Nusinersen in adults with type 3 spinal muscular atrophy: long-term outcomes on motor and respiratory function.

INTRODUCTION: Nusinersen was approved for 5q spinal muscular atrophy (SMA), irrespective of age, SMA type or functional status. Nonetheless, long-term data on adults with milder phenotypes are scarce. We aimed to characterize evolution on motor and respiratory function in our cohort of adults with type 3 SMA. METHODS: We conducted a longitudinal retrospective single-center study, including adults (≥18 years) with type 3 SMA under nusinersen for > 22 months. We reported on motor scores and spirometry parameters. RESULTS: Ten patients were included, with a median follow-up of 34 months (range = 22-46). Four patients (40%) were walkers. None used non-invasive ventilation. In Revised Upper Limb Module (RULM) and Expanded Hammersmith Functional Motor Scale (HFMSE), difference of medians increased at 6, 22 and 46 months comparing to baseline (-0.5 vs. + 1.5 vs. + 2.5 in RULM; + 4.0 vs. + 7.5 vs. + 6.0 in HFMSE). Two (50%) walkers presented a clinically meaningful improvement in 6-min walk distance. We did not report any clinically meaningful decrement in motor scores. Spirometry parameters showed an increasing difference of medians in maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) (-3 vs. + 13.4 vs. + 28.7 percentage points of predicted value for MIP; + 11.8 vs. + 13.1 vs. 13.3 percentage points of predicted value for MEP). DISCUSSION: Our cohort supports a sustained benefit of nusinersen in adults with type 3 SMA, in motor and respiratory function. Multicentric studies are still warranted.

Diaphragm weakness in late-onset Pompe disease: A complex interplay between lower motor neuron and muscle fibre degeneration.

BACKGROUND: Late-onset Pompe disease (LOPD) patients may still need ventilation support at some point of their disease course, despite regular recombinant human alglucosidase alfa treatment. This suggest that other pathophysiological mechanisms than muscle fibre lesion can contribute to the respiratory failure process. We investigate through neurophysiology whether spinal phrenic motor neuron dysfunction could contribute to diaphragm weakness in LOPD patients. MATERIAL AND METHODS: A group of symptomatic LOPD patients were prospectively studied in our centre from January 2022 to April 2023. We collected both demographic and clinical data, as well as neurophysiological parameters. Phrenic nerve conduction studies and needle EMG sampling of the diaphragm were perfomed. RESULTS: Eight treated LOPD patients (3 males, 37.5%) were investigated. Three patients (37.5%) with no respiratory involvement had normal phrenic nerve motor responses [median phrenic compound muscle action potential (CMAP) amplitude of 0.49 mV; 1st-3rd interquartile range (IQR), 0.48-0.65]. Those with respiratory failure (under nocturnal non-invasive ventilation) had abnormal phrenic nerve motor responses (median phrenic CMAP amplitude of 0 mV; 1st-3rd IQR, 0-0.15), and were then investigated with EMG. Diaphragm needle EMG revealed both myopathic and neurogenic changes in 3 (60%) and myopathic potentials in 1 patient. In the last one, no motor unit potentials could be recruited. CONCLUSIONS: Our study provide new insights regarding respiratory mechanisms in LOPD, suggesting a contribution of spinal phrenic motor neuron dysfunction for diaphragm weakness. If confirmed in further studies, our results recommend the need of new drugs crossing the blood-brain barrier.

Multisystemic RFC1-Related Disorder: Expanding the Phenotype Beyond Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome.

Background and Objectives: The RFC1 spectrum has become considerably expanded as multisystemic features beyond the triad of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) have started to be unveiled, although many still require clinical replication. Here, we aimed to clinically characterize a cohort of RFC1-positive patients by addressing both classic and multisystemic features. In a second part of this study, we prospectively assessed small nerve fibers (SNF) and autonomic function in a subset of these RFC1-related patients. Methods: We retrospectively enrolled 67 RFC1-positive patients from multiple neurologic centers in Portugal. All patients underwent full neurologic and vestibular evaluation, as well as neuroimaging and neurophysiologic studies. For SNF and autonomic testing (n = 15), we performed skin biopsies, quantitative sensory testing, sudoscan, sympathetic skin response, heart rate deep breathing, and tilt test. Results: Multisystemic features beyond CANVAS were present in 82% of the patients, mainly chronic cough (66%) and dysautonomia (43%). Other features included motor neuron (MN) affection and motor neuropathy (18%), hyperkinetic movement disorders (16%), sleep apnea (6%), REM and non-REM sleep disorders (5%), and cranial neuropathy (5%). Ten patients reported an inverse association between cough and ataxia severity. A very severe epidermal denervation was found in skin biopsies of all patients. Autonomic dysfunction comprised cardiovascular (67%), cardiovagal (54%), and/or sudomotor (50%) systems. Discussion: The presence of MN involvement, motor neuropathy, small fiber neuropathy, or extrapyramidal signs should not preclude RFC1 testing in cases of sensory neuronopathy. Indeed, the RFC1 spectrum can overlap not only with multiple system atrophy but also with hereditary motor and sensory neuropathy, hereditary sensory and autonomic neuropathy, and feeding dystonia phenotypes. Some clinical-paraclinical dissociations can pose diagnostic challenges, namely large and small fiber neuropathy and sudomotor dysfunction which are usually subclinical.

