Analysis of biodiversity in natural environments based on environmental DNA (eDNA) has been applied to a wide range of ecosystems and species. The combination of high-throughput sequencing technologies and eDNA analysis is a powerful tool that enables comprehensive non-invasive monitoring of species present in the environment. Quantitative data of the eDNA from each species is essential for understanding species abundance but until recently required individual assays targeting each species. Recently developed quantitative sequencing (qSeq) allows simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. The eDNA extracted from aquaria with five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus) across 4 days was quantified by microfluidic digital PCR using a TaqMan probe and qSeq. The eDNA abundance quantified by qSeq was consistent with dPCR for each fish species at each sampling time. However, the relative abundances of sequences obtained from high throughput sequencing did not follow the same trend as the quantitative analyses, probably due to different PCR amplification efficiencies for each species. The correlation coefficients between qSeq and dPCR were 1.052, 1.074, and 1.114 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA. The application of qSeq to eDNA of other species will provide comprehensive quantitative data that could deepen our knowledge of natural ecosystems.

N6-methylated adenosine (m6A) and N1-methylated adenosine (m1A) are two epi-transcriptomic modifications on eukaryotic mRNA which have recently been rediscovered and are generating considerable interest. M6A methylation impacts on all aspects of cellular RNA metabolism and numerous physiological processes. Although less abundant than the m6A epitranscriptomic mark, m1A methylation has recently also attracted interest due to its dynamic nature in response to physiological changes. We investigated the role of the m6A and m1A methylation regulators on the expression of a transgene in Chinese Hamster Ovary (CHO) cells - the host cell of choice in producing biopharmaceutical proteins commercially. Using siRNA-mediated gene depletion and methylation-specific RNA immunoprecipitation with anti-m6A or m1A-antibodies, we show that (i) knock-down of the m6A ‘reader’ YTHDF2 or the m1A ‘eraser’ ALKBH3 dramatically impacts transgene expression; (ii) the effects of YTHDF2 and ALKBH3 depletion on transgene expression are m6A- and m1A-mediated. We conclude that the expression of transgenes in CHO cells can be subjected to regulation by both m6A and m1A regulators. These findings open up the prospect of previously unexplored epi-transcriptomic-based approaches to CHO cell line engineering for improved recombinant protein production.

The cleavage of heparin by heparin lyases showed great potential as a cost-effective and innoxious method for producing heparin with low molecular weight (LMWH). One of the most studied and sought heparin lyase is heparinase I (HepI). However, the industrial use of HepI was largely hampered by its low specific activity and thermal stability. In this article we describe increasing in specific heparinase I activity by stepwise site-directed mutagenesis. Thus after two cycles of mutagenesis, we obtained mutant heparinase I Flavobacterium heparinum with significantly increased specific activity (25%).

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell selection. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.

Objectives: To investigate how surgeons interpret the ATA 2015 and BTA 2014 guidelines for low risk well differentiated thyroid cancers (LRDTCs) and how they impact patient experiences across the UK. Design: Three nationally disseminated anonymised questionnaires. Setting: A nationwide snapshot of LRDTC management. Participants: Thyroid surgeons and their respective thyroid cancer multidisciplinary teams (MDTs) and thyroid cancer patients. Main outcome measures: The outcomes of interest were how surgeons/MDTs are managing LRDTCs and patient perspectives on ‘shared-decision-making’ and their ideal surgical management for LRDTCs. Results: 74 surgeons responded. 88% utilised BTA guidelines to assess recurrence risk. Tumour size, histology, stage T3b and central nodal involvement were important for >85%, but age (>45 years) only for 50%. In T1 (2cm), Thy5 solitary nodule, 58% supported hemi-thyroidectomy (HT), with 33% for total thyroidectomy (TT). In T2 (3cm) PTC, 54% opted for TT, with 24% favouring HT. Over 90% recommended TT for any incidentally excised microscopically positive lymph nodes. In T1a(m) multifocal micro-PTC, 63% suggested HT, but with contralateral benign nodules, 66% supported TT. 40% of patients felt ‘pros and cons’ of different managements were not fully explained. 47% felt they didn’t have significant input in their management, with 53% feeling final management was clinician’s choice. 60% preferred TT, with 80% wanting to ensure there was no cancer left and avoid recurrence. 20% preferred HT, with 46% wishing to avoid lifelong thyroxine. Conclusions: There is variation in risk assessment and management of LRDTCs nationally, with contrasting views of optimum treatment between patients and clinicians. These variations in practice are affecting patient experiences nationally.

