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Near-surface seafloor properties affect offshore mining and infrastructure engineering. Shallow seafloor sediments experience extremely low effective stress, and consequently, these sediments exhibit very low in-situ density, shear wave velocity, and shear stiffness. We combined data extracted from the literature with new laboratory and field results to develop a comprehensive understanding of shallow seafloor sediments. First, we explored the sediment-dependent self-compaction characteristics starting with the asymptotic void ratio at the interface between the water column and the sediment column. The asymptotic void ratio depends on the particle size and shape in coarse-grained sediments and on mineralogy and pore fluid chemistry in fine-grained clayey sediments; overall, the asymptotic void ratio correlates with the sediment-specific surface and compressibility. Second, we developed a fork-type insertion probe to measure shear wave velocity profiles with depth. Detailed data analyses confirm the prevalent role of effective stress on shear wave velocity Vs=α(σ′m/kPa)β, and the inverse relationship between α and β parameters reveals that electrical interactions alter the velocity profile only in very high specific surface area sediments at very low effective stress and shows that ray bending affects the computed velocities only in the upper few centimeters (for the probe geometry used in this study). Probe insertion causes excess pore fluid pressure and effective stress changes; the ensuing time-dependent diffusion detected through shear wave velocity changes can be analyzed to estimate the coefficient of consolidation. Shear wave velocity profiles and velocity transients after insertion provide valuable information for sediment preclassification and engineering design.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Leveraging spatial molecular information to construct a phylogeographic map of tumor evolution can reveal individualized growth trajectories with diagnostic and therapeutic potential. Integrative analysis of spatial multi-omic data from 31 colorectal specimens revealed simultaneous microenvironmental and clonal alterations as a function of progression. Copy number variation served to re-stratify microsatellite stable and unstable tumors into chromosomally unstable (CIN+) and hypermutated (HM) classes. Phylogeographical maps classified tumors by their evolutionary dynamics, and clonal regions were placed along a global pseudotemporal progression trajectory. Cell-state discovery from a single-cell cohort revealed recurring epithelial gene signatures and infiltrating immune states in spatial transcriptomics. Charting these states along progression pseudotime, we observed a transition to immune exclusion in CIN+ tumors as characterized by a novel gene expression signature comprised ofDDR1, TGFBI, PAK4,andDPEP1. We demonstrated how these genes and their protein products are key regulators of extracellular matrix components, are associated with lower cytotoxic immune infiltration, and show prognostic value in external cohorts. Through high-dimensional data integration, this atlas provides insights into co-evolution of tumors and their microenvironments, serving as a resource for stratification and targeted treatment of CRC.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer’s disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases., Zhang et al. identify and characterize supermeres as extracellular nanoparticles that exhibit unique biological and functional properties with potential prognostic and therapeutic value across distinct diseases.

Background Little is known about how benign prostatic hyperplasia (BPH) develops and why patients respond differently to medical therapy designed to reduce lower urinary tract symptoms (LUTS). The Medical Therapy of Prostatic Symptoms (MTOPS) trial randomized men with symptoms of BPH and followed response to medical therapy for up to 6 years. Treatment with a 5α-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (α-blocker) reduced the risk of clinical progression, while men treated with combination therapy showed a 66% decrease in risk of progressive disease. However, medical therapies for BPH/LUTS are not effective in many patients. The reasons for nonresponse or loss of therapeutic response in the remaining patients over time are unknown. A better understanding of why patients fail to respond to medical therapy may have a major impact on developing new approaches for the medical treatment of BPH/LUTS. Prostaglandins (PG) act on G-protein-coupled receptors (GPCRs), where PGE2 and PGF2 elicit smooth muscle contraction. Therefore, we measured PG levels in the prostate tissue of BPH/LUTS patients to assess the possibility that this signaling pathway might explain the failure of medical therapy in BPH/LUTS patients. Method Surgical BPH (S-BPH) was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy and underwent surgical intervention to relieve LUTS. Control tissue was termed Incidental BPH (I-BPH). I-BPH was TZ obtained from men undergoing radical prostatectomy for low-volume, low-grade prostatic adenocarcinoma (PCa, Gleason score ≤ 7) confined to the peripheral zone. All TZ tissue was confirmed to be cancer-free. S-BPH patients divided into four subgroups: patients on α-blockers alone, 5ARI alone, combination therapy (α-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on α-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF2α , PGI2 , PGE2 , PGD2 , and TxA2 ), quantitative polymerase chain reaction levels of mRNAs encoding enzymes within the PG synthesis pathway, cellular distribution of COX1 (PTGS1) and COX2 (PTGS2), and tested the ability of PGs to contract bladder smooth muscle in an in vitro assay. Results All PGs were significantly elevated in TZ tissues from S-BPH patients (n = 36) compared to I-BPH patients (n = 15), regardless of the treatment subgroups. In S-BPH versus I-BPH, mRNA for PG synthetic enzymes COX1 and COX2 were significantly elevated. In addition, mRNA for enzymes that convert the precursor PGH2 to metabolite PGs were variable: PTGIS (which generates PGI2 ) and PTGDS (PGD2 ) were significantly elevated; nonsignificant increases were observed for PTGES (PGE2 ), AKR1C3 (PGF2α ), and TBxAS1 (TxA2 ). Within the I-BPH group, men responding to α-blockers for symptoms of BPH but requiring prostatectomy for PCa did not show elevated levels of COX1, COX2, or PGs. By immunohistochemistry, COX1 was predominantly observed in the prostatic stroma while COX2 was present in scattered luminal cells of isolated prostatic glands in S-BPH. PGE2 and PGF2α induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that α-blockers that inhibit alpha-adrenergic receptors do not appear to inhibit PG stimulation of GPCRs in bladder muscle. Only patients who required surgery to relieve BPH/LUTS symptoms showed significantly increased tissue levels of PGs and the PG synthetic enzymes. Conclusions Treatment of BPH/LUTS by inhibition of alpha-adrenergic receptors with pharmaceutical α-blockers or inhibiting androgenesis with 5ARI may fail because of elevated paracrine signaling by prostatic PGs that can cause smooth muscle contraction. In contrast to patients who fail medical therapy for BPH/LUTS, control I-BPH patients do not show the same evidence of elevated PG pathway signaling. Elevation of the PG pathway may explain, in part, why the risk of clinical progression in the MTOPS study was only reduced by 34% with α-blocker treatment.

