Search Results (4)

A powerful strategy to accelerate bioprocess development is to complement parallel bioreactor systems with an automated approach, often achieved using liquid handling stations. The benefit of such high-throughput experiments is determined by the employed monitoring procedures. To gain a molecular understanding of the microbial production strains in miniaturized parallel single-use bioreactors, we extended the at-line monitoring procedures to transcriptome analysis in a parallel approach using RNA-Seq. To perform automated RNA-Seq experiments, we developed a sample preparation workflow consisting of at-line cell disruption by enzymatic cell lysis, total RNA extraction, nucleic acid concentration normalization, and Nanopore cDNA Library preparation. The pH-controlled aerobic batch growth of Saccharomyces cerevisiae was studied with six different carbon sources (glucose, pyruvate, fructose, galactose, sucrose, and mannose) on a 11 mL scale using 24 parallel stirred tank bioreactors integrated into a liquid handling station while performing at-line sample preparation for RNA-Seq on the same deck. With four biