Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart.

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.

Ribosomes are macromolecular machines responsible for protein synthesis in all living beings. Recent studies have shown that ribosomes can be heterogeneous in their structure, possibly leading to a specialized function. Here, we focus on RPL3L, a ribosomal protein expressed exclusively in striated muscles. We find that the deletion of the Rpl3l gene in a mouse model triggers a compensation mechanism, in which the missing RPL3L protein is replaced by its paralogue, RPL3. Furthermore, we find that RPL3-containing ribosomes establish closer interactions with mitochondria, cellular organelles responsible for energy production, leading to higher energy production when compared with RPL3L-containing ribosomes. Finally, we show that the RPL3­RPL3L compensation mechanism is also triggered in heart disease conditions, such as hypertrophy and myocardial infarction.

Efficacy of an AAV vector encoding a thermostable form of glucocerebrosidase in alleviating symptoms in a Gaucher disease mouse model.

Almost all attempts to date at gene therapy approaches for monogenetic disease have used the amino acid sequences of the natural protein. In the current study, we use a designed, thermostable form of glucocerebrosidase (GCase), the enzyme defective in Gaucher disease (GD), to attempt to alleviate neurological symptoms in a GD mouse that models type 3 disease, i.e. the chronic neuronopathic juvenile subtype. Upon injection of an AAVrh10 (adeno-associated virus, serotype rh10) vector containing the designed GCase (dGCase) into the left lateral ventricle of Gba-/-;Gbatg mice, a significant improvement in body weight and life-span was observed, compared to injection of the same mouse with the wild type enzyme (wtGCase). Moreover, a reduction in levels of glucosylceramide (GlcCer), and an increase in levels of GCase activity were seen in the right hemisphere of Gba-/-;Gbatg mice, concomitantly with a significant improvement in motor function, reduction of neuroinflammation and a reduction in mRNA levels of various genes shown previously to be elevated in the brain of mouse models of neurological forms of GD. Together, these data pave the way for the possible use of modified proteins in gene therapy for lysosomal storage diseases and other monogenetic disorders.

Proteomics analysis of the brain from a Gaucher disease mouse identifies pathological pathways including a possible role for transglutaminase 1.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid ß-glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra-peritoneal injection of conduritol B-epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non-targeted, mass spectrometry-based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE-injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up-regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE-injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology.

Analysis of inflammatory markers and tau deposits in an autopsy series of nine patients with anti-IgLON5 disease.

Anti-IgLON5 disease is a rare neurological, probably autoimmune, disorder associated in many cases with a specific tauopathy. Only a few post-mortem neuropathological studies have been reported so far. Little is known about the pathogenic mechanisms that result in neurodegeneration. We investigated the neuropathology of anti-IgLON5 disease and characterized cellular and humoral inflammation. We included nine cases (six of them previously published). Median age of patients was 71 years (53-82 years), the median disease duration was 6 years (0.5-13 years), and the female to male ratio was 5:4. Six cases with a median disease duration of 9 years presented a prominent tauopathy. Five of them had a classical anti-IgLON5-related brainstem tauopathy and another presented a prominent neuronal and glial 4-repeat tauopathy, consistent with progressive supranuclear palsy (PSP). Three cases with short disease duration (median 1.25 years) only showed a primary age-related neurofibrillary pathology. Inflammatory infiltrates of T and B cells were mild to moderate and did not significantly differ between anti-IgLON5 disease cases with or without tauopathy. In contrast, we found an extensive neuropil deposition of IgG4 in the tegmentum of the brainstem, olivary nucleus, and cerebellar cortex that was most prominent in two patients with short disease duration without the typical IgLON5-related tauopathy. The IgG4 deposits were particularly prominent in the cerebellar cortex and in these regions accompanied by mild IgG1 deposits. Activated complement deposition (C9neo) was absent. Our study indicates that IgLON5-related tau pathology occurs in later disease stages and may also present a PSP-phenotype with exclusively 4-repeat neuronal and glial tau pathology. The prominent deposition of anti-IgLON5 IgG4 at predilection sites for tau pathology suggests that anti-IgLON5 antibodies precede the tau pathology. Early start of immunotherapy might prevent irreversible neuronal damage and progression of the disease, at least in a subgroup of patients.

Pulse-synchronous torsional nystagmus.

Purely torsional spontaneous nystagmus almost always has a central vestibular cause. We describe a man with spontaneous pulse-synchronous torsional nystagmus in which the clockwise component corresponded to his pulse upswing, in keeping with a peripheral vestibular cause; following imaging we diagnosed left-sided superior canal dehiscence syndrome. Identifying pulse synchronicity of spontaneous nystagmus may help to distinguish central from peripheral vestibular torsional nystagmus, and is readily confirmed at the bedside using Frenzel's glasses and a pulse oximeter.

Integrated Proteomic and Metabolomic Profiling of Phytophthora cinnamomi Attack on Sweet Chestnut (Castanea sativa) Reveals Distinct Molecular Reprogramming Proximal to the Infection Site and Away from It.

Phytophthora cinnamomi is one of the most invasive tree pathogens that devastates wild and cultivated forests. Due to its wide host range, knowledge of the infection process at the molecular level is lacking for most of its tree hosts. To expand the repertoire of studied Phytophthora-woody plant interactions and identify molecular mechanisms that can facilitate discovery of novel ways to control its spread and damaging effects, we focused on the interaction between P. cinnamomi and sweet chestnut (Castanea sativa), an economically important tree for the wood processing industry. By using a combination of proteomics, metabolomics, and targeted hormonal analysis, we mapped the effects of P. cinnamomi attack on stem tissues immediately bordering the infection site and away from it. P. cinnamomi led to a massive reprogramming of the chestnut proteome and accumulation of the stress-related hormones salicylic acid (SA) and jasmonic acid (JA), indicating that stem inoculation can be used as an easily accessible model system to identify novel molecular players in P. cinnamomi pathogenicity.

Hippocampal Radial Glial Subtypes and Their Neurogenic Potential in Human Fetuses and Healthy and Alzheimer's Disease Adults.

Neuropathological conditions might affect adult granulogenesis in the adult human dentate gyrus. However, radial glial cells (RGCs) have not been well characterized during human development and aging. We have previously described progenitor and neuronal layer establishment in the hippocampal pyramidal layer and dentate gyrus from embryonic life until mid-gestation. Here, we describe RGC subtypes in the hippocampus from 13 gestational weeks (GW) to mid-gestation and characterize their evolution and the dynamics of neurogenesis from mid-gestation to adulthood in normal and Alzheimer's disease (AD) subjects. In the pyramidal ventricular zone (VZ), RGC density declined with neurogenesis from mid-gestation until the perinatal period. In the dentate area, morphologic and antigenic differences among RGCs were observed from early ages of development to adulthood. Density and proliferative capacity of dentate RGCs as well as neurogenesis were strongly reduced during childhood until 5 years, few DCX+ cells are seen in adults. The dentate gyrus of both control and AD individuals showed Nestin+ and/or GFAPδ+ cells displaying different morphologies. In conclusion, pools of morphologically, antigenically, and topographically diverse neural progenitor cells are present in the human hippocampus from early developmental stages until adulthood, including in AD patients, while their neurogenic potential seems negligible in the adult.

The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.

Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.

A novel nonsense autosomal dominant mutation in the GLRA1 gene causing hyperekplexia.

We present a family with two members affected by hyperekplexia and two unaffected members. All exons in the glycine receptor alpha 1 subunit gene (GLRA1) were sequenced in all four family members. Our index patient harbored a novel nonsense mutation (p.Trp314*; rs867618642) in the transmembrane domain three of the GLRA1 and a novel missense variant in the NH2-terminal part (p.Val67Met; rs142888296). After development of tolerance for the effective treatment with clobazam a drug holiday led to a sustained restoration of the treatment response.

Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP.

KEY POINTS: Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell-specific and tonotopically-determined manner. Developmental down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high-to-low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP-AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors. ABSTRACT: Synaptic refinement and strengthening are activity-dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell-specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high-frequency regions, ATP responsiveness diminished before hearing onset. In low-to-mid frequency regions, ATP modulation persisted after hearing onset in a subset of low-frequency bushy cells (characteristic frequency< 10 kHz). Down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high-to-low frequency maturation pattern. Facilitated AP generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R.

Validation of dopamine receptor DRD1 and DRD2 antibodies using receptor deficient mice.

Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.

Aging-related tau astrogliopathy (ARTAG): harmonized evaluation strategy.