espanolEn el presente trabajo se tiene como objetivo disenar e implementar una herramienta tecnologica para el control y seguimiento de las buenas practicas agricolas en los pequenos agricultores de frutas y hortalizas en el Valle del Cauca. Se presenta el desarrollo de una investigacion del tipo aplicada, que se apoyo en la revision de la literatura sobre buenas practicas agricolas (BPA), el estudio de las entidades y estandares que existian para la certificacion de BPA, y encuestas los agricultores sobre el conocimiento y apropiacion de herramientas tecnologicas para facilitar las BPA. Se siguieron los pasos de metodologia de desarrollo de software ICONIX, que involucra las etapas de analisis de requerimientos, analisis y diseno preliminar, analisis detallado, codificacion y pruebas al sistema. Finalmente se desarrollo una aplicacion web facilitadora de las BPA que permite el seguimiento a las practicas agricolas, incluyendo un registro constante de las tareas que los agricultores realizan sobre sus cultivos, condicion indispensable en estos procesos de certificacion. La herramienta permite a los pequenos agricultores llevar un mejor manejo de la informacion de los procesos sobre los cultivos, tener disponibles consultas sobre la trazabilidad de los productos y el historico de los lotes de la finca. EnglishThe objective of this work is to design and implement a technological tool for the control and monitoring of good agricultural practices in small fruit and vegetable farmers in Valle del Cauca. It shows the development of applied research, supported by a literature review on good agricultural practices (GAP), a study of the entities and standards related to GAP certification, and surveys of farmers on the knowledge and appropriation of technological tools to facilitate GAP. The steps of the ICONIX software development methodology were followed, which involve the stages of requirements analysis, preliminary analysis and design, detailed analysis, coding, and system testing. Finally, a GAP facilitating web application was developed that allows monitoring of agricultural practices, including a constant record of the tasks that farmers perform on their crops, an essential condition in these certification processes. The application allows small farmers to better manage the information of the processes on the crops and have data available on the traceability of the products and the history of the farm's lands.

Extracellular small RNAs (sRNAs) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDLs) in mice were enriched with multiple classes of sRNAs derived from the endogenous transcriptome, but also from exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and nonhost species, the profiles of which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and that these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL binds to tDRs and other single-stranded sRNAs with strong affinity but did not bind to double-stranded RNA or DNA. Furthermore, we show that natural and synthetic RNA modifications influenced tDR binding to HDL. We demonstrate that reconstituted HDL bound to tDRs only in the presence of apoA-I, and purified apoA-I alone were able to bind sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, we conclude that HDL binds to single-stranded sRNAs likely through nonionic interactions with apoA-I. These results highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA interactions for therapeutic approaches to prevent or treat disease.

Objective Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes β-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance β-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). Methods Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and β-cell proliferation and β-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. Results EP3 blockade increased β-cell mass in db/db mice through enhanced β-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. Conclusions The current study suggests that EP3 blockade promotes β-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage., Highlights • Systemic blockade of EP3 in vivo in db/db mice increase beta-cell proliferation and mass. • EP3 blockade has no effect on alpha-cell proliferation. • EP3 blockade restores islet architecture and beta-cell identity genes in db/db mice. • Beta cell GLP-1 receptor expression is increased by EP3 blockade. • EP3 blockade activates the Nrf2 anti-oxidant pathway.

