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.
Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work we derived the PA5 cells, a polyclonal population that retains functional attributes of primary OMCs. Microcarrier suspension culture is an alternative to planar 2D culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on Plastic compared to Plastic Plus microcarriers with upregulation of neuronal markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4x105 cells/mL and 4.9x104 cells/mL for Plastic and Plastic Plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, co-culture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study presents the successful expansion of PA5 cells using microcarrier suspension culture and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.
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.
In hemophilia A and B, analysis of the F8 and F9 variants has become standard over recent decades, giving information on the severity of hemophilia, inhibitor formation and allowing counseling for the families. The PedNet Registry collects data on hemophilia in children and has more than 2000 children listed. Genetic reports are collected uniformly and re-evaluated following international guidelines. We report 90 novel variants in the F8 and F9 gene, respectively, causing hemophilia with detailed information on severity, factor level and inhibitor formation. This will lead to further guidance for genetic laboratories and the treating physician. These findings can be implemented in hemophilia variant databases. The study highlights the need to re-evaluate and update earlier genetic reports in hemophilia both locally but also in variant databases in the light of changed nomenclature, the use of in silico prediction and new sequencing techniques.
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.
Most cell penetrating peptides (CPPs) are unstructured and susceptible to proteolytic degradation. One alternative is to incorporate D-chirality amino acids into unstructured CPPs to allow for enhanced uptake and intracellular stability. This work investigates CPP internalization using a series of time, concentration, temperature, and energy dependent studies, resulting in a three-fold increase in uptake and 50-fold increase in stability of D-chirality peptides over L-chirality counterparts. CPP internalization occurred via a combination of direct penetration and endocytosis, with a percentage of internalized CPP expelling from cells in a time-dependent manner. Mechanistic studies identified that cells exported the intact internalized D-chirality CPPs via an exocytosis independent pathway, analogous to a direct penetration method out of the cells. These findings highlight the potential of D-chirality CPPs as bio-vectors in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics
The ongoing wide spread of Covid-19, also referred to as 2019-nCoV or SARS-CoV-2, is undoubtedly one of the deadliest zoonotic diseases the whole world has grappled with. As of April 10, 2020, this disease has infected above 1.6 million people in over 200 countries worldwide, and claimed the lives of more than 96,000.1 Figure 1 shows the distribution of confirmed cases in the first 20 nations with the highest number of Covid-19 patients as of April 10, 2020, the least of which has over 9,000 cases.**Figure 1**With the increasing trend of daily new cases and daily death from the beginning of March to April 9, 2020, as displayed on Figure 2, it could be predicted that this fatal pandemic could last well beyond a year. According to the modeling completed by pandemic intelligence experts at the Imperial College, London, the 2019 novel coronavirus is likely to remain for another 12-18 months.2 Typically, past pandemics have lasted between one to three years.3Examples of such recent pandemics are the H1N1 influenza,4 SARS-CoV,5Ebola,6 and MERS-CoV,7 which all lasted for more than 12 months. As the emergence of these novel viruses keep increasing, how could biosafety and biosecurity measures guard against the introduction of the harmful causative organisms to humans in the future?**Figure 2**The terms biosafety and biosecurity are broadly used in diverse frameworks and refer not only to protection of humans and their surrounding environment against lethal biological agents, but also to global deactivation of arms of mass destruction.8 In the concept of biorisk management, these two terms refer to best practices that prevent the spillover of toxic organisms to human beings and into the environment.9,10 Although these two terms have been used interchangeably and often denoted with similar meanings, scientists have distinguished between the two concepts. According to Zaki,11 biosafety involves all the preventive measures undertaken to eradicate strains of pathogenic microorganisms and their potential toxins. On the other hand, biosecurity includes a set of preventive strategies intended to reduce the risk of transmission of infectious diseases in humans, crops, livestock, isolated pests and genetically modified organisms.12The World Health Organization (WHO) has classified disease-causing microorganisms into four different groups based on their principal characteristics, hazardous threat to individuals and the community, and their route of transmission.13,14 Table 1 presents the four different groups with their associated risk levels.**Table 1**Concluding from the descriptions for the various pathogenic organisms and their risk levels to individuals and the community in Table 1, the novel coronaviruses that cause Covid-19, SARS-CoV, MERS-CoV, and many other pandemic-causing pathogens, could be classified as risk level 4 pathogens. As spillover events keep occurring in recent years, and more of these high-risk emerging infectious diseases (EIDs) are likely to be introduced into the environment, it is necessary for the general public and stakeholders around the globe to institute biosafety and biosecurity measures in preventing the transmission of these biological toxins to mankind, livestock and their inhabitations. Among the core elements of the principles of biosafety measures and biosecurity strategies, the following are principal in guiding against EIDs:Effective regulations have to be put in place to avoid and manage intentional exposures to the sources and hosts of pathogenic organisms of medium to high risk levels. Strict observation of such policies around the world will minimize human activities that have caused several spillover events in the past.As the sources of novel pathogens have been identified in the past, it is necessary for scientists to inform the general public about the sources and hosts of such toxic organisms. Further research should also be targeted towards identifying these pathogens in animals and plants which are usually exposed to the environment. Such knowledge will help stakeholders and policymakers to notify the general public about the potential spillover events that are likely from contacts with identified sources and hosts of these pathogens.The risk assessment of pathogenic diseases of the past and a predicted assessment of likely EIDs in the future should be made available to the general public. Such awareness will inform the mass about the economic, social, and health impacts of these diseases. This will reinforce public adherence to policies and regulations which are instituted to limit contacts with pathogenic sources as people are aware of the potential losses to national and global economy, and the adverse effects on social life and health facilities.As health personnel, scientists, and leaders in various capacities often find themselves in the frontline during the emergence of epidemic and pandemic diseases, they need to be trained adequately to guarantee proper apprehension and execution of biosafety procedures to ensure the maintenance of a safe working environment for individuals and the wider community. This will ensure appropriate measures are taken to limit the spread of infections.Finally, routine upgrades are required to render instituted biosafety and biosecurity measures more effective and efficient in this ever-changing world. As the etiology and epidemiology of EIDs keep evolving, constant updates to safe practices in health centers around the world are required to adapt these practices to effectively manage the emerging diseases.As various country-based mitigation measures are being implemented around the world to contain and control the course of Covid-19,15 it is essential that the above biosafety and biosecurity measures are adopted and implemented to effectively manage the ongoing outbreak, and prevent future emerging infections.
In the present investigation temperature dependence fatigue strength behaviour of Inconel 825 super alloys is investigated. Based on the experimental results different S-N models have been derived and suitable model for the prediction of fatigue strength have been proposed. An inverse power and exponential relation between fatigue strength and absolute temperature is demonstrated. The proposed models are used to predict the fatigue life using well known Palmgren-Miner rule. Based on high to low and low to high load steps test data sets under identical test conditions, Miner rule based statistical damage constant is stochastically modeled for fatigue life prediction at different level of probability and validated. The modeling process combines a probabilistic fatigue damage accumulation and a stress-life-temperature relation technique.
Measuring biological diversity is a crucial but difficult undertaking, as exemplified in oaks where complex morphological, ecological, biogeographic and genetic differentiation patterns collide with traditional taxonomy that measures biodiversity in number of species (or higher taxa). In this pilot study, we generated High-Throughput Sequencing (HTS) amplicon data of the intergenic spacer of the 5S nuclear ribosomal DNA cistron (5S-IGS) in oaks, using six mock samples that differ in geographic origin, species composition, and pool complexity. The potential of the marker for automated geno-taxonomy applications was assessed using a reference dataset of 1770 5S-IGS cloned sequences, covering the entire taxonomic breadth and distribution range of western Eurasian Quercus, and applying similarity (BLAST) and evolutionary approaches (ML trees and EPA). Both methods performed equally well, with correct identification of species in sections Ilex and Cerris in the pure and mixed samples and main genotypes shared by species of sect. Quercus. Application of different cut-off thresholds revealed that medium-high abundance sequences (>10 or 25) suffice for a net species identification of samples containing one or few individuals. Lower thresholds identify phylogenetic correspondence with all target species in highly mixed samples (analogue to environmental bulk samples) and include rare variants pointing towards reticulation, incomplete lineage sorting, pseudogenic 5S units, and in-situ (natural) contamination. Our pipeline is highly promising for future assessments of intra-specific and inter-population diversity, and of the genetic resources of natural ecosystems, which are fundamental to empower fast and solid biodiversity conservation programs worldwide.