Dynamic Regulation of Grapevine's microRNAs in Response to Mycorrhizal Symbiosis and High Temperature.

MicroRNAs (miRNAs) are non-coding small RNAs that play crucial roles in plant development and stress responses and can regulate plant interactions with beneficial soil microorganisms such as arbuscular mycorrhizal fungi (AMF). To determine if root inoculation with distinct AMF species affected miRNA expression in grapevines subjected to high temperatures, RNA-seq was conducted in leaves of grapevines inoculated with either Rhizoglomus irregulare or Funneliformis mosseae and exposed to a high-temperature treatment (HTT) of 40 °C for 4 h per day for one week. Our results showed that mycorrhizal inoculation resulted in a better plant physiological response to HTT. Amongst the 195 identified miRNAs, 83 were considered isomiRs, suggesting that isomiRs can be biologically functional in plants. The number of differentially expressed miRNAs between temperatures was higher in mycorrhizal (28) than in non-inoculated plants (17). Several miR396 family members, which target homeobox-leucine zipper proteins, were only upregulated by HTT in mycorrhizal plants. Predicted targets of HTT-induced miRNAs in mycorrhizal plants queried to STRING DB formed networks for Cox complex, and growth and stress-related transcription factors such as SQUAMOSA promoter-binding-like-proteins, homeobox-leucine zipper proteins and auxin receptors. A further cluster related to DNA polymerase was found in R. irregulare inoculated plants. The results presented herein provide new insights into miRNA regulation in mycorrhizal grapevines under heat stress and can be the basis for functional studies of plant-AMF-stress interactions.

Updated Evaluation of the Safety, Efficacy and Tolerability of Tafamidis in the Treatment of Hereditary Transthyretin Amyloid Polyneuropathy.

Hereditary amyloid transthyretin (ATTRv) amyloidosis is a devastating hereditary multisystemic disease affecting predominantly the peripheral and autonomic nervous systems and the heart. ATTRv is caused by mutations in the transthyretin (TTR) gene, leading to extracellular deposition of amyloid fibrils in multiple organs including the peripheral nervous system. If untreated, it is associated with a fatal outcome 10-12 years after disease onset. Different treatments are available for patients with ATTRv polyneuropathy. Tafamidis 20 mg is approved in Europe since 2011 for early stages of ATTRv polyneuropathy (stage I - able to walk without support) and it is recommended as first-line therapy in these patients. Tafamidis is a TTR stabilizer that selectively binds to TTR and kinetically stabilizes both wild-type native TTR and mutant TTR. Consequently, it has the potential to prevent the amyloidogenic cascade initiated by TTR tetramer dissociation into its monomers and subsequent misfolding and aggregation. Tafamidis is an oral drug, taken once per day, with proved efficacy, safety and tolerability in ATTRv-PN patients as demonstrated in different clinical trials and open-label extension studies as well in clinical practice setting with around 10 years of experience. Tafamidis treatment started in the earliest stages of the disease is associated with better neurological outcomes. A multidisciplinary approach in referral centres is also fundamental for monitoring patients to assess individual response to treatment.

A TaqMan® Assay Allows an Accurate Detection and Quantification of Fusarium spp., the Causal Agents of Tomato Wilt and Rot Diseases.

