Can Grapevine Leaf Water Potential Be Modelled from Physiological and Meteorological Variables? A Machine Learning Approach.

Climate change is affecting global viticulture, increasing heatwaves and drought. Precision irrigation, supported by robust water status indicators (WSIs), is inevitable in most of the Mediterranean basin. One of the most reliable WSIs is the leaf water potential (Ψleaf), which is determined via an intrusive and time-consuming method. The aim of this work is to discern the most effective variables that are correlated with plants' water status and identify the variables that better predict Ψleaf. Five grapevine varieties grown in the Alentejo region (Portugal) were selected and subjected to three irrigation treatments, starting in 2018: full irrigation (FI), deficit irrigation (DI), and no irrigation (NI). Plant monitoring was performed in 2023. Measurements included stomatal conductance (gs), predawn water potential Ψpd, stem water potential (Ψstem), thermal imaging, and meteorological data. The WSIs, namely Ψpd and gs, responded differently according to the irrigation treatment. Ψstem measured at mid-morning (MM) and mid-day (MD) proved unable to discern between treatments. MM measurements presented the best correlations between WSIs. gs showed the best correlations between the other WSIs, and consequently the best predictive capability to estimate Ψpd. Machine learning regression models were trained on meteorological, thermal, and gs data to predict Ψpd, with ensemble models showing a great performance (ExtraTrees: R2=0.833, MAE=0.072; Gradient Boosting: R2=0.830; MAE=0.073).

Novel systemic delivery of a peptide-conjugated antisense oligonucleotide to reduce α-synuclein in a mouse model of Alzheimer's disease.

Neurodegenerative disorders of aging are characterized by the progressive accumulation of proteins such as α-synuclein (α-syn) and amyloid beta (Aß). Misfolded and aggregated α-syn has been implicated in neurological disorders such as Parkinson's disease, and Dementia with Lewy Bodies, but less so in Alzheimer's Disease (AD), despite the fact that accumulation of α-syn has been confirmed in over 50% of postmortem brains neuropathologically diagnosed with AD. To date, no therapeutic strategy has effectively or consistently downregulated α-syn in AD. Here we tested the hypothesis that by using a systemically-delivered peptide (ApoB11) bound to a modified antisense oligonucleotide against α-syn (ASO-α-syn), we can downregulate α-syn expression in an AD mouse model and improve behavioral and neuropathologic phenotypes. Our results demonstrate that monthly systemic treatment with of ApoB11:ASO α-syn beginning at 6 months of age reduces expression of α-synuclein in the brains of 9-month-old AD mice. Downregulation of α-syn led to reduction in Aß plaque burden, prevented neuronal loss and astrogliosis. Furthermore, we found that AD mice treated with ApoB11:ASO α-syn had greatly improved hippocampal and spatial memory function in comparison to their control counterparts. Collectively, our data supports the reduction of α-syn through use of systemically-delivered ApoB11:ASO α-syn as a promising future disease-modifying therapeutic for AD.

Validation of automated image co-registration integrated into in-house software for voxel-based internal dosimetry on single-photon emission computed tomography images.

Objective: To develop an automated co-registration system and test its performance, with and without a fiducial marker, on single-photon emission computed tomography (SPECT) images. Materials and Methods: Three SPECT/CT scans were acquired for each rotation of a Jaszczak phantom (to 0°, 5°, and 10° in relation to the bed axis), with and without a fiducial marker. Two rigid co-registration software packages-SPM12 and NMDose-coreg-were employed, and the percent root mean square error (%RMSE) was calculated in order to assess the quality of the co-registrations. Uniformity, contrast, and resolution were measured before and after co-registration. The NMDose-coreg software was employed to calculate the renal doses in 12 patients treated with 177Lu-DOTATATE, and we compared those with the values obtained with the Organ Level INternal Dose Assessment for EXponential Modeling (OLINDA/EXM) software. Results: The use of a fiducial marker had no significant effect on the quality of co-registration on SPECT images, as measured by %RMSE (p = 0.40). After co-registration, uniformity, contrast, and resolution did not differ between the images acquired with fiducial markers and those acquired without. Preliminary clinical application showed mean total processing times of 9 ± 3 min/patient for NMDose-coreg and 64 ± 10 min/patient for OLINDA/EXM, with a strong correlation between the two, despite the lower renal doses obtained with NMDose-coreg. Conclusion: The use of NMDose-coreg allows fast co-registration of SPECT images, with no loss of uniformity, contrast, or resolution. The use of a fiducial marker does not appear to increase the accuracy of co-registration on phantoms.

Objetivo: Desenvolver corregistro automático e testar seu desempenho com ou sem marcador fiducial em imagens de tomografia computadorizada de emissão de fóton único (SPECT). Materiais e Métodos: Três SPECT/CTs foram adquiridas para cada rotação de um simulador de Jaszczak em relação ao eixo da maca (0°, 5° e 10°), com e sem fiducial. Dois métodos de corregistro inelástico foram aplicados - SPM12 e NMDose-coreg -, e a porcentagem do erro quadrático médio (%RMSE) foi usada para analisar a qualidade do corregistro. Uniformidade, contraste e resolução foram medidos antes e após o corregistro. NMDose com corregistro automático foi usado para calcular a dose renal de 12 pacientes tratados com 177Lu-DOTATATE e comparado com OLINDA/EXM. Resultados: A marcação fiducial não modificou a qualidade do corregistro das imagens SPECT, medida pela %RMSE (p = 0,40). Não houve impacto na uniformidade, contraste e resolução após o corregistro de imagens adquiridas com ou sem fiduciais. Aplicação clínica preliminar mostrou tempo total de processamento de 9 ± 3 min/paciente para NMDose e 64 ± 10 min/paciente para OLINDA/EXM, com alta correlação entre ambos, apesar de menor dose renal em NMDose. Conclusão: NMDose-coreg permite o corregistro rápido de imagens SPECT, sem perda de uniformidade, contraste ou resolução. O uso da marcação fiducial não aumentou a precisão do corregistro em fantomas.

Hippocampal Reduction of α-Synuclein via RNA Interference Improves Neuropathology in Alzheimer's Disease Mice.

BACKGROUND: Alzheimer's disease (AD) cases are often characterized by the pathological accumulation of α-synuclein (α-syn) in addition to amyloid-ß (Aß) and tau hallmarks. The role of α-syn has been extensively studied in synucleinopathy disorders, but less so in AD. Recent studies have shown that α-syn may also play a role in AD and its downregulation may be protective against the toxic effects of Aß accumulation. OBJECTIVE: We hypothesized that selectively knocking down α-syn via RNA interference improves the neuropathological and biochemical findings in AD mice. METHODS: Here we used amyloid precursor protein transgenic (APP-Tg) mice to model AD and explore pathologic and behavioral phenotypes with knockdown of α-syn using RNA interference. We selectively reduced α-syn levels by stereotaxic bilateral injection of either LV-shRNA α-syn or LV-shRNA-luc (control) into the hippocampus of AD mice. RESULTS: We found that downregulation of α-syn results in significant reduction in the number of Aß plaques. In addition, mice treated with LV-shRNA α-syn had amelioration of abnormal microglial activation (Iba1) and astrocytosis (GFAP) phenotypes in AD mice. CONCLUSION: Our data suggests a novel link between Aß and α-syn pathology as well as a new therapeutic angle for targeting AD.

