Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Synthetic Corpus Generation for Deep Learning-Based Translation of Spanish Sign Language.

Sign language serves as the primary mode of communication for the deaf community. With technological advancements, it is crucial to develop systems capable of enhancing communication between deaf and hearing individuals. This paper reviews recent state-of-the-art methods in sign language recognition, translation, and production. Additionally, we introduce a rule-based system, called ruLSE, for generating synthetic datasets in Spanish Sign Language. To check the usefulness of these datasets, we conduct experiments with two state-of-the-art models based on Transformers, MarianMT and Transformer-STMC. In general, we observe that the former achieves better results (+3.7 points in the BLEU-4 metric) although the latter is up to four times faster. Furthermore, the use of pre-trained word embeddings in Spanish enhances results. The rule-based system demonstrates superior performance and efficiency compared to Transformer models in Sign Language Production tasks. Lastly, we contribute to the state of the art by releasing the generated synthetic dataset in Spanish named synLSE.

Enrichment of Immune Dysregulation Disorders in Adult Patients with Human Inborn Errors of Immunity.

Human inborn errors of immunity (IEI) comprise a group of diseases resulting from molecular variants that compromise innate and adaptive immunity. Clinical features of IEI patients are dominated by susceptibility to a spectrum of infectious diseases, as well as autoimmune, autoinflammatory, allergic, and malignant phenotypes that usually appear in childhood, which is when the diagnosis is typically made. However, some IEI patients are identified in adulthood due to symptomatic delay of the disease or other reasons that prevent the request for a molecular study. The application of next-generation sequencing (NGS) as a diagnostic technique has given rise to an ever-increasing identification of IEI-monogenic causes, thus improving the diagnostic yield and facilitating the possibility of personalized treatment. This work was a retrospective study of 173 adults with IEI suspicion that were sequenced between 2005 and 2023. Sanger, targeted gene-panel, and whole exome sequencing were used for molecular diagnosis. Disease-causing variants were identified in 44 of 173 (25.43%) patients. The clinical phenotype of these 44 patients was mostly related to infection susceptibility (63.64%). An enrichment of immune dysregulation diseases was found when cohorts with molecular diagnosis were compared to those without. Immune dysregulation disorders, group 4 from the International Union of Immunological Societies Expert Committee (IUIS), were the most prevalent among these adult patients. Immune dysregulation as a new item in the Jeffrey Model Foundation warning signs for adults significantly increases the sensitivity for the identification of patients with an IEI-producing molecular defect.

Artificial nighttime lighting impacts Plasmodium falciparum mature stage V gametocytes infectivity in Anopheles stephensi.

BACKGROUND: Malaria is one of the most important vector-borne diseases of humans with an estimated 241 million cases worldwide in 2020. As an urban and periurban mosquito species, Anopheles stephensi is exposed to artificial human stimuli like light that can alter many aspects of mosquito behaviour, physiology and metabolism. Therefore, fluctuations in the light environment may influence the host, parasite and/or mosquito biology and hence modulate risk for disease transmission. In this study, the effect of artifitial light at night on mosquito infectivity by Plasmodium falciparum during the first hours of blood digestion was tested. METHODS: A total of three independent standard membrane feeding assays were performed to artificially fed septic and aseptic mosquitoes with P. falciparum infected blood. After blood feeding, females were transferred to incubators with different photoperiod cycles, so digestion occurred under day artificial light or dark. At 7 and 16 days post blood feeding, mosquitoes were dissected for midguts and salivary glands, respectively. Percentage of mosquitoes fed, percentage of prevalence and P. falciparum oocyst intensity between septic and aseptic mosquitoes in the two different photoperiod regimes, were compared using a Kruskal-Wallis test followed by a Dunn´s multiple comparison test . RESULTS: The exposition of mosquitoes to light after they took an infected blood meal has a negative effect on the successful progression of P. falciparum in the mosquito midgut. Antibiotic treatment significantly incremented the number of oocysts per midgut. Photophase significantly reduced the median oocyst intensity in both septic and aseptic mosquitoes. The percentage of oocyst reduction, understood as the percentage of reduction in the mean oocyst intensity of the parasite in the mosquito midgut between photophase and scotophase, was 51% in the case of aseptic mosquitoes and 80% for septic mosquitoes, both in the photophase condition. CONCLUSION: Although there are still many gaps in the understanding of parasite-mosquito interactions, these results support the idea that light can, not only, influence mosquito biting behaviour but also parasite success in the mosquito midgut. Hence, light can be considered an interesting additional mosquito-control strategy to reduce mosquito-borne diseases.

