Search

Summary: 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.

Abstract 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-3β (GSK3β). 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 GSK3β. Thus, correcting damage to the VAPB-PTPIP51 tethers may have therapeutic value for FTD/ALS and other age-related neurodegenerative diseases.

Abstract 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.

Abstract 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.

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.

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.

Abstract 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.

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.

Abstract Anti-vascular endothelial growth factor (anti-VEGF) therapies have become the standard of care in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME), resulting in a remarkable decrease in disease-related vision loss. However, the need for regular injections places a significant burden on patients, caregivers, and the healthcare system and improvements in vision may not be maintained long term. As a result of its drying potency and duration of action, brolucizumab, an intravitreal anti-VEGF therapy approved for the treatment of nAMD and DME, could decrease injection frequency for patients and provide an efficacious treatment; however, balancing its benefits and risks can be challenging. There have been reports of intraocular inflammation (IOI) in patients treated with brolucizumab, which, if left untreated, may result in severe vision loss. Recent evidence, however, indicates that early recognition of IOI and prompt and aggressive systemic corticosteroid treatment in response to posterior segment involvement can lead to favorable outcomes in these relatively rare but severe cases. A series of consensus meetings were conducted in 2022 between Swiss medical retina experts and diabetologists, discussing the current data for brolucizumab and exploring various challenges to its use, including the associated risk of IOI. The outcome is a collation of practical insights and guidance for ophthalmologists on the use of brolucizumab in patients with nAMD and DME, including patient selection and assessment, treatment regimen and monitoring, and the recognition and management of adverse events.

Fungal endophytes can improve plant tolerance to abiotic stress conditions. Dark septate endophytes (DSEs) belong to phylogenetically non-related groups of root colonizing fungi among the Ascomycota with high melanin-producing activities. They can be isolated from roots of more than 600 plant species in diverse ecosystems. Still the knowledge about their interaction with host plants and their contribution to stress alleviation is limited. The current work aimed to test the abilities of three DSEs (Periconia macrospinosa, Cadophora sp., Leptodontidium sp.) to alleviate moderate and high salt stress in tomato plants. By including an albino mutant, the role of melanin for the interaction with plants and salt stress alleviation could also be tested. P. macrospinosa and Cadophora sp. improved shoot and root growth 6 weeks after inoculation under moderate and high salt stress conditions. No matter how much salt stress was applied, macroelement (P, N, and C) contents were unaffected by DSE inoculation. The four tested DSE strains successfully colonized the roots of tomato, but the colonization level was clearly reduced in the albino mutant of Leptodontidium sp. Any difference in the effects on plant growth between the Leptodontidium sp. wild type strain and the albino mutant could, however, not be observed. These results show that particular DSEs are able to increase salt tolerance as they promote plant growth specifically under stress condition. Increased plant biomasses combined with stable nutrient contents resulted in higher P uptake in shoots of inoculated plants at moderate and high salt conditions and higher N uptake in the absence of salt stress in all inoculated plants, in P. macrospinosa-inoculated plants at moderate salt condition and in all inoculated plants except the albino mutants at high salt condition. In summary, melanin in DSEs seems to be important for the colonization process, but does not influence growth, nutrient uptake or salt tolerance of plants.

Abstract RecQ helicases—also known as the “guardians of the genome”—play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to affect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Human Bloom’s syndrome (BLM) helicase has a role in DNA repair, and BLM deficiency in humans is associated with chromosomal abnormalities. Here the authors employ solution biophysical assays to show BLM maintains a balance for disruption and stabilization of oligonucleotide-based D-loops. Interaction with the Topoisomerase IIIalpha-RMI1-RMI2 complex orients the activity toward D-loop disruption.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates many neuronal functions that are perturbed in amyotrophic lateral sclerosis (ALS) and perturbation to ER-mitochondria signaling is seen in cell and transgenic models of ALS. However, there is currently little evidence that ER-mitochondria signaling is altered in human ALS. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and “tether” regions of ER to the mitochondrial surface. The VAPB-PTPI51 tethers are now known to regulate a number of ER-mitochondria signaling functions. These include delivery of Ca2+ from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and ALS spinal cords. We show that VAPB protein levels are reduced in ALS. Proximity ligation assays were then used to quantify the VAPB-PTPIP51 interaction in spinal cord motor neurons in control and ALS cases. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in ALS. Thus, we identify a new pathogenic event in post-mortem ALS.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of fundamental physiological processes. This signaling involves close physical contacts between the two organelles that are mediated by the VAPB-PTPIP51 ″tethering” proteins. The VAPB-PTPIP51 tethers facilitate inositol 1,4,5-trisphosphate (IP3) receptor delivery of Ca2+ from ER to mitochondria. Damage to the tethers is seen in Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia with related amyotrophic lateral sclerosis (FTD/ALS). Understanding the mechanisms that regulate the VAPB‐PTPIP51 interaction thus represents an important area of research. Recent studies suggest that an FFAT motif in PTPIP51 is key to its binding to VAPB but this work relies on in vitro studies with short peptides. Cellular studies to support this notion with full-length proteins are lacking. Here we address this issue. Immunoprecipitation assays from transfected cells revealed that deletion of the PTPIP51 FFAT motif has little effect on VAPB binding. However, mutation and deletion of a nearby coiled-coil domain markedly affect this binding. Using electron microscopy, we then show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on ER-mitochondria contacts. Finally, we show that deletion of the coiled-coil domain but not the FFAT motif abrogates the effect of PTPIP51 on the IP3 receptor-mediated delivery of Ca2+ to mitochondria. Thus, the coiled-coil domain is essential for PTPIP51 ER-mitochondria signaling functions.

