Challenges and Opportunities Arising from Host-Botrytis cinerea Interactions to Outline Novel and Sustainable Control Strategies: The Key Role of RNA Interference.

The necrotrophic plant pathogenic fungus Botrytis cinerea (Pers., 1794), the causative agent of gray mold disease, causes significant losses in agricultural production. Control of this fungal pathogen is quite difficult due to its wide host range and environmental persistence. Currently, the management of the disease is still mainly based on chemicals, which can have harmful effects not only on the environment and on human health but also because they favor the development of strains resistant to fungicides. The flexibility and plasticity of B. cinerea in challenging plant defense mechanisms and its ability to evolve strategies to escape chemicals require the development of new control strategies for successful disease management. In this review, some aspects of the host-pathogen interactions from which novel and sustainable control strategies could be developed (e.g., signaling pathways, molecules involved in plant immune mechanisms, hormones, post-transcriptional gene silencing) were analyzed. New biotechnological tools based on the use of RNA interference (RNAi) are emerging in the crop protection scenario as versatile, sustainable, effective, and environmentally friendly alternatives to the use of chemicals. RNAi-based fungicides are expected to be approved soon, although they will face several challenges before reaching the market.

Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea.

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.

Limited evidence for neuropsychological dysfunction in patients initially affected by severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in coronavirus disease 2019 (COVID-19), has caused a pandemic. There is now considerable evidence that neuropsychological functions could be affected. We further tested this hypothesis on a sample of post COVID-19 patients, who, initially, had been severely affected. METHODS: We tested 22 post COVID-19 patients who, after the intensive care unit (all but one), were admitted to our rehabilitation unit to be treated for severe post COVID-19 sequelae. All patients were administered a comprehensive neuropsychological battery including measures assessing perceptual, attentive, mnestic, linguistic, and executive functions, and overall cognitive status. The patients were also administered rehabilitation measures including scales for investigating aerobic capacity/endurance deficits, dyspnea, and fatigue. RESULTS: Our findings revealed that evidence of neuropsychological disorders in post COVID-19 patients was very limited. Furthermore, COVID-19 severity and other relevant variables were not correlated with patients' scores on the neuropsychological tests. CONCLUSIONS: We suggest that the relation between COVID-19 and neuropsychological disorders is unclear. New studies and metanalyses are highly required to shed light on this highly complex issue.

Psychological impact of COVID-19 after hospital discharge: A follow-up study on Italian recovered patients.

BACKGROUND: Since COVID-19 outbreak, clinical experience on its management during the acute phase has rapidly grown, including potential effects on the psychopathological dimension. However, still few data are available regarding the impact on survivors' mental health over the long-term. METHODS: A sample of 1457 COVID-19 patients underwent a multidisciplinary follow-up protocol, approximately 3 months after hospital discharge, including a psychological evaluation. The primary outcomes were anxiety, depression, resilience, post-traumatic symptoms, and health-related quality of life. Furthermore, we examined the potential role of hospitalization and delay in the follow-up assessment on the increased burden of illness. RESULTS: Although a general high level of resilience emerged, suggesting most patients relied on their individual and interpersonal resources to face difficulties related to the pandemic, almost one third of the sample reported signs of psychological distress over time, especially post-traumatic symptoms, with anxiety being more represented than depression. Furthermore, hospitalization - regardless of the setting of care - and promptness in follow-up evaluation were found to play a protective role on patients' recovery and mental wellbeing. LIMITATIONS: Selection bias of patients exclusively admitted to the hospital; absence of a control group; psychological assessment relying on self-reported instruments. CONCLUSIONS: The current crisis demands resilience and adjustment resources, either in the acute and post-acute phase. Thus, the clinical effort should aim at relieving the traumatic impact of such condition through timely interventions. Further investigation may address potential predictors of developing a traumatic stress response, in order to identify and promptly treat at-risk subpopulations.

Fine-Tuning Plant Gene Expression with Synthetic Trans-Acting Small Interfering RNAs.

