Macromolecular crowding in equine bone marrow mesenchymal stromal cell cultures using single and double hyaluronic acid macromolecules.

Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition in eukaryotic cell culture. Single hyaluronic acid (HA) molecules have not induced a notable increase in the amount and rate of deposited ECM. Thus, herein we assessed the physicochemical properties and biological consequences in equine bone marrow mesenchymal stromal cell cultures of single and mixed HA molecules and correlated them to the most widely used MMC agents, the FicollⓇ cocktail (FC) and carrageenan (CR). Dynamic light scattering analysis revealed that all HA cocktails had significantly higher hydrodynamic radius than the FC and CR; the FC and the 0.5 mg/ml 100 kDa and 500 kDa single HA molecules had the highest charge; and, in general, all molecules had high polydispersity index. Biological analyses revealed that none of the MMC agents affected cell morphology and basic cell functions; in general, CR outperformed all other macromolecules in collagen type I and V deposition; FC, the individual HA molecules and the HA cocktails outperformed CR in collagen type III deposition; FC outperformed CR and the individual HA molecules and the HA cocktails outperformed their constituent HA molecules in collagen type IV deposition; FC and certain HA cocktails outperformed CR and constituent HA molecules in collagen type VI deposition; and all individual HA molecules outperformed FC and CR and the HA cocktails outperformed their constituent HA molecules in laminin deposition. With respect to tri-lineage analysis, CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. This work opens new avenues in mixed MMC in eukaryotic cell culture. STATEMENT OF SIGNIFICANCE: Mixed macromolecular crowding (MMC) in eukaryotic cell culture is still under-investigated. Herein, single and double hyaluronic acid (HA) macromolecules, along with the traditional MMC agents FicollⓇ cocktail (FC) and carrageenan (CR), were used as MMC agents in equine mesenchymal stromal cell cultures. Biological analysis showed that none of the MMC agents affected cell morphology and basic cell functions. Protein deposition analysis made apparent that CR outperformed all other macromolecules in collagen type I and collagen type V deposition, whilst FC, the individual HA macromolecules and the HA cocktails outperformed CR in collagen type III deposition. Tri-lineage analysis revealed that CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. These data illustrate that MMC agents are not inert macromolecules.

Impact of COVID-19 on pediatric asthma-related healthcare utilization in New York City: a community-based study.

BACKGROUND: COVID-19 disproportionately affects families of low socioeconomic status and may worsen health disparities that existed prior to the pandemic. Asthma is a common chronic disease in children exacerbated by environmental exposures. METHODS: A cross-sectional survey was conducted to understand the impact of the initial stage of the pandemic on environmental and social conditions, along with access to care for children with asthma in New York City (NYC). Participants were recruited from a community-based organization in East Harlem and a nearby academic Pediatric Pulmonary clinic and categorized as having either public or private insurance (n = 51). RESULTS: Factors significantly associated with public compared to private insurance respectively were: increased reports of indoor asthma triggers (cockroach 76% vs 23%; mold 40% vs 12%), reduced income (72% vs 27%), and housing insecurity (32% vs 0%). Participants with public insurance were more likely to experience conditions less conducive to social distancing compared to respondents with private insurance, such as remaining in NYC (92% vs 38%) and using public transportation (44% vs 4%); families with private insurance also had greater access to remote work (81% vs 8%). Families with public insurance were significantly more likely to test positive for SARS-CoV-2 (48% vs 15%) but less likely to have gotten tested (76% vs 100%). Families with public insurance also reported greater challenges accessing office medical care and less access to telehealth, although not statistically significant (44% vs 19%; 68% vs 85%, respectively). CONCLUSIONS: Findings highlight disproportionate burdens of the pandemic, and how these disparities affect children with asthma in urban environments.

Capturing Wheat Phenotypes at the Genome Level.

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world's most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public-private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.

Variability of three activated clotting time point-of-care systems in cardiac surgery: reinforcing available evidence.

BACKGROUND: Cardiac surgery with extracorporeal circulation (ECC) requires the administration of anticoagulant drugs to maintain ACT ranges 400-600 seconds, which requires exhaustive coagulation monitoring for which various point-of-care devices are available. However, there is variability between them, so we aimed to compare the values in ACT measurement. METHODS: Simultaneous ACT measurements were performed with the Hemochron Response®, Hemostasis Management System Plus® (HMS Plus®) and Hemochron Signature® systems. RESULTS: A total of 255 simultaneous measurements were taken, the mean and standard deviation (SD) of each device were: Hemochron Signature® 361.1 seconds (SD: 156.9), HMS Plus® 412.8 seconds (SD: 180.9) and Hemochron Response® 422.8 seconds (SD: 187.9), being these differences statistically significant (Fridman's test p < 0.01). For comparisons the Bland-Altman method was used, resulting the Hemochron Response® has 61.7 seconds higher mean values than the Hemochron Signature®, the Hemochron Response® 10 seconds higher than the HMS Plus® and the HMS Plus® 51.7 seconds higher than the Hemochron Signature®. CONCLUSION: The differences found in comparisons are considered to be clinically relevant, which is why it is considered important to make the variability of the different monitoring systems known and to take them into account for optimal control of this parameter and its clinical repercussions.

Analyzing Modern Biomolecules: The Revolution of Nucleic-Acid Sequencing - Review.

Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.

Hyaluronic Acid as Macromolecular Crowder in Equine Adipose-Derived Stem Cell Cultures.

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll®, the most widely used macromolecular crowding molecules, in enhanced and accelerated collagen I, collagen III and collagen IV deposition.

BLVector: Fast BLAST-Like Algorithm for Manycore CPU With Vectorization.

New High-Performance Computing architectures have been recently developed for commercial central processing unit (CPU). Yet, that has not improved the execution time of widely used bioinformatics applications, like BLAST+. This is due to a lack of optimization between the bases of the existing algorithms and the internals of the hardware that allows taking full advantage of the available CPU cores. To optimize the new architectures, algorithms must be revised and redesigned; usually rewritten from scratch. BLVector adapts the high-level concepts of BLAST+ to the x86 architectures with AVX-512, to harness their capabilities. A deep comprehensive study has been carried out to optimize the approach, with a significant reduction in time execution. BLVector reduces the execution time of BLAST+ when aligning up to mid-size protein sequences (∼750 amino acids). The gain in real scenario cases is 3.2-fold. When applied to longer proteins, BLVector consumes more time than BLAST+, but retrieves a much larger set of results. BLVector and BLAST+ are fine-tuned heuristics. Therefore, the relevant results returned by both are the same, although they behave differently specially when performing alignments with low scores. Hence, they can be considered complementary bioinformatics tools.

Normothermic Regional Perfusion and Donation After Circulatory Death (Controlled and Uncontrolled): Metabolic Differences and Kidney Transplantation Evolution.

OBJECTIVE: To analyze metabolic differences during normothermic regional perfusion (NRP) between the dissimilar types of donation after circulatory death, uncontrolled (uDCD) and controlled (cDCD), and the evolution of the transplanted kidneys. METHODS: Observational, prospective, cohort study. We included patients from uDCD and cDCD maintained with NRP in 2017. Six consecutive blood gases were collected with determination of pH and lactic acid. Creatinine levels were monitored at 24 hours, 3 months, and 6 months after transplant and the need for renal replacement therapy was evaluated. Descriptive statistical analysis was performed, presenting the qualitative variables as frequencies and percentages, and quantitative as mean ± SD or median (interquartile range [IQR]). We used χ2 testing for bivariate analysis of qualitative variables. RESULTS: We collected 18 donors. Fifteen out of 18 (83.3%) were men with a median of 51 years (IQR, 46-60). Eleven out of 18 (61.1%) were cDCD and 7 out of 18 (38.9%) were uDCD. The blood gas results are illustrated in Table 1. A total of 28 renal transplants were obtained with a median age of 47 years (IQR, 45-57); 83% were male. Ten out of 28 (35.7%) came from uDCD and 18 out of 28 (64.7%) from cDCD. Table 2 shows the monitoring of the creatinine values of the recipients after the transplantation. CONCLUSIONS: There are more metabolic disorders in our series in uDCD organ donation compared with cDCD. The recovery of the renal function of organs from uDCD is slower than that of cDCD, however; the tendency is toward normality.

Preparation and Characterization of Tissue Surrogates Rich in Extracellular Matrix Using the Principles of Macromolecular Crowding.

Tissue engineering by self-assembly allows for the fabrication of living tissue surrogates by taking advantage of the cell's inherent ability to produce and deposit tissue-specific extracellular matrix. However, the long culture periods required to build a tissue substitute in conducive to phenotypic drift in vitro microenvironments result in phenotype and function losses. Although several biophysical microenvironmental modulators (e.g., surface topography, substrate stiffness, mechanical stimulation) have been used to address these issues, slow extracellular matrix deposition remains a limiting factor in clinical translation and commercialization of such therapies. Macromolecular crowding is an alternative in vitro microenvironment modulator that has been shown to accelerate extracellular matrix deposition by several orders of magnitude, thereby decreasing culture periods required for the development of an implantable device, while maintaining cell phenotype and function. Herein, we provide protocols for the production of tissue surrogates rich in extracellular matrix from human dermal fibroblasts, equine tenocytes, and equine adipose-derived stem cells using the principles of macromolecular crowding and the subsequent characterization thereof by means of immunofluorescent staining and complementary fluorescence intensity analysis.

Genetic dissection of agronomic and quality traits based on association mapping and genomic selection approaches in durum wheat grown in Southern Spain.

Climatic conditions affect the growth, development and final crop production. As wheat is of paramount importance as a staple crop in the human diet, there is a growing need to study its abiotic stress adaptation through the performance of key breeding traits. New and complementary approaches, such as genome-wide association studies (GWAS) and genomic selection (GS), are used for the dissection of different agronomic traits. The present study focused on the dissection of agronomic and quality traits of interest (initial agronomic score, yield, gluten index, sedimentation index, specific weight, whole grain protein and yellow colour) assessed in a panel of 179 durum wheat lines (Triticum durum Desf.), grown under rainfed conditions in different Mediterranean environments in Southern Spain (Andalusia). The findings show a total of 37 marker-trait associations (MTAs) which affect phenotype expression for three quality traits (specific weight, gluten and sedimentation indexes). MTAs could be mapped on the A and B durum wheat subgenomes (on chromosomes 1A, 1B, 2A, 2B and 3A) through the recently available bread wheat reference assembly (IWGSC RefSeqv1). Two of the MTAs found for quality traits (gluten index and SDS) corresponded to the known Glu-B1 and Glu-A1 loci, for which candidate genes corresponding to high molecular weight glutenin subunits could be located. The GS prediction ability values obtained from the breeding materials analyzed showed promising results for traits as grain protein content, sedimentation and gluten indexes, which can be used in plant breeding programs.

Hotspots in the genomic architecture of field drought responses in wheat as breeding targets.

Wheat can adapt to most agricultural conditions across temperate regions. This success is the result of phenotypic plasticity conferred by a large and complex genome composed of three homoeologous genomes (A, B, and D). Although drought is a major cause of yield and quality loss in wheat, the adaptive mechanisms and gene networks underlying drought responses in the field remain largely unknown. Here, we addressed this by utilizing an interdisciplinary approach involving field water status phenotyping, sampling, and gene expression analyses. Overall, changes at the transcriptional level were reflected in plant spectral traits amenable to field-level physiological measurements, although changes in photosynthesis-related pathways were found likely to be under more complex post-transcriptional control. Examining homoeologous genes with a 1:1:1 relationship across the A, B, and D genomes (triads), we revealed a complex genomic architecture for drought responses under field conditions, involving gene homoeolog specialization, multiple gene clusters, gene families, miRNAs, and transcription factors coordinating these responses. Our results provide a new focus for genomics-assisted breeding of drought-tolerant wheat cultivars.