Salud sexual y reproductiva en población adolescente y adulta en México, 2022.

OBJETIVO: Caracterizar la situación de la salud sexual y reproductiva (SSR) en población adolescente (10-19) y adulta (20-49). Material y métodos. Utilizando la Encuesta Nacional de Salud y Nutrición 2022, se estimaron prevalencias e intervalos de confianza al 95% para indicadores de SSR. RESULTADOS: En adolescentes, 73.2% ha escuchado hablar de anticonceptivos, 88.1% saben que el condón se usa una sola vez y 60.4% que previene embarazos e infecciones de transmisión sexual; 22.8% ha iniciado vida sexual y 73.2% usaron condón en la primera relación sexual. En la población adulta, 42.7 y 38.0% no usaron protección en la primera y última relación sexual y 53.4 y 40.7% usaron condón en la primera y última relación sexual. Se encontraron diferencias en la atención de la salud materna entre adolescentes y adultas. Conclusión. Contar con información actualizada en SSR permite focalizar la atención en las necesidades de las personas.

Patient-derived lymphoma spheroids integrating immune tumor microenvironment as preclinical follicular lymphoma models for personalized medicine.

BACKGROUND: Follicular lymphoma (FL), the most common indolent non-Hodgkin's Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses. METHODS: To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability. RESULTS: FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3. CONCLUSIONS: In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.

Genetic mapping of the p47 L.3.2 mutation in Drosophilamelanogaster.

An EMS-based forward genetic screen was conducted in an apoptotic null background to identify genetic aberrations that contribute to regulation of cell growth in Drosophila melanogaster . The current work maps the genomic location of one of the identified mutants, L.3.2 . Genetic crosses conducted through the Fly-CURE consortium determined that the gene locus for the L.3.2 mutation is p47 on chromosome 2R.

Ocular Surface Microbiota in Naïve Keratoconus: A Multicenter Validation Study.

In the field of Ophthalmology, the mNGS 16S rRNA sequencing method of studying the microbiota and ocular microbiome is gaining more and more weight in the scientific community. This study aims to characterize the ocular microbiota of patients diagnosed with keratoconus who have not undergone any prior surgical treatment using the mNGS 16S rRNA sequencing method. Samples of naïve keratoconus patients were collected with an eNAT with 1 mL of Liquid Amies Medium (Copan Brescia, Italy), and DNA was extracted and analyzed with 16S NGS. The microbiota analysis showed a relative abundance of microorganisms at the phylum level in each sample collected from 38 patients with KC and 167 healthy controls. A comparison between healthy control and keratoconus samples identified two genera unique to keratoconus, Pelomonas and Ralstonia. Our findings suggest that alterations in the microbiota may play a role in the complex scenario of KC development.

The role of matrix metalloproteinases in infectious corneal ulcers.

During infectious keratitis, the production of collagenolytic and inflammatory substances, along with increased corneal matrix metalloproteinase (MMP) activity, induces the degradation of corneal collagen and may cause postkeratitis complications, such as opacity, thinning, and corneal perforation. MMPs, especially MMP-2 and MMP-9, are overexpressed in infectious keratitis and sustained over time by inflammatory and nonmicrobial mechanisms. The high MMP levels are correlated with excessive corneal destruction in bacterial, herpetic, fungal, and acanthamoeba infections. Nonspecific treatments, such as tetracyclines, particularly doxycycline, or corticosteroids, are used as adjuvants to antimicrobials to alleviate the disproportionate degradation and inflammation of the corneal layers caused by corneal MMPs and decrease the recruitment and infiltration of inflammatory cells. Treatments showing inhibition of specific MMPs (Galardin, ZHAWOC7726), interfering with pro-MMP activation (EDTA, ascorbic acid), or showing anticytokine effect (epigallocatechin-2-gallate, TRAM-34) have been reported. Other treatments show a direct action over corneal collagen structure such as corneal cross-linking or have been associated with reduction of MMP levels such as amniotic membrane grafting. Although the use of these drugs has been shown in studies to be effective in controlling inflammation, especially in experimental ones, robust studies are still needed based on randomized and randomized clinical trials to demonstrate their potential effect as adjuvants in the management of infectious keratitis.