In tomato plants, Fusarium spp. have been increasingly associated with several wilt and rot diseases that are responsible for severe yield losses. Here, we present a real-time PCR TaqMan® MGB (Minor Groove Binder) assay to detect and discriminate Fusarium spp. from other fungal species that affect tomato plants. The methodology used is based on the selective amplification of the internal transcribed spacer (ITS) region of Fusarium spp. This assay revealed to be highly specific and sensitive for Fusarium species, targeting only the 29 Fusarium isolates from the 45 tested isolates associated to tomato diseases. Sensitivity was assessed with serial dilutions of Fusarium genomic DNA, with the limit of detection of 3.05 pg. An absolute DNA quantification method was also established, based on the determination of the absolute number of target copies. Finally, the effectiveness of the assay was successfully validated with the detection and quantification of Fusarium spp. in potentially infected tomato plants from an experimental field and in control plants grown under controlled conditions. The established methodology allows a reliable, sensitive, and reproducible estimation of Fusarium accumulation in infected tomato plants, gaining new insights for disease control and providing an additional tool in the screening of resistant plants.

TaqMan Probes for Plant Species Identification and Quantification in Food and Feed Traceability.

In the last few years, the traceability and labeling of processed food and feeds have gained increasing importance due to the impact that mislabeling and product fraud may have on human/animal health or on the quality of final products, such as milk, cheese, and meat, as a consequence of animal dietary. The presence of contaminants or possible frauds due to the use of alternative plant materials in food and feeds can greatly impact the economy; therefore, they are becoming important targets for product certification by competent institutional services. This is especially relevant when complex matrixes are considered, in which the visual identification of the different components is quite difficult or even impossible. Despite the existence of mandatory traceability requirements for the analysis of feed/food composition addressed by European Community regulations, the labels do not always provide a sufficient guarantee about the ingredients and additive composition of those products. In this sense, the development of new methodologies that aim to assess the traceability of feed and food complex matrixes is crucial. In this chapter, a general protocol is presented for the establishment of quantitative real-time PCR-based techniques based on TaqMan assays applied to feed/food traceability, with a special focus on applications in the areas of food and feed security (e.g., for the detection of plant species involved in allergenic reactions), fraud detection (e.g., genetically modified organisms), and certification (e.g., protected denomination of origin).

Understanding the Role of PIN Auxin Carrier Genes under Biotic and Abiotic Stresses in Olea europaea L.

The PIN-FORMED (PIN) proteins represent the most important polar auxin transporters in plants. Here, we characterized the PIN gene family in two olive genotypes, the Olea europaea subsp. europaea var. sylvestris and the var. europaea (cv. 'Farga'). Twelve and 17 PIN genes were identified for vars. sylvestris and europaea, respectively, being distributed across 6 subfamilies. Genes encoding canonical OePINs consist of six exons, while genes encoding non-canonical OePINs are composed of five exons, with implications at protein specificities and functionality. A copia-LTR retrotransposon located in intron 4 of OePIN2b of var. europaea and the exaptation of partial sequences of that element as exons of the OePIN2b of var. sylvestris reveals such kind of event as a driving force in the olive PIN evolution. RNA-seq data showed that members from the subfamilies 1, 2, and 3 responded to abiotic and biotic stress factors. Co-expression of OePINs with genes involved in stress signaling and oxidative stress homeostasis were identified. This study highlights the importance of PIN genes on stress responses, contributing for a holistic understanding of the role of auxins in plants.

Psychosis treatment using clozapine in velocardiofacial syndrome

Introducción: El síndrome velocardiovacial (velocardiofacial syndrome - VCFS) es una entidad clínica heterogénea causada, en su mayoría, por una microdeleción en el cromosoma 22. Las principales características clínicas de este síndrome son: cambios en la morfología facial y del paladar, anomalías cardíacas congénitas, hipoplasia del timo, inmunodeficiencias con mayor riesgo de infección, hipoparatiroidismo, retraso mental y trastornos psiquiátricos. En la mayoría de los casos, la patología mental comórbida suele estar dentro del rango de los trastornos psicóticos.Métodos: Presentamos el caso clinico de una paciente de 34 años diagnosticada con SVCF con patologia cardíaca (comunicación interventricular con cierre espontáneo desde la infancia) y psicosis refractaria a varios antipsicóticos. Por tratarse de una paciente con mala respuesta al uso de varios antipsicóticos, inició tratamiento con clozapina. Teniendo en consideración los posibles efectos adversos del fármaco sobre la comorbilidad de VCFS, se realizó una revisión bibliográfica no sistemática en Pubmed sobre el tratamiento de la psicosis en VCFS con clozapina, con las palabras clave: síndrome velocardiofacial; clozapina.Resultados: No hay muchos estudios sobre el uso de psicofármacos en el tratamiento de la comorbilidad psiquiátrica en pacientes con VCFS. El tratamiento con clozapina en pacientes con VCFS debe considerarse teniendo en cuenta las particularidades de cada caso, sobre todo en casos con una mayor propensión a la psicosis refractaria, y, sobre todo, evaluando los riesgos y beneficios de su administración, siempre teniendo en cuenta que existe una mayor vulnerabilidad a los efectos adversos en la administración de fármacos en esta población.Discusión: En base al presente trabajo, se concluyó que el tratamiento de la psicosis en pacientes con VCFS sigue siendo un gran desafío dadas las vulnerabilidades clínicas de esta población. (AU)

Olive Fruit Fly Symbiont Population: Impact of Metamorphosis.