Speeding-up Bioinformatics Algorithms with Heterogeneous Architectures: Highly Heterogeneous Smith-Waterman (HHeterSW).

The Smith-Waterman algorithm has a great sensitivity when used for biological sequence-database searches, but at the expense of high computing-power requirements. To overcome this problem, there are implementations in literature that exploit the different hardware-architectures available in a standard PC, such as GPU, CPU, and coprocessors. We introduce an application that splits the original database-search problem into smaller parts, resolves each of them by executing the most efficient implementations of the Smith-Waterman algorithms in different hardware architectures, and finally unifies the generated results. Using non-overlapping hardware allows simultaneous execution, and up to 2.58-fold performance gain, when compared with any other algorithm to search sequence databases. Even the performance of the popular BLAST heuristic is exceeded in 78% of the tests. The application has been tested with standard hardware: Intel i7-4820K CPU, Intel Xeon Phi 31S1P coprocessors, and nVidia GeForce GTX 960 graphics cards. An important increase in performance has been obtained in a wide range of situations, effectively exploiting the available hardware.

MC64-ClustalWP2: a highly-parallel hybrid strategy to align multiple sequences in many-core architectures.

We have developed the MC64-ClustalWP2 as a new implementation of the Clustal W algorithm, integrating a novel parallelization strategy and significantly increasing the performance when aligning long sequences in architectures with many cores. It must be stressed that in such a process, the detailed analysis of both the software and hardware features and peculiarities is of paramount importance to reveal key points to exploit and optimize the full potential of parallelism in many-core CPU systems. The new parallelization approach has focused into the most time-consuming stages of this algorithm. In particular, the so-called progressive alignment has drastically improved the performance, due to a fine-grained approach where the forward and backward loops were unrolled and parallelized. Another key approach has been the implementation of the new algorithm in a hybrid-computing system, integrating both an Intel Xeon multi-core CPU and a Tilera Tile64 many-core card. A comparison with other Clustal W implementations reveals the high-performance of the new algorithm and strategy in many-core CPU architectures, in a scenario where the sequences to align are relatively long (more than 10 kb) and, hence, a many-core GPU hardware cannot be used. Thus, the MC64-ClustalWP2 runs multiple alignments more than 18x than the original Clustal W algorithm, and more than 7x than the best x86 parallel implementation to date, being publicly available through a web service. Besides, these developments have been deployed in cost-effective personal computers and should be useful for life-science researchers, including the identification of identities and differences for mutation/polymorphism analyses, biodiversity and evolutionary studies and for the development of molecular markers for paternity testing, germplasm management and protection, to assist breeding, illegal traffic control, fraud prevention and for the protection of the intellectual property (identification/traceability), including the protected designation of origin, among other applications.

Next-generation sequencing and syntenic integration of flow-sorted arms of wheat chromosome 4A exposes the chromosome structure and gene content.

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding improved varieties to cope with increasing food demand under the conditions of changing global climate. However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated construction of an ordered gene map of chromosome 4A, embracing ≥85% of its total gene content, and have enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5 and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics between homoeologous chromosomes and syntenic chromosomal regions.

Next-generation bioinformatics: using many-core processor architecture to develop a web service for sequence alignment.

MOTIVATION: Bioinformatics algorithms and computing power are the main bottlenecks for analyzing huge amount of data generated by the current technologies, such as the 'next-generation' sequencing methodologies. At the same time, most powerful microprocessors are based on many-core chips, yet most applications cannot exploit such power, requiring parallelized algorithms. As an example of next-generation bioinformatics, we have developed from scratch a new parallelization of the Needleman-Wunsch (NW) sequence alignment algorithm for the 64-core Tile64 microprocessor. The unprecedented performance it offers for a standalone personal computer (PC) is discussed, optimally aligning sequences up to 20 times faster than the non-parallelized version, thus saving valuable time. AVAILABILITY: This algorithm is available as a free web service for the scientific community at http://www.sicuma.uma.es/multicore. The open source code is also available on such site.