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen‐based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next‐generation sequencing combined with lesion‐specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one‐polymerase branch, while it is less suited to investigations of homology‐related repair processes, such as TSw. Cis‐syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion‐free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη‐PCNA interaction interface. TLS on 6–4 pyrimidine–pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin‐binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two major neurodegenerative diseases. FTD is the second most common cause of dementia and ALS is the most common form of motor neuron disease. These diseases are now known to be linked. There are no cures or effective treatments for FTD or ALS and so new targets for therapeutic intervention are required but this is hampered by the large number of physiological processes that are damaged in FTD/ALS. Many of these damaged functions are now known to be regulated by signaling between the endoplasmic reticulum (ER) and mitochondria. This signaling is mediated by “tethering” proteins that serve to recruit ER to mitochondria. One tether strongly associated with FTD/ALS involves an interaction between the ER protein VAPB and the mitochondrial protein PTPIP51. Recent studies have shown that ER-mitochondria signaling is damaged in FTD/ALS and that this involves breaking of the VAPB-PTPIP51 tethers. Correcting disrupted tethering may therefore correct many other downstream damaged features of FTD/ALS. Here, we review progress on this topic with particular emphasis on targeting of the VAPB-PTPIP51 tethers as a new drug target.

Because it is impossible to comprehensively characterize biodiversity at all levels of organization, conservation prioritization efforts need to rely on surrogates. As species distribution maps of relished groups as well as high-resolution remotely sensed data increasingly become available, both types of surrogates are commonly used. A good surrogate should represent as much of biodiversity as possible, but it often remains unclear to what extent this is the case. Here, we aimed to address this question by assessing how well bird species and habitat diversity represent one another. We conducted our study in Romania, a species-rich country with high landscape heterogeneity where bird species distribution data have only recently started to become available. First, we prioritized areas for conservation based on either 137 breeding bird species or 36 habitat classes, and then evaluated their reciprocal surrogacy performance. Second, we examined how well these features are represented in already existing protected areas. Finally, we identified target regions of high conservation value for the potential expansion of the current network of reserves (as planned under the new EU Biodiversity Strategy for 2030). We found a limited reciprocal surrogacy performance, with bird species performing slightly better as a conservation surrogate for habitat diversity than vice versa. We could also show that areas with a high conservation value based on habitat diversity were represented better in already existing protected areas than areas based on bird species, which varied considerably between species. Our results highlight that taxonomic and environmental (i.e., habitat types) data may perform rather poorly as reciprocal surrogates, and multiple sources of data are required for a full evaluation of protected areas expansion.

We aimed to test the hypothesis that treatment with an aboveground plant conditioner has an effect on important vegetable crops inoculated with arbuscular mycorrhizal fungi (AMF) and on their colonization by AMF. Potting experiments were set with pepper and tomato plants inoculated with commercial AMF inoculum and plants were treated with an aboveground plant conditioner. After harvesting, the dry weight of shoots and roots were measured, and the AMF colonization of the roots was quantified. We found a significant effect of the treatment on fungal colonization: the AMF colonization, the hyphal colonization rate and the frequency of the arbuscules in the roots of both vegetables were lower when aboveground plant conditioner was applied. Although the two species differed, no significant effect of the treatment on the growth of the plants was detected. Based on our findings we assumed that the lower AMF colonization more greatly influenced the growth of the pepper cultivar studied. We demonstrated that treatment with a commercial aboveground plant conditioner had an antagonistic effect on AMF colonization, which, in addition to many other effects, might influence the growth vegetable crops. The interaction of different practices applied simultaneously should be tested to effectively help the development suitable agriculture systems.

