Crystal structure of Cmr5 from Pyrococcus furiosus and its functional implications

AbstractThe bacterial acquired immune system consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRIPSR-associated (Cas) genes, which include Cas-module repeat-associated mysterious proteins (Cmr). The six Cmr proteins of Pyrococcus furiosus (pfCmr1–pfCmr6) form a Cmr effector complex that functions against exogenous nucleic acid. Among the Cmr proteins, the role of pfCmr5 and its involvement in the complex’s cleavage activity have been obscure. The elucidated pfCmr5 structure has two inserted α-helices compared with the other trimeric Cmr5 structure. However, pfCmr5 exists as a monomeric protein both in the crystalline state and in solution. In vitro assays indicate that pfCmr5 interacts with pfCmr4. These structural and biophysical data might help in understanding the complicated and ill-characterized Cmr effector complex.Structured summary of protein interactionspfCmr4 and pfCmr5 bind by molecular sieving (View interaction)pfCmr4 and pfCmr4 bind by molecular sieving (View interaction)pfCmr5 and pfCmr4 bind by ion exchange chromatography (View interaction

Utility of targeted deep sequencing for detecting circulating tumor DNA in pancreatic cancer patients.

Targeted deep sequencing across broad genomic regions has been used to detect circulating tumor DNA (ctDNA) in pancreatic ductal adenocarcinoma (PDAC) patients. However, since most PDACs harbor a mutation in KRAS, sequencing of broad regions needs to be systemically compared to analyzing only KRAS mutations for PDAC. Using capture-based targeted deep sequencing, we detected somatic tumor mutations in 17 fine needle aspiration biopsy and 69 longitudinal cell-free DNA (cfDNA) samples from 17 PDAC patients. KRAS mutations were detected in 10 out of 17 pretreatment patient plasma samples. Next, interrogation of genetic alterations in matched primary tumor samples detected ctDNA in 12 of 17 pretreatment plasma samples and cfDNA sequencing across the 83 target genes identified ctDNA in 15 of 17 cases (88.2% sensitivity). This improved sensitivity of ctDNA detection resulted in enhanced tumor burden monitoring when we analyzed longitudinal plasma samples. We found that cfDNA sequencing detected the lowest mutant allelic fractions and number of variants when complete response or partial response to chemotherapy was achieved. We demonstrated that ctDNA levels measured by targeted deep sequencing sensitively indicate the presence of cancer and correlate well with clinical responses to therapy and disease progression in PDAC patients

Controllable modification of transport properties of single-walled carbon nanotube field effect transistors with in situ Al decoration

We use an in situ Al decoration technique to control the transport characteristics of single-walled carbon nanotube field effect transistors (SWNT-FETs). Al nanoparticle decoration in a high vacuum caused the devices to change from p -type to n -type FETs, and subsequent exposure to the ambient atmosphere induced a gradual recovery of p -type character. In comparison with the bare SWNT-FETs under high vacuum, the channel-open devices with decorated Al particles exhibited reduced current under ambient conditions. However, selective Al decoration only at the contact resulted in an improved p -type current in ambient air.open182

Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth

Arabidopsis U2AF65 Regulates Flowering Time and the Growth of Pollen Tubes

During pre-mRNA splicing, U2 small nuclear ribonucleoprotein auxiliary factor 65 (U2AF65) interacts with U2AF35 and splicing factor 1 (SF1), allowing for the recognition of the 3′-splice site by the ternary complex. The functional characterization of U2AF65 homologs has not been performed in Arabidopsis thaliana yet. Here, we show that normal plant development, including floral transition, and male gametophyte development, requires two Arabidopsis U2AF65 isoforms (AtU2AF65a and AtU2AF65b). Loss-of-function mutants of these two isoforms displayed opposite flowering phenotypes: atu2af65a mutants showed late flowering, whereas atu2af65b mutants were characterized by slightly early flowering, as compared to that in the wild-type (Col-0) plants. These abnormal flowering phenotypes were well-correlated with the expression patterns of the flowering time genes such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). However, the two atu2af65 mutants did not display any morphological abnormalities or alterations in abiotic stress tests. Double mutation of the AtU2AF65a and AtU2AF65b genes resulted in non-viable seeds due to defective male gametophyte. In vitro pollen germination test revealed that mutations in both AtU2AF65a and AtU2AF65b genes significantly impaired pollen tube growth. Collectively, our findings suggest that two protein isoforms of AtU2AF65 are differentially involved in regulating flowering time and display a redundant role in pollen tube growth

