Basics of high throughput sequencing Summary

Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.

Study on the expression of TOP2A in hepatocellular carcinoma and its relationship with patient prognosis

Background Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate. However, the molecular mechanism of HCC formation remains to be explored and studied. Objective To investigate the expression of TOP2A in hepatocellular carcinoma (HCC) and its prognosis. Methods The data set of hepatocellular carcinoma was downloaded from GEO database for differential gene analysis, and hub gene was identified by Cytoscape. GEPIA was used to verify the expression of HUB gene and evaluate its prognostic value. Then TOP2A was selected as the research object of this paper by combining literature and clinical sample results. Firstly, TIMER database was used to study TOP2A, and the differential expression of TOP2A gene between normal tissues and cancer tissues was analyzed, as well as the correlation between TOP2A gene expression and immune infiltration of HCC cells. Then, the expression of top2a-related antibodies was analyzed using the Human Protein Atlas database, and the differential expression of TOP2A was verified by immunohistochemistry. Then, SRTING database and Cytoscape were used to establish PPI network for TOP2A and protein–protein interaction analysis was performed. The Oncomine database and cBioPortal were used to express and identify TOP2A mutation-related analyses. The expression differences of TOP2A gene were identified by LinkedOmics, and the GO and KEGG pathways were analyzed in combination with related genes. Finally, Kaplan–Meier survival analysis was performed to analyze the clinical and prognosis of HCC patients. Results TOP2A may be a new biomarker and therapeutic target for hepatocellular carcinoma.

Effects of episodic future thinking on reinforcement pathology during smoking cessation treatment among individuals with substance use disorders

Rationale Reinforcer pathology (RP) is a theoretical model based on two processes: delay discounting (DD) and drug demand. Given that RP has been shown to have a predictive value on smoking behaviors, several studies have explored which interventions can reduce RP. Consistent with the RP framework, episodic future thinking (EFT) has shown effects on treatment outcomes and RP processes. The vast majority of studies that assess the effects of EFT on RP consist of experimental studies, and no previous research has tested these effects in a clinical sample of smokers. Objectives The primary aim of this study was to assess the effects of EFT on RP throughout the course of a smoking cessation intervention in smokers with substance use disorders (SUDs). Methods Participants were randomized to cognitive behavior therapy (CBT) + EFT (n = 39) or CBT + EFT + contingency management (n = 33). Cotinine, frequency of EFT practices, cigarette purchase task (CPT), and DD were evaluated in treatment sessions. Mixed-effects model repeated measures analysis was used to explore DD and CPT in-treatment changes as a function of EFT practices and cotinine levels. Results Greater practice of the EFT component significantly reduced cigarette demand (p < .020) as well as DD (p = .003). Additionally, a greater reduction in cotinine levels coupled with greater EFT practice led to a greater decrease in cigarette demand (p < .014). Conclusions EFT reduced the two facets of RP in treatment-seeking smokers with SUDs.

Review of vision screening referrals in children

Background: Childhood vision screening is aimed at the detection of reduced vision due to amblyopia, thus enabling early diagnosis and timely intervention. The purpose of the study was to review the demographics and visual parameters of children referred to the ophthalmologist at Community Health Centre Ljubljana from Slovenian community-based vision screening program and define the visual outcome after treatment in children with amblyopia. Methods: Retrospective medical records review of children referred from community-based vision screening program for further assessment. Medical records were reviewed to determine findings from ophthalmic assessments, treatment received, and visual acuity at the final visit. The main outcome measures were the cause of visual impairment and the visual acuity at the final follow-up visit. Results: From 439 children (mean age 7.3 +/- 3.7 years) referred from community-based vision screening program, 75 children (17%; mean age 5.3 +/- 2.6 years) had amblyopia and received treatment. They had amblyogenic refractive error (3.67 +/- 2.44 diopters of sphere and 1.86 +/- 1.23 diopters of astigmatism) with uncorrected visual acuity on average 0.32 +/- 0.28 logMAR in the worse eye. Visual outcomes after treatment were good with a visual acuity of 0.04 +/- 0.07 logMAR in the worse eye, 60% of them had a visual acuity of 0.00 logMAR (40/40). Conclusion: Children with amblyopia in the presented clinical sample were mostly diagnosed and treated before the school-age. These children showed significant improvement in visual acuity in the amblyopic eye.

