Droplet Microfluidics for the ex Vivo Expansion of Human Primary Multiple Myeloma Cells.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device We demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double layered bead composed by an inner layer of alginate and an outer layer of puramatrix constructed using a soft technology without the use of any external force. In this work, we use this micro manipulation technique to build a 3D scaffold as a biomimetic model to recapitulate the niche of patient-derived multiple myeloma cells (MM cell) using a multilayered 3D tissue scaffold constructed in a microfluidic device and cultured in 10% FBS culture medium. In the current study, we included the use of this biomimetic model comprising supporting human Mesenchymal stem cells to show the mid-term survival of MM cells in the proposed structures. We found that the generated microniches were suitable for the maintenance of MM cells with and without supporting cells. Additionally, cultured MM cells in droplets were exposed to both Bortezomib and Lenalidomide to test their toxicity in the cultured patient derived cells. Results indicate that the maintained MM cells were consistently responding to the applied medication, opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening.This research was funded by European Commission H2020 Marie Curie Research Grants Scheme MSCA-IF-GF (705163).S

DNA microarray analysis of genes differentially expressed in diet-induced (cafeteria) obese rats.

Objective: To better understand the molecular basis of dietary obesity, we examined adipose tissue genes differentially expressed in an obesity model using DNA microarray analysis. Research Methods and Procedures: We assessed the expression level of over 12,500 transcripts in epididymal fat pads from (cafeteria) obese and control rats with the aid of the array technology. Results: Cafeteria (obese) rats weighed 50% more and had 2.5-fold higher levels of epididymal fat and elevated levels of circulating leptin. Adipose genes differentially expressed in obese and control rats were categorized into five groups: macronutrient metabolism, transcription factors, hormone receptor and signal transduction, redox and stress proteins, and cellular cytoskeleton. Interestingly, the expression levels of a number of genes involved in lipid metabolism such as glycerol-3-phosphate dehydrogenase, stearoyl coenzyme A desaturase, together with the transcription factors implicated in adipocyte differentiation (CAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma), were significantly increased in obese animals compared with control. The most up-regulated transcripts were the ob (49.2-fold change) and the fatty acid-binding protein genes (15.7- fold change). In contrast, genes related to redox and stress protein were generally down-regulated in obese animals compared with the control. Discussion: Our study showed that in diet-induced obesity, the expression levels of some important genes implicated in lipid metabolism were up-regulated, whereas those related to redox and stress protein were down-regulated in obese animals compared with control. This pattern of gene expression may occur in human obesity cases after high-fat intake

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Application of Entropy and Fractal Dimension Analyses to the Pattern Recognition of Contaminated Fish Responses in Aquaculture

The objective of the work was to develop a non-invasive methodology for image acquisition, processing and nonlinear trajectory analysis of the collective fish response to a stochastic event. Object detection and motion estimation were performed by an optical flow algorithm in order to detect moving fish and simultaneously eliminate background, noise and artifacts. The Entropy and the Fractal Dimension (FD) of the trajectory followed by the centroids of the groups of fish were calculated using Shannon and permutation Entropy and the Katz, Higuchi and Katz-Castiglioni’s FD algorithms respectively. The methodology was tested on three case groups of European sea bass (Dicentrarchus labrax), two of which were similar (C1 control and C2 tagged fish) and very different from the third (C3, tagged fish submerged in methylmercury contaminated water). The results indicate that Shannon entropy and Katz-Castiglioni were the most sensitive algorithms and proved to be promising tools for the non-invasive identification and quantification of differences in fish responses. In conclusion, we believe that this methodology has the potential to be embedded in online/real time architecture for contaminant monitoring programs in the aquaculture industry

Hematological- and Immunological-Related Biomarkers to Characterize Patients with COVID-19 from Other Viral Respiratory Diseases

COVID-19 has overloaded health system worldwide; thus, it demanded a triage method for an efficient and early discrimination of patients with COVID-19. The objective of this research was to perform a model based on commonly requested hematological variables for an early featuring of patients with COVID-19 form other viral pneumonia. This investigation enrolled 951 patients (mean of age 68 and 56% of male) who underwent a PCR test for respiratory viruses between January 2019 and January 2020, and those who underwent a PCR test for detection of SARS-CoV-2 between February 2020 and October 2020. A comparative analysis of the population according to PCR tests and logistic regression model was performed. A total of 10 variables were found for the characterization of COVID-19: age, sex, anemia, immunosuppression, C-reactive protein, chronic obstructive pulmonary disease, cardiorespiratory disease, metastasis, leukocytes and monocytes. The ROC curve revealed a sensitivity and specificity of 75%. A deep analysis showed low levels of leukocytes in COVID-19-positive patients, which could be used as a primary outcome of COVID-19 detection. In conclusion, this investigation found that commonly requested laboratory variables are able to help physicians to distinguish COVID-19 and perform a quick stratification of patients into different prognostic categories

Clinical Score to Predict Recurrence in Patients with Stage II and Stage III Colon Cancer

Background: The prognosis of patients with stage II and stage III colon cancer is heterogeneous. Clinical and pathological characteristics, such as tumor budding, may help to further refine the recurrence risk. Methods: We included all the patients with localized colon cancer at Hospital Universitario La Paz from October 2016 to October 2021. We built a prognostic score for recurrence in the training cohort based on multivariate cox regression analysis and categorized the patients into two risk groups. Results: A total of 440 patients were included in the training cohort. After a median follow-up of 45 months, 81 (18%) patients had a first tumor recurrence. T4, N2, and high tumor budding remained with a p value <0.05 at the last step of the multivariate cox regression model for time to recurrence (TTR). We assigned 2 points to T4 and 1 point to N2 and high tumor budding. Forty-five percent of the patients were assigned to the low-risk group (score = 0). Compared to the high-risk group (score 1–4), patients in the low-risk group had a significantly longer TTR (hazard ratio for disease recurrence of 0.14 (95%CI: 0.00 to 0.90; p < 0.045)). The results were confirmed in the validation cohort. Conclusions: In our study, we built a simple score to predict tumor recurrence based on T4, N2, and high tumor budding. Patients in the low-risk group, that comprised 44% of the cohort, had an excellent prognosis

Differential neuroprotective effects of 5'-Deoxy-5'-Methylthioadenosine

Background: 59-deoxy-59-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. Methods: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson’s disease and epilepsy. Results: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson’s disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. Conclusion: MTA may potentially offer therapeutic neuroprotection

Novel genes and sex differences in COVID-19 severity.

Here we describe the results of a genome-wide study conducted in 11 939 COVID-19 positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (p < 5x10-8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (p = 1.3x10-22 and p = 8.1x10-12, respectively), and for variants in 9q21.32 near TLE1 only among females (p = 4.4x10-8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (p = 2.7x10-8) and ARHGAP33 (p = 1.3x10-8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, p = 4.1x10-8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥ 60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old