Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA.

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.

Mutations causing premature termination codons discriminate and generate cellular and clinical variability in HHT.

ABSTRACT: For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we subcategorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense-mediated decay. In 3 patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from patients with ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC genotypes, PTC-containing RNA transcripts persisted at low levels (8%-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni P < .05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (isopeptide-bond/ubiquitin-like conjugation) and pulse-chase experiments detected subtle protein maturation differences but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of near-invariant housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor 4 (ATF4), which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modeled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other less rare ENG nonsense variants but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.

A new updated prognostic index for patients with brain metastases (BMs) treated with palliative whole brain radiotherapy (WBRT) in the era of precision oncology. METASNCore project.

INTRODUCTION: Palliative WBRT is the main treatment for multiple BMs. Recent studies report no benefit in survival after WBRT compared to palliative supportive care in patients (pts) with poor prognosis. A new era of systemic treatment strategies based on targeted therapies are improving the prognosis of patients with BMs. The purpose of this study is to develop a prognostic score in palliative pts with BMs who undergo WBRT in this new setting. METHODS: 239 pts with BMs who received palliative WBRT between 2013-2022 in our center were analyzed retrospectively. The score was designed according to the value of the ß coefficient of each variable with statistical significance in the multivariate model using Cox regression. Once the score was established, a comparison was performed according to Kaplan-Meier and was analyzed by log-rank test. RESULTS: 149 pts (62.3%) were male and median (m) age was 60 years. 139 (58,2%) were lung cancer and 35 (14,6%) breast cancer. All patients received 30Gys in 10 sessions. m overall survival (OS) was 3,74 months (ms). 37 pts (15,5%) had a specific target mutation. We found that 62 pts were in group < 4 points with mOS 6,89 ms (CI 95% 3,18-10,62), 84 in group 4-7 points with mOS 4,01 ms (CI 95% 3,40-4,62) and 92 pts in group > 7 points with mOS 2,72 ms (CI 95% 1,93-3,52) (p < 0,001). CONCLUSIONS: METASNCore items are associated with OS and they could be useful to select palliative pts to receive WBRT. More studies are necessary to corroborate our findings.

A Computer Vision-Based System to Help Health Professionals to Apply Tests for Fall Risk Assessment.

The increase in life expectancy, and the consequent growth of the elderly population, represents a major challenge to guarantee adequate health and social care. The proposed system aims to provide a tool that automates the evaluation of gait and balance, essential to prevent falls in older people. Through an RGB-D camera, it is possible to capture and digitally represent certain parameters that describe how users carry out certain human motions and poses. Such individual motions and poses are actually related to items included in many well-known gait and balance evaluation tests. According to that information, therapists, who would not need to be present during the execution of the exercises, evaluate the results of such tests and could issue a diagnosis by storing and analyzing the sequences provided by the developed system. The system was validated in a laboratory scenario, and subsequently a trial was carried out in a nursing home with six residents. Results demonstrate the usefulness of the proposed system and the ease of objectively evaluating the main items of clinical tests by using the parameters calculated from information acquired with the RGB-D sensor. In addition, it lays the future foundations for creating a Cloud-based platform for remote fall risk assessment and its integration with a mobile assistant robot, and for designing Artificial Intelligence models that can detect patterns and identify pathologies for enabling therapists to prevent falls in users under risk.

Safe Procedure for Efficient Hydrodynamic Gene Transfer to Isolated Porcine Liver in Transplantation.