The Gut Cell Atlas (GCA), an initiative funded by the Helmsley Charitable Trust, seeks to create a reference platform to understand the human gut, with a specific focus on Crohn’s disease. Although a primary focus of the GCA is on focusing on single-cell profiling, we seek to provide a framework to integrate other analyses on multi-modality data such as electronic health record data, radiological images, and histology tissues/images. Herein, we use the research electronic data capture (REDCap) system as the central tool for a secure web application that supports protected health information (PHI) restricted access. Our innovations focus on addressing the challenges with tracking all specimens and biopsies, validating manual data entry at scale, and sharing organizational data across the group. We present a scalable, cross-platform barcode printing/record system that integrates with REDCap. The central informatics infrastructure to support our design is a tuple table to track longitudinal data entry and sample tracking. The current data collection (by December 2020) is illustrated with types and formats of the data that the system collects. We estimate that one terabyte is needed for data storage per patient study. Our proposed data sharing informatics system addresses the challenges with integrating physical sample tracking, large files, and manual data entry with REDCap.

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single cell-based RNA-seq of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) has not been previously compared to that of T1DN. We analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26fsTRAP; eNOS-/-; db/db mice and compared it to streptozotocin-induced T1DN in podocin-Cre; Rosa26fsTRAP; eNOS-/- mice utilizing Translating Ribosome Affinity Purification (TRAP) and RNA-seq. Over 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN compared to T1DN. TGF-β signaling pathway genes were upregulated while MAPK pathway genes were downregulated only in T2DN while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number diabetes-induced genes not previously been linked to podocyte injury were confirmed in both mouse and human DN. Differences and similarities in the podocyte translatome in T2DN and T1DN can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.

Ricinus communis is a plant that is characterized by its different applications, in addition to being a wild plant favors its inclusion in agroecological practices because it does not represent high costs for producers. The aim of the work was to evaluate the bacterial isolates of R. communis with respect to its inhibitory activity, chitinolytic, DNase, solubilization of phosphate, biofilm formation and growth promoting effect in Allium sativum and Medicago sativa. The collection of R. communis leaves was carried, which were washed and disinfected with sodium hypochlorite solution in order to isolate bacteria in nutritive agar. The isolates obtained were identified by PCR amplification with the oligonucleotides RM and RB and the amplified products were sequenced for identification. The following tests were carried out on each of the isolates: growth inhibition vs Fusarium oxysporum, chitinolytic activity, DNase, solubilization of phosphate, biofilm formation and growth evaluation in A. sativum and M. sativa. Ten isolates were obtained that were identified as Pseudomonas 50%, Enterobacter 30% and Bacillus 20%. Growth inhibition evaluations against F. oxysporum showed that Bacillus showed significant inhibitory activity. The total isolates were negative for chitinolytic activity, 50% were positive for DNase, 40% positive for solubilization of phosphate and for biofilm formation 25% promoted weak formation and 10% moderate. Both plant models showed an increase in the biomass of fresh weight and dry weight of their roots when they were inoculated with the bacterial consortium at 50%, 75% and 100%.

The novelty of this study was to investigate for the first time in literature the influence of Bombyx mori silkworm cocoon’s age on the properties of silk fibroin-based materials, during all stages of cocoon processing to obtain the fibroin film. The study started with the premise that the cocoon age could cause modifications on fibroin properties during processing and, consequently, a possible interference on the characteristics of the final product. Characterizations were performed using batches of cocoons produced in different years, named C0 (fresh cocoons) and C6 (six-year-old cocoons). The influence of cocoon’s aging was observed on dialyzed dispersion regarding the molecular weight, particle size, and conformation. C6 films showed a more crystalline structure and higher thermal resistance than C0 films. The findings reported in this work are relevant for the reproducibility of fibroin-based materials, and the cocoon age is a key factor that should be considered in the preparation of fibroin-based materials.

Background and aims Cardiovascular disease (CVD) is the leading cause of death in chronic kidney disease (CKD) patients, however, the underlying mechanisms that link CKD and CVD are not fully understood and limited treatment options exist in this high-risk population. microRNAs (miRNA) are critical regulators of gene expression for many biological processes in atherosclerosis, including endothelial dysfunction and inflammation. We hypothesized that renal injury-induced endothelial miRNAs promote atherosclerosis. Here, we demonstrate that dual inhibition of endothelial miRNAs inhibits atherosclerosis in the setting of renal injury. Methods Aortic endothelial miRNAs were analyzed in apolipoprotein E-deficient (Apoe−/-) mice with renal damage (5/6 nephrectomy, 5/6Nx) by real-time PCR. Endothelial miR-92a-3p and miR-489-3p were inhibited by locked-nucleic acid (LNA) miRNA inhibitors complexed to HDL. Results Renal injury significantly increased endothelial miR-92a-3p levels in Apoe−/-;5/6Nx mice. Dual inhibition of miR-92a-3p and miR-489-3p in Apoe−/−;5/6Nx with a single injection of HDL + LNA inhibitors significantly reduced atherosclerotic lesion area by 28.6% compared to HDL + LNA scramble (LNA-Scr) controls. To examine the impact of dual LNA treatment on aortic endothelial gene expression, total RNA sequencing was completed, and multiple putative target genes and pathways were identified to be significantly altered, including the STAT3 immune response pathway. Among the differentially expressed genes, Tgfb2 and Fam220a were identified as putative targets of miR-489-3p and miR-92a-3p, respectively. Both Tgfb2 and Fam220a were significantly increased in aortic endothelium after miRNA inhibition in vivo compared to HDL + LNA-Scr controls. Furthermore, Tgfb2 and Fam220a were validated with gene reporter assays as direct targets of miR-489-3p and miR-92a-3p, respectively. In human coronary artery endothelial cells, over-expression and inhibition of miR-92a-3p decreased and increased FAM220A expression, respectively. Moreover, miR-92a-3p overexpression increased STAT3 phosphorylation, likely through direct regulation of FAM220A, a negative regulator of STAT3 phosphorylation. Conclusions These results support endothelial miRNAs as therapeutic targets and dual miRNA inhibition as viable strategy to reduce CKD-associated atherosclerosis.