Prevalence and clinical significance in our setting of incidental uptake in the thyroid gland found on 18F-fluordeoxyglucose positron emission tomography-computed tomography (PET-CT).

INTRODUCTION: The expanding use of 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) has resulted in an increased frequency of incidentally discovered areas of FDG uptake within the thyroid gland. In these incidentalomas, high malignancy rates are reported. The study aimed, on the one hand, to determine the prevalence in our setting of thyroid incidentalomas in patients with no previous history of thyroid cancer undergoing an FDG PET-CT as well as the risk of malignancy and, on the other hand, to evaluate the usefulness of the maximum standard uptake value (SUVmax) for detecting thyroid cancer. MATERIAL AND METHODS: The FDG PET-CT scans performed at our hospital between June 2013 and December 2020 were retrospectively reviewed. In those incidentalomas with sufficient additional investigation, a diagnosis of benign or malignant was established based on the complementary tests. RESULTS: From the 21,594 PET-CT scans performed, 398 (1.8%) patients had an incidental FDG uptake, either focal (n=324) or diffuse (n=74). Among incidentalomas with further investigation, the rate of malignancy was higher in patients with focal FDG uptake than in those with diffuse uptake (26.5% versus 4%, respectively, p<0.05). The SUVmax value was significantly lower in benign focal lesions (5.7 [range: 2.3-66] than in malignant ones 10.6 [range: 3.1-51.2]; p<0.05). Nearly a quarter of malignant diagnoses (23.3%) were related to potentially aggressive tumours. CONCLUSION: The high rate of malignant tumours found among PET-CT incidentalomas and the high proportion of aggressive tumours demonstrate the need for a standardised approach in the investigation of incidental focal FDG uptake in the thyroid gland.

A novel patient-derived 3D model recapitulates mantle cell lymphoma lymph node signaling, immune profile and in vivo ibrutinib responses.

Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Prediction of Smoking Habits From Class-Imbalanced Saliva Microbiome Data Using Data Augmentation and Machine Learning.

Human microbiome research is moving from characterization and association studies to translational applications in medical research, clinical diagnostics, and others. One of these applications is the prediction of human traits, where machine learning (ML) methods are often employed, but face practical challenges. Class imbalance in available microbiome data is one of the major problems, which, if unaccounted for, leads to spurious prediction accuracies and limits the classifier's generalization. Here, we investigated the predictability of smoking habits from class-imbalanced saliva microbiome data by combining data augmentation techniques to account for class imbalance with ML methods for prediction. We collected publicly available saliva 16S rRNA gene sequencing data and smoking habit metadata demonstrating a serious class imbalance problem, i.e., 175 current vs. 1,070 non-current smokers. Three data augmentation techniques (synthetic minority over-sampling technique, adaptive synthetic, and tree-based associative data augmentation) were applied together with seven ML methods: logistic regression, k-nearest neighbors, support vector machine with linear and radial kernels, decision trees, random forest, and extreme gradient boosting. K-fold nested cross-validation was used with the different augmented data types and baseline non-augmented data to validate the prediction outcome. Combining data augmentation with ML generally outperformed baseline methods in our dataset. The final prediction model combined tree-based associative data augmentation and support vector machine with linear kernel, and achieved a classification performance expressed as Matthews correlation coefficient of 0.36 and AUC of 0.81. Our method successfully addresses the problem of class imbalance in microbiome data for reliable prediction of smoking habits.

Opioids and Ocular Surface Pathology: A Literature Review of New Treatments Horizons.