Although calcineurin inhibitors are very effective as immunosuppressants in organ transplantation, complete graft acceptance remains as a challenge. Transfer of genes with immunosuppressant functions could contribute to improving the clinical evolution of transplantation. In this sense, hydrodynamic injection has proven very efficacious for liver gene transfer. In the present work, the hIL-10 gene was hydrofected 'ex vivo' to pig livers during the bench surgery stage, to circumvent the cardiovascular limitations of the procedure, in a model of porcine orthotopic transplantation with a 10-day follow-up. We used IL-10 because human and porcine proteins can be differentially quantified and for its immunomodulatory pleiotropic functions. Safety (biochemical parameters and histology), expression efficacy (RNA transcription and blood protein expression), and acute inflammatory response (cytokines panel) of the procedure were evaluated. The procedure proved safe as no change in biochemical parameters was observed in treated animals, and human IL-10 was efficaciously expressed, with stationary plasma protein levels over 20 pg/mL during the follow-up. Most studied cytokines showed increments (interferon-α, IFN-α; interleukin-1ß, IL-1ß; tumor necrosis factor α, TNFα; interleukin-6, IL-6; interleukin-8, IL-8; interleukin-4, IL-4; and transforming growth factor-ß, TGF-ß) in treated animals, without deleterious effects on tissue. Collectively, the results support the potential clinical interest in this gene therapy model that would require further longer-term dose-response studies to be confirmed.

Fundamental neurochemistry review: Old brain stories - Influence of age and sex on the neurodegeneration-associated lipid changes.

Brain aging is a naturally occurring process resulting in the decline of cognitive functions and increased vulnerability to develop age-associated disorders. Fluctuation in lipid species is crucial for normal brain development and function. However, impaired lipid metabolism and changes in lipid composition in the brain have been increasingly recognized to play a crucial role in physiological aging, as well as in several neurodegenerative diseases. In the last decades, the role of sexual dimorphism in the vulnerability to develop age-related neurodegeneration has increased. However, further studies are warranted for detailed assessment of how age, sex, and additional non-biological factors may influence the lipid changes in brains. The aim of this work is to address the presence of sex differences in the brain lipid changes that occur along aging, and in the two most common age-related neurodegenerative disorders (Alzheimer's and Parkinson's diseases). We included the studies that assessed lipid-related alterations in the brain of both humans and experimental models. Additionally, we explored the influence of sex on lipid-lowering therapies. We conclude that sex exerts a notable effect on lipid modifications occurring with age and neurodegeneration, and in lipid-reducing interventions. Therefore, the application of sex as an experimental variable is strongly encouraged for future research in the field of precision medicine approach.

Aminochrome Induces Neuroinflammation and Dopaminergic Neuronal Loss: A New Preclinical Model to Find Anti-inflammatory and Neuroprotective Drugs for Parkinson's Disease.

Studies have suggested aminochrome as an endogenous neurotoxin responsible for the dopaminergic neuron degeneration in Parkinson's disease (PD). However, neuroinflammation, an important alteration in PD pathogenesis, has been strictly induced in vitro by aminochrome. The aim of this study was to characterize the neuroinflammation induced in vivo by aminochrome. Wistar rats (male, 250-270 g) received a unilateral single dose by stereotaxic injection of saline into three sites in the striatum in the negative control group, or 32 nmol 6-hydroxydopamine (6-OHDA) in the positive control, or 6 nmol aminochrome. After 14 days, histological and molecular analyses were performed. We observed by immunofluorescence that aminochrome, as well as 6-OHDA, induced an increase in the number of Iba-1+ cells and in the number of activated (Iba-1+/ CD68+) microglia. An increase in the number of S100b+ cells and in the GFAP expression were also evidenced in the striatum and the SNpc of animals from aminochrome and positive control group. Dopaminergic neuronal loss was marked by reduction of TH+ cells and confirmed with reduction in the number of Nissl-stained neurons in the SNpc of rats from aminochrome and positive control groups. In addition, we observed by qPCR that aminocrhome induced an increase in the levels of IL-1ß, TNF-α, NLRP3, CCL5 and CCR2 mRNA in the SNpc. This work provides the first evidence of microgliosis, astrogliosis and neuroinflammation induced by aminochrome in an in vivo model. Since aminochrome is an endogenous molecule derived from dopamine oxidation present in the targeted neurons in PD, these results reinforce the potential of aminochrome as a useful preclinical model to find anti-inflammatory and neuroprotective drugs for PD. Aminochrome induced dopaminergic neuronal loss, microglial activation, astroglial activation and neuroinflammation marked by an increase in NLRP3, IL1ß, TNF-α, CCL2, CCL5 and CCR2.