Abstract Damage to axonal transport is an early pathogenic event in Alzheimer’s disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer’s disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer’s disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in Drosophila neurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer’s disease.

Abstract Cyclin dependent kinase-5 (cdk5)/p35 is a neuronal kinase that regulates key axonal and synaptic functions but the mechanisms by which it is transported to these locations are unknown. Lemur tyrosine kinase-2 (LMTK2) is a binding partner for p35 and here we show that LMTK2 also interacts with kinesin-1 light chains (KLC1/2). Binding to KLC1/2 involves a C-terminal tryptophan/aspartate (WD) motif in LMTK2 and the tetratricopeptide repeat (TPR) domains in KLC1/2, and this interaction facilitates axonal transport of LMTK2. Thus, siRNA loss of KLC1 or mutation of the WD motif disrupts axonal transport of LMTK2. We also show that LMTK2 facilitates the formation of a complex containing KLC1 and p35 and that siRNA loss of LMTK2 disrupts axonal transport of both p35 and cdk5. Finally, we show that LMTK2 levels are reduced in Alzheimer’s disease brains. Damage to axonal transport and altered cdk5/p35 are pathogenic features of Alzheimer’s disease. Thus, LMTK2 binds to KLC1 to direct axonal transport of p35 and its loss may contribute to Alzheimer’s disease.

Abstract Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by “tethering proteins” that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson’s disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson’s disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS.

Papaya (Carica papaya L.) is an important fruit crop in many tropical and subtropical countries. Powdery mildew commonly affects this host, causing premature leaf loss, reduced yields and poor fruit quality. At least fifteen different fungi have been identified as the causal agents of papaya powdery mildew. Powdery mildew symptoms were detected on potted papaya plants growing in two locations in Hungary. This study aimed to identify the causal agents. Morphology of powdery mildew samples was examined, and sequences of two loci were used for molecular taxonomic identifications. Only anamophs were detected in all samples, and four morphological types were distinguished. Most samples had Pseudoidium anamorphs, while some were of the Fibroidium anamorph. Based on morphology and molecular taxonomy, the Fibroidium anamorph was identified as Podosphaera xanthii. The Pseudoidium anamorphs corresponded to three different Erysiphe species: E. cruciferarum, E. necator and an unidentified Erysiphe sp., for which molecular phylogenetic analyses showed it belonged to an unresolved species complex of E. malvae, E. heraclei and E. betae. Infectivity of P. xanthii and E. necator on papaya was verified with cross inoculations. A review of previous records of powdery mildew fungi infecting papaya is also provided. Podosphaera xanthii was known to infect, and E. cruciferarum was suspected to infect Carica papaya, while E. necator was recorded on this host only once previously. No powdery mildew fungus belonging to the E. malvae/E. heraclei/E. betae species complex is known to infect papaya or any other plants in the Caricaceae, so the unidentified Erysiphe sp. is a new record on papaya and the Caricaceae. This study indicates host range expansion of this powdery mildew fungus onto papaya.

Fungal diseases in agronomically important plants such as grapevines result in significantly reduced production, pecuniary losses, and increased use of environmentally damaging chemicals. Beside the well-known diseases, there is an increased interest in wood-colonizing fungal pathogens that infect the woody tissues of grapevines. In 2015, a traditional isolation method was performed on grapevine trunks showing symptoms of trunk diseases in Hungary. One isolate (T15142) was identified as Kalmusia longispora (formerly Dendrothyrium longisporum) according to morphological and phylogenetic analyses. To evaluate the pathogenicity of this fungus on grapevines, artificial infections were carried out under greenhouse and field conditions, including the CBS 824.84 and ex-type CBS 582.83 strains. All isolates could be re-isolated from inoculated plants; however, varying virulence was observed among them in terms of the vascular necrosis caused. The incidence and severity of this symptom seemed to be congruent with the laccase-producing capabilities of the isolates. This is the first report on the ability of Kalmusia longispora to cause symptoms on grapevines, and on its possible dependence on laccase secretion.