Pathological accumulation of abnormally phosphorylated tau protein in astrocytes is a frequent, but poorly characterized feature of the aging brain. Its etiology is uncertain, but its presence is sufficiently ubiquitous to merit further characterization and classification, which may stimulate clinicopathological studies and research into its pathobiology. This paper aims to harmonize evaluation and nomenclature of aging-related tau astrogliopathy (ARTAG), a term that refers to a morphological spectrum of astroglial pathology detected by tau immunohistochemistry, especially with phosphorylation-dependent and 4R isoform-specific antibodies. ARTAG occurs mainly, but not exclusively, in individuals over 60 years of age. Tau-immunoreactive astrocytes in ARTAG include thorn-shaped astrocytes at the glia limitans and in white matter, as well as solitary or clustered astrocytes with perinuclear cytoplasmic tau immunoreactivity that extends into the astroglial processes as fine fibrillar or granular immunopositivity, typically in gray matter. Various forms of ARTAG may coexist in the same brain and might reflect different pathogenic processes. Based on morphology and anatomical distribution, ARTAG can be distinguished from primary tauopathies, but may be concurrent with primary tauopathies or other disorders. We recommend four steps for evaluation of ARTAG: (1) identification of five types based on the location of either morphologies of tau astrogliopathy: subpial, subependymal, perivascular, white matter, gray matter; (2) documentation of the regional involvement: medial temporal lobe, lobar (frontal, parietal, occipital, lateral temporal), subcortical, brainstem; (3) documentation of the severity of tau astrogliopathy; and (4) description of subregional involvement. Some types of ARTAG may underlie neurological symptoms; however, the clinical significance of ARTAG is currently uncertain and awaits further studies. The goal of this proposal is to raise awareness of astroglial tau pathology in the aged brain, facilitating communication among neuropathologists and researchers, and informing interpretation of clinical biomarkers and imaging studies that focus on tau-related indicators.

Dynamic fidelity control to the central auditory system: synergistic glycine/GABAergic inhibition in the cochlear nucleus.

GABA and glycine are the major inhibitory transmitters that attune neuronal activity in the CNS of mammals. The respective transmitters are mostly spatially separated, that is, synaptic inhibition in the forebrain areas is mediated by GABA, whereas glycine is predominantly used in the brainstem. Accordingly, inhibition in auditory brainstem circuits is largely mediated by glycine, but there are few auditory synapses using both transmitters in maturity. Little is known about physiological advantages of such a two-transmitter inhibitory mechanism. We explored the benefit of engaging both glycine and GABA with inhibition at the endbulb of Held-spherical bushy cell synapse in the auditory brainstem of juvenile Mongolian gerbils. This model synapse enables selective in vivo activation of excitatory and inhibitory neuronal inputs through systemic sound stimulation and precise analysis of the input (endbulb of Held) output (spherical bushy cell) function. The combination of in vivo and slice electrophysiology revealed that the dynamic AP inhibition in spherical bushy cells closely matches the inhibitory conductance profile determined by the glycine-R and GABAA-R. The slow and potent glycinergic component dominates the inhibitory conductance, thereby primarily accounting for its high-pass filter properties. GABAergic transmission enhances the inhibitory strength and shapes its duration in an activity-dependent manner, thus increasing the inhibitory potency to suppress the excitation through the endbulb of Held. Finally, in silico modeling provides a strong link between in vivo and slice data by simulating the interactions between the endbulb- and the synergistic glycine-GABA-conductances during in vivo-like spontaneous and sound evoked activities.

Patterns of hippocampal tau pathology differentiate neurodegenerative dementias.

BACKGROUND/AIMS: Deposits of phosphorylated tau protein and convergence of pathology in the hippocampus are the hallmarks of neurodegenerative tauopathies. Thus we aimed to evaluate whether regional and cellular vulnerability patterns in the hippocampus distinguish tauopathies or are influenced by their concomitant presence. METHODS: We created a heat map of phospho-tau (AT8) immunoreactivity patterns in 24 hippocampal subregions/layers in individuals with Alzheimer's disease (AD)-related neurofibrillary degeneration (n = 40), Pick's disease (n = 8), progressive supranuclear palsy (n = 7), corticobasal degeneration (n = 6), argyrophilic grain disease (AGD, n = 18), globular glial tauopathy (n = 5), and tau-astrogliopathy of the elderly (n = 10). AT8 immunoreactivity patterns were compared by mathematical analysis. RESULTS: Our study reveals disease-specific hot spots and regional selective vulnerability for these disorders. The pattern of hippocampal AD-related tau pathology is strongly influenced by concomitant AGD. Mathematical analysis reveals that hippocampal involvement in primary tauopathies is distinguishable from early-stage AD-related neurofibrillary degeneration. CONCLUSION: Our data demonstrate disease-specific AT8 immunoreactivity patterns and hot spots in the hippocampus even in tauopathies, which primarily do not affect the hippocampus. These hot spots can be shifted to other regions by the co-occurrence of tauopathies like AGD. Our observations support the notion that globular glial tauopathies and tau-astrogliopathy of the elderly are distinct entities.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

Climate acts as an environmental filter to plant pathogens.