Summary Single-cell RNA sequencing data require several processing procedures to arrive at interpretable results. While commercial platforms can serve as “one-stop shops” for data analysis, they relinquish the flexibility required for customized analyses and are often inflexible between experimental systems. For instance, there is no universal solution for the discrimination of informative or uninformative encapsulated cellular material; thus, pipeline flexibility takes priority. Here, we demonstrate a full data analysis pipeline, constructed modularly from open-source software, including tools that we have contributed. For complete details on the use and execution of this protocol, please refer to Petukhov et al. (2018), Heiser et al. (2020), and Heiser and Lau (2020)., Graphical Abstract, Highlights • An open-source, containerized pipeline for processing scRNA-seq data • The provided Singularity and Conda files permit rapid software installation • Multiple methods detailed for matrix quality control are provided for modular usage • Data entry points are highlighted for flexibility with other pipelines, Single-cell RNA sequencing data require several processing procedures to arrive at interpretable results. While commercial platforms can serve as “one-stop shops” for data analysis, they relinquish the flexibility required for customized analyses and are often inflexible between experimental systems. For instance, there is no universal solution for the discrimination of informative or uninformative encapsulated cellular material; thus, pipeline flexibility takes priority. Here, we demonstrate a full data analysis pipeline, constructed modularly from open-source software, including tools that we have contributed.

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the natural factors that instigate this polarization are largely unknown. Here, we demonstrate that microbial small RNAs (msRNA) are enriched on LDL and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the ribonucleic acid sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids (nt-LNA) was found to prevent nLDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.

El sobrepeso y obesidad toma atención en estudios epidemiológicos y los resultados después de la cirugía. Se plantea como objetivo general determinar la relación entre el tiempo de ayuno y el estado nutricional en pacientes quirúrgicos. Estudio descriptivo y transversal, se realizó con 244 pacientes con cirugía abdominal y de pared (128 mujeres y 116 varones), con edades entre 15 – 65 años, seleccionados de manera no probabilística por conveniencia. Se revisó la historia clínica para medir el tiempo de ayuno en postoperatorio y al alta y, la evolución metabólica nutricional. Los resultados muestran a un 70,5% de pacientes con un ayuno < 24h, (14,8%; ayuno 24 a 36h); (14,7%; ayuno >36h) pacientes con sobrepeso (40,6%), obesos (15,2%), la estancia hospitalaria fue de 6,28 días como promedio. Se concluye que el ayuno se correlaciona con el IMC durante el posoperatorio y con el IMC durante el alta (p=0,000). Las variables sexo y ocupación también se asociaron al diagnóstico nutricional. Se requiere mayor atención al paciente con sobrepeso y obesidad en el perioperatorio. Palabras clave: Ayuno, desnutrición, periodo postoperatorio, obesidad, sobrepeso.

ObjectivesTo determine if plasma microbial small RNAs (sRNAs) are altered in patients with rheumatoid arthritis (RA) compared with control subjects, associated with RA disease-related features, and altered by disease-modifying antirheumatic drugs (DMARDs).MethodssRNA sequencing was performed on plasma from 165 patients with RA and 90 matched controls and a separate cohort of 70 patients with RA before and after starting a DMARD. Genome alignments for RA-associated bacteria, representative bacterial and fungal human microbiome genomes and environmental bacteria were performed. Microbial genome counts and individual sRNAs were compared across groups and correlated with disease features. False discovery rate was set at 0.05.ResultsGenome counts of Lactobacillus salivarius, Anaerobaculum hydrogeniformans, Staphylococcus epidermidis, Staphylococcus aureus, Paenisporosarcina spp, Facklamia hominis, Sphingobacterium spiritivorum, Lentibacillus amyloliquefaciens, Geobacillus spp, and Pseudomonas fluorescens were significantly decreased in the plasma of RA compared with control subjects. Three microbial transfer RNA-derived sRNAs were increased in RA versus controls and inversely associated with disease activity. Higher total microbial sRNA reads were associated with lower disease activity in RA. Baseline total microbial sRNAs were threefold higher among patients who improved with DMARD versus those who did not but did not change significantly after 6 months of treatment.ConclusionPlasma microbial sRNA composition is altered in RA versus control subjects and associated with some measures of RA disease activity. DMARD treatment does not alter microbial sRNA abundance or composition, but increased abundance of microbial sRNAs at baseline was associated with disease activity improvement at 6 months.