RNAi-based tools are widely used in gene function studies and for crop improvement. However, no effective methods for precisely controlling the degree of induced silencing have been reported until recently. Here we report a detailed protocol for designing and generating synthetic trans-acting small interfering RNA (syn-tasiRNA) constructs for fine-tuning gene expression in plants. Recently developed high-throughput AtTAS1c-D2-B/c-based vectors are used to clone and express syn-tasiRNAs that possess different efficacies depending on their precursor location and on their degree of base-pairing with the 5' end of target RNAs.

Silencing of the Slt2-Type MAP Kinase Bmp3 in Botrytis cinerea by Application of Exogenous dsRNA Affects Fungal Growth and Virulence on Lactuca sativa.

Botrytis cinerea can attack over 500 genera of vascular plants and is considered the second phytopathogen in the 'top ten' for its economic importance. Traditional fungicides can be ineffective and with increasing fungicide resistance, new sustainable technologies are required. Lately, RNA interference-based fungicides are emerging for their potential uses in crop protection. Therefore, we assessed the potential of this innovative approach targeting the MAP kinase Bmp3 in B. cinerea, a gene involved in saprophytic growth, response to low osmolarity, conidiation, surface sensing, host penetration and lesion formation. After performing a prediction analysis of small interfering RNAs, a 427 nucleotides long dsRNA was selected as construct. We tested the effect of topical applications of dsRNA construct both in vitro by a fungal growth assay in microtiter plates and in vivo on detached lettuce leaves artificially inoculated. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strong reduction of necrotic lesions on leaves. These results correlated with a strongly reduced expression of Bmp3 gene. In accordance to these findings, the Bmp3 gene could be a promising target for the development of an RNAi-based fungicide against B. cinerea.

Remote support by multidisciplinary teams: A crucial means to cope with the psychological impact of the SARS-COV-2 pandemic on patients with cystic fibrosis and inflammatory bowel disease in Lombardia.

BACKGROUND: During Coronavirus Disease 2019 (COVID-19) outbreak in Lombardia, people were recommended to avoid visiting emergency departments and attending routine clinic visits. In this context, it was necessary to understand the psychological reactions of patients with chronic diseases. We evaluated the psychological effects on patients with chronic respiratory conditions and inflammatory bowel disease (IBD) through the analysis of their spontaneous contacts with their referral centres. METHODS: Cross-sectional study was conducted from February 23 to April 27, 2020 in patients, or their parents, who contacted their multidisciplinary teams (MDT). E-mails and phone calls directed to the MDT of the centre for cystic fibrosis (CF) in Milano and for paediatric IBD in Bergamo, were categorised according to their contents as information on routine disease management, updates on the patient's health status, COVID-19 news monitoring, empathy towards health professionals, positive feedback and concern of contagion during the emergency. RESULTS: One thousand eight hundred and sixteen contacts were collected during the study period. In Milano, where the majority of patients were affected by CF, 88.7% contacted health professionals by e-mail, with paediatricians receiving the largest volume of emails and phone calls compared with other professionals (P< .001). Compared with Milano, the centre for IBD in Bergamo recorded more expression of empathy towards health professionals and thanks for their activity in the COVID-19 emergency (52.4% vs 12.7%, P< .001), as well as positive feedback (64.3% vs 2.7%, P = .003). CONCLUSION: One of the most important lessons we can learn from COVID-19 is that it is not the trauma itself that can cause psychological consequences but rather the level of balance, or imbalance, between fragility and resources. To feel safe, people need to be able to count on the help of those who represent a bulwark against the threat. This is the role played, even remotely, by health professionals.

Surviving COVID-19 in Bergamo province: a post-acute outpatient re-evaluation.

