qRAT: an R-based stand-alone application for relative expression analysis of RT-qPCR data.

BACKGROUND: Reverse transcription quantitative real-time PCR (RT-qPCR) is a well-established method for analysing gene expression. Most RT-qPCR experiments in the field of microbiology aim for the detection of transcriptional changes by relative quantification, which means the comparison of the expression level of a specific gene between different samples by the application of a calibration condition and internal reference genes. Due to the numerous data processing procedures and factors that can influence the final result, relative expression analysis and interpretation of RT-qPCR data are still not trivial and often necessitate the use of multiple separate software packages capable of performing specific functions. RESULTS: Here we present qRAT, a stand-alone desktop application based on R that automatically processes raw output data from any qPCR machine using well-established and state-of-the-art statistical and graphical techniques. The ability of qRAT to analyse RT-qPCR data was evaluated using two example datasets generated in our laboratory. The tool successfully completed the procedure in both cases, returning the expected results. The current implementation includes functionalities for parsing, filtering, normalizing and visualisation of relative RT-qPCR data, like the determination of the relative quantity and the fold change of differentially expressed genes as well as the correction of inter-plate variation for multiple-plate experiments. CONCLUSION: qRAT provides a comprehensive, straightforward, and easy-to-use solution for the relative quantification of RT-qPCR data that requires no programming knowledge or additional software installation. All application features are available for free and without requiring a login or registration.

Video Watermarking Algorithm Based on NSCT, Pseudo 3D-DCT and NMF.

Video watermarking is an important means of video and multimedia copyright protection, but the current watermarking algorithm is difficult to ensure high robustness under various attacks. In this paper, a video watermarking algorithm based on NSCT, pseudo 3D-DCT and NMF has been proposed. Combined with NSCT, 3D-DCT and NMF, the algorithm embeds the encrypted QR code copyright watermark into the NMF base matrix to improve the anti-attack ability of the watermark under the condition of invisibility. The experimental results show that the algorithm ensures the invisibility of the watermark with a high signal-to-noise ratio of the video, and meanwhile has high ability and robustness against common single and combined attacks, such as filtering, noise, compression, shear, rotation and so on. The issue that the video watermarking algorithm has poor resistance to various attacks, especially the shearing attack, has been solved in this paper; thus, it can be used for digital multimedia video copyright protection.

Relationship between the Viral Load in Patients with Different COVID-19 Severities and SARS-CoV-2 Variants.

SARS-CoV-2 has spread throughout the world since 2019, changing in its genome and leading to the appearance of new variants. This gave it different evolutionary advantages, such as greater infectivity and/or a greater ability to avoid the immune response, which could lead to an increased severity of COVID-19 cases. There is no consistent information about the viral load that occurs in infection with the different SARS-CoV-2 variants, hence, in this study we quantify the viral load of more than 16,800 samples taken from the Mexican population with confirmed diagnosis of COVID-19 and we analyze the relation between different demographic and disease variables. We detected that the viral load caused by different variants differs only in the first two days after the onset of symptoms, being higher when infections are caused by the delta variant and lower when caused by omicron. Furthermore, the viral load appears to be higher in outpatients compared to hospitalized patients or in cases of death. On the other hand, no differences were found in the viral load produced in vaccinated and unvaccinated patients, nor did it differ between genders.

An Asymptomatic Patient with Fatal Infertility Carried a Swedish Strain of Chlamydia trachomatis with Additional Deletion in The Plasmid orf1 that Belonged to A Different MLST Sequence Type.

