-VEGF for at least six months before receiving DEX therapy. Data were extracted from electronic patient records, focusing on best-corrected visual acuity (BCVA), central subfield thickness (CST), and injection frequency. RESULTS: A total of 95 eyes from 89 patients (38.8% females, mean age 65.6 ± 9.1 years, follow-up time 80.6 ± 38.5 [13.5-166.7] months) were analyzed. Prior to the first DEX implant, eyes had undergone an average of 16.0 ± 13.3 anti-VEGF injections over 32.5 ± 22.4 months. Post-DEX treatment, 22.1% of eyes received DEX monotherapy, 44.2% received a combination of DEX and anti-VEGF, 25.3% continued with anti-VEGF monotherapy, and 8.4% received no further treatment. The number of anti-VEGF injections decreased significantly from 6.4 ± 3.1 in the year before DEX to 1.6 ± 2.4 in the year after DEX (p < 0.001). BCVA remained stable (0.4 ± 0.3 logMAR at baseline, 0.4 ± 0.5 logMAR at 24 months, p = 0.2), while CST improved from 477.7 ± 141.0 to 320.4 ± 125.5 µm (p < 0.001), and the presence of retinal fluid decreased from 98.0% to 61.1% (p = 0.021). During follow-up, 26.3% of eyes required glaucoma medication, 4.2% underwent glaucoma surgery, and 1.1% needed cataract surgery. CONCLUSIONS: In real-world clinical settings, the addition of DEX to anti-VEGF therapy in DME patients significantly reduces treatment burden and retinal fluid while maintaining visual function. Treatment decisions should balance anatomical and functional outcomes, considering individual patient needs.

Redox-dependent condensation and cytoplasmic granulation by human ssDNA-binding protein-1 delineate roles in oxidative stress response.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in DNA repair. Here, we show that purified hSSB1 undergoes redox-dependent liquid-liquid phase separation (LLPS) in the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions in response to treatment modeling cellular oxidative stress. hSSB1's intrinsically disordered region is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we detected cytoplasmic hSSB1 condensates in various cell lines colocalizing with stress granules upon oxidative stress, implying extranuclear function in cellular stress response. Our results suggest condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.

Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

Werner helicase interacting protein 1 contributes to G-quadruplex processing in human cells.

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

Development and Implementation of a Novel CAPS Assay Reveals High Prevalence of a Boscalid Resistance Marker and Its Co-Occurrence with an Azole Resistance Marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a nonsynonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

Trans-kingdom fungal pathogens infecting both plants and humans, and the problem of azole fungicide resistance.

Azole antifungals are abundantly used in the environment and play an important role in managing fungal diseases in clinics. Due to the widespread use, azole resistance is an emerging global problem for all applications in several fungal species, including trans-kingdom pathogens, capable of infecting plants and humans. Azoles used in agriculture and clinics share the mode of action and facilitating cross-resistance development. The extensive use of azoles in the environment, e.g., for plant protection and wood preservation, contributes to the spread of resistant populations and challenges using these antifungals in medical treatments. The target of azoles is the cytochrome p450 lanosterol 14-α demethylase encoded by the CYP51 (called also as ERG11 in the case of yeasts) gene. Resistance mechanisms involve mainly the mutations in the coding region in the CYP51 gene, resulting in the inadequate binding of azoles to the encoded Cyp51 protein, or mutations in the promoter region causing overexpression of the protein. The World Health Organization (WHO) has issued the first fungal priority pathogens list (FPPL) to raise awareness of the risk of fungal infections and the increasingly rapid spread of antifungal resistance. Here, we review the main issues about the azole antifungal resistance of trans-kingdom pathogenic fungi with the ability to cause serious human infections and included in the WHO FPPL. Methods for the identification of these species and detection of resistance are summarized, highlighting the importance of these issues to apply the proper treatment.

Stimulating VAPB-PTPIP51 ER-mitochondria tethering corrects FTD/ALS mutant TDP43 linked Ca2+ and synaptic defects.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are clinically linked major neurodegenerative diseases. Notably, TAR DNA-binding protein-43 (TDP43) accumulations are hallmark pathologies of FTD/ALS and mutations in the gene encoding TDP43 cause familial FTD/ALS. There are no cures for FTD/ALS. FTD/ALS display damage to a broad range of physiological functions, many of which are regulated by signaling between the endoplasmic reticulum (ER) and mitochondria. This signaling is mediated by the VAPB-PTPIP51 tethering proteins that serve to recruit regions of ER to the mitochondrial surface so as to facilitate inter-organelle communications. Several studies have now shown that disrupted ER-mitochondria signaling including breaking of the VAPB-PTPIP51 tethers are features of FTD/ALS and that for TDP43 and other familial genetic FTD/ALS insults, this involves activation of glycogen kinase-3ß (GSK3ß). Such findings have prompted suggestions that correcting damage to ER-mitochondria signaling and the VAPB-PTPIP51 interaction may be broadly therapeutic. Here we provide evidence to support this notion. We show that overexpression of VAPB or PTPIP51 to enhance ER-mitochondria signaling corrects mutant TDP43 induced damage to inositol 1,4,5-trisphosphate (IP3) receptor delivery of Ca2+ to mitochondria which is a primary function of the VAPB-PTPIP51 tethers, and to synaptic function. Moreover, we show that ursodeoxycholic acid (UDCA), an FDA approved drug linked to FTD/ALS and other neurodegenerative diseases therapy and whose precise therapeutic target is unclear, corrects TDP43 linked damage to the VAPB-PTPIP51 interaction. We also show that this effect involves inhibition of TDP43 mediated activation of GSK3ß. Thus, correcting damage to the VAPB-PTPIP51 tethers may have therapeutic value for FTD/ALS and other age-related neurodegenerative diseases.