The high genetic diversity of rear-edge refugia has been predicted to result from repeated retreats of species’ ranges to low latitudes during glacial periods in the course of Quaternary climate change. However, a few recent empirical studies of cold tolerant plants revealed an opposite pattern. We investigate whether current populations of the cold-adapted and range-restricted Bupleurum euphorbioides in the Baekdudaegan, South Korea and North Korea, could be interglacial refugia, and document how their rear-edge populations differ genetically from those of typical temperate species. Phylogeographic analysis and ecological niche modeling (ENM) were used. Genetic structure was analyzed using microsatellite markers and chloroplast DNA sequences. The congener B. longiradiatum was included as a typical temperate plant species. Despite having almost identical life history traits, these congeneric species exhibited contrasting patterns of genetic diversity. ENM revealed a wide range expansion along the Korean mountains to northern Far East Russia (Primorsky) during the Last Glacial Maximum, but not during the last interglacial. Thus, we hypothesize that B. euphorbioides retreated to refugia following maximum range contraction during interglacial periods. Unlike populations in the central region, the rear-edge populations were genetically impoverished and uniform, both within populations and in pooled regional populations. The rear-edge has endured at least one past interglacial, contributing to the species’ genetic diversity. We believe that such genetic variation in the cold-adapted B. euphorbioides gives it the necessary adaptations that will enable it to survive an upcoming favorable environment (the next glacial), unless there is artificial environmental change.

Herein, we report an unusual case of metallic foreign body consisting of blades of scissors in the maxillary sinus for about 40 years. Even though there are potential risk for sinus infection, not all retained foreign body require prompt removal.

Purpose: High altitude pulmonary edema (HAPE) has rapid onset and development, and may be life-threatening if not treated in time. It is important to correctly identify and recognize the characteristics of HAPE and to provide timely treatment. We aimed to summarize the characteristics of patients with HAPE by analyzing their general information, clinical symptoms, examination and laboratory results. Methods: 429 HAPE patients were enrolled in our study, and 200 of them were divided into three groups according to the period from their arrival in Naqu to the onset of the disease. We collected and analyzed the demographic information, results of laboratory tests, imaging and electrocardiography of all the participants at admission. The patients’ results of laboratory tests and imaging at admission were compared with those at discharge. The results of blood routine were compared among different groups. Results: Most of the HAPE patients were male (90.21%). The average white blood cell and neutrophil counts, alanine aminotransferase and aspartate aminotransferase levels, uric acid level, lactic dehydrogenase and creatine kinase levels were increased among HAPE patients at admission. The counts of white blood cell, neutrophil and lymphocyte, and the concentration of hemoglobin in HAPE patients at admission were higher than those at discharge (p<0.05).The counts of white blood cell and neutrophil were higher in the patients who developed the disease within 1 day than in those who developed the disease in more than 7 days (p<0.05). Conclusion: The proportion of males in HAPE patients was higher. Hepatocyte and myocardium in HAPE patients were more likely to be damaged. The white blood cell and neutrophil counts were significantly higher than normal range, and their increment was reduced as the incubation period extended.

Immune tissue associated with the major histocompatibility complex (MHC) is widely present in vertebrates highly polymorphic gene cluster. However, available published data about how amphibians MHCI genes react to pathogen infections are very few. The present study reports MHCI molecule in Rana dybowskii, and its differential expression in immunologically relevant tissues post-infection with lipopolysaccharide (LPS). The results showed that cDNA sequence of MHCIα contained 1047bp nucleotides encoding putative 348 amino acids. The phylogenetic analysis exhibited its evolutionary conservation within amphibians and formed a different clade with vertebrates. Morover, quantitative reverse transcription PCR analyses demonstrated that the MHCI gene was transcribed in the seven tested tissues, and analysis of immunologically relevant tissues of MHCI gene from the infected Rana dybowskii exhibited differential transcriptional activities. The expression of MHCI in the heart, liver, spleen, lung, kidneys, skin and muscular reached peak levels at 72, 24, 48, 12, 12, 12 and 72h post-infection(hpi), respectively. These findings indicate that MHCI is an indispensable part of the immune system. This is one of the first studies to investigate MHCIα in Rana dybowskii and can provide the foundation for further study of the immune function of MHC molecules in anti-infection.

