An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9
Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.
Meng et al. (2020) An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9. PeerJ 8:e10077 https://doi.org/10.7717/peerj.10077
Integrating multiple microarray dataset analysis and machine learning methods to reveal the key genes and regulatory mechanisms underlying human intervertebral disc degeneration
Intervertebral disc degeneration (IDD), a major cause of lower back pain, has multiple contributing factors including genetics, environment, age, and loading history. Bioinformatics analysis has been extensively used to identify diagnostic biomarkers and therapeutic targets for IDD diagnosis and treatment. However, multiple microarray dataset analysis and machine learning methods have not been integrated. In this study, we downloaded the mRNA, microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) expression profiles (GSE34095, GSE15227, GSE63492 GSE116726, GSE56081 and GSE67566) associated with IDD from the GEO database. Using differential expression analysis and recursive feature elimination, we extracted four optimal feature genes. We then used the support vector machine (SVM) to make a classification model with the four optimal feature genes. The ROC curve was used to evaluate the model’s performance, and the expression profiles (GSE63492, GSE116726, GSE56081, and GSE67566) were used to construct a competitive endogenous RNA (ceRNA) regulatory network and explore the underlying mechanisms of the feature genes. We found that three miRNAs (hsa-miR-4728-5p, hsa-miR-5196-5p, and hsa-miR-185-5p) and three circRNAs (hsa_circRNA_100723, hsa_circRNA_104471, and hsa_circRNA_100750) were important regulators with more interactions than the other RNAs across the whole network. The expression level analysis of the three datasets revealed that BCAS4 and SCRG1 were key genes involved in IDD development. Ultimately, our study proposes a novel approach to determining reliable and effective targets in IDD diagnosis and treatment.
Chang et al. (2020) Integrating multiple microarray dataset analysis and machine learning methods to reveal the key genes and regulatory mechanisms underlying human intervertebral disc degeneration. PeerJ 8:e10120 https://doi.org/10.7717/peerj.10120
Upper-room ultraviolet air disinfection might help to reduce COVID-19 transmission in buildings: a feasibility study
As the world’s economies come out of the lockdown imposed by the COVID-19 pandemic, there is an urgent need for technologies to mitigate COVID-19 transmission in confined spaces such as buildings. This feasibility study looks at one such technology, upper-room ultraviolet (UV) air disinfection, that can be safely used while humans are present in the room space, and which has already proven its efficacy as an intervention to inhibit the transmission of airborne diseases such as measles and tuberculosis. Using published data from various sources, it is shown that the SARS-CoV-2 virus, the causative agent of COVID-19, is highly likely to be susceptible to UV-C damage when suspended in air, with a UV susceptibility constant likely to be in the region 0.377–0.590 m2/J, similar to that for other aerosolised coronaviruses. As such, the UV-C flux required to disinfect the virus is expected to be acceptable and safe for upper-room applications. Through analysis of expected and worst-case scenarios, the efficacy of the upper-room UV-C approach for reducing COVID-19 transmission in confined spaces (with moderate but sufficient ceiling height) is demonstrated. Furthermore, it is shown that with SARS-CoV-2, it should be possible to achieve high equivalent air change rates using upper-room UV air disinfection, suggesting that the technology might be particularly applicable to poorly ventilated spaces.
Environmental Contamination and Remediation
Beggs et al. (2020) Upper-room ultraviolet air disinfection might help to reduce COVID-19 transmission in buildings: a feasibility study. PeerJ 8:e10196 https://doi.org/10.7717/peerj.10196
Soil carbon, nitrogen and phosphorus ecological stoichiometry shifts with tree species in subalpine plantations
Understanding ecological stoichiometric characteristics of soil nutrient elements, such as carbon (C), nitrogen (N) and phosphorus (P) is crucial to guide ecological restoration of plantations in ecologically vulnerable areas, such as alpine and subalpine regions. However, there has been only a few related studies, and thus whether and how different tree species would affect soil C:N:P ecological stoichiometry remains unclear. We compared soil C:N:P ecological stoichiometry of Pinus tabulaeformis, Larix kaempferi and Cercidiphyllum japonicum to primary shrubland in a subalpine region. We observed strong tree-specific and depth-dependent effects on soil C:N:P stoichiometry in subalpine plantations. In general, the C:N, C:P and N:P of topsoil (0–10 cm) are higher than subsoil (>10 cm) layer at 0–30 cm depth profiles. The differences in C:N, N:P and C:P at the topsoil across target tree species were significantly linked to standing litter stock, tree biomass/total aboveground biomass and Margalef’s index of plant community, respectively, whereas the observed variations of C:N, N:P and C:P ratio among soil profiles are closely related to differences in soil bulk density, soil moisture, the quantity and quality of aboveground litter inputs as well as underground fine root across plantations examined. Our results highlight that soil nutrients in plantation depend on litter quantity and quality of selected tree species as well as soil physical attributes. Therefore, matching site with trees is crucial to enhance ecological functioning in degraded regions resulting from human activity.