[Characteristics and factors associated with mortality in patients receiving mechanical ventilation: first Chilean multicenter study]. / Características de los pacientes que reciben ventilación mecánica en unidades de cuidados intensivos: primer estudio multicéntrico chileno.

BACKGROUND: The outcome of mechanically ventilated patients can be influenced by factors such as the indication of mechanical ventilation (MV) and ventilator parameters. AIM: To describe the characteristics of patients receiving MV in Chilean critical care units. MATERIAL AND METHODS: Prospective cohort of consecutive adult patients admitted to 19 intensive care units (ICU) from 9 Chilean cities who received MV for more than 12 hours between September lst, 2003, and September 28th, 2003. Demographic data, severity of illness, reason for the initiation of MV, ventilation modes and settings as well as weaning strategies were registered at the initiation and then, daily throughout the course of MV for up to 28 days. ICU and hospital mortality were recorded. RESULTS: Of 588 patients admitted, 156 (26.5%) received MV (57% males). Mean age and Simplified Acute Physiology Score-II (SAPS II) were 54.6+/-18 years and 40.6+/-16.4 points respectively The most common indications for MV were acute respiratory failure (71.1%) and coma (22.4%). Assist-control mode (71.6%) and synchronized intermittent mandatory ventilation (SIMV) (14,2%) were the most frequently used. T-tube was the main weaning strategy. Mean duration of MV and length of stay in ICU were 7.8+/-8.7 and 11.1+/- 14 days respectively. Overall ICU mortality was 33.9% (53 patients). The main factors independently associated with increased mortality were (1) SAPS II > or =60 points (Odds Ratio (OR), 10.5; 95% CI, 1.04-106.85) and (2) plateau pressure > or =30 cm H2O at second day (OR, 3.9; 95% CI, 1.17-12.97). CONCLUSIONS: Conditions present at the onset of MV and ventilator management were similar to those reported in the literature. Magnitude of multiorgan dysfunction and high plateau pressures are the most important factors associated with mortality.

Características de los pacientes que reciben ventilación mecánica en unidades de cuidados intensivos: primer estudio multicéntrico chileno / Characteristics and factors associated with mortality in patients receiving mechanical ventilation

Background: The outcome oí' mechanically ventilated patients can be inñuenced byfactors such as the indication of mechanical ventilation (MV) and ventilator parameters. Aim: To describe the characterístics of patients receiving MV in Chilean critical care uníts. Material and methods: Prospective cohort of consecutive adult patients admitted to 19 intensive care uníts ([CU) from 9 Chilean cities who received MV for more than 12 hours between September lst, 2003, and September 28th, 2003. Demographic data, severity of illness, reason for the initiation of MV, ventilation modes and settings as well as weaning strategies were registered at the initiation and then, daily throughout the course of MV for up to 28 days. ¡CU and hospital mortality were recorded. Resulte: Of 588 patients admitted, 156 (26.5 percent) received MV (57 percent males). Mean age and Simplified Acute Physiology Score-II (SAPSII) were 54.6±18years and 40.6±16.4 points respectively The most common indications for MV were acute respiratory failure (71.1 percent) and coma (22.4 percent). Assist-control mode (71.6 percent) and synchronized intermittent mandatory ventilation (SIMV) (14,2 percent) were the most frequently used. T-tube was the main weaning strategy. Mean duration of MV and length of stay in ICU were 7.8±8.7 and 11.1± 14 days respectively. OverallICUmortality was 33.9 percent (53patients). The main factors independently associated with increased mortality were (1) SAPS II ≥ 60 points (Odds Patio (OR), 10.5; 95 percent CI, 1.04-106.85) and (2) plateaupressure ≥ 30 cm Hfi atsecond day (OR, 3.9; 95 percent CI, 1.17-12.97). Conclusions: Conditionspresent at the onsetofMVand ventilator management were similar to those reported in the literature. Magnitude ofmultiorgan dysfunction and high plateau pressures are the most important factors associated with mortality).