Validation of automated image co-registration integrated into in-house software for voxel-based internal dosimetry on single-photon emission computed tomography images / Validação do corregistro automático de imagem integrado a software de análise de dosimetria interna baseada em voxel para tomografia computadorizada de emissão de fóton único

Abstract Objective: To develop an automated co-registration system and test its performance, with and without a fiducial marker, on single-photon emission computed tomography (SPECT) images. Materials and Methods: Three SPECT/CT scans were acquired for each rotation of a Jaszczak phantom (to 0°, 5°, and 10° in relation to the bed axis), with and without a fiducial marker. Two rigid co-registration software packages-SPM12 and NMDose-coreg-were employed, and the percent root mean square error (%RMSE) was calculated in order to assess the quality of the co-registrations. Uniformity, contrast, and resolution were measured before and after co-registration. The NMDose-coreg software was employed to calculate the renal doses in 12 patients treated with 177Lu-DOTATATE, and we compared those with the values obtained with the Organ Level INternal Dose Assessment for EXponential Modeling (OLINDA/EXM) software. Results: The use of a fiducial marker had no significant effect on the quality of co-registration on SPECT images, as measured by %RMSE (p = 0.40). After co-registration, uniformity, contrast, and resolution did not differ between the images acquired with fiducial markers and those acquired without. Preliminary clinical application showed mean total processing times of 9 ± 3 min/patient for NMDose-coreg and 64 ± 10 min/patient for OLINDA/EXM, with a strong correlation between the two, despite the lower renal doses obtained with NMDose-coreg. Conclusion: The use of NMDose-coreg allows fast co-registration of SPECT images, with no loss of uniformity, contrast, or resolution. The use of a fiducial marker does not appear to increase the accuracy of co-registration on phantoms.

Resumo Objetivo: Desenvolver corregistro automático e testar seu desempenho com ou sem marcador fiducial em imagens de tomografia computadorizada de emissão de fóton único (SPECT). Materiais e Métodos: Três SPECT/CTs foram adquiridas para cada rotação de um simulador de Jaszczak em relação ao eixo da maca (0°, 5° e 10°), com e sem fiducial. Dois métodos de corregistro inelástico foram aplicados - SPM12 e NMDose-coreg -, e a porcentagem do erro quadrático médio (%RMSE) foi usada para analisar a qualidade do corregistro. Uniformidade, contraste e resolução foram medidos antes e após o corregistro. NMDose com corregistro automático foi usado para calcular a dose renal de 12 pacientes tratados com 177Lu-DOTATATE e comparado com OLINDA/EXM. Resultados: A marcação fiducial não modificou a qualidade do corregistro das imagens SPECT, medida pela %RMSE (p = 0,40). Não houve impacto na uniformidade, contraste e resolução após o corregistro de imagens adquiridas com ou sem fiduciais. Aplicação clínica preliminar mostrou tempo total de processamento de 9 ± 3 min/paciente para NMDose e 64 ± 10 min/paciente para OLINDA/EXM, com alta correlação entre ambos, apesar de menor dose renal em NMDose. Conclusão: NMDose-coreg permite o corregistro rápido de imagens SPECT, sem perda de uniformidade, contraste ou resolução. O uso da marcação fiducial não aumentou a precisão do corregistro em fantomas.

Influence on voxel-based dosimetry: noise effect on absorbed dose dosimetry at single time-point versus sequential single-photon emission computed tomography.

OBJECTIVE: The purpose of this study was to evaluate how statistical fluctuation in single-photon emission computed tomography (SPECT) images propagate to absorbed dose maps. METHODS: SPECT/computed tomography (CT) images of iodine-131 filled phantoms, using different acquisition and processing protocols, were evaluated using STRATOS software to assess the absorbed dose distribution at the voxel level. Absorbed dose values and coefficient of variation (COV) were analyzed for dosimetry based on single time-point SPECT images and time-integrated activities of SPECT sequences with low and high counts. RESULTS: Considering dosimetry based on a single time-point, the mean absorbed dose was not significantly affected by total counts or reconstruction parameters, but the uniformity of the absorbed dose maps had an almost linear correlation with SPECT noise. When high- and low-count SPECT sequences were used to generate an absorbed dose map, the absorbed dose COV for each of the temporal sequences was slightly lower than the absorbed dose COV based on the single SPECT image with the highest count included in the sequence. CONCLUSION: The impact of changes in SPECT counts and reconstruction parameters is almost linear when dosimetry is based on isolated SPECT images, but less pronounced when dosimetry is based on sequential SPECTs.

Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy.

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimer's disease (AD) in PD patients.

Endosomal sorting drives the formation of axonal prion protein endoggresomes.

The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.

Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein.

The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson's Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by ß-Synuclein.

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and ß-synuclein (ßS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of ßS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of ßS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that ßS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of ßS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with ßS.

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome.

The assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals.

Observed reduction in recovery of Corynebacterium spp. from bovine milk samples by use of a teat cannula.

Although Corynebacterium bovis and coagulase-negative staphylococci are frequently the most commonly isolated bacteria from milk samples submitted for identification of pathogens causing intramammary infection, the individual quarter somatic cell count (SCC) from those samples is most often low. The present study aimed at evaluating the difference in bacteriology results from milk sampled by the standard technique (as recommended by the National Mastitis Council) and by the use of a teat cannula surpassing the teat canal, since C. bovis is often only found in the teat canal. Single quarter milk samples were collected in duplicate from 132 dairy cows on a commercial dairy farm using the standard milk sampling technique and also using a cannula introduced into the teat. Two groups of quarters were sampled: a group that was selected randomly at cow and quarter level and a group that was selected based on having SCC >200,000 cells/ml at the previous milk recording at cow level and on California mastitis test result at quarter level. Bacteriological culture performed on the samples yielded 29 Corynebacterium spp. isolates from the samples collected with the standard technique and 6 isolates from the samples collected with a cannula. Bacteriological culture yielded 73 and 100 culture negative samples respectively with the standard and the alternative sampling technique. A significant difference between the two sampling techniques was observed for recovery of Corynebacterium spp. and for no-growth samples. There was no significant difference in the isolation of Corynebacterium spp. or other bacterial species when using the standard technique before or after sampling with the cannula; thus the observed difference in bacteriology results could not be attributed to a particular sampling order. No significant change was observed overall in individual quarter SCC measured on the sampling day and 7 d later. Our results agree with several studies showing that Corynebacterium bovis often colonizes the teat canal, without causing true intramammary infection.

Mutations of the GLA gene in young patients with stroke: the PORTYSTROKE study--screening genetic conditions in Portuguese young stroke patients.

BACKGROUND AND PURPOSE: Fabry disease is an X-linked monogenic disorder caused by mutations in the GLA gene. Recent data suggest that stroke in young adults may be associated with Fabry disease. We aimed to ascertain the prevalence of this disorder among young adult patients with stroke in Portugal by GLA genotyping. METHODS: During 1 year, all patients aged 18 to 55 years with first-ever stroke, who were admitted into any of 12 neurology hospital departments in Portugal, were prospectively enrolled (n=625). Ischemic stroke was classified according to Trial of Org 10172 in Acute Stroke Treatment criteria. Alpha-galactosidase activity was further assayed in all patients with GLA mutations. RESULTS: Four hundred ninety-three patients (mean age, 45.4 years; 61% male) underwent genetic analyses: 364 with ischemic stroke, 89 with intracerebral hemorrhage, 26 with subarachnoid hemorrhage, and 14 with cerebral venous thrombosis. Twelve patients had missense GLA mutations: 9 with ischemic stroke (p.R118C: n=4; p.D313Y: n=5), including 5 patients with an identified cause of stroke (cardiac embolism: n=2; small vessel disease: n=2; other cause: n=1), 2 with intracerebral hemorrhage (p.R118C: n=1; p.D313Y: n=1), and one with cerebral venous thrombosis (p.R118C: n=1). Leukocyte alpha-galactosidase activity was subnormal in the hemizygous males and subnormal or low-normal in the heterozygous females. Estimated prevalence of missense GLA mutations was 2.4% (95% CI, 1.3% to 4.1%). CONCLUSIONS: Despite a low diagnostic yield, screening for GLA mutations should probably be considered in different types of stroke. Restricting investigation to patients with cryptogenic stroke may underestimate the true prevalence of Fabry disease in young patients with stroke.