Here, we present the first case of asymptomatic genital Chlamydial infection caused by the new emerging Chlamydia trachomatis (C.t.) ST13 strain genovar E, which has a double deletion of 377 bp and 17 bp in orf1 gene of the cryptic plasmid (ddCT). This case occurred in an infertile patient (case-patient) with a detectable level of Chlamydial antibodies and a spermatozoa deficiency known as azoospermia. Additionally, the ddCT strain showed the presence of a duplication of 44 bp in the plasmid orf3 and SNP in orf4, which were known as the typical characteristics of the Swedish variant of C.t. (nvCT) genovar E. Multilocus sequence typing (MLST) determined a significant difference between ddCT and nvCT in four alleles (oppA, hfiX, gitA and enoA). Both ddCT and nvCT were assigned to different genetic lineages and could be allocated to two different non-overlapping clonal complexes. Furthermore, ddCT demonstrated a considerable difference among 4-5 alleles in comparison with other C.t. strains of genovar E of ST4, ST8, ST12, and ST94, including the founder of a single relevant cluster, wtCT E/SW3 (Swedish genetic lineage). In contrast to other genovar E strains, ddCT had identical alleles with seven out of seven loci found in ST13 strains of genovars D and G, including the founder for this clonal group, D/UW-3/CX, and six out of seven loci found in its derivatives, such as ST6, ST10, and ST95 of genovars G and H. Nevertheless, MSTree V2 showed that ddCT and nvCT could have a common early ancestor, which is a parental C.t. G/9301 strain of ST9. A significant difference between ddCT and nvCT of genovar D (nvCT-D) that was recently found in Mexico was also determined as: (i) ddCT belonged to genovar E but not to genovar D; (ii) ddCT had a 44 bp duplication within the orf3 of the plasmid typical for nvCT; (iii) ddCT possessed an additional 17 bp deletion in the orf1. In conclusion, improved case management should include the clinical physician's awareness of the need to enhance molecular screening of asymptomatic Chlamydia patients. Such molecular diagnostics might be essential to significantly reducing the global burden of Chlamydial infection on international public health.

Comparative Transcriptomics of Ex Vivo, Patient-Derived Endothelial Cells Reveals Novel Pathways Associated With Type 2 Diabetes Mellitus.

In this study low-input RNA-sequencing was used to annotate the molecular identity of endothelial cells isolated and immunopurified with CD144 microbeads. Using this technique, comparative gene expression profiling from healthy subjects and patients with type 2 diabetes mellitus identified both known and novel pathways linked with EC dysfunction. Modeling of diabetes by treating cultured ECs with high glucose identified shared changes in gene expression in diabetic cells. Overall, the data demonstrate how purified ECs from patients can be used to generate new hypotheses about mechanisms of human vascular disease.

Leukocyte Dynamics Influence Reference Gene Stability in Whole Blood: Data-Driven qRT-PCR Normalization Is a Robust Alternative for Measurement of Transcriptional Biomarkers.

BACKGROUND: The use of reference genes for normalization of whole blood qRT-PCR data may be problematic in conditions such as stroke which induce alterations in white blood cell differential. In this study, we assessed the influence of stroke on the stability of commonly employed reference genes, and we evaluated data-driven normalization as an alternative. METHODS: Peripheral whole blood was sampled from 33 stroke patients and 29 controls, and qRT-PCR was used to measure the expression levels of 10 target genes whose transcripts are known stroke biomarkers. Target gene expression levels were normalized via those of 2 frequently cited reference genes (ACTB and B2M) as well as with the NORMA-Gene data-driven normalization algorithm. RESULTS: Whole blood expression levels of reference genes were significantly altered in stroke patients relative to controls. In comparison to normalization via reference genes, NORMA-Gene produced more robust target gene expression data in terms of differential expression dynamics, variance properties, and diagnostic performance. CONCLUSIONS: Our findings suggest that whole blood expression levels of commonly used reference genes may be sensitive to changes in white blood cell differential, and that data-driven qRT-PCR normalization approaches offer a powerful alternative.

Glioblastomas with copy number gains in EGFR and RNF139 show increased expressions of carbonic anhydrase genes transformed by ENO1.

BACKGROUND: Prominence of glycolysis in glioblastomas may be non-specific or a feature of oncogene-related subgroups (i.e. amplified EGFR, etc.). Relationships between amplified oncogenes and expressions of metabolic genes associated with glycolysis, directly or indirectly via pH, were therefore investigated. METHODS: Using multiplex ligation-dependent probe amplification, copy numbers (CN) of 78 oncogenes were quantified in 24 glioblastomas. Related expressions of metabolic genes encoding lactate dehydrogenases (LDHA, LDHC), carbonic anhydrases (CA3, CA12), monocarboxylate transporters (SLC16A3 or MCT4, SLC16A4 or MCT5), ATP citrate lyase (ACLY), glycogen synthase1 (GYS1), hypoxia inducible factor-1A (HIF1A), and enolase1 (ENO1) were determined in 22 by RT-qPCR. To obtain supra-glycolytic levels and adjust for heterogeneity, concurrent ENO1 expression was used to mathematically transform the expression levels of metabolic genes already normalized with delta-delta crossing threshold methodology. RESULTS: Positive correlations with EGFR occurred for all metabolic genes. Significant differences (Wilcoxon Rank Sum) for oncogene CN gains in tumors of at least 2.00-fold versus less than 2.00-fold occurred for EGFR with CA3's expression (p < 0.03) and for RNF139 with CA12 (p < 0.004). Increased CN of XIAP associated negatively. Tumors with less than 2.00-fold CN gains differed from those with gains for XIAP with CA12 (p < 0.05). Male gender associated with CA12 (p < 0.05). CONCLUSIONS: Glioblastomas with CN increases in EGFR had elevated CA3 expression. Similarly, tumors with RNF149 CN gains had elevated CA12 expression. GENERAL SIGNIFICANCE: In larger studies, subgroups of glioblastomas may emerge according to oncogene-related effects on glycolysis, such as control of pH via effects on carbonic anhydrases, with prognostic and treatment implications.

Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case.

Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems. This study expands the spectrum of RAD21 mutations and emphasizes the clinical utility of performing RAD21 mutation analysis in patients presenting with atypical forms of CdLS. Moreover, the variability of clinical presentation within families and low penetrance of mutations as well as the significance of performing molecular genetic testing in mildly affected patients are discussed.

Human apolipoprotein E4 modulates the expression of Pin1, Sirtuin 1, and Presenilin 1 in brain regions of targeted replacement apoE mice.

The apolipoprotein E4 (apoE4) allele is consistently associated with increased risk for Alzheimer's disease (AD). We investigated the molecular mechanism of this susceptibility by analyzing the levels of genes involved in AD pathogenesis in transgenic mice expressing human apoE3 or apoE4 isoforms. mRNA and protein levels of Pin1, Sirtuin 1 (Sirt1), Presenilin 1 (PS1), and pro-Brain-derived Neurotrophic Factor (BDNF) were analyzed in brain regions affected by neuropathological changes in AD. Pin1 mRNA was significantly higher in the hippocampus of apoE4 mice than in apoE3 controls, whereas lower expression was detected in the entorhinal and parietal cortices. Reduced Pin1 levels may increase neurofibrillary degeneration and amyloidogenic processes, while compensatory mechanisms may take place in the hippocampus to balance spatial memory deficits. Sirt1 levels were significantly reduced in the frontal cortex of apoE4 mice. Sirt1 reduction may hinder its protective role against the formation of plaques and tangles and diminish its anti-inflammatory actions. Sirt1 decrease may also play a role in apoE4-associated memory impairments. Moreover, in apoE4 mice PS1 mRNA levels were lower in the frontal cortex. Lower PS1 expression may hamper γ-secretase function, thus affecting amyloid precursor protein processing. Pro-BDNF mRNA levels did not differ between apoE3 and apoE4 mice in any region analyzed. This study showed dysregulated expression of Pin1, Sirt1, and PS1 genes in different cerebral areas of apoE4 mice, suggesting that these changes may play a role in the mechanism of AD vulnerability.

LukS-PV induces mitochondrial-mediated apoptosis and G0/G1 cell cycle arrest in human acute myeloid leukemia THP-1 cells.

The S component (LukS-PV) is one of the two components of Panton-Valentine leukocidin (PVL), which is a pore-forming cytotoxin secreted by Staphylococcus aureus, with the ability to lyse leukocytes. In this study, LukS-PV had the ability to induce apoptosis in the human acute myeloid leukemia (AML) cell line THP-1. Therefore, we investigated the mechanisms of LukS-PV-induced apoptosis in THP-1 cells. THP-1 cells treated with LukS-PV, resulted in a significant inhibition of proliferation in a dose- and time-dependent manner, and induced G0/G1 arrest associated with an inhibition of cell cycle arrest regulatory protein (cyclin D1) in a dose- and time-dependent manner, as measured by flow cytometry (FCM). After 12h exposure to LukS-PV (1.00 µM), annexin V-EGFP/propidium iodide (PI) FCM revealed that 19.5±3.6% of THP-1 cells were apoptotic, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining also revealed THP-1 cells were apoptotic. Chip analysis of 84 apoptosis-related genes demonstrated that 9 genes were up-regulated at least 2-fold and that 5 genes were down-regulated at least 2-fold in the treatment group when compared with levels in the control group. Western blotting reveled that the expression of caspase-8 increased significantly (approximately 4-fold). The levels of caspase-9, -3 and Bax increased significantly, and levels of Bcl-2 decreased rapidly with LukS-PV treatment. These data suggest that LukS-PV acts as an anti-leukemia agent and activates AML cell apoptosis via the mitochondrial pathway. Therefore, LukS-PV may be a multi-targeting drug candidate for the prevention and therapy of AML.