Fluctuating levels of reprogramming factor expression in cultured human undifferentiated keratinocytes

Although human undifferentiated keratinocytes (HUKs) can be reprogrammed to become induced pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the endogenous expression of reprogramming factors in cultured HUKs is not clear at different stages. In this study, keratinocytes were isolated from foreskin of adult subject (18 years old) and cultured on collagen type IV-coated culture dishes in a low-calcium, serum-free medium (Epilife, Invitrogen). In order to clarify the expression patterns of RFs and other stem cell markers in cultured human keratinocytes, total RNA was extracted using Trizol reagent, and polymerase chain reaction (PCR) was performed using established GenBank sequences to design primers. The subsequent PCR analysis was carried out by agarose gel electrophoresis. The expression levels of RFs and other stem cell markers in human HUKs clearly fluctuated during culturing, which supports the hypothesis that HUKs might be reprogrammed into a pluripotent state when the maximum levels of RFs expression are maintained by appropriate culture conditions.Keywords: Human undifferentiated keratinocytes, reprogramming factors, expression fluctuationAfrican Journal of Biotechnology Vol. 12(35), pp. 5389-539

Brassinosteroids affect wood development and properties of Fraxinus mandshurica

IntroductionXylem development plays a crucial role in wood formation in woody plants. In recent years, there has been growing attention towards the impact of brassinosteroids (BRs) on this xylem development. In the present study, we evaluated the dynamic variation of xylem development in Fraxinus mandshurica (female parent, M8) and a novel interspecific hybrid F. mandshurica × Fraxinus sogdiana (1601) from May to August 2020.MethodsWe obtained RNA-Seq transcriptomes of three tissue types (xylem, phloem, and leaf) to identify the differences in xylem-differentially expressed genes (X-DEGs) and xylem-specifically expressed genes (X-SEGs) in M8 and 1601 variants. We then further evaluated these genes via weighted gene co-expression network analysis (WGCNA) alongside overexpressing FmCPD, a BR biosynthesis enzyme gene, in transient transgenic F. mandshurica.ResultsOur results indicated that the xylem development cycle of 1601 was extended by 2 weeks compared to that of M8. In addition, during the later wood development stages (secondary wall thickening) of 1601, an increased cellulose content (14%) and a reduced lignin content (11%) was observed. Furthermore, vessel length and width increased by 67% and 37%, respectively, in 1601 compared with those of M8. A total of 4589 X-DEGs were identified, including enzymes related to phenylpropane metabolism, galactose metabolism, BR synthesis, and signal transduction pathways. WGCNA identified hub X-SEGs involved in cellulose synthesis and BR signaling in the 1601 wood formation–related module (CESA8, COR1, C3H14, and C3H15); in contrast, genes involved in phenylpropane metabolism were significantly enriched in the M8 wood formation–related module (CCoAOMT and CCR). Moreover, overexpression of FmCPD in transient transgenic F. mandshurica affected the expression of genes associated with lignin and cellulose biosynthesis signal transduction. Finally, BR content was determined to be approximately 20% lower in the M8 xylem than in the 1601 xylem, and the exogenous application of BRs (24-epi brassinolide) significantly increased the number of xylem cell layers and altered the composition of the secondary cell walls in F. mandshurica.DiscussionOur findings suggest that BR biosynthesis and signaling play a critical role in the differing wood development and properties observed between M8 and 1601 F. mandshurica

The impact of empirical Marshall vein ethanol infusion as a first-choice intraoperative strategy on the long-term outcomes in patients with persistent atrial fibrillation undergoing mitral isthmus ablation