Qi et al. (2020) Soil carbon, nitrogen and phosphorus ecological stoichiometry shifts with tree species in subalpine plantations. PeerJ 8:e9702 https://doi.org/10.7717/peerj.9702
Genetic characterization of feline panleukopenia virus from dogs in Vietnam reveals a unique Thr101 mutation in VP2
Background Canine parvovirus type 2 (CPV-2) and feline parvovirus (FPV) are known as the main causes of several serious diseases and have a severe impact on puppies and kittens, respectively. FPV and new CPV-2 variants are all able to infect cats, causing diseases indistinguishable from feline panleukopenia. However, FPV only replicates efficiently in feline cells in vitro and replicates in dogs in the thymus and bone marrow without being shed in feces. In our previous study, the genotypes of six parvoviral isolates were unable to be identified using a SimpleProbe® real-time PCR assay. Methods In the present study, we characterized previously unidentified FPV-like viruses isolated from dogs in Vietnam. The six isolates were utilized to complete VP2 gene sequencing and to conduct phylogenetic analyses. Results Sequence analysis of the six parvoviral strains identified the species as being similar to FPV. Phylogenetic analysis demonstrated that the complete VP2 genes of the strains are similar to those of FPV. The FPV-like strains contain a Thr101 mutation in the VP2 protein, which is different from prototype FPV strains. Discussion Our data provide evidence for the existence of changes in the charge, protein contact potential and molecular surface of the core of the receptor-binding size with an Ile101 to Thr101 mutation. This is also the first study to provide reliable evidence that FPV may be a threat to the Vietnamese dog population.
Hoang et al. (2020) Genetic characterization of feline panleukopenia virus from dogs in Vietnam reveals a unique Thr101 mutation in VP2. PeerJ 8:e9752 https://doi.org/10.7717/peerj.9752
Physiological aspects and energetic contribution in 20s:10s high-intensity interval exercise at different intensities
Background One of the most popular high-intensity interval exercises is the called “Tabata Protocol”. However, most investigations have limitations in describing the work intensity, and this fact appears to be due to the protocol unfeasibility. Furthermore, the physiological demands and energetic contribution during this kind of exercise remain unclear. Methods Eight physically active students (21.8 ± 3.7 years) and eight well-trained cycling athletes (27.8 ± 6.4 years) were enrolled. In the first visit, we collected descriptive data and the peak power output (PPO). On the next three visits, in random order, participants performed interval training with the same time structure (effort:rest 20s:10s) but using different intensities (115%, 130%, and 170% of PPO). We collected the number of sprints, power output, oxygen consumption, blood lactate, and heart rate. Results The analysis of variance for multivariate test (number of sprints, power output, blood lactate, peak heart rate and percentage of maximal heart rate) showed significant differences between groups (F = 9.62; p = 0.001) and intensities (F = 384.05; p < 0.001), with no interactions (F = 0.94; p = 0.57). All three energetic contributions and intensities were different between protocols. The higher contribution was aerobic, followed by alactic and lactic. The aerobic contribution was higher at 115%PPO, while the alactic system showed higher contribution at 130%PPO. In conclusion, the aerobic system was predominant in the three exercise protocols, and we observed a higher contribution at lower intensities.
Protzen et al. (2020) Physiological aspects and energetic contribution in 20s:10s high-intensity interval exercise at different intensities. PeerJ 8:e9791 https://doi.org/10.7717/peerj.9791
Genomic signatures of globally enhanced gene duplicate accumulation in the megadiverse higher Diptera fueling intralocus sexual conflict resolution
Gene duplication is an important source of evolutionary innovation. To explore the relative impact of gene duplication during the diversification of major insect model system lineages, we performed a comparative analysis of lineage-specific gene duplications in the fruit fly Drosophila melanogaster (Diptera: Brachycera), the mosquito Anopheles gambiae (Diptera: Culicomorpha), the red flour beetle Tribolium castaneum (Coleoptera), and the honeybee Apis mellifera (Hymenoptera). Focusing on close to 6,000 insect core gene families containing maximally six paralogs, we detected a conspicuously higher number of lineage-specific duplications in Drosophila (689) compared to Anopheles (315), Tribolium (386), and Apis (223). Based on analyses of sequence divergence, phylogenetic distribution, and gene ontology information, we present evidence that an increased background rate of gene duplicate accumulation played an exceptional role during the diversification of the higher Diptera (Brachycera), in part by providing enriched opportunities for intralocus sexual conflict resolution, which may have boosted speciation rates during the early radiation of the megadiverse brachyceran subclade Schizophora.