Dark septate endophytes (DSEs) represent a diverse group of root-endophytic fungi that have been isolated from plant roots in many different natural and anthropogenic ecosystems. Melanin is widespread in eukaryotic organisms and possesses various functions such as protecting human skin from UV radiation, affecting the virulence of pathogens, and playing a role in development and physiology of insects. Melanin is a distinctive feature of the cell walls of DSEs and has been thought to protect these fungi from abiotic stress. Melanin in DSEs is assumed to be synthesized via the 1,8-dihydroxynaphthalene (DHN) pathway. Its function in alleviation of salt stress is not yet known. The aims of this study were: (i) investigating the growth responses of three DSEs (Periconia macrospinosa, Cadophora sp., and Leptodontidium sp.) to salt stress, (ii) analyzing melanin production under salt stress and, (iii) testing the role of melanin in salt stress tolerance of DSEs. The study shows that the three DSE species can tolerate high salt concentrations. Melanin content increased in the hyphae of all DSEs at 100 mM salt, but decreased at 500 mM. This was not reflected in the RNA accumulation of the gene encoding scytalone dehydratase which is involved in melanin biosynthesis. The application of tricyclazole, a DHN-melanin biosynthesis inhibitor, did not affect either salt stress tolerance or the accumulation of sodium in the hyphae. In addition, melanin biosynthesis mutants of Leptodontidium sp. did not show decreased growth performance compared to the wild-type, especially not at high salt concentrations. This indicates that DSEs can live under salt stress and withstand these conditions regardless of melanin accumulation.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions, many of which are perturbed in Alzheimer's disease. Moreover, damage to ER-mitochondria signaling is seen in cell and transgenic models of Alzheimer's disease. However, as yet there is little evidence that ER-mitochondria signaling is altered in human Alzheimer's disease brains. ER-mitochondria signaling is mediated by interactions between the integral ER protein VAPB and the outer mitochondrial membrane protein PTPIP51 which act to recruit and “tether” regions of ER to the mitochondrial surface. The VAPB-PTPIP51 tethers are now known to regulate a number of ER-mitochondria signaling functions including delivery of Ca2+from ER stores to mitochondria, mitochondrial ATP production, autophagy and synaptic activity. Here we investigate the VAPB-PTPIP51 tethers in post-mortem control and Alzheimer's disease brains. Quantification of ER-mitochondria signaling proteins by immunoblotting revealed loss of VAPB and PTPIP51 in cortex but not cerebellum at end-stage Alzheimer's disease. Proximity ligation assays were used to quantify the VAPB-PTPIP51 interaction in temporal cortex pyramidal neurons and cerebellar Purkinje cell neurons in control, Braak stage III-IV (early/mid-dementia) and Braak stage VI (severe dementia) cases. Pyramidal neurons degenerate in Alzheimer's disease whereas Purkinje cells are less affected. These studies revealed that the VAPB-PTPIP51 tethers are disrupted in Braak stage III-IV pyramidal but not Purkinje cell neurons. Thus, we identify a new pathogenic event in post-mortem Alzheimer's disease brains. The implications of our findings for Alzheimer's disease mechanisms are discussed.

DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.

Abstract Dark septate endophytes (DSE) are a form-group of root endophytic fungi with elusive functions. Here, the genomes of two common DSE of semiarid areas, Cadophora sp. and Periconia macrospinosa were sequenced and analyzed with another 32 ascomycetes of different lifestyles. Cadophora sp. (Helotiales) and P. macrospinosa (Pleosporales) have genomes of 70.46 Mb and 54.99 Mb with 22,766 and 18,750 gene models, respectively. The majority of DSE-specific protein clusters lack functional annotation with no similarity to characterized proteins, implying that they have evolved unique genetic innovations. Both DSE possess an expanded number of carbohydrate active enzymes (CAZymes), including plant cell wall degrading enzymes (PCWDEs). Those were similar in three other DSE, and contributed a signal for the separation of root endophytes in principal component analyses of CAZymes, indicating shared genomic traits of DSE fungi. Number of secreted proteases and lipases, aquaporins, and genes linked to melanin synthesis were also relatively high in our fungi. In spite of certain similarities between our two DSE, we observed low levels of convergence in their gene family evolution. This suggests that, despite originating from the same habitat, these two fungi evolved along different evolutionary trajectories and display considerable functional differences within the endophytic lifestyle.

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.

Abstract In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.