A revised nomenclature for the lemur family of protein kinases.

The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.

Two New Families and a Literature Review of ELOVL4-Associated Spinocerebellar Ataxia Type 34.

Autosomal dominant variants in ELOVL4 cause spinocerebellar ataxia type 34 (SCA34; ATX-ELOVL4), classically associated with a skin condition known as erythrokeratoderma. Here, we report a large Italian-Maltese-Australian family with spinocerebellar ataxia. Notably, while there were dermatological manifestations (eczema), erythrokeratoderma was not present. Using a next-generation sequencing panel, we identified a previously reported ELOVL4 variant, NM_022726.4: c.698C > T p.(Thr233Met). The variant was initially classified as a variant of uncertain significance; however, through segregation studies, we reclassified the variant as likely pathogenic. We next identified an individual from another family (Algerian-Maltese-Australian) with the same ELOVL4 variant with spinocerebellar ataxia but without dermatological manifestations. We subsequently performed the first dedicated literature review of ELOVL4-associated ataxia to gain further insights into genotype-phenotype relationships. We identified a total of 60 reported cases of SCA34 to date. The majority had gait ataxia (88.3%), limb ataxia (76.7%), dysarthria (63.3%), and nystagmus (58.3%). Of note, skin lesions related to erythrokeratoderma were seen in a minority of cases (33.3%). Other extracerebellar manifestations included pyramidal tract signs, autonomic disturbances, retinitis pigmentosa, and cognitive impairment. For brain MRI data, cerebellar atrophy was seen in all cases (100%), whereas the hot cross bun sign (typically associated with multiple system atrophy type C) was seen in 32.4% of cases. Our family study and literature review highlight the variable phenotypic spectrum of SCA34. Importantly, it shows that erythrokeratoderma is not found in most cases and that, while a dermatological assessment may be helpful in these patients, SCA34 diagnosis should be considered irrespective of dermatological manifestations.

Exploring diversity within the genus Tulostoma (Basidiomycota, Agaricales) in the Pannonian sandy steppe: four fascinating novel species from Hungary.

Steppe vegetation on sandy soil in Hungary has recently been revealed as one of the hot spots in Europe for the stalked puffballs (genus Tulostoma). In the framework of the taxonomic revision of gasteroid fungi in Hungary, four Tulostoma species are described here as new to science: T.dunense, T.hungaricum, T.sacchariolens and T.shaihuludii. The study is based on detailed macro- and micromorphological investigations (including light and scanning electron microscopy), as well as a three-locus phylogeny of nrDNA ITS, nrDNA LSU and tef1-α sequences. The ITS and LSU sequences generated from the type specimen of T.cretaceum are provided and this resolved partly the taxonomy of the difficult species complex of T.aff.cretaceum.

Video head impulse and suppression head impulse test in vestibular migraine.

BACKGROUND: Vestibular Migraine (VM) is a frequent cause of recurrent spontaneous vertigo. While some report a normal Video Head Impulse Test (vHIT) in VM, others observed abnormal results on this test. Whether or not methodological discrepancies could be the cause of these differences is not known. There are 2 vHIT methods: subjects fixating an earth-fixed target (HIMP paradigm) or a head-fixed target, the suppression head impulse test (SHIMP paradigm). OBJECTIVES: The present study aimed to compare VM patients against healthy controls on both HIMP and SHIMP in order to unravel any differences between them. METHODS: Forty-eight VM patients and 27 healthy controls tested with both the HIMP and SHIMP paradigm. Results: Our 48 VM patients showed mean VOR normal range gains in both the HIMP and SHIMP paradigms, although there were some VOR impairments in individual semicircular SCCs. VM patients with motion sickness had lower horizontal VOR gain than those without motion sickness, with the HIMP, but not the SHIMP paradigm. CONCLUSION: VM patients have normal VOR gain with either vHIT paradigm. SIGNIFICANCE: The clinical significance of this observation is that a definitely abnormal vHIT with either method is unlikely to be due to vestibular migraine and an alternative diagnosis should be sought.

Untargeted metabolomic analyses support the main phylogenetic groups of the common plant-associated Alternaria fungi isolated from grapevine (Vitis vinifera).

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)-high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations.