Background The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Results Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. Conclusions Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Background & Aims Countries endemic for parasitic infestations have a lower incidence of Crohn’s disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. Methods We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn’s-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. Results Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. Conclusions We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

INTRODUCTION AND OBJECTIVE:Approximately 30% of men with lower urinary tract symptoms (LUTS) are resistant to medical therapies and progress to surgical intervention. The pathological changes produ...

Extra-enteric infections by Blastocystis spp. have rarely been documented. Here, we report a case of extra-enteric blastocystosis in a patient with minimal cervicitis symptoms. A 47-year-old Hispanic female patient was attended in a primary health centre in Michoacan state, Mexico, for her routine gynaecological medical examination. As only symptom, she referred to a slight vaginal itching. The presence of several vacuolar-stages of Blastocystis spp. were identified by Papanicolaou staining; molecular identification was attempted by culture-PCR sequencing of a region of 18S gene from cervical and faecal samples obtained 2 months after cytological examination, even when patient declared that she tried self-medicating with vaginal ovules. Blastocystis ST1 was identified only in the faecal sample. The presence of Blastocystis spp. in the cervix of a patient with scarce symptomatology, demonstrates the extraordinary flexibility of this microorganism to adapt to new environments and niches.

Additional file 2: Supplementary Table 1. Cost of Sequencing for inDrop. Supplementary Table 2. Evaluation of two TruDrop libraries’ raw yield and quality in low-throughput sequencing run on the iSeq 100. Supplementary Table 3. 24 TruDrop libraries raw data yield and quality in combined high-throughput sequencing run on the NovaSeq. Supplementary Table 4. 37 inDrop library quality scores from TruDrop on NovaSeq and V2 on NextSeq. Supplementary Table 5. inDrop library alignment metrics from TruDrop on NovaSeq and V2 on NextSeq. Supplementary Table 6. Diversity of UMI’s and genes expressed for cells sequenced with the TruDrop structure.

Additional file 5: Supplementary file 3.

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

Additional file 1: Supplementary Figure 1. Comparison of amplification of TruDrop and inDrop V2 primers during library preparation. (A) Diagnostic qPCR amplification curves comparing performance of all TruDrop primer pairs to V2 primers, all performed on the same sample. (B) Ct values of A. Supplementary Figure 2. Comparison of the library size distributions for TruDrop and inDrop V2 structured libraries during library preparation. (A) A BioAnalyzer profile of the size distribution of a V2 structured library. The spikes at 35 bp and 10,380 bp are controls. The numbers above the profile indicate the timepoints at which the various peaks were measured. (B) A BioAnalyzer profile of the size distribution of a TruDrop structured library. The spikes at 35 bp and 10,380 bp are controls. The numbers above the profile indicate the timepoints at which the various peaks were measured. (C) Plot of the average library size as determined via a BioAnalyzer for inDrop V2 libraries and TruDrop libraries. The median value is marked with a dotted line and a 95% confidence interval for the median is shown. Supplementary Figure 3. Comparison of sequence alignment metrics of inDrop V2 on NextSeq and TruDrop on NovaSeq. (A) Plot of the percent of reads with Valid Barcodes in 11 inDrop V2 mouse libraries and 23 TruDrop mouse libraries. The Median value is marked with a dotted line and a 95% confidence interval for the median is shown. (B) Plot of the percent of reads that uniquely align to a section of the mouse genome for inDrop V2 libraries and TruDrop libraries. The median value is marked with a dotted line and a 95% confidence interval for the median is shown. (C) Plot of the percent of reads that contain valid cell barcodes and a transcript that uniquely aligns to a section of the mouse genome for inDrop V2 libraries and TruDrop libraries. The median value is marked with a dotted line and a 95% confidence interval for the median is shown. Supplementary Figure 4. Another comparison of cell types identified between inDrop V2 on NextSeq and TruDrop on NovaSeq. (A) t-SNE and (B) sc-UniFrac analysis as performed in Fig. 5.

Colesevelam is a bile acid sequestrant approved to treat both hyperlipidemia and type 2 diabetes, but the mechanism for its glucose-lowering effects is not fully understood. The aim of this study was to investigate the role of hepatic microRNAs (miRNAs) as regulators of metabolic disease and to investigate the link between the cholesterol and glucose-lowering effects of colesevelam. To quantify the impact of colesevelam treatment in rodent models of diabetes, metabolic studies were performed in Zucker diabetic fatty (ZDF) rats and db/db mice. Colesevelam treatments significantly decreased plasma glucose levels and increased glycolysis in the absence of changes to insulin levels in ZDF rats and db/db mice. High-throughput sequencing and real-time PCR were used to quantify hepatic miRNA and mRNA changes, and the cholesterol-sensitive miR-96/182/183 cluster was found to be significantly increased in livers from ZDF rats treated with colesevelam compared with vehicle controls. Inhibition of miR-182 in vivo attenuated colesevelam-mediated improvements to glycemic control in db/db mice. Hepatic expression of mediator complex subunit 1 (MED1), a nuclear receptor coactivator, was significantly decreased with colesevelam treatments in db/db mice, and MED1 was experimentally validated to be a direct target of miR-96/182/183 in humans and mice. In summary, these results support that colesevelam likely improves glycemic control through hepatic miR-182–5p, a mechanism that directly links cholesterol and glucose metabolism. NEW & NOTEWORTHY Colesevelam lowers systemic glucose levels in Zucker diabetic fatty rats and db/db mice and increases hepatic levels of the sterol response element binding protein 2-responsive microRNA cluster miR-96/182/183. Inhibition of miR-182 in vivo reverses the glucose-lowering effects of colesevelam in db/db mice. Mediator complex subunit 1 (MED1) is a novel, direct target of the miR-96/182/183 cluster in mice and humans.