Bao et al. (2020) Genomic signatures of globally enhanced gene duplicate accumulation in the megadiverse higher Diptera fueling intralocus sexual conflict resolution. PeerJ 8:e10012 https://doi.org/10.7717/peerj.10012
Instrumental validity and intra/inter-rater reliability of a novel low-cost digital pressure algometer
Background Pain assessment is a key measure that accompanies treatments in a wide range of clinical settings. A low-cost valid and reliable pressure algometer would allow objective assessment of pressure pain to assist a variety of health professionals. However, the pressure algometer is often expensive, which limits its daily use in both clinical and research settings. Objectives This study aimed to assess the instrumental validity, and the intra- and inter-rater reliability of an inexpensive digital adapted pressure algometer. Methods A single rater applied 60 random compressions on a force platform. The pressure pain thresholds of 20 volunteers were collected twice (3 days apart) by two raters. The main outcome measurements were as follows: the maximal peak force (in kPa) and the pressure pain threshold (adapted pressure algometer vs. force platform). Cronbach’s α test was used to assess internal consistency. The standard error of measurement provided estimates of measurement error, and the measurement bias was estimated with the Bland–Altman method, with lower and upper limits of agreement. Results No differences were observed when comparing the compression results (P = 0.51). The validity and internal intra-rater consistencies ranged from 0.84 to 0.99, and the standard error of measurement from 0.005 to 0.04 kPa. Very strong (r = 0.73–0.74) to near-perfect (r = 0.99) correlations were found, with a low risk of bias for all measurements. The results demonstrated the validity and intra-rater reliability of the digitally adapted pressure algometer. Inter-rater reliability results were moderate (r = 0.55–0.60; Cronbach’s α = 0.71–0.75). Conclusion The adapted pressure algometer provide valid and reliable measurements of pressure pain threshold. The results support more widespread use of the pressure pain threshold method among clinicians.
Jerez-Mayorga et al. (2020) Instrumental validity and intra/inter-rater reliability of a novel low-cost digital pressure algometer. PeerJ 8:e10162 https://doi.org/10.7717/peerj.10162
Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load
SARS-CoV-2 is a betacoronavirus responsible for COVID-19, a pandemic with global impact that first emerged in late 2019. Since then, the viral genome has shown considerable variance as the disease spread across the world, in part due to the zoonotic origins of the virus and the human host adaptation process. As a virus with an RNA genome that codes for its own genomic replication proteins, mutations in these proteins can significantly impact the variance rate of the genome, affecting both the survival and infection rate of the virus, and attempts at combating the disease. In this study, we analyzed the mutation densities of viral isolates carrying frequently observed mutations for four proteins in the RNA synthesis complex over time in comparison to wildtype isolates. Our observations suggest mutations in nsp14, an error-correcting exonuclease protein, have the strongest association with increased mutation load without selective pressure and across the genome, compared to nsp7, nsp8 and nsp12, which form the core polymerase complex. We propose nsp14 as a priority research target for understanding genomic variance rate in SARS-CoV-2 isolates and nsp14 mutations as potential predictors for high mutability strains.
Eskier et al. (2020) Mutations of SARS-CoV-2 nsp14 exhibit strong association with increased genome-wide mutation load. PeerJ 8:e10181 https://doi.org/10.7717/peerj.10181
Convolutional neural network in upper limb functional motion analysis after stroke
In this work, implementation of Convolutional Neural Network (CNN) for the purpose of analysis of functional upper limb movement pattern was applied. The main aim of the study was to compare motion of selected activities of daily living of participants after stroke with the healthy ones (in similar age). The optical, marker-based motion capture system was applied for the purpose of data acquisition. There were some attempts made in order to find the existing differences in the motion pattern of the upper limb. For this purpose, the motion features of dominant and non-dominant upper limb of healthy participants were compared with motion features of paresis and non-paresis upper limbs of participants after stroke. On the basis of the newly collected data set, a new CNN application was presented to the classification of motion data in two different class label configurations. Analyzing individual segments of the upper body, it turned out that the arm was the most sensitive segment for capturing changes in the trajectory of the lifting movements of objects.
Szczęsna et al. (2020) Convolutional neural network in upper limb functional motion analysis after stroke. PeerJ 8:e10124 https://doi.org/10.7717/peerj.10124