This review discusses the role of opioids in the corneal surface and the different pathways and therapeutic methods of management. A literature review was performed using PubMed database. For the database search, the main searching words "opioid" and "topical opioid treatment" were used with the descriptors "cornea", "ocular surface", "neuropathic corneal pain", "corneal sensitivity" and "naltrexone"; original scientific articles and reviews were included to achieve the purpose of the review. The endogenous opioid system has relevant functions in the organism, and in daily use, opioids are used as painkillers. However, these drugs may be employed for other indications as opioid pathways have a wide spectrum. The corneal surface for topical treatment is easily accessible, hence sparing the side effects of systemic opioids. Instillation of opioid antagonist substances, such as naltrexone, increases corneal healing rates and stimulates the division of corneal epithelium cells without deleterious effects. The natural modulation of endogenous opioids controls different forms of pain, including inflammatory and neuropathic pain, both in the ocular surface and in the central nervous system. There are diverse methods in controlling pain using opioids, especially in refractory forms. This review attempts to collect the literature about corneal surface and opioid pathways to provide an overview image and a possible direction of the news treatments.

Is perioperative brachytherapy effective in carcinoma of the tongue?

Purpose: To analyze the results of patients treated with perioperative interstitial brachytherapy (ISBT) in tongue carcinoma (TC). Material and methods: From April 2009 to May 2015, 43 squamous cell carcinoma consecutive patients diagnosed with TC were treated with limited partial glossectomy and perioperative ISBT, using high-dose-rate (HDR). Twenty- seven patients were treated by brachytherapy (BT), and sixteen received BT as a complement to subsequent external beam radiotherapy (EBRT) after results of lymph node dissection. Median age was 66 years. Distribution by stage, included 10 patients stage I, 14 stage II, 10 stage III, and 9 stage IV. Eighteen patients had negative margins, nineteen margin involvement, and in six cases, the margin was < 5 mm. Results: With a median follow-up of 54 months, LC at 3 and 5 years was 87% and 84%, respectively. LC was 95% at five years in patients with clear margins, and 75% with involved margins. LC in N0 patients treated with BT was 83% at 5 years, and in patients N+ with posterior EBRT treatment, LC was 86%. By tumor size, we found one local relapse in 13 cases T1, in 5 of 27 patients T2 was found, and no local relapse T3 with LC of 87%, 70%, and 100% respectively at five years. Regional control (RC) was 81% at 3 and 5 years. We found a metastasis-free survival of 91% at 3- and 5-year. Twenty-three patients have died, 11 of them due to other causes, with overall survival of 56% at three years and 53% at five years. Conclusions: Combined treatment with conservative surgery and ISBT shows similar results to radical surgery or RT alone, allowing a more patient-tailored approach, with good organ function preservation and cosmetic outcomes.

Integrating the human microbiome in the forensic toolkit: Current bottlenecks and future solutions.

Over the last few years, advances in massively parallel sequencing technologies (also referred to next generation sequencing) and bioinformatics analysis tools have boosted our knowledge on the human microbiome. Such insights have brought new perspectives and possibilities to apply human microbiome analysis in many areas, particularly in medicine. In the forensic field, the use of microbial DNA obtained from human materials is still in its infancy but has been suggested as a potential alternative in situations when other human (non-microbial) approaches present limitations. More specifically, DNA analysis of a wide variety of microorganisms that live in and on the human body offers promises to answer various forensically relevant questions, such as post-mortem interval estimation, individual identification, and tissue/body fluid identification, among others. However, human microbiome analysis currently faces significant challenges that need to be considered and overcome via future forensically oriented human microbiome research to provide the necessary solutions. In this perspective article, we discuss the most relevant biological, technical and data-related issues and propose future solutions that will pave the way towards the integration of human microbiome analysis in the forensic toolkit.

High Incidence of Cataracts in the Follow-Up of Patients Undergoing Percutaneous Coronary Intervention for Chronic Coronary Total Occlusion.

Development of cataracts is a well-known adverse effect of ionizing radiation, but little information is available on their incidence in patients after other medical procedures, such as cardiac catheterizations. The study objective was to determine the incidence of cataracts in a cohort of patients undergoing percutaneous coronary intervention (PCI) for chronic coronary total occlusion (CTO) and its association with radiation dose. The study analyzed the incidence of cataracts during the follow-up of 126 patients who underwent chronic total coronary PCI, using Cox regression to identify predictive factors of cataract development. The study included 126 patients, 86.9% male, with a mean age of 60.5 years (range, 55.0-68.0 years). Twenty-three (18.2% n = 23) developed cataracts during a mean follow-up of 49.5 months (range 37.3-64.5 months). A higher incidence was observed in patients who received more than 5 Gy (29.0% vs. 14.7%, Hazard ratio (HR = 2.84 [1.19-6.77]). Multivariate analysis revealed a relationship between cataract development during the follow-up and a receipt of radiation dose >5 Gy (HR = 2.60, 95% confidence interval [CI 1.03-6.61]; p = 0.03), presence or history of predisposing eye disease (HR = 4.42, CI:1.57-12.40), diabetes (HR = 3.33 [1.22-9.24]), and older age, as in >57 (HR, 6.40 [1.81-22.61]). An elevated incidence of cataracts was observed in patients after PCI for CTO. The onset of cataracts is related to the radiation dose during catheterization, which is a potentially avoidable effect of which operators should be aware.