Growing evidence shows that a homozygous 6.7-kb deletion of the novel anti-inflammatory molecule leukocyte immunoglobulin like receptor A3 (LILRA3) is associated with many autoimmune disorders. However, its effects on pathogenesis of inflammatory bowel disease (IBD) have not been clarified yet. LILRA3 is mainly expressed in monocytes, whereas its effects on biological behaviors of monocytes have not been systematically reported. To investigate the association between LILRA3 polymorphism and IBD susceptibility, LILRA3 polymorphism was assessed in 378 IBD patients and 509 healthy controls in our study, quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC) were employed to detect the LILRA3 expression in IBD patient blood and intestinal samples. Despite no association of the polymorphism with IBD development was found, LILRA3 expression was markedly increased in IBD patients compared with healthy controls. Human U937 monocyte cell line was employed to establish LILRA3-overexpressing cells and the effects of LILRA3 on the biological behaviors of U937 cells were systematically explored. We found that overexpression of LILRA3 in monocytes led to significant decreases in secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Additionally, LILRA3 abated monocyte migration by reducing the expression of several chemokines and enhanced monocyte phagocytosis by increasing CD36 expression. Furthermore, LILRA3 promoted monocyte proliferation through a combination of Akt and MEK/Erk signaling pathways. We report for the first time that LILRA3 is related to IBD and functions as an anti-inflammatory modulator in U937 cells.
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.
Tree canopies provide habitats for diverse and until now, still poorly characterised communities of microbial eukaryotes. One of the most general patterns in community ecology is the increase in species richness with increasing habitat diversity. Thus, environmental heterogeneity of tree canopies should be an important factor governing community structure and diversity in this subsystem of forest ecosystems. Nevertheless, it is unknown if similar patterns are reflected at the microbial scale within unicellular eukaryotes (protists). In this study, high-throughput sequencing of two prominent protistan taxa, Cercozoa and Oomycota, was performed. For a comprehensive assessment of their diversity across all ecological compartments from forest soils to the canopy, group specific primers were used. When taking OTU abundances into account, our results showed highly dissimilar protistan communities within the investigated microhabitats. We observed no pattern of nestedness, because the majority of OTUs was present in all sampled microhabitats. According to the microbiological tenet ‘Everything is everywhere, but, the environment selects’, habitat diversity strongly favoured distinct protistan taxa in terms of abundance, but due to their almost ubiquitous distribution the effect of species richness on community composition was negligible.
Ecological theory postulates that the niche of co-occurring species must differ along some ecological dimensions in order to allow their stable coexistence. Yet, many biological systems challenge this competitive exclusion principle. For instance, insectivorous bats from the Northern Hemisphere typically form local assemblages of multiple species sharing highly similar functional traits and pertaining to identical feeding guilds. Although their trophic niche can be accessed with unprecedented details using genetic identification of prey, the underlying mechanisms of resource partitioning remain vastly unexplored. Here, we studied the differential diet of three phenotypically and phylogenetically closely-related bat species of the genus Plecotus in an area of sympatry and throughout their entire breeding season (April-October) using DNA metabarcoding. Even at such a small geographic scale, we identified strong seasonal and spatial variation of their trophic niche at both intra- and inter-specific levels. Indeed, while the different bats fed on a distinct array of prey during spring, they showed higher niche overlap during summer and fall, when all three bat species switched their hunting behavior to feed on few temporarily abundant moths. Furthermore, by considering the ecological traits of prey species, we inferred from the menu of each bat species that feeding grounds and hunting techniques differed suggesting that niche partitioning was primarily habitat-driven. As predicted by their phylogenetic relationships, the two most-closely related bat species exhibited the most distinct foraging habitat preferences, while the third, more distantly-related species was more generalist. These results highlight the need of extensive samples to fully understand species coexistence.