Climate shapes the distribution of plant-associated microbes such as mycorrhizal and endophytic fungi. However, the role of climate in plant pathogen community assembly is less understood. Here, we explored the role of climate in the assembly of Phytophthora communities at >250 sites along a latitudinal gradient from Spain to northern Sweden and an altitudinal gradient from the Spanish Pyrenees to lowland areas. Communities were detected by ITS sequencing of river filtrates. Mediation analysis supported the role of climate in the biogeography of Phytophthora and ruled out other environmental factors such as geography or tree diversity. Comparisons of functional and species diversity showed that environmental filtering dominated over competitive exclusion in Europe. Temperature and precipitation acted as environmental filters at different extremes of the gradients. In northern regions, winter temperatures acted as an environmental filter on Phytophthora community assembly, selecting species adapted to survive low minimum temperatures. In southern latitudes, a hot dry climate was the main environmental filter, resulting in communities dominated by drought-tolerant Phytophthora species with thick oospore walls, a high optimum temperature for growth, and a high maximum temperature limit for growth. By taking a community ecology approach, we show that the establishment of Phytophthora plant pathogens in Europe is mainly restricted by cold temperatures.

Large-scale proteomics analysis of five brain regions from Parkinson's disease patients with a GBA1 mutation.

Despite being the second most common neurodegenerative disorder, little is known about Parkinson's disease (PD) pathogenesis. A number of genetic factors predispose towards PD, among them mutations in GBA1, which encodes the lysosomal enzyme acid-ß-glucosidase. We now perform non-targeted, mass spectrometry based quantitative proteomics on five brain regions from PD patients with a GBA1 mutation (PD-GBA) and compare to age- and sex-matched idiopathic PD patients (IPD) and controls. Two proteins were differentially-expressed in all five brain regions whereas significant differences were detected between the brain regions, with changes consistent with loss of dopaminergic signaling in the substantia nigra, and activation of a number of pathways in the cingulate gyrus, including ceramide synthesis. Mitochondrial oxidative phosphorylation was inactivated in PD samples in most brain regions and to a larger extent in PD-GBA. This study provides a comprehensive large-scale proteomics dataset for the study of PD-GBA.

Encapsulated Thuja plicata essential oil into biopolymer matrix as a potential pesticide against Phytophthora root pathogens.

A new formulation that gradually released encapsulated Thuja plicata essential oil (TPEO) as an active component from a biopolymer matrix within a given period was obtained. Antimicrobial activity was determined in in-vitro tests where pure TPEO successfully inhibited the development of different Phytophthora species. The TPEO essential oil was encapsulated into the biopolymer matrix and an oil-in-water emulsion was formed. FTIR spectra analysis confirmed the formation of electrostatic interaction between these polymers, and hydrogen interactions between active components of TPEO and polymer chains. The stability of the emulsions was confirmed by zeta potential measurements, with a value of about 30 mV, even after 14 days of aging. UV-Vis spectra analysis revealed that >60 % of TPEO remained in the emulsion after 14 days of exposure to ambient conditions, whereas pure TPEO evaporated faster, and around 20 % remained after 6 days. Encapsulated TPEO almost completely inhibited the growth of Phytophthora species during the ten-day day's exposition being statistically significantly improved compared to fungicide treatment. It was demonstrated that the emulsion exhibited a prolonged antimicrobial effect and successfully suppressed the growth of Phytophthora species, and can be considered as a means of protection in forests and crops.

Next-generation sequencing and comprehensive data reassessment in 263 adult patients with neuromuscular disorders: insights into the gray zone of molecular diagnoses.

BACKGROUND: Neuromuscular disorders (NMDs) are heterogeneous conditions with a considerable fraction attributed to monogenic defects. Despite the advancements in genomic medicine, many patients remain without a diagnosis. Here, we investigate whether a comprehensive reassessment strategy improves the diagnostic outcomes. METHODS: We analyzed 263 patients with NMD phenotypes that underwent diagnostic exome or genome sequencing at our tertiary referral center between 2015 and 2023. We applied a comprehensive reassessment encompassing variant reclassification, re-phenotyping and NGS data reanalysis. Multivariable logistic regression was performed to identify predictive factors associated with a molecular diagnosis. RESULTS: Initially, a molecular diagnosis was identified in 53 cases (20%), while an additional 23 (9%) had findings of uncertain significance. Following comprehensive reassessment, the diagnostic yield increased to 23%, revealing 44 distinct monogenic etiologies. Reasons for newly obtained molecular diagnoses were variant reclassifications in 7 and NGS data reanalysis in 3 cases including one recently described disease-gene association (DNAJB4). Male sex reduced the odds of receiving a molecular diagnosis (OR 0.42; 95%CI 0.21-0.82), while a positive family history (OR 5.46; 95%CI 2.60-11.76) and a myopathy phenotype (OR 2.72; 95%CI 1.11-7.14) increased the likelihood. 7% were resolved through targeted genetic testing or classified as acquired etiologies. CONCLUSION: Our findings reinforce the use of NGS in NMDs of suspected monogenic origin. We show that a comprehensive reassessment enhances diagnostic accuracy. However, one needs to be aware that genetic diagnoses are often made with uncertainty and can even be downgraded based on new evidence.