Summary Single cell RNA sequencing is a revolutionary technique to characterize inter-cellular transcriptomics heterogeneity. However, the data are noise-prone because gene expression is often driven by both technical artifacts and genuine biological variations. Proper disentanglement of these two effects is critical to prevent spurious results. While several tools exist to detect and remove low-quality cells in one single cell RNA-seq dataset, there is lack of approach to examining consistency between sample sets and detecting systematic biases, batch effects and outliers. We present scRNABatchQC, an R package to compare multiple sample sets simultaneously over numerous technical and biological features, which gives valuable hints to distinguish technical artifact from biological variations. scRNABatchQC helps identify and systematically characterize sources of variability in single cell transcriptome data. The examination of consistency across datasets allows visual detection of biases and outliers. Availability and implementation scRNABatchQC is freely available at https://github.com/liuqivandy/scRNABatchQC as an R package. Supplementary information Supplementary data are available at Bioinformatics online.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

ObjectiveThe Latin American Network of Congenital Malformations: ReLAMC was established in 2017 to provide accurate congenital anomaly surveillance. This study used data from ReLAMC registries to quantify the prevalence of microcephaly from 2010 to 2017 (before, during and after the Zika virus epidemic).DesignNine ReLAMC congenital anomaly registries provided case-level data or aggregate data for any live births, still births or terminations of pregnancy with microcephaly. Births to pregnant women infected with Zika virus first occurred in Brazil in 2015, and in the remaining registry areas in 2016 with the exception of Chile that did not experience Zika virus. Therefore the prevalence of microcephaly for 2010–2014 and individual years 2015, 2016 and 2017 was estimated using multilevel random effect Poisson models. Clinical classification and characteristics of the cases were compared pre and post Zika for all centres providing individual case-level data.ResultsThe prevalence of microcephaly for all registries excluding Brazil was 2.3 per 10 000 (95% CI 2.0 to 2.6) for 2010–2014 rising to 5.4 (95% CI 4.8 to 6.0) in 2016 and 5.9 (95% CI 5.3 to 6.6) in 2017. Brazil had a prevalence of 0.6 per 10 000 (95% CI 0.5 to 0.6) in 2010–2014, rising to 5.8 (95% CI 5.6 to 6.1) in 2015, 8.0 (95% CI 7.6 to 8.3) in 2016 and then falling in 2017. Only 29 out of 687 cases of microcephaly were reported as congenital Zika syndrome in countries excluding Brazil.ConclusionsThe prevalence of microcephaly was influenced both by Zika causing congenital Zika syndrome and by increased reporting awareness.

Specific surface area is more informative than grain size for fine-grained sediments where Ss > 1 m2/g. In fact, specific surface area plays a central role in engineering properties and processes in fine-grained soils, including pore size and bioactivity; fabric, plasticity, and rheology; hydraulic and electrical conductivity; compressibility and residual friction angle; and all forms of coupled processes. This research advanced a dye adsorption method using digital image colorimetry implemented with smartphone technology. In particular, this research adopted a water-based approach to reach internal surfaces in platy phyllosilicates, selected short dye molecules to reduce the range of potential molecular contact area, and developed a physics-based adsorption model to analyze the complete data set to minimize the uncertainty in specific surface area determinations. The study involved fine-grained soils with distinct mineralogy and specific surface area (from 1 to 600 m2/g) and various cationic dyes and a protein to explore the effect of molecular size, shape, and pH. Crystal violet emerged as a reliable dye for soil characterization. Time-dependent measurements confirmed second-order kinetics and highlight the importance of adsorption time.