Genetically distinct groups of Erysiphe necator, the fungus causing grapevine powdery mildew infect grapevine in Europe, yet the processes sustaining stable genetic differences between those groups are less understood. Genotyping of over 2000 field samples from six wine regions in Hungary collected between 2017 and 2019 was conducted to reveal E. necator genotypes and their possible differentiation. The demethylase inhibitor (DMI) fungicide resistance marker A495T was detected in all wine regions, in 16% of the samples. Its occurrence differed significantly among wine regions and grape cultivars, and sampling years, but it did not differ between DMI-treated and untreated fields. Multilocus sequence analyses of field samples and 59 in vitro maintained isolates revealed significant genetic differences among populations from distinct wine regions. We identified 14 E. necator genotypes, of which eight were previously unknown. In contrast to the previous concept of A and B groups, European E. necator populations should be considered genetically more complex. Isolation by geographic distance, growing season, and host variety influence the genetic structuring of E. necator, which should be considered both during diagnoses and when effective treatments are planned.

Wilson Disease: A Case Report of Psychosis Preceding Parkinsonism.

BACKGROUND A first psychotic episode requires the exclusion of toxic-metabolic, inflammatory, infective, and neoplastic causes. Wilson disease is a rare, autosomal recessive disorder of copper metabolism and can present with neuropsychiatric symptoms secondary to copper accumulation in the brain. CASE REPORT We describe the case of a 48-year-old man with parkinsonism on a background of longstanding schizophrenia and psychotic depression in the setting of previously undiagnosed Wilson disease. The common history of neuropsychiatric disturbance and neuroleptic use complicated the assessment of parkinsonism. However, close attention to the temporal appearance of symptoms and signs differentiated his case from drug-induced parkinsonism, which commonly develops hours to weeks after commencement or uptitration of antipsychotic medication. The early features of sialorrhea and dysarthria were also atypical for idiopathic Parkinson disease. The diagnosis was confirmed by serum copper testing and supported by Kayser-Fleischer rings on bedside ophthalmological examination. Magnetic resonance imaging (MRI) of the brain demonstrated copper accumulation in the basal ganglia and pons, contributing to the characteristic neurological manifestations of an akinetic-rigid syndrome with dysarthria. CONCLUSIONS Serum copper testing is easily obtained and should be considered as part of the first-line investigations for new neuropsychiatric disturbances. Although rare, Wilson disease, if diagnosed early, is a potentially treatable and reversible cause of psychosis. With advanced disease, extrapyramidal findings on examination correlate with MRI brain changes, aiding the clinical assessment in differentiating the disease from drug-induced parkinsonism.

Neurological update: neuro-otology 2023.

Much has changed since our last review of recent advances in neuro-otology 7 years ago. Unfortunately there are still not many practising neuro-otologists, so that most patients with vestibular problems need, in the first instance, to be evaluated and treated by neurologists whose special expertise is not neuro-otology. The areas we consider here are mostly those that almost any neurologist should be able to start managing: acute spontaneous vertigo in the Emergency Room-is it vestibular neuritis or posterior circulation stroke; recurrent spontaneous vertigo in the office-is it vestibular migraine or Meniere's disease and the most common vestibular problem of all-benign positional vertigo. Finally we consider the future: long-term vestibular monitoring and the impact of machine learning on vestibular diagnosis.

Mitochondrial DNA 13513G>A Mutation Causing Leber Hereditary Optic Neuropathy Associated With Adult-Onset Renal Failure.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is one of the more common mitochondrial diseases and is rarely associated with mitochondrial renal disease. We report 3 unrelated patients with a background of adult-onset renal failure who presented to us with LHON and were shown to have a heteroplasmic mitochondrial DNA mutation (m.13513G>A). METHODS: Retrospective chart review. RESULTS: All 3 patients had a background of chronic renal failure and presented to us with bilateral optic neuropathy (sequential in 2) and were found to have heteroplasmic m.13513G>A mutations in the MT-ND5 gene. Two of the patients were females (aged 30 and 45 years) with chronic kidney disease from their 20s, attributed to pre-eclampsia, one of whom also had diabetes and sudden bilateral hearing loss. One patient was a male (aged 54 years) with chronic kidney disease from his 20s attributed to IgA nephropathy. His mother had diabetes and apparently sudden bilateral blindness in her 70s. Renal biopsy findings were variable and included interstitial fibrosis, acute tubular necrosis, focal segmental glomerulosclerosis, and IgA/C3 tubular casts on immunofluorescence. Mild improvements in vision followed treatment with either idebenone or a combination supplement including coenzyme Q10, alpha-lipoic acid, and B vitamins. CONCLUSIONS: Our cases expand the clinical syndromes associated with m.13513G>A to include bilateral optic neuropathy and adult-onset renal disease. This highlights that in patients with bilateral, especially sequential, optic neuropathy a broad approach to mitochondrial testing is more useful than a limited LHON panel. Mitochondrial diseases present a diagnostic challenge because of their clinical and genetic variability.