In several terrestrial ecosystems such as grasslands, plants live together with various root-colonizing dark septate endophytes (DSEs), fungi that are relatively frequent colonizers of healthy belowground tissues of plants in these environments. They are important members of the plant microbiota and may have various effects on plant survival under different stress conditions; however, their general functions in relation to plants and the greater ecosystem remain elusive. Although an increasing number of studies has been published focusing on DSEs in Asian grasslands, our knowledge is limited. Especially in Mongolia, where the steppe region represents a significant area, information is not available on these root colonizers. In this study, we aimed to characterize DSEs of a common dominant gramineous plant species, Stipa krylovii in a semiarid grassland of Mongolia. Root samples were collected in a natural steppe and were processed for isolation of fungal endophytes. For molecular identification of the isolates, the internal transcribed spacer (ITS) region of the nrDNA was obtained for all the isolates investigated; furthermore, the partial translation elongation factor 1-α (TEF) gene and large subunit (LSU) and small subunit (SSU) of rDNA were also amplified and sequenced in case of representative isolates. In vitro tests were used to examine the rough symbiotic nature of the fungi, and root colonization was visualized. A majority of the 135 isolates examined in detail was found to belong to several orders of Ascomycota (110 isolates) and some to Basidiomycota (25 isolates). A significant number of the isolates represented presumably novel taxa, and dominant similarities of the lineages have been found with relatively frequent and known grass root endophytes of semiarid areas in other geographic regions. These endophytes included Periconia macrospinosa, Microdochium bolley, and Darksidea, the genus of which comprised one fourth of the isolates. We found numerous lineages, which have been detected not only from Asian steppe ecosystems, but also from prairies in North America and sandy grasslands in Europe. Therefore, our results strengthen the hypothesized worldwide presence of a common and dominant core group of a DSE community in arid and semiarid grasslands.

RecQ helicases promote genomic stability through their unique ability to suppress illegitimate recombination and resolve recombination intermediates. These DNA structure-specific activities of RecQ helicases are mediated by the helicase-and-RNAseD like C-terminal (HRDC) domain, via unknown mechanisms. Here, employing single-molecule magnetic tweezers and rapid kinetic approaches we establish that the HRDC domain stabilizes intrinsic, sequence-dependent, pauses of the core helicase (lacking the HRDC) in a DNA geometry-dependent manner. We elucidate the core unwinding mechanism in which the unwinding rate depends on the stability of the duplex DNA leading to transient sequence-dependent pauses. We further demonstrate a non-linear amplification of these transient pauses by the controlled binding of the HRDC domain. The resulting DNA sequence- and geometry-dependent pausing may underlie a homology sensing mechanism that allows rapid disruption of unstable (illegitimate) and stabilization of stable (legitimate) DNA strand invasions, which suggests an intrinsic mechanism of recombination quality control by RecQ helicases.

Expression of the frontotemporal dementia-related tau mutation, P301L, at physiological levels in adult mouse brain (KI-P301L mice) results in overt hypophosphorylation of tau and age-dependent alterations in axonal mitochondrial transport in peripheral nerves. To determine the effects of P301L tau expression in the central nervous system, we examined the kinetics of mitochondrial axonal transport and tau phosphorylation in primary cortical neurons from P301L knock-in (KI-P301L) mice. We observed a significant 50% reduction in the number of mitochondria in the axons of cortical neurons cultured from KI-P301L mice compared to wild-type neurons. Expression of murine P301L tau did not change the speed, direction of travel or likelihood of movement of mitochondria. Notably, the angle that defines the orientation of the mitochondria in the axon, and the volume of individual moving mitochondria, were significantly increased in neurons expressing P301L tau. We found that murine tau phosphorylation in KI-P301L mouse neurons was diminished and the ability of P301L tau to bind to microtubules was also reduced compared to tau in wild-type neurons. The P301L mutation did not influence the ability of murine tau to associate with membranes in cortical neurons or in adult mouse brain. We conclude that P301L tau is associated with mitochondrial changes and causes an early reduction in murine tau phosphorylation in neurons coupled with impaired microtubule binding of tau. These results support the association of mutant tau with detrimental effects on mitochondria and will be of significance for the pathogenesis of tauopathies.

Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.

Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.

The objective of this study was to apply high resolution melting analysis in the detection of complex vertebral malformation (CVM) carriers in Hosltein cattle. A total of 47 animals of Holstein cattle were included in this study. Genomic DNA was extracted from blood, hair follicles and sperm by commercial QIAamp® DNA Mini kit. The amplification and high resolution melting analysis (HRMA) was done by commercial SensiMixTM HRM kit. The confirmation of sensitivity of this method was done by PCR-PIRA method and sequencing. Four samples of heterozygous genotype GT for causal mutation in the bovine solute carrier family 35 member 3 gene (SLC35A3), which is responsible for CVM disease, were detected. Our results demostrated that the use of HRMA for genotyping of mutant allele T for SLC35A3 gene in Holstein cattle is an effective method for the selection of carriers of CVM disease.