Bergamo province was badly hit by the coronavirus disease 2019 (COVID-19) epidemic. We organised a public-funded, multidisciplinary follow-up programme for COVID-19 patients discharged from the emergency department or from the inpatient wards of 'Papa Giovanni XXIII' Hospital, the largest public hospital in the area. As of 31 July, the first 767 patients had completed the first post-discharge multidisciplinary assessment. Patients entered our programme at a median time of 81 days after discharge. Among them, 51.4% still complained of symptoms, most commonly fatigue and exertional dyspnoea, and 30.5% were still experiencing post-traumatic psychological consequences. Impaired lung diffusion was found in 19%. Seventeen per cent had D-dimer values two times above the threshold for diagnosis of pulmonary embolism (two unexpected and clinically silent pulmonary thrombosis were discovered by investigating striking D-dimer elevation). Survivors of COVID-19 exhibit a complex array of symptoms, whose common underlying pathology, if any, has still to be elucidated: a multidisciplinary approach is fundamental, to address the different problems and to look for effective solutions.

COVID-19 presenting with agraphia and conduction aphasia in a patient with left-hemisphere ischemic stroke.

COVID-19 following infection by SARS-CoV-2 can affect the brain causing confusion, depression, and dementia-like signs. Nonetheless, the presence of more specific neuropsychological signs because of COVID-19 remains unexplored. We report on LA, a patient who was affected by a left-hemisphere ischemic stroke, probably because of SARS-CoV-2. The patient showed a highly specific neuropsychological profile characterized by severe agraphia and some signs of conduction aphasia. All other cognitive and sensorimotor functions remained intact. We sustain that specific neuropsychological signs can be observed in patients with COVID-19. Therefore, in-depth and comprehensive neuropsychological assessment should be included to better explore and qualify the neuropsychological consequences of COVID-19. This is a new challenge for diagnosis and rehabilitation, with important consequences for the involved neuropsychological services.

Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs.

Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3' end of the syn-tasiRNA and the 5' end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.

A home-based prism adaptation training for neglect patients.

Spatial neglect is a debilitating disorder frequently observed after damage to the right cerebral hemisphere. Previous investigations have revealed that prism adaptation (PA) therapy can lead to improvements in neglect-related symptoms. In the typical PA protocol patients repeatedly point toward a visual target while wearing prism goggles. A few years ago, a novel PA procedure, involving a variety of more "ecological" visuo-motor activities during adaptation, less repetitive than a sequence of pointings, was introduced by our research group, and shown to be able to improve neglect-related symptoms to the same extent as the standard pointing task. The ecological procedure was easy to administer and pleasant for the patients. In all previous studies, patients were treated by specialized personnel during hospitalization. In the current study, we investigated the effectiveness of the ecological PA method when performed in a home-based setting, with the help of caregivers and family members. Seven right-brain-damaged patients with chronic left spatial neglect underwent a two-week ecological PA treatment, extended, for two extra weeks, in 6 patients, who were available for this additional rehabilitation session. As a control treatment, patients performed the same activities while wearing neutral goggles, before the PA procedure. Two weeks of ecological PA training proved to be able to significantly improve performance in neuropsychological tests (BIT, Cancellation tasks), a neurological scale (NIH), and functional abilities (CBS), when compared to both the baseline and the neutral control treatment, with improvements being maintained over 6 months. The ecological home-based PA training is effective in alleviating signs of spatial neglect. Importantly, this training is affordable, pleasant, and feasible to be performed in the comfort of the patient's home. Easily extendable to larger patient populations and prolonged periods, this method has a real potential to benefit the quality of life of brain-damaged patients with left spatial neglect.

Are Moral and Socio-conventional Knowledge Impaired in Severe Traumatic Brain Injury?

OBJECTIVE: The aim of this study was to investigate explicit moral and socio-conventional knowledge in Traumatic Brain Injury (TBI) patients. METHOD: A group of 28 TBI patients was tested on a new set of moral and socio-conventional items. Responses of TBI patients were compared with those of 28 matched controls. Participants had to report how hard would be to perform specific moral or socio-conventional transgressions, using a 10-point Likert scale. We analyzed our data through mixed-effects models, to jointly assess by-participants and by-items variance. The factors considered were Type of Item (Moral vs. Socio-conventional) and Group (TBI vs. Controls). RESULTS: Results revealed a significant interaction between Type of Item and Group (χ2[1] = 25.5, p < .001). Simple-effects analyses showed that TBI, as Controls, were able to differentiate moral and socio-conventional transgressions (χ2[1] = 72.3, p < .001), as they deemed the former as more difficult to enact. TBI patients, however, evaluated moral transgressions as easier to fulfill (χ2[1] = 12.2, p = .001). CONCLUSIONS: TBI patients can clearly differentiate moral and socio-conventional transgressions, suggesting that the explicit knowledge of these two dimensions is spared. TBI patients, however, considered moral transgressions as easier to fulfill with respect to Controls. This finding may suggest a tendency in TBI patients to underestimate the weight of moral transgressions.

Genetic epidemiology of autosomal recessive hypercholesterolemia in Sicily: Identification by next-generation sequencing of a new kindred.

BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) is a rare inherited lipid disorder. In Sardinia, differently from other world regions, the mutated allele frequency is high. It is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 gene. Fourteen different mutations have been reported so far; in Sardinia, 2 alleles (ARH1 and ARH2) explain most of the cases. Four ARH patients, all carriers of the ARH1 mutation, have been identified in mainland Italy and 2 in Sicily. OBJECTIVE: The objectives of the study were to improve the molecular diagnosis of familial hypercholesterolemia (FH) and to estimate the frequency of the ARH1 allele in 2 free-living Sicilian populations. METHODS: We sequenced by targeted next-generation sequencing 20 genes related to low-density lipoprotein metabolism in 50 hypercholesterolemic subjects. Subjects from 2 free-living populations from Northern (Ventimiglia Heart Study, 848 individuals) and Southern Sicily (Zabut Zabùt Aging Project, 1717 individuals) were genotyped for ARH1 allele. RESULTS: We identified 1 homozygous carrier of the ARH1 mutation among the 50 hypercholesterolemic outpatients. Population-based genotyping of ARH1 in 2565 subjects allowed the identification of 1 heterozygous carrier. The overall estimated allele frequency of ARH1 in Sicily was 0.0002 (0.02%). CONCLUSIONS: The identification of a new case of ARH in Sicily among 50 clinically diagnosed FH highlights the importance of next-generation sequencing analysis as tool to improve the FH diagnosis. Our results also indicate that ARH1 carrier status is present in ∼1:2500 of Sicilian inhabitants, confirming that ARH is extremely rare outside Sardinia.

Left neglect dyslexia: Perseveration and reading error types.

Right-brain-damaged patients may show a reading disorder termed neglect dyslexia. Patients with left neglect dyslexia omit letters on the left-hand-side (the beginning, when reading left-to-right) part of the letter string, substitute them with other letters, and add letters to the left of the string. The aim of this study was to investigate the pattern of association, if any, between error types in patients with left neglect dyslexia and recurrent perseveration (a productive visuo-motor deficit characterized by addition of marks) in target cancellation. Specifically, we aimed at assessing whether different productive symptoms (relative to the reading and the visuo-motor domains) could be associated in patients with left spatial neglect. Fifty-four right-brain-damaged patients took part in the study: 50 out of the 54 patients showed left spatial neglect, with 27 of them also exhibiting left neglect dyslexia. Neglect dyslexic patients who showed perseveration produced mainly substitution neglect errors in reading. Conversely, omissions were the prevailing reading error pattern in neglect dyslexic patients without perseveration. Addition reading errors were much infrequent. Different functional pathological mechanisms may underlie omission and substitution reading errors committed by right-brain-damaged patients with left neglect dyslexia. One such mechanism, involving the defective stopping of inappropriate responses, may contribute to both recurrent perseveration in target cancellation, and substitution errors in reading. Productive pathological phenomena, together with deficits of spatial attention to events taking place on the left-hand-side of space, shape the manifestations of neglect dyslexia, and, more generally, of spatial neglect.

'I still have difficulties feeling like a mother': The transition to motherhood of preterm infants mothers.

OBJECTIVE: The premature birth of their infant can constitute a sudden interruption of the transition to motherhood that requires a reorganisation of the process. The present study aimed to analyse the experience of the transition to motherhood of preterm infants' mothers, framing it within Stern's transition to motherhood theory. METHOD: A semi-structured interview was administered to 30 mothers during the recovery of the infant in the Neonatal Intensive Care Unit. The interview explored the experience of mothers related to pregnancy, the infant's birth and recovery. RESULTS: Thematic Analyses evidenced four interrelated themes: disconnection from the child, perception of maternal inadequacy, loss of parental role and temporal suspension. The themes showed that the mothers' experience of preterm birth not only concerns the traumatic delivery, but is also embedded in the entire process of becoming a mother within an institutional context. CONCLUSIONS: Results were connected to Stern's theory. Findings revealed difficulties for preterm mothers that could affect the development of the maternal constellation and thus their transition to motherhood. These difficulties may influence the construction of maternal identity, mother's representation of their child and the bond with their child.

Colistin-associated Acute Kidney Injury in Severely Ill Patients: A Step Toward a Better Renal Care? A Prospective Cohort Study.

BACKGROUND: Critically ill patients with severe sepsis or septic shock may need relatively high colistin daily doses for efficacy against multidrug-resistant and extensively drug-resistant gram-negative rods. However, acute kidney injury (AKI) may represent a major dose-limiting adverse effect of colistin. We sought to determine AKI occurrence and to identify factors influencing AKI risk in severely ill patients receiving colistin according to a recently proposed dosing strategy. METHODS: A prospective, observational, cohort study involving patients with severe sepsis or septic shock who received colistin was performed. AKI was defined according to Acute Kidney Injury Network criteria. Colistin administration was driven by a modified pharmacokinetics-pharmacodynamics (PK/PD)-based dosing approach. RESULTS: Of 70 patients who received colistin at a median daily dose of 9 million IU (MIU; interquartile range, 5.87-11.1 MIU), 31 (44%) developed AKI. In univariate analysis, age, Acute Physiology and Chronic Health Evaluation (APACHE) II score, Sequential Organ Failure Assessment (SOFA), score and baseline renal impairment were significantly associated with AKI. Moreover, patients with AKI were less frequently treated with adjuvant ascorbic acid (P = .003). In multivariate analysis, independent predictors of AKI were baseline renal impairment (adjusted hazard ratio, 4.15; 95% confidence interval, 1.9-9.2; P < .001) and age (1.03; 1.0-1.05; P = .028), whereas a strong independent renal-protective role emerged for ascorbic acid (0.27; .12-.57; P < .001). CONCLUSIONS: In severely ill patients receiving colistin according to a PK/PD-driven dosing approach, baseline renal impairment and older age strongly predict AKI occurrence, but concomitant administration of ascorbic acid markedly reduces AKI risk, allowing safer use of colistin.

(Un)awareness of unilateral spatial neglect: a quantitative evaluation of performance in visuo-spatial tasks.

Right-brain-damaged patients with unilateral spatial neglect are usually unaware (anosognosic) about their spatial deficits. However, in the scientific literature there is a lack of systematic and quantitative evaluation of this kind of unawareness, despite the negative impact of anosognosia on rehabilitation programs. This study investigated anosognosia for neglect-related impairments at different clinical tasks, by means of a quantitative assessment. Patients were tested in two different conditions (before and after execution of each task), in order to evaluate changes in the level of awareness of neglect-related behaviours triggered by task execution. Twenty-nine right-brain-damaged patients (17 with left spatial neglect) and 27 neurologically unimpaired controls entered the study. Anosognosia for spatial deficits is not pervasive, with different tasks evoking different degrees of awareness about neglect symptoms. Indeed, patients showed a largely preserved awareness about their performance in complex visuo-motor spatial and reading tasks; conversely, they were impaired in evaluating their spatial difficulties in line bisection and drawing from memory, showing over-estimation of their performance. The selectivity of the patients' unawareness of specific manifestations of spatial neglect is further supported by their preserved awareness of performance at a linguistic task, and by the absence of anosognosia for hemiplegia. This evidence indicates that discrete processes are involved in the aware monitoring of cognitive and motor performance, which can be selectively compromised by brain damage. Awareness of spatial difficulties is supported by a number of distinct components, and influenced by the specific skills required to perform a given task.