Estimating the Time Since Deposition of Saliva Stains With a Targeted Bacterial DNA Approach: A Proof-of-Principle Study.

Information on the time when a stain was deposited at a crime scene can be valuable in forensic investigations. It can link a DNA-identified stain donor with a crime or provide a post-mortem interval estimation in cases with cadavers. The available methods for estimating stain deposition time have limitations of different types and magnitudes. In this proof-of-principle study we investigated for the first time the use of microbial DNA for this purpose in human saliva stains. First, we identified the most abundant and frequent bacterial species in saliva using publicly available 16S rRNA gene next generation sequencing (NGS) data from 1,848 samples. Next, we assessed time-dependent changes in 15 identified species using de-novo 16S rRNA gene NGS in the saliva stains of two individuals exposed to indoor conditions for up to 1 year. We selected four bacterial species, i.e., Fusobacterium periodonticum, Haemophilus parainfluenzae, Veillonella dispar, and Veillonella parvula showing significant time-dependent changes and developed a 4-plex qPCR assay for their targeted analysis. Then, we analyzed the saliva stains of 15 individuals exposed to indoor conditions for up to 1 month. Bacterial counts generally increased with time and explained 54.9% of the variation (p = <2.2E-16). Time since deposition explained ≥86.5% and ≥88.9% of the variation in each individual and species, respectively (p = <2.2E-16). Finally, based on sample duplicates we built and tested multiple linear regression models for predicting the stain deposition time at an individual level, resulting in an average mean absolute error (MAE) of 5 days (ranging 3.3-7.8 days). Overall, the deposition time of 181 (81.5%) stains was correctly predicted within 1 week. Prediction models were also assessed in stains exposed to similar conditions up to 1 month 7 months later, resulting in an average MAE of 8.8 days (ranging 3.9-16.9 days). Our proof-of-principle study suggests the potential of the DNA profiling of human commensal bacteria as a method of estimating saliva stains time since deposition in the forensic scenario, which may be expanded to other forensically relevant tissues. The study considers practical applications of this novel approach, but various forensic developmental validation and implementation criteria will need to be met in more dedicated studies in the future.

Estudio epidemiológico sobre la práctica clínica de la psicoterapia psicoanalítica en España: pacientes y terapeutas / Epidemiological study on the clinical practice of psychoanalytic psychotherapy in Spain: patients and therapists

Sabemos poco sobre los dispositivos asistenciales de salud mental que tratan mediante psicoterapia psicoanalítica en España. Presentamos los resultados de un estudio epidemiológico que recoge la información proporcionada por 97 psicoterapeutas con orientación psicoanalítica pertenecientes a la Federación Española de Asociaciones de Psicoterapeutas (FEAP). Se describen los perfiles de los terapeutas, de los pacientes atendidos (n=1862) y de sus tratamientos. Los resultados son discutidos a la luz de otros estudios y representan un 7% de la actividad psicoanalítica de FEAP. (AU)

We do not know much about mental health care centers that treat through psychoanalytic psychotherapy in Spain. We present the results of an epidemiological study that collects the information provided by 97 psychoanalytic-oriented psychotherapists belonging to the Spanish Federation of Psychotherapist Associations (FEAP). Therapists’ profiles are described, as well as the patients attended (n=1862) and their treatments. The results are discussed in the light of other studies and represent 7% of the psychoanalytic activity of FEAP. (AU)

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαß+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors' laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

3D Model Characterization by 2D and 3D Imaging in t(14;18)-Positive B-NHL: Perspectives for In Vitro Drug Screens in Follicular Lymphoma.

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.