BackgroundMarshall vein ethanol infusion (MVEI) as an additional therapy to conventional catheter ablation (CA) has been proved to be efficacious in patients with persistent atrial fibrillation (PeAF). However, whether empirical MVEI could be the first-line strategy in mitral isthmus (MI) ablation has seldom been investigated. Here, we aim to compare the efficacy, safety, and long-term outcomes between provisional and empirical MVEI in PeAF patients undergoing the index MI ablation procedure.MethodsWe enrolled 133 patients with PeAF either in the provisional group (n = 38, MVEI was performed when conventional endocardial and/or epicardial ablation procedures were inadequate to achieve bidirectional MI block) or in the empirical group (n = 95, MVEI was performed empirically before MI CA).ResultsAll of the baseline characteristics were comparable. Less spontaneous or inducible atrial tachycardias (ATs) were encountered in the empirical group of patients (P < 0.001). More epicardial ablations were applied (26.3% vs. 9.5%, P = 0.016) and a higher incidence of CA-facilitated restoration of sinus rhythm was recorded (86.8% vs. 11.7%, P < 0.001) in the provisional group of patients. Although more fluoroscopy time (6.4[4.2, 9.3] vs. 9.5[5.9, 11.6] min, P = 0.019) and radiation exposure (69.0[25.3, 160.2] vs. 122.0[62.5, 234.1] mGy, P = 0.010) were documented in the empirical group with comparable procedure time, less time (455.9 ± 192.2 vs. 366.5 ± 161.3 s, P = 0.038) was consumed to achieve bidirectional MI block during endocardial ablation in the provisional group. Incidences of procedure-related complications were similar between the two groups. During a 16.5 ± 4.4-month follow-up, the empirical group of patients showed a significantly higher rate of freedom from AT recurrence (95.8% vs. 81.6%, log-rank P = 0.003), while the rate of freedom from AF or atrial tachyarrhythmias (combining AF and AT) was similar. Both univariate (HR 0.19, 95% CI 0.05–0.64, P = 0.008) and multivariate (HR 0.25, 95% CI 0.07–0.92, P = 0.037) Cox regression analyses indicated that empirical MVEI was independently associated with lower long-term AT recurrence.ConclusionAmong patients with PeAF who underwent the index MI ablation procedure, empirical MVEI could reduce endocardial MI ablation time and provide greater long-term freedom from AT recurrence

A High-Precision Machine Learning Algorithm to Classify Left and Right Outflow Tract Ventricular Tachycardia

Introduction: Multiple algorithms based on 12-lead ECG measurements have been proposed to identify the right ventricular outflow tract (RVOT) and left ventricular outflow tract (LVOT) locations from which ventricular tachycardia (VT) and frequent premature ventricular complex (PVC) originate. However, a clinical-grade machine learning algorithm that automatically analyzes characteristics of 12-lead ECGs and predicts RVOT or LVOT origins of VT and PVC is not currently available. The effective ablation sites of RVOT and LVOT, confirmed by a successful ablation procedure, provide evidence to create RVOT and LVOT labels for the machine learning model. Methods: We randomly sampled training, validation, and testing data sets from 420 patients who underwent successful catheter ablation (CA) to treat VT or PVC, containing 340 (81%), 38 (9%), and 42 (10%) patients, respectively. We iteratively trained a machine learning algorithm supplied with 1,600,800 features extracted via our proprietary algorithm from 12-lead ECGs of the patients in the training cohort. The area under the curve (AUC) of the receiver operating characteristic curve was calculated from the internal validation data set to choose an optimal discretization cutoff threshold. Results: The proposed approach attained the following performance: accuracy (ACC) of 97.62 (87.44–99.99), weighted F1-score of 98.46 (90–100), AUC of 98.99 (96.89–100), sensitivity (SE) of 96.97 (82.54–99.89), and specificity (SP) of 100 (62.97–100). Conclusions: The proposed multistage diagnostic scheme attained clinical-grade precision of prediction for LVOT and RVOT locations of VT origin with fewer applicability restrictions than prior studies

Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3

The clinical relevance of human papillomavirus type 16 (HPV16) DNA methylation has not been well documented, although its role in modulation of viral transcription is recognized.Study subjects were 211 women attending Planned Parenthood clinics in Western Washington for routine Papanicolaou screening who were HPV16 positive at the screening and/or subsequent colposcopy visit. Methylation of 11 CpG dinucleotides in the 3' end of the long control region of the HPV16 genome was examined by sequencing the cloned polymerase chain reaction products. The association between risk of CIN2/3 and degree of CpG methylation was estimated using a logistic regression model.CIN2/3 was histologically confirmed in 94 (44.5%) of 211 HPV16 positive women. The likelihood of being diagnosed as CIN2/3 increased significantly with decreasing numbers of methylated CpGs (meCpGs) in the 3' end of the long control region (P(for trend) = 0.003). After adjusting for HPV16 variants, number of HPV16-positive visits, current smoking status and lifetime number of male sex partners, the odds ratio for the association of CIN2/3 with ≥4 meCpGs was 0.31 (95% confidence interval, 0.12-0.79). The proportion of ≥4 meCpGs decreased appreciably as the severity of the cervical lesion increased (P(for trend) = 0.001). The inverse association remained similar when CIN3 was used as the clinical endpoint. Although not statistically significant, the ≥4 meCpGs-related risk reduction was more substantial among current, as compared to noncurrent, smokers.Results suggest that degree of the viral genome methylation is related to the outcome of an HPV16 cervical infection

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Molecular Cloning and Expression Analysis of Cyclin H from Black Tiger Shrimp (Penaeus monodon)

Cyclin H is an important cell protein that plays a crucial role in cell division. In the present study, the cDNA of cyclin H (designated as Pmcyclin H) was identified from black tiger shrimp (Penaeus monodon) by expressed sequence tag (EST) analysis, and RACE techniques. The full length cDNA of Pmcyclin H is 1280bp, including a 5’-terminal un-translated region (5’UTR) of 63 bp, a 3’UTR of 218 bp with a poly (A) tail, and an open reading frame (ORF) of 999 bp encoding a polypeptide of 332 amino acids with a predicted molecular weight of 39 kDa, predicted pI of 6.39. Both Blast, and phylogenetic analysis, confirmed that Pmcyclin H is a new member of the shrimp cyclin H family. Using real-time PCR the mRNA expression of Pmcyclin H in eight tissues was examined, and mRNA transcript of Pmcyclin H was predominantly detectable in ovarian tissue, and to a lesser degree in the tissues of intestine, testis, stomach, and heart, but almost undetectable in the tissues of liver (hepar), brain, and muscle. The temporal expression of Pmcyclin H in different developmental stages of the ovaries was investigated by real-time PCR. During the six stages of ovarian development, one peak expression of Pmcyclin H was detected in stage II. All these results indicated that Pmcyclin H might be involved in the regulation of cell cycle and ovarian development of P. monodon

Molecular Analysis of a Selenoprotein M Gene from Penaeus monodon and its Expression at Different Ovarian Stages of Development

In the present study, a Selenoprotein M (SelM) gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of Penaeus monodon. Its cDNA sequence consisted of 901 nucleotides including an open reading frame (ORF) 396bp. The open reading frame encoded a peptide of 131 amino acids. Putative SECIS element stem-loop structure of PmSelM mRNA was predicted by SECISearch software. Like the SECIS elements in vertebrate selenoprotein mRNAs, PmSelM SECIS element contains several highly conserved sequences, including a quartet containing non-Watson-Crick base pairs (5′-UGAN and NGAN-3′), and an unpaired AA motif in the apical loop that is separated from the quartet by 11 nucleotides. Analysis of the tissue expression pattern of the PmSelM gene showed that the PmSelM mRNA was expressed in all tested tissues however there was a considerable difference in level of expression in the tissues. High expression levels were observed in the ovary and hepatopancreas of both female and male P. mondon respectively. The expression of PmSelM increased significantly from stage 1 ovary to stage 2-4 ovaries, which belong to rapid development stage ovaries, followed by a significant decrease in the stage 5 ovary (mature ovary). The results indicated PmSelM might play an important role in ovarian development

Continuous cleaning process of molten copper slags in a channel reactor under the external electric field

Continuous cleaning process of the molten copper slags in a channel reactor under a vertical electric field is proposed. The copper-matte settling velocity under the vertical electric field is much faster than that only under the gravity in the molten slags, and the electric field can also accelerate the magnetite reduction. Cu content in the slag can fall below 0.35 wt. % during the continuous cleaning process, which exceeds much more existing slag treatment process. In addition, the behaviours of some other heavy metal elements in the molten slags during the continuous process are also discussed