Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets.

Numerous methodologies are used for blood RNA extraction, and large quantitative differences in recovered RNA content are reported. We evaluated three archived data sets to determine how extraction methodologies might influence mRNA and lncRNA sequencing results. The total quantity of RNA recovered /ml of blood affects RNA sequencing by impacting the recovery of weakly expressed mRNA, and lncRNA transcripts. Transcript expression (TPM counts) plotted in relation to transcript size (base pairs, bp) revealed a 30% loss of short to midsized transcripts in some data sets. Quantitative recovery of RNA is of considerable importance, and it should be viewed more judiciously. Transcripts common to the three data sets were subsequently normalized and transcript mean TPM counts and TPM count coefficient of variation (CV) were plotted in relation to increasing transcript size. Regression analysis of mean TPM counts versus transcript size revealed negative slopes in two of the three data sets suggesting a reduction of TPM transcript counts with increasing transcript size. In the third data set, the regression slope line of mRNA transcript TPM counts approximates zero and TPM counts increased in proportion to transcript size over a range of 200 to 30,000 bp. Similarly, transcript TPM count CV values also were uniformly distributed over the range of transcript sizes. In the other data sets, the regression CV slopes increased in relation to transcript size. The recovery of weakly expressed and /or short to midsized mRNA and lncRNA transcripts varies with different RNA extraction methodologies thereby altering the fundamental sequencing relationship between transcript size and TPM counts. Our analysis identifies differences in RNA sequencing results that are dependent upon the quantity of total RNA recovery from whole blood. We propose that incomplete RNA extraction directly impacts the recovery of mRNA and lncRNA transcripts from human blood and speculate these differences contribute to the "batch" effects commonly identified between sequencing results from different archived data sets.

Risk Factors of Early Adolescence in the Criminal Career of Polish Offenders in the Light of Life Course Theory.

Life course theory (LCT) diagnoses childhood and adolescent factors that determine an individual's involvement in crime in the future. Farrington lists eight key correlates identified by empirical analyses of criminal careers. In this paper, we seek to discuss the inconsistencies with LCT that we observed in our three empirical studies of the criminal careers of Polish offenders. During 12 years of qualitative research, we conducted direct observations and in-depth interviews in juvenile correction institutions (21) and prisons (8) across the country. We gained access to incarcerated (102) and released (30) juvenile offenders, as well as to incarcerated (68) and released (28) adult offenders. We also conducted in-depth interviews (92) with experts working with young and adult offenders. We similarly accessed some offenders' criminal records and psychological opinions. Our study revealed the strong presence of family and neighborhood influences on early criminality. Contrary to LCT assumptions, state-dependent institutions (military, work, family) were not strong enough determinants of delinquency. Polish offenders generally experience criminal onset later than LCT-oriented criminologists indicate. Based on our data, we also agree with the thesis that the onset of crime should be discussed as different age-related periods rather than just a general onset.

Age-Graded Transitions and Turning Points in Polish Offenders' Criminal Careers from the Standpoint of Life Course Theory.

Both juvenile and adult criminal careers show regularities in the origins of delinquency, the dynamics of the criminal pathway, and the turning points that lead to desistance/persistence in crime. Research shows that family, education, and friendship environments contribute significantly to the individual choices that create criminal biographies. Our aim was to apply core aspects of life course theory (LCT): trajectory, the aged-graded process, transitions, institutions, and ultimately how desistance/persistence factor into explaining the criminal careers of Polish offenders. The research is based on in-depth interviews (130) carried out with both offenders (90) and experts (40). The offenders were divided into two groups: 30 were juveniles, and 60 were adults of whom half were sentenced for the first time (30) and half were recidivists (30) located in correctional institutions or released. The experts group (40) includes psychologists, educators, social rehabilitators, and prison and juvenile detention personnel working with offenders. We used triangulation of researcher, data, and methodology. Our data revealed that similar biographical experiences characterized by an early socialization, family and friends-based circles laid the groundwork for their entry and continued participation in criminal activity. Juvenile and adult first-time sentenced offenders led criminal careers significantly different from those of recidivists, who faced problems with social adaptation caused by lack of family and institutional support.