Captive Breeding and Trichomonas gallinae Alter the Oral Microbiome of Bonelli's Eagle Chicks.

Bonelli's eagle (Aquila fasciata) is an endangered raptor species in Europe, and trichomonosis is one of the menaces affecting chicks at nest. In this paper, we attempt to describe the oral microbiome of Bonelli's eagle nestlings and evaluate the influence of several factors, such as captivity breeding, Trichomonas gallinae infection, and the presence of lesions at the oropharynx. The core oral microbiome of Bonelli's eagle is composed of Firmicutes, Bacteroidota, Fusobacteria and Proteobacteria as the most abundant phyla, and Megamonas and Bacteroides as the most abundant genera. None of the factors analysed showed a significant influence on alfa diversity, but beta diversity was affected for some of them. Captivity breeding exerted a high influence on the composition of the oral microbiome, with significant differences in the four most abundant phyla, with a relative increase of Proteobacteria and a decrease of the other three phyla in comparison with chicks bred at nest. Some genera were more abundant in captivity bred chicks, such as Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium and Staphylococcus, while Bacteroides, Oceanivirga, Peptostreptococcus, Gemella, Veillonella, Mycoplasma, Suttonella, Alloscardovia, Varibaculum and Campylobacter were more abundant in nest raised chicks. T. gallinae infection slightly influenced the composition of the microbiome, but chicks displaying trichomonosis lesions had a higher relative abundance of Bacteroides and Gemella, being the last one an opportunistic pathogen of abscess complications in humans. Raptor's microbiomes are scarcely studied. This is the first study on the factors that influence the oral microbiome of Bonelli's eagle.

Brain injections of glial cytoplasmic inclusions induce a multiple system atrophy-like pathology.

Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.

Use of bioelectrical impedance analysis in centenarians: a systematic review.

BACKGROUND: Centenarians often represent one of the best examples of aging successfully. However, the role of body composition or hydration status assessed with bioelectrical impedance analysis (BIA) is poorly explored in this population. Therefore, the aim of this systematic review was to better understand the use and the role of BIA for evaluating body composition and hydration status in centenarians. METHODS: We conducted a systematic review of the literature up to the 1st of May, 2022 for published articles providing data on BIA to evaluate body composition parameters or hydration status in centenarians. Data were summarized descriptively because a meta-analysis was not possible due to the scarcity of available studies. RESULTS: Among 2222 articles screened, four were eligible including 291 centenarians (mean age: 100.5 years) who were mainly women (88%). In one study, BIA overestimated fat-free mass and underestimated fat mass when compared to deuterium oxide dilution. Another study carried out in Italy including 14 centenarians found a significant correlation between BIA and fat-free mass evaluated using anthropometric tools. In one study, BIA showed a significant agreement with anthropometric measures of fat mass. In the same sample, sarcopenia and dehydration, evaluated with BIA, had a high prevalence. CONCLUSION: BIA may be used for assessing body composition in centenarians, but research is limited to a few studies suggesting the need of future research in this area.

JUNO Project: Deployment and Validation of a Low-Cost Cloud-Based Robotic Platform for Reliable Smart Navigation and Natural Interaction with Humans in an Elderly Institution.

This paper describes the main results of the JUNO project, a proof of concept developed in the Region of Murcia in Spain, where a smart assistant robot with capabilities for smart navigation and natural human interaction has been developed and deployed, and it is being validated in an elderly institution with real elderly users. The robot is focused on helping people carry out cognitive stimulation exercises and other entertainment activities since it can detect and recognize people, safely navigate through the residence, and acquire information about attention while users are doing the mentioned exercises. All the information could be shared through the Cloud, if needed, and health professionals, caregivers and relatives could access such information by considering the highest standards of privacy required in these environments. Several tests have been performed to validate the system, which combines classic techniques and new Deep Learning-based methods to carry out the requested tasks, including semantic navigation, face detection and recognition, speech to text and text to speech translation, and natural language processing, working both in a local and Cloud-based environment, obtaining an economically affordable system. The paper also discusses the limitations of the platform and proposes several solutions to the detected drawbacks in this kind of complex environment, where the fragility of users should be also considered.