The motor domain of myosin is the core element performing mechanochemical energy transduction. This domain contains the actin and ATP binding sites and the base of the force-transducing lever. Coordinated subdomain movements within the motor are essential in linking the ATPase chemical cycle to translocation along actin filaments. A dynamic subdomain interface located at the base of the lever was previously shown to exert an allosteric influence on ATP hydrolysis in the non-processive myosin 2 motor. By solution kinetic, spectroscopic and ensemble and single-molecule motility experiments, we determined the role of a class-specific adaptation of this interface in the mechanochemical mechanism of myosin 5a, a processive intracellular transporter. We found that the introduction of a myosin 2-specific repulsive interaction into myosin 5a via the I67K mutation perturbs the strong-binding interaction of myosin 5a with actin, influences the mechanism of ATP binding and facilitates ATP hydrolysis. At the same time, the mutation abolishes the actin-induced activation of ADP release and, in turn, slows down processive motility, especially when myosin experiences mechanical drag exerted by the action of multiple motor molecules bound to the same actin filament. The results highlight that subtle structural adaptations of the common structural scaffold of the myosin motor enable specific allosteric tuning of motor activity shaped by widely differing physiological demands.

Dark septate endophytic (DSE) fungi represent a frequent root-colonizing fungal group common in environments with strong abiotic stress, such as (semi)arid ecosystems. This work aimed to study the DSE fungi colonizing the plants of semiarid sandy grasslands with wood steppe patches on the Great Hungarian Plain. As we may assume that fungi colonizing both invasive and native species are generalists, root associated fungi (RAF) were isolated from eight native and three invasive plant species. The nrDNA sequences of the isolates were used for identification. To confirm that the fungi were endophytes an artificial inoculation system was used to test the isolates: we considered a fungus as DSE if it colonized the roots without causing a negative effect on the plant and formed microsclerotia in the roots. According to the analyses of the ITS sequence of nrDNA the 296 isolates clustered into 41 groups. We found that 14 of these 41 groups were DSE, representing approximately 60% of the isolates. The main DSE groups were generalist and showed no specificity to area or season and colonized both native and invasive species, demonstrating that exotic plants are capable of using the root endophytic fungi of the invaded areas. The DSE community of the region shows high similarity to those found in arid grasslands of North America. Taking into account a previous hypothesis about the common root colonizers of those grasslands and our results reported here, we hypothesize that plants of (semi)arid grasslands share common dominant members of the DSE fungal community on a global scale.

Rapidly evolving, indel-rich phylogenetic markers play a pivotal role in our understanding of the relationships at multiple levels of the tree of life. There is extensive evidence that indels provide conserved phylogenetic signal, however, the range of phylogenetic depths for which gaps retain tree signal has not been investigated in detail. Here we address this question using the fungal internal transcribed spacer (ITS), which is central in many phylogenetic studies, molecular ecology, detection and identification of pathogenic and non-pathogenic species. ITS is repeatedly criticized for indel-induced alignment problems and the lack of phylogenetic resolution above species level, although these have not been critically investigated. In this study, we examined whether the inclusion of gap characters in the analyses shifts the phylogenetic utility of ITS alignments towards earlier divergences. By re-analyzing 115 published fungal ITS alignments, we found that indels are slightly more conserved than nucleotide substitutions, and when included in phylogenetic analyses, improved the resolution and branch support of phylogenies across an array of taxonomic ranges and extended the resolving power of ITS towards earlier nodes of phylogenetic trees. Our results reconcile previous contradicting evidence for the effects of data exclusion: in the case of more sophisticated indel placement, the exclusion of indel-rich regions from the analyses results in a loss of tree resolution, whereas in the case of simpler alignment methods, the exclusion of gapped sites improves it. Although the empirical datasets do not provide to measure alignment accuracy objectively, our results for the ITS region are consistent with previous simulations studies alignment algorithms. We suggest that sophisticated alignment algorithms and the inclusion of indels make the ITS region and potentially other rapidly evolving indel-rich loci valuable sources of phylogenetic information, which can be exploited at multiple taxonomic levels.