The current symbiotic view of the organisms also calls for new approaches in the way we perceive and manage our pest species. The olive fruit fly, the most important olive tree pest, is dependent on an obligate bacterial symbiont to its larvae development in the immature fruit. This symbiont, Candidatus (Ca.) Erwinia dacicola, is prevalent throughout the host life stages, and we have shown significant changes in its numbers due to olive fruit fly metamorphosis. The olive fruit fly microbiota was analyzed through 16S metabarcoding, at three development stages: last instar larvae, pupae, and adult. Besides Ca. E. dacicola, the olive fruit flies harbor a diverse bacterial flora of which 13 operational taxonomic units (grouped in 9 genera/species) were now determined to persist excluding at metamorphosis (Corynebacterium sp., Delftia sp., Enhydrobacter sp., Kocuria sp., Micrococcus sp., Propionibacterium sp., Pseudomonas sp., Raoultella sp., and Staphylococcus sp.). These findings open a new window of opportunities in symbiosis-based pest management.

Trends in the diagnostic delay and pathway for amyotrophic lateral sclerosis patients across different countries.

Background: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with a median survival of 2-5 years. An early diagnosis is essential for providing ALS patients the finest management possible. Studies from different countries report a similar median diagnostic delay of around 12 months, which is still far from desirable. We analyzed the diagnostic pathway in different countries in order to identify the major challenges. Methods: We studied a cohort of 1,405 ALS patients from five different centers, in four different countries (Turkey, Germany, Poland, and Portugal), which collaborated in a common database. Demographic, disease and sociocultural factors were collected. Time from first symptom onset to first medical evaluation and to diagnosis, the specialist assessment and investigations requested were analyzed. Factors contributing to diagnostic delay were evaluated by multivariate linear regression. Results: The median diagnostic delay from first symptom onset was 11 months and was similar between centers. Major differences were seen in the time from symptom onset to first medical evaluation. An earlier first medical evaluation was associated with a longer time to diagnosis, highlighting that ALS diagnosis is not straightforward in the early stages of the disease. The odds for ALS diagnosis were superior when evaluated by a neurologist and increased over time. Electromyography was decisive in establishing the diagnosis. Conclusions: We suggest that a specific diagnostic test for ALS-a specific biomarker-will be needed to achieve early diagnosis. Early referral to a neurologist and to electromyography is important for early ALS diagnosis.

Carrot AOX2a Transcript Profile Responds to Growth and Chilling Exposure.

Alternative oxidase (AOX) is a key enzyme of the alternative respiration, known to be involved in plant development and in response to various stresses. To verify the role of DcAOX1 and DcAOX2a genes in carrot tap root growth and in response to cold stress, their expression was analyzed in two experiments: during root growth for 13 weeks and in response to a cold challenge trial of 7 days, in both cases using different carrot cultivars. Carrot root growth is initially characterized by an increase in length, followed by a strong increase in weight. DcAOX2a presented the highest expression levels during the initial stages of root growth for all cultivars, but DcAOX1 showed no particular trend in expression. Cold stress had a negative impact on root growth, and generally up-regulated DcAOX2a with no consistent effect on DcAOX1. The identification of cis-acting regulatory elements (CAREs) located at the promoters of both genes showed putative sequences involved in cold stress responsiveness, as well as growth. However, DcAOX2a promoter presented more CAREs related to hormonal pathways, including abscisic acid and gibberellins synthesis, than DcAOX1. These results point to a dual role of DcAOX2a on carrot tap root secondary growth and cold stress response.

Delayed Diagnosis and Diagnostic Pathway of ALS Patients in Portugal: Where Can We Improve?