Role of Damage-Associated Molecular Patterns (DAMPS) in the Postoperative Period after Colorectal Surgery.

Anastomotic leakage (AL) is a defect of the intestinal wall at the anastomotic site and is one of the most severe complications in colorectal surgery. Previous studies have shown that the immune system response plays a significant role in the development of AL. In recent years, DAMPs (damage-associated molecular patterns) have been identified as cellular compounds with the ability to activate the immune system. The NLRP3 inflammasome plays an important role in the inflammatory responses which are mediated by DAMPs such as ATP, HSP proteins or uric acid crystals, when found in extracellular environments. Recent publications suggest that systemic concentration of DAMPs in patients with colorectal surgery may determine the inflammatory process and have a role in the occurrence of AL and other post-surgery complications. This review provides valuable knowledge about the current evidence supporting this hypothesis and highlights the possible role of these compounds in postoperative processes, which could open a new path to explore new strategies to prevent possible post-surgical complications.

Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

Role of Microgliosis and NLRP3 Inflammasome in Parkinson's Disease Pathogenesis and Therapy.

Parkinson's disease (PD) is a neurodegenerative disorder marked primarily by motor symptoms such as rigidity, bradykinesia, postural instability and resting tremor associated with dopaminergic neuronal loss in the Substantia Nigra pars compacta (SNpc) and deficit of dopamine in the basal ganglia. These motor symptoms can be preceded by pre-motor symptoms whose recognition can be useful to apply different strategies to evaluate risk, early diagnosis and prevention of PD progression. Although clinical characteristics of PD are well defined, its pathogenesis is still not completely known, what makes discoveries of therapies capable of curing patients difficult to be reached. Several theories about the cause of idiopathic PD have been investigated and among them, the key role of inflammation, microglia and the inflammasome in the pathogenesis of PD has been considered. In this review, we describe the role and relation of both the inflammasome and microglial activation with the pathogenesis, symptoms, progression and the possibilities for new therapeutic strategies in PD.

SERS-Based Methodology for the Quantification of Ultratrace Graphene Oxide in Water Samples.

The extensive use of graphene materials in real-world applications has increased their potential release into the environment. To evaluate their possible health and ecological risks, there is a need for analytical methods that can quantify these materials at very low concentrations in environmental media such as water. In this work, a simple, reproducible, and sensitive method to detect graphene oxide (GO) in water samples using the surface-enhanced Raman spectroscopy (SERS) technique is presented. The Raman signal of graphene is enhanced when deposited on a substrate of gold nanoparticles (AuNPs), thus enabling its determination at low concentrations with no need for any preconcentration step. The practical limit of quantification achieved with the proposed method was 0.1 ng mL-1, which is lower than the predicted concentrations for graphene in effluent water reported to date. The optimized procedure has been successively applied to the determination of ultratraces of GO in water samples.

Atomic shell structure from Born probabilities: Comparison to other shell descriptors and persistence in molecules.

Real space chemical bonding descriptors, such as the electron localization function or the Laplacian of the electron density, have been widely used in electronic structure theory thanks to their power to provide chemically intuitive spatial images of bonded and non-bonded interactions. This capacity stems from their ability to display the shell structure of atoms and its distortion upon molecular formation. Here, we examine the spatial position of the N electrons of an atom at the maximum of the square of the wavefunction, the so-called Born maximum, as a shell structure descriptor for ground state atoms with Z = 1-36, comparing it to other available indices. The maximization is performed with the help of variational quantum Monte Carlo calculations. We show that many electron effects (mainly Pauli driven) are non-negligible, that Born shells are closer to the nucleus than any other of the examined descriptors, and that these shells are very well preserved in simple molecules.