Orthorexia nervosa and dieting in a non-clinical sample: a prospective study

Purpose Orthorexia Nervosa (ON) is characterised by excessive attention to a dietary regimen perceived as healthy. A critical factor in the distinction between ON and other eating disorders (EDs) is the dichotomy of quality-versus-quantity of food intake. We investigated whether specific types of diet or dieting frequency are associated with orthorexic features, explored the overlap between ON and EDs symptoms, and examined which constructs are predictive of ON after 6 months. Methods A total of 1075 students (75.1% female, mean age 20.9) completed a set of questionnaires assessing Orthorexia, Eating Disorders, Obsessions and Compulsions, Anxiety and Depression; 358 individuals (79.9 female, mean age 20.9) agreed to participate in the study and completed the same questionnaires after 6 months. Different regression models were defined to investigate our hypothesis. Results Findings suggest that ON is associated with the number and type of diets followed over a lifetime. Moreover, participants with EDs, body dissatisfaction, or a dysfunctional idea of thinness are more likely to report a greater degree of ON features. After 6 months, the best predictors of ON characteristics are the same ON characteristics assessed at the first administration, with a significant role in the ideal of thinness. Conclusions ON is more frequent in individuals with a previous diagnosis of EDs and in individuals who followed a restrictive diet or a vegan/vegetarian one; the number of lifetime diets, beliefs, and behaviors related to the ideals of thinness or body dissatisfaction is common features of ON. Moreover, considering that having ON features in the past is the best ON predictor in the present, we can presume that ON is a construct stable over time. Level of evidence Level IV: Evidence obtained from multiple time series analysis such as case studies. (NB: Dramatic results in uncontrolled trials might also be regarded as this type of evidence).

New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing

Background Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. Methods In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. Results We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. Conclusions By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

Measurement Invariance of Alabama Parenting Questionnaire Across Age, Gender, Clinical Status, and Informant

This study aimed to investigate the measurement invariance of the Alabama Parenting Questionnaire (for both long [APQ-lg] and short [APQ-9] forms) across age, gender, clinical status, and informant (i.e., parent vs. child reports). The sample was composed of adolescents (community sample: N = 1,746; clinical sample: N = 166) and parents ( N = 149). The analyses were conducted in R. Measurement invariance was assessed via multi-group confirmatory factor analysis, equivalence test, and subsampling approach. The original model of APQ-lg (five factors) showed a significantly better fit than other concurrent models (five concurrent models were specified, based on prior literature). For APQ-lg, we found measurement invariance across gender and partial measurement invariance across age, clinical status, and informant. For APQ-9, we confirmed the measurement invariance across gender and clinical status, while across age and informant partial measurement invariance was attested. Overall, our study indicated that APQ-lg and APQ-9 are two valid tools for measuring parenting practices with some caveats.

Confirmatory Factor Analysis of Three Versions of the Depression Anxiety Stress Scale (DASS-42, DASS-21, and DASS-12) in Polish Adults

Background: The Depression Anxiety Stress Scales (DASS) are designed to identify quickly and differentiate between the symptoms of depression and anxiety in the non-clinical population. Different versions (original and short) were validated in many cultures. Nevertheless, there are no data of factorial validity of the different versions of this scale in Polish culture. Thus, the aim of this study was to evaluate the factor structure using confirmatory factor analysis (CFA) and internal consistency of DASS-42 (original version) and two short versions (DASS-21 items and DASS-12 items) in the Polish population.Methods: The DASS-42 was administered to a non-clinical sample, broadly representative of the general Polish adult population (n = 1,021) in terms of demographic variables. The DASS-21 and DASS-12 version used in this study comprise seven and four items from each of the following corresponding three subscales of the Polish version of DASS-42.Results: There were two models that fitted best for DASS-42: (a) modified three correlated factors (depression, anxiety, and stress) with cross-loadings and (b) second order (general factor of psychological distress) and three factors with cross-loadings. There were also two models that fitted best for DASS-21 and DASS-12: (a) modified three correlated factors (depression, anxiety, and stress) and (b) second order (general factor of psychological distress) and three factors.Conclusions: All three versions of DASS appear to have an acceptable factorial structure. However, the shorter versions (DASS-21 and DASS-12) may be more feasible to use in general medical practice and also be less burdensome to participants.

Genome Analysis of the Enterococcus faecium Entfac.YE Prophage

Background: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. Methods: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. Results: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. Conclusion: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.