Mental and Physical Health Problems as Conditions of Ex-Prisoner Re-Entry.

The article focuses on a less-discussed issue of social marginalization of people leaving penitentiaries, which is the prevalence of multifaceted health problems experienced by people in this category. It includes poor health status, resulting from, among others, poor housing conditions, harmful or risky lifestyle, and lack of access to medical services. Data from the District Inspectorate of the Prison Service in Lodz, Poland on the health conditions of inmates was accessed. These data were supplemented by qualitative research conducted in 21 juvenile detention centers and 8 prisons across the country, conducting direct observations and In-Depth Interviews (IDI). A total of 198 IDIs were conducted with incarcerated (72) and released (30) juvenile offenders, and incarcerated (68) and released (28) adult offenders. These were complemented by IDIs with experts (50) and Focus Group Interviews (FGIs; 8) with male and female inmates in 4 Polish prisons. The study revealed that mental and physical health is a serious obstacle to social reintegration of ex-prisoners. It is rarely addressed by state institutions. There are strong associations between neglect of health issues in the prison population and increasing social exclusion after leaving prison. As Poland has a restrictive penal policy, former prisoners remain a group with social stigma and little support.

Strategies for detecting and identifying biological signals amidst the variation commonly found in RNA sequencing data.

BACKGROUND: RNA sequencing analysis focus on the detection of differential gene expression changes that meet a two-fold minimum change between groups. The variability present in RNA sequencing data may obscure the detection of valuable information when specific genes within certain samples display large expression variability. This paper develops methods that apply variance and dispersion estimates to intra-group data to identify genes with expression values that diverge from the group envelope. STRING database analysis of the identified genes characterize gene affiliations involved in physiological regulatory networks that contribute to biological variability. Individuals with divergent gene groupings within network pathways can thereby be identified and judiciously evaluated prior to standard differential analysis. RESULTS: A three-step process is presented for evaluating biological variability within a group in RNA sequencing data in which gene counts were: (1) scaled to minimize heteroscedasticity; (2) rank-ordered to detect potentially divergent "trendlines" for every gene in the data set; and (3) tested with the STRING database to identify statistically significant pathway associations among the genes displaying marked trendline variability and dispersion. This approach was used to identify the "trendline" profile of every gene in three test data sets. Control data from an in-house data set and two archived samples revealed that 65-70% of the sequenced genes displayed trendlines with minimal variation and dispersion across the sample group after rank-ordering the samples; this is referred to as a linear trendline. Smaller subsets of genes within the three data sets displayed markedly skewed trendlines, wide dispersion and variability. STRING database analysis of these genes identified interferon-mediated response networks in 11-20% of the individuals sampled at the time of blood collection. For example, in the three control data sets, 14 to 26 genes in the defense response to virus pathway were identified in 7 individuals at false discovery rates ≤1.92 E-15. CONCLUSIONS: This analysis provides a rationale for identifying and characterizing notable gene expression variability within a study group. The identification of highly variable genes and their network associations within specific individuals empowers more judicious inspection of the sample group prior to differential gene expression analysis.

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR.

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

Coordinated analysis of exon and intron data reveals novel differential gene expression changes.

RNA-Seq expression analysis currently relies primarily upon exon expression data. The recognized role of introns during translation, and the presence of substantial RNA-Seq counts attributable to introns, provide the rationale for the simultaneous consideration of both exon and intron data. We describe here a method for the coordinated analysis of exon and intron data by investigating their relationship within individual genes and across samples, while taking into account changes in both variability and expression level. This coordinated analysis of exon and intron data offers strong evidence for significant differences that distinguish the profiles of the exon-only expression data from the combined exon and intron data. One advantage of our proposed method, called matched change characterization for exons and introns (MEI), is its straightforward applicability to existing archived data using small modifications to standard RNA-Seq pipelines. Using MEI, we demonstrate that when data are examined for changes in variability across control and case conditions, novel differential changes can be detected. Notably, when MEI criteria were employed in the analysis of an archived data set involving polyarthritic subjects, the number of differentially expressed genes was expanded by sevenfold. More importantly, the observed changes in exon and intron variability with statistically significant false discovery rates could be traced to specific immune pathway gene networks. The application of MEI analysis provides a strategy for incorporating the significance of exon and intron variability and further developing the role of using both exons and intron sequencing counts in studies of gene regulatory processes.