Cotton producers aim for optimal nitrogen (N) fertilizer application rates that are correctly timed. Supplemental N is only used at the growth stage when and where it can be most effective. One management strategy monitors N levels using leaf spectral reflectance throughout the growing season. However, when the canopy is sparse or absent, a mobile implement-mounted spectral sensor is subject to influence from the exposed soil and stubble background. The results are misleading readings. This exploratory research project evaluated two vegetation index (VI) algorithms. The optimal soil-adjusted vegetation index (OSAVI) and normalized difference vegetation index (NDVI) values were selectively trimmed for improving a commercial NDVI sensing system‘s ability to discriminate between plant biomass and the ground surface. Varying plant populations, especially in early growth, can lead to erroneous real-time readings during mobile applications when passing over exposed ground surfaces. The goal of this study was to improve the real-time monitoring of cotton N status, both spatially and at the field scale. A large-scale field experiment statistically established a distribution of small contiguous plots in rows. A plot unit was a combination of one of three seeding rates, one of three cotton varieties, and one of four N application rates. Plant height, leaf N, and VI values were collected and analyzed. Both VI algorithms were found to reduce the varying plant population effect on NDVI. Plant population affected raw NDVI values throughout the season, which confirmed the effect of soil background exposure on NDVI values. Research findings suggest that after applying the two algorithms and comparing results with the non-filtered data analysis, both algorithms detected the differences due to variety, seeding rate, and N rate treatments. However, the N effect was detected earliest in the season by NDVI.

BackgroundMale lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells.MethodsImmunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. surgical BPH (S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging system and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN.ResultsOPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression ofCXCL1,CXCL2,CXCL8,PTGS2andIL6in BHPrS-1, but not in epithelial cell lines.ConclusionsOPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Here, we engineered a new dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. We overcame the index-hopping issue, demonstrated significant improvements in base-calling accuracy, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior library structures. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Extracellular microRNAs (miRNAs) are a new class of biomarkers for cellular phenotypes and disease, and are bioactive signals within intercellular communication networks. Previously, we reported that miRNAs are secreted from macrophage to high-density lipoproteins (HDL) and delivered to recipient cells to regulate gene expression. Despite the potential importance of HDL-miRNAs, regulation of HDL-miRNA export from cells has not been fully studied. Here, we report that pancreatic islets and beta cells abundantly export miR-375-3p to HDL and this process is inhibited by cellular mechanisms that promote insulin secretion. Small RNA sequencing and PCR approaches were used to quantify beta cell miRNA export to HDL. Strikingly, high glucose conditions were found to inhibit HDL-miR-375-3p export, which was dependent on extracellular calcium. Likewise, stimulation of cAMP was found to repress HDL-miR-375-3p export. Furthermore, we found that beta cell ATP-sensitive potassium channel (KATP) channels are required for HDL-miRNA export as chemical inhibition (tolbutamide) and global genetic knockout (Abcc8−/−) approaches inhibited HDL-miR-375-3p export. This process is not likely associated with cholesterol flux, as gain-of-function and loss-of-function studies for cholesterol transporters failed to alter HDL-miR-375-3p export. In conclusion, results support that pancreatic beta cells export miR-375-3p to HDL and this process is inversely regulated to insulin secretion.

Cellular microRNAs (miRNA) have proven to be critical regulators of inflammatory gene expression across many pathways within autoimmunity. Circulating miRNAs serve as a new class of disease biomarkers. Nevertheless, the functional roles of miRNAs, particularly extracellular miRNAs, in systemic lupus erythematosus (SLE) remain poorly understood. Therefore, we aimed to link changes in extracellular miRNAs to lymphocyte gene regulation and the pathophysiology of SLE. Here, we demonstrate that circulating miR-22-3p levels are associated with SLE, and miR-22-3p regulates T and B cell function and SLE-associated kidney disease. Based on high-throughput small RNA sequencing and real-time PCR, extracellular miR-22-3p levels were found to be significantly increased in whole plasma in human SLE subjects. To determine the functional impact of miR-22-3p in SLE, miR-22-3p loss-of-function studies were performed in a mouse model of SLE (B6.SLE1.2.3). We found that in vivo administration of locked-nucleic acid inhibitors of miR-22-3p (LNA-22) reduced lymphocyte accumulation in both the spleen and lymph nodes compared to LNA scramble (LNA-Scr) control-treated mice. Strikingly, LNA-22-3p treatments reduced kidney disease pathology and glomerular IgG deposition compared to LNA-Scr treatments in SLE mice. Moreover, miR-22-3p inhibition reduced the proportion of T effector memory IFN-γ producing CD4+ T cells, suggesting that miR-22-3p regulates Th1 T cell differentiation. We also found that miR-22 inhibition in mice reduced STAT1 phosphorylation in the kidney which was correlated with loss of IFN-γ production by splenic CD4+ T cells. In conclusion, our findings suggest that miR-22-3p is a critical regulator of SLE-associated CD4+ T cell immunity and kidney disease. These results provide therapeutic potential for limiting splenic Th1 signaling and preventing the progression of lupus nephritis.Key FindingsExtracellular miR-22-3p levels are significantly increased in plasma from human SLE subjects.Inhibition of miR-22-3p in vivo significantly reduced lymphocyte accumulation in both the spleen and lymph nodes in a mouse model of SLE, thus reducing splenomegaly and lymphadenopathy.miR-22-3p inhibition significantly reduced IFN-γ expression and secretion from splenic T cell subsets.Inhibition of miR-22-3p in vivo resulted in decreased IgG deposition in the kidney, decreased STAT1 phosphorylation, and decreased kidney disease in a mouse model of SLE.