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with unsatisfactory treatment options. Best management and recruitment into clinical trials requires early diagnosis. However, diagnosis is often delayed. Analysis of the diagnostic pathway and identification of the causes of diagnostic delay are imperative. Methods: We studied a cohort of 580 ALS patients followed up in our ALS clinic in Lisbon. Demographic, disease, and sociocultural factors were collected. Time from first symptom onset to diagnosis, the specialist's assessment, and investigations requested were analyzed. Predictors of diagnostic delay were evaluated by multivariate linear regression, adjusting for potential confounders. Results: The median diagnostic delay from first symptom onset was 10 months. Spinal-onset, slower disease progression, cognitive symptoms at onset, and lower income were associated with increased diagnostic delay. Most patients were first assessed by general practitioners. Patients who were first evaluated by a neurologist were more likely to be correctly diagnosed, decreasing time to diagnosis. Electromyography was decisive in establishing the diagnosis. Conclusions: Late referral from non-neurologists to a neurologist is a potentially modifiable factor contributing to significant diagnostic delay. Educational interventions targeted to non-neurologists physicians, in order to increase awareness of ALS and, consequently, promote early referral to a neurologist at a tertiary center, will be important in reducing diagnostic delay.

High-Throughput Sequencing Identifies 3 Novel Susceptibility Genes for Hereditary Melanoma.

Cutaneous melanoma is one of the most aggressive human cancers due to its high invasiveness. Germline mutations in high-risk melanoma susceptibility genes have been associated with development hereditary melanoma; however, most genetic culprits remain elusive. To unravel novel susceptibility genes for hereditary melanoma, we performed whole exome sequencing (WES) on eight patients with multiple primary melanomas, high number of nevi, and negative for high and intermediate-risk germline mutations. Thirteen new potentially pathogenic variants were identified after bioinformatics analysis and validation. CDH23, ARHGEF40, and BRD9 were identified as the most promising susceptibility genes in hereditary melanoma. In silico analysis of CDH23 and ARHGEF40 variants provided clues for altered protein structure and function associated with the identified mutations. Then, we also evaluated the clinical value of CDH23, ARHGEF40, and BRD9 expression in sporadic melanoma by using the TCGA dataset (n = 461). No differences were observed in BRD9 expression between melanoma and normal skin samples, nor with melanoma stage, whereas ARHGEF40 was found overexpressed, and CDH23 was downregulated and its loss was associated with worse survival. Altogether, these results reveal three novel genes with clinical relevance in hereditary and sporadic melanoma.

Transcriptome Analysis of Wheat Roots Reveals a Differential Regulation of Stress Responses Related to Arbuscular Mycorrhizal Fungi and Soil Disturbance.

Symbioses with soil microorganisms are central in shaping the diversity and productivity of land plants and provide protection against a diversity of stresses, including metal toxicity. Arbuscular mycorrhizal fungi (AMF) can form extensive extraradical mycelial networks (ERM), which are very efficient in colonizing a new host. We quantified the responses of transcriptomes of wheat and one AMF partner, Rhizoglomus irregulare, to soil disturbance (Undisturbed vs. Disturbed) and to two different preceding mycotrophic species (Ornithopus compressus and Lolium rigidum). Soil disturbance and preceding plant species engender different AMF communities in wheat roots, resulting in a differential tolerance to soil manganese (Mn) toxicity. Soil disturbance negatively impacted wheat growth under manganese toxicity, probably due to the disruption of the ERM, and activated a large number of stress and starvation-related genes. The O. compressus treatment, which induces a greater Mn protection in wheat than L. rigidum, activated processes related to cellular division and growth, and very few related to stress. The L. rigidum treatment mostly induced genes that were related to oxidative stress, disease protection, and metal ion binding. R. irregulare cell division and molecular exchange between nucleus and cytoplasm were increased by O. compressus. These findings are highly relevant for sustainable agricultural systems, when considering a fit-for-purpose symbiosis.

Mitochondrial Neurogastrointestinal Encephalomyopathy: Novel Pathogenic Mutation in Thymidine Phosphorylase Gene in a Patient from Cape Verde Islands.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is a rare autosomal recessive disorder caused by mutations in the gene encoding the Thymidine Phosphorylase (TP). It is clinically characterized by severe gastrointestinal dysmotility, cachexia, palpebral ptosis, ophthalmoparesis, sensorimotor polyneuropathy and leukoencephalopathy. The diagnosis is established by the presence of typical clinical and neuroimaging features, positive family history, and abnormal genetic test. A 19-year-old Cape Verdean patient with a history since childhood of recurrent episodes of nausea, vomiting, diarrhoea and painful abdominal distension associated with progressive motor disability with difficulty in climbing stairs and running and clumsiness with her hands. The diagnostic workup was suggestive of MNGIE. Genetic screening of the TYMP gene identified a novel mutation (c. 1283 G>A). Patients with MNGIE have significant comorbidity and mortality, and they are frequently misdiagnosed. A better acknowledgment of this disorder is essential to permit an earlier diagnosis and to improve disease management.