An underdeveloped but potentially valuable molecular method in ecology is the ability to quantify the frequency with which foraging pollinators carry different plant pollens. Thus far, DNA metabarcoding has only reliably identified the presence/absence of a plant species in a pollen sample, but not its relative abundance in a mixed sample. Here we use a system of four congeneric, co-flowering plants in the genus Clarkia and their bee pollinators to (1) develop a molecular method to quantify different Clarkia pollens found on foraging bees; and (2) determine if bee pollinators carry Clarkia pollens in predictable ways, based on knowledge of their foraging behaviors. We develop a molecular method we call quantitative amplicon sequencing (qAMPseq) which varies cycling number (20, 25, 30, and 35 cycles) in polymerase chain reaction (PCR), individually indexing the same samples in different cycle treatments, and sequencing the resulting amplicons. These values are used to approximate an amplification curve for each Clarkia species in each sample, similar to the approach of quantitative PCR, which can then be used to estimate the relative abundance of the different Clarkia species in the sample. Using this method, we determine that bee visitation behaviors are generally predictive of the pollens that bees carry while foraging. We also show that some bees carry multiple species of Clarkia at the same time, indicating that Clarkia likely compete via interspecific pollen transfer. In addition to adding a ‘missing link’ between bee visitation behavior and actual pollen transfer, we suggest qAMPseq as another molecular method to add to the developing molecular ecology and pollination biology toolbox.

Heterologous production of 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new ganoderic acid (GA) 3,28-dihydroxy-lanosta-8, 24-dien-26 oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by LC-MS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmid pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR and hygromycin resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418 resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work not only enriches the library of GAs and GA biosynthetic enzymes, but also helps to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum.

Many salmonid species exist in highly structured and isolated populations, and are susceptible to habitat fragmentation and disturbances. Gila Trout (Oncorhynchus gilae) is a threatened species found in the Southwestern United States. Gila trout is managed to preserve remnant populations (i.e. lineages) distributed across a fragmented landscape. We evaluated genomic variation within and among remaining lineages of Gila Trout using RADseq to assess how drift and selection have structured populations using neutral and outlier loci. We also examined whether a signature of hybridization was evident in relict populations. Despite Gila Trout lineages being significantly differentiated and highly structured with low effective population sizes, we found that most lineages maintained genomic diversity and were potentially locally adapted. Hybridization with non-native Rainbow Trout (O. mykiss) was not detected in any lineage. Some lineages may have experienced recent population bottlenecks perhaps associated with mortality from drought and severe wildfires. Current management strategies should be reevaluated and adapted to better account for long-term effects of climate change. Specifically, we suggest reconnecting some populations via dendritic stream networks to facilitate natural dispersal in a metapopulation context. This would allow natural genetic mixing on the landscape and potentially increase adaptive potential. Furthermore, genetic rescue should be implemented to preserve integrity of the unique Spruce Creek lineage that is currently compromised by extremely low diversity.

Introduction: muscle pain/fibromyalgia (FM) is common among individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We recently demonstrated that during orthostatic stress testing, adults with ME/CFS reported increased pain. In the current study, we hypothesized that pain pressure thresholds (PPT) would decrease and temporal summation (windup) would increase after head-up tilt testing (HUT), and that the presence of co-morbid FM would be associated with greater change in both measures. Methods and Results: 248 ME/CFS patients (164 with FM and84 without FM), and 22 healthy controls (HC) were analyzed. In HC there were no significant differences in PPT between pre- and post-HUT (finger: from 4.7(1.6) to 4.4(1.5); shoulder: from 2.8(1.0) to 2.9(1.0)). In ME/CFS patients with and without FM, a significant decrease in PPT post-HUT was found compared to HC (both p<0.0001). Patients with FM had a lower PPT pre- and post-HUT (finger: from 2.0(0.9) to 1.5(0.8); shoulder: from 1.2(0.5) to 1.0(0.5) compared to patients without FM (finger: from 5.0(1.6) to 3.3(1.5); shoulder: from 2.2(0.9) to1.9(1.0) (p ranging from 0.001 to <0.0001). In contrast to HC in ME/CFS patients windup was increased compared to HC pre-HUT (both p<0.0001), but did not significantly change post-HUT. Conclusions: Pressure pain threshold decreased in ME/CFS patients with or without fibromyalgia after head-up tilt test (HUT), compared to healthy controls. Windup pre-and post-HUT was significantly higher compared to healthy controls, but did not change from pre- to post-HUT. These results demonstrate that, like exercise, orthostatic stress can negatively influence the physiology of pain perception in ME/CFS.

This work aimed to test innovations for the diagnosis of agricultural properties at Rio do Peixe Watershed, to locate erosions and to correct them by changing soil management, aiming at recovering degraded areas, rehabilitating them and promoting conservation agriculture, evaluating the results by remote sensing and water quality indicators. In 2019, it turned twelve years of inspection/monitoring at Rio do Peixe Watershed. From 2007 to 2017, using the Conventional CDA methodology, 14,076 ha were inspected at Vera Cruz sector, 94 properties were notified and in Ocauçu, 82 properties, located in 9,027 ha. As a work strategy, in Marília, the Innovative CDA Methodology was used, which allowed the inspection and rehabilitation of 52 properties in 27,775 ha, from 2017 to 2018. After the notifications, the owners presented the conservationist technical projects for each property, which were implemented, using conservation practices such as improving vegetation cover and crop rotation to control laminar erosion and agricultural terracing, divergent channels and containment basin to control gullies erosions. This work promoted a transformation from degrading agriculture to conservation agriculture, having degraded pastures transformed into an agricultural area, implementing the No-tillage system. Pastures were recovered by implementing the Integrated Crop-Livestock System; it was possible to increase the occupancy rate by 31% comparing to the original situation. This is a great work that benefits Watershed farmers, increasing productivity and consequently the profit, as well as for the local people improving the quality of water that supplies the region of Marília.