La presente investigación tuvo como objetivo evaluar la ecoeficiencia en la Municipalidad Distrital de La Peca, provincia de Bagua, departamento de Amazonas, Perú. Dicho trabajo se realizó en la Municipalidad del distrito La Peca a 484 msnm y se encuentra dentro de las coordenadas 5°36´6” S 78°26´06” O. Se realizó el inventario de equipos y dispositivos relacionados con el consumo de agua y energía eléctrica, se determinó el consumo de agua y energía eléctrica, se caracterizó los residuos sólidos generados y se identificó la ecoeficiencia respecto al consumo de energía eléctrica, agua, y manejo de residuos sólidos. Según el inventario que se realizó, en la municipalidad existen 2 grifos, 2 inodoros, un urinario, 75 equipos y dispositivos ofimáticos, 87 de iluminación y 5 de ventilación. El consumo mensual de agua (CM) es 44.448 m3 (44448 litros), y el consumo Per cápita diario (CPD) es 0.099 m3 (99 litros). El consumo de energía eléctrica más elevado fue en agosto del 2018, con 18.97 kWh/trabajador/mes, mientras que el menor fue en marzo, con 5.25 kWh/trabajador/mes. Los residuos sólidos recogidos durante los días de evaluación pesó 8.17 kg, el cual fueron caracterizados en residuos aprovechables (86 %), residuos no aprovechables (14 %), de acuerdo a este resultado el promedio diario y mensual fue de 1.17 kg/día (35.1 kg/mensual). La Municipalidad de la Peca presentó una ecoeficiencia regular en la energía eléctrica, ecoeficiencia óptima en el consumo de agua y deficiente en los residuos sólidos.

Atherosclerosis is a chronic inflammatory disease, and despite resounding success in lipid management to reduce cardiovascular events, most heart attacks in the US occur in patients with clinically normal LDL-C levels. The next generation of therapies for cardiovascular disease (CVD) will include inflammation-based targets; however, systemic immune suppression has limitations. Thus, the critical barrier for successful targeting of inflammation in CVD is the identification of vascular ligands that drive immune cell activation. Previously, we demonstrated that low-density lipoproteins (LDL) transport microRNAs. Here, we report that LDL traffic a wide-diversity of small non-coding RNAs (sRNA) in circulation, many of which are derived from exogenous species, e.g. bacteria and fungi. Moreover, using high-throughput sRNA sequencing, we found that LDL-sRNA signatures are influenced primarily by environmental microbiota, as opposed to commensal bacteria in the gut microbiome. Using a mouse model of compromised mucosal immunity, we identified a process by which LDL accumulates bacterial sRNAs. Next, we demonstrated that native LDL potently stimulated cytokine release from macrophages through activation of single-stranded RNA (ssRNA)-sensing toll-like receptor (TLR) signaling, which was blunted by partial silencing of ssRNA-sensing TLRs in macrophages. Most importantly, preliminary results in mice suggest that targeting ssRNA-sensing TLRs is a potential strategy to suppress atherosclerosis. Taken together, we put forth a novel paradigm in which, LDL scavenge microbial sRNAs to promote clearance during health, yet serve as TLR-ligands that propagate macrophage inflammation in hypercholesterolemia, which likely contributes to the underlying inflammation in the pathogenesis of CVD.

High-throughput small RNA sequencing (sRNA-seq) has facilitated the discovery of many classes of small RNAs (sRNA) and helped establish the field of extracellular RNA (exRNA). Although several tools are available for sRNA-seq analysis, exRNAs present unique analytical challenges that are not met by current software. Therefore, we developed a novel data analysis pipeline specifically for exRNAs entitled, Tools for Integrative Genome analysis of Extracellular sRNAs (TIGER). To demonstrate the power of this tool, sRNA-seq was performed on high-density lipoproteins (HDL), apolipoprotein B-containing particles (APOB), bile, urine, and liver samples collected from wild-type (WT) and scavenger receptor BI knockout (SR-BI KO) mice. TIGER was able to account for approximately 60% of reads on lipoproteins and >85% of reads in liver, bile, and urine, a significant advance compared to existing software, largely due to the identification of non-host sRNAs in these datasets. A key advance for the TIGER pipeline is the ability to analyze host and non-host sRNAs across many classes at the genome, parent RNA, and individual fragment levels. Moreover, disparate sample types were compared at each level using hierarchical clustering, correlations, betadispersions, principal coordinate analysis, and permutational multivariate analysis of variance. TIGER analysis was also used to quantify distinct features of exRNAs, including 5-prime microRNA (miRNA) variants, 3-prime miRNA non-templated additions, parent RNA positional coverage, and length distributions by RNA class. Results suggest that the majority of sRNAs on lipoproteins are non-host sRNAs derived from bacterial sources in the microbiome and environment, specifically rRNA-derived sRNAs from proteobacteria. Here, we report novel discoveries of lipoprotein sRNAs that were facilitated by the new sRNA-seq analysis pipeline, TIGER, which has tremendous applicability for the field of exRNA.