Efficient transgene expression system using a cumate-inducible promoter and Cre-loxP recombination in avian cells

Objective Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression

The association of asthma and its subgroups with osteoporosis: a cross-sectional study using KoGES HEXA data

Background A few studies have reported the association between asthma and osteoporosis. We aimed to analyze the association of asthma and its subgroups with osteoporosis in the Korean adult population. Methods We used the health examinee (HEXA) data from the Korean Genome and Epidemiology Study (KoGES) obtained between 2004 and 2016. We included 162,579 participants (n = 3,160 with asthma; n = 159,419 controls) who reported their previous histories of asthma and osteoporosis. The participants were categorized into 3 groups based on asthma management: participants who did not need further treatment due to controlled symptoms (well controlled); participants with ongoing treatment (being treated); participants who were not treated even though they had symptoms (not being treated). Multiple logistic regression analyses were used to calculate the adjusted odds ratios (aORs) with 95% confidence intervals (CIs) for osteoporosis. Subgroup analyses for age and sex were conducted. Results The prevalence of osteoporosis was higher in patients with asthma (13.6%) than in controls (6.8%). In the full-adjusted model, the aORs for osteoporosis were 1.74 (95% CI 1.55–1.94, P < 0.001) in patients with asthma compared to controls. There were consistent findings across the age and sex subgroups. The aORs for osteoporosis were 1.43 (95% CI 1.10–1.86, P = 0.008) in the well-controlled asthma group; 1.55 (95% CI 1.28–1.89, P < 0.001) in the being treated asthma group; and 1.96 (95% CI 1.66–2.31, P < 0.001) in the not being treated asthma group compared to the control group. Conclusion Asthma was associated with osteoporosis in the Korean adult population. Patients with asthma not being treated showed the highest ORs for osteoporosis.This research was funded by National Research Foundation (NRF) of Korea, grant number (NRF-2018-R1D1A1A0-2085328 by Hyo Geun Choi, NRF-2020R1G1A1005390 by Jee Hye Wee). The funding organization did not contribute to the design or conduct of this study, preparation, review, approval, or decision to submit this manuscript for publication

Feasibility of video-based real-time nystagmus tracking: a lightweight deep learning model approach using ocular object segmentation

BackgroundEye movement tests remain significantly underutilized in emergency departments and primary healthcare units, despite their superior diagnostic sensitivity compared to neuroimaging modalities for the differential diagnosis of acute vertigo. This underutilization may be attributed to a potential lack of awareness regarding these tests and the absence of appropriate tools for detecting nystagmus. This study aimed to develop a nystagmus measurement algorithm using a lightweight deep-learning model that recognizes the ocular regions.MethodThe deep learning model was used to segment the eye regions, detect blinking, and determine the pupil center. The model was trained using images extracted from video clips of a clinical battery of eye movement tests and synthesized images reproducing real eye movement scenarios using virtual reality. Each eye image was annotated with segmentation masks of the sclera, iris, and pupil, with gaze vectors of the pupil center for eye tracking. We conducted a comprehensive evaluation of model performance and its execution speeds in comparison to various alternative models using metrics that are suitable for the tasks.ResultsThe mean Intersection over Union values of the segmentation model ranged from 0.90 to 0.97 for different classes (sclera, iris, and pupil) across types of images (synthetic vs. real-world images). Additionally, the mean absolute error for eye tracking was 0.595 for real-world data and the F1 score for blink detection was ≥ 0.95, which indicates our model is performing at a very high level of accuracy. Execution speed was also the most rapid for ocular object segmentation under the same hardware condition as compared to alternative models. The prediction for horizontal and vertical nystagmus in real eye movement video revealed high accuracy with a strong correlation between the observed and predicted values (r = 0.9949 for horizontal and r = 0.9950 for vertical; both p &lt; 0.05).ConclusionThe potential of our model, which can automatically segment ocular regions and track nystagmus in real time from eye movement videos, holds significant promise for emergency settings or remote intervention within the field of neurotology