Background: Early extubation after cardiac surgery results in better postoperative outcomes but the optimal time for extubation remains unclear. Premature extubation may lead to unplanned reintubation that may result in adverse outcomes. The present study was undertaken to compare preoperative and operative risks and outcomes of patients with prolonged intubation >48 hours and reintubation after cardiac surgery. Aims: This is a retrospective chart review of 1259 patients who had cardiac surgery at a community hospital. After excluding patients with either operating room death, extubation <48 hours without reintubation or extubation >48 hours with reintubation, the final sample included 83 patients (6.6%) requiring reintubation after extubation in <48 hours and 100 (8.0%) with prolonged intubation without requiring reintubation. Results: Bivariate analyses revealed few statistically different preoperative and operative risks between patients with reintubation and prolonged intubation. Reintubation patients were older (p = .033) and had lower body mass index (p = .000), higher preoperative hematocrit (p = .021), and more chronic kidney disease stages >2 (p = .046) but lower odds for intra-aortic balloon pump (p = .006) and emergency surgery (p = .005). Reintubation led to worse postoperative outcomes than prolonged intubation: more acute kidney injury stage 1-3 (p = .014), coma/encephalopathy (p = .004), postoperative transfusion (p = .003), increased intensive care unit length of stay (p = .001) and hospital mortality (p = .007). Based on binary and ordinal logistic regression analyses, the differences in preoperative and operative risks were either inconsistent or trivial contributors and reintubation appears to make the largest independent contribution to poor postoperative outcomes. Conclusion: While early extubation remains the goal, patients with marginal weaning readiness may benefit from more recovery time before extubation. This study suggests that premature extubation may increase unplanned reintubation that could adversely affect postoperative outcomes after cardiac surgery.

Vitamin D insufficiency is closely related to various kinds of metabolic diseases. Acted as a marker of vitamin D status, 25-HydroxyvitaminD3 detection possesses important practical significance. In this study, highly sensitive fluorescent detection of 25-HydroxyvitaminD3 using truncated affinity-improved aptamers were developed, based on fluorescence intensity changes of PicoGreen (PG) generated upon binding to double-stranded DNA (dsDNA) formed through hybridization of aptamer and corresponding complementary strand. Four truncated aptamers were obtained by intercepting the small hairpin loop as the functional domain and retaining double helix structural domains of different lengths that exist in the selected original 25-HydroxyvitaminD3 aptamer. Under the optimized PG concentration, we conducted comparison experiments for affinity and specificity of these four truncated aptamers. Among them, the shortest aptamer with only 21 bp, D3-4, was found to show the highest affinity and specificity to 25-HydroxyvitaminD3, with the limit of detection of 0.04μg/mL, which solved the problem that original long aptamer could not applied for this fluorescent detection of 25-HydroxyvitaminD3. The truncated 25-HydroxyvitaminD3-specific aptamer with highly enhanced affinity performs promising application in sensitive detection of 25-HydroxyvitaminD3.

Methods This prospective observational study included 44 patients who underwent isolated ASD closures in the department of cardiac surgery at the National Heart Foundation Hospital and Research Institute (NHFH & RI) from July 2014 to June 2016. After fulfilling the enrolment criteria, participants were divided into two groups.

Tissue engineering (TE) constitutes a multidisciplinary scientific discipline focused on the construction of artificial tissues to regenerate end-stage organs. The impact of TE has led to a clinical revolution since novel therapies are available to attend several conditions. In this sense, its onset has supposed the communication of innovative discoveries in the age of social and mass media. This study aims to evaluate the global online dimension of TE from 2012 to 2018 by using data from the Web of Science (WoS) and Altmetrics. We have analysed 23,719 documents through descriptive and statistical methodologies. First, the descriptive analysis showed the evolution of TE original articles in five online platforms (Twitter, Patents, Facebook, Mendeley readers and News) and compared the most relevant TE documents ranked by their traditional and alternative metrics of impact. Secondly, we carried out a correlation and factorial analysis and then constructed a linear regression model to define a mathematical equation for the prediction of future TE citations counts from Altmetric scores. The obtained results suggest a growing presence of TE in the online social web and the feasibility in the context of global science to anticipate TE traditional academic impact by using social media