Let food be your medicine: nutraceutical properties of lycopene.

Currently, an increase in the awareness of a healthy lifestyle has been observed in society. People are seeking added health benefits from their dietary intake. Thus, functional foods with supplemented components that promote wellness are becoming popular. Lycopene is a carotenoid that gives vegetables and fruits their red color. Due to its chemical structure, lycopene acts as an antioxidant, which is the basis for its health-promoting properties. Oxidative stress is recognized as an important agent of many chronic diseases; thus, lycopene appears to be a universal medicine. Lycopene has the greatest antioxidant potential among carotenoids. Nutraceutical effects of lycopene have been reported for patients with cancer, infertility, metabolic syndrome and liver damage. Therefore, its supplementation can function as a proper causative treatment of disease. In this review, we highlight primary research and clinical trials involving lycopene and its impact on human health.

Inter-Individual Differences in RNA Levels in Human Peripheral Blood.

Relatively little is known about the range of RNA levels in human blood. This report provides assessment of peripheral blood RNA level and its inter-individual differences in a group of 35 healthy humans consisting of 25 females and 10 males ranging in age from 50 to 89 years. In this group, the average total RNA level was 14.59 µg/ml of blood, with no statistically significant difference between females and males. The individual RNA level ranged from 6.7 to 22.7 µg/ml of blood. In healthy subjects, the repeated sampling of an individual's blood showed that RNA level, whether high or low, was stable. The inter-individual differences in RNA level in blood can be attributed to both, differences in cell number and the amount of RNA per cell. The 3.4-fold range of inter-individual differences in total RNA levels, documented herein, should be taken into account when evaluating the results of quantitative RT-PCR and/or RNA sequencing studies of human blood. Based on the presented results, a comprehensive assessment of gene expression in blood should involve determination of both the amount of mRNA per unit of total RNA (U / ng RNA) and the amount of mRNA per unit of blood (U / ml blood) to assure a thorough interpretation of physiological or pathological relevance of study results.

[Internal capsule integrity and its sex-related structural differences in early-onset schizophrenia - diffusion tensor imaging study]. / Zaburzenia integralnosci torebki wewnetrznej oraz róznice strukturalne zalezne od plci w schizofrenii o wczesnym poczatku - badanie DTI.

OBJECTIVES: In schizophrenia, the most repeatable DTI findings concern reduced FA in temporal and frontal lobes with associated abnormalities in connecting neural fibers. The goal of study was to evaluate the differences in FA of the internal capsule in EOS-patients and healthy controls and to place emphasis on the sex as a potential factor determining a predominant pathological pattern of described changes. METHODS: 30 EOS patients and 30 healthy controls were studied using DTI. FA measures within internal capsules were performed in selected ROIs. For statistical analyses the one-way ANOVA test was used (p<0.05). RESULTS: Significant differences of FA between EOS-patients and controls in the right ALIC with lower values of FA in EOS were observed. Within the women sub-groups, statistical differences of FA were observed only for the right ALIC. There were no statistically significant differences within men sub-groups. CONCLUSIONS: 1. Statistically significant differences were found between EOS - subjects (subgroups of woman only) and the control group within the WM diffusivity of the brain in the right ALIC. 2. These results indicate possible involvement of the structures of internal capsule in the EOS development.

[Lycopene--occurrence, properties and applications]. / Likopen--wystepowanie, wlasciwosci oraz potencjalne zastosowanie.

Natural products from plants, fungi and higher animals are valuable sources of attractive alternatives for therapeutics. One of them, lycopene is a bright red carotene found in several fruits and vegetables. Tomato, tomato-based sauces and juices are the most abundant sources of this compound for human. There is a positive correlation between lycopene intake and health. It plays an important role in preventing several diseases, inclusing cancers. Lycopene is the most efficient oxygen and free radicals scavenger. Moreover it controls cell cycle and activates phase II detoxification enzymes. Epidemiological studies confirm its significant role in preventing diseases. Constant progress on this field makes lycopene an interesting object of researches.

The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.

Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.