A programme for early diagnosis of atrial fibrillation: a multi-centre study in primary care.

BACKGROUND: Atrial fibrillation (AF) is a morbid disease whose complications can be prevented if prompt and correctly treated. OBJECTIVE: To assess the usefulness of an early AF diagnosis programme in at-risk individuals in primary care centres. METHODS: In an open-label, multi-centre, controlled interventional study, individuals with one or more risk factors for AF but without known AF were enrolled. They were allocated to intervention and control groups in a 1:2 ratio. Participants in the intervention group had three clinical and educational visits (0, 6 and 12 months). In intervention subgroup A, an electrocardiogram (ECG) was performed at each visit and in subgroup B, only if arrhythmia was detected on auscultation. After 2 years, the medical records of all participants were reviewed. Participants diagnosed with AF were followed for two additional years. RESULTS: Of the total 2231 participants enrolled, 1503 (67.36%) were allocated to the control group and 728 (32.63%) to the intervention groups (355 in subgroup A, 373 subgroup B). The groups showed similar clinical characteristics. New-onset AF was diagnosed in 38 patients. Early detection in subgroup B was similar to subgroup A and superior to control group (3.2% versus 1.2%, hazard ratio 3.149, 95% confidence interval 1.503-6.597, P = 0.002). AF patients in subgroups A and B had similar long-term complications and a tendency for fewer complications than AF patients in the control group. CONCLUSIONS: An intervention programme consisting of health education, systematic auscultation and opportunistic ECG by a primary care provider is a useful method for the early diagnosis of AF.

The Urinary Level of Injury Biomarkers Is Not Univocally Reflective of the Extent of Toxic Renal Tubular Injury in Rats.

Nephrotoxicity is a major cause of intrinsic acute kidney injury (AKI). Because renal tissue damage may occur independently of a reduction in glomerular filtration rate and of elevations in plasma creatinine concentration, so-called injury biomarkers have been proposed to form part of diagnostic criteria as reflective of tubular damage independently of renal function status. We studied whether the urinary level of NGAL, KIM-1, GM2AP, t-gelsolin, and REGIIIb informed on the extent of tubular damage in rat models of nephrotoxicity, regardless of the etiology, moment of observation, and underlying pathophysiology. At a time of overt AKI, urinary biomarkers were measured by Western blot or ELISA, and tubular necrosis was scored from histological specimens stained with hematoxylin and eosin. Correlation and regression studies revealed that only weak relations existed between biomarkers and tubular damage. Due to high interindividual variability in the extent of damage for any given biomarker level, urinary injury biomarkers did not necessarily reflect the extent of the underlying tissue injury in individual rats. We contended, in this work, that further pathophysiological contextualization is necessary to understand the diagnostic significance of injury biomarkers before they can be used for renal tubular damage severity stratification in the context of nephrotoxic and, in general, intrinsic AKI.

Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells.

Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up 'in vitro'. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2' O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.

Invasive Fusariosis in Nonneutropenic Patients, Spain, 2000-2015.

Invasive fusariosis (IF) is associated with severe neutropenia in patients with concurrent hematologic conditions. We conducted a retrospective observational study to characterize the epidemiology of IF in 18 Spanish hospitals during 2000-2015. In that time, the frequency of IF in nonneutropenic patients increased from 0.08 cases per 100,000 admissions in 2000-2009 to 0.22 cases per 100,000 admissions in 2010-2015. Nonneutropenic IF patients often had nonhematologic conditions, such as chronic cardiac or lung disease, rheumatoid arthritis, history of solid organ transplantation, or localized fusariosis. The 90-day death rate among nonneutropenic patients (28.6%) and patients with resolved neutropenia (38.1%) was similar. However, the death rate among patients with persistent neutropenia (91.3%) was significantly higher. We used a multivariate Cox regression analysis to characterize risk factors for death: persistent neutropenia was the only risk factor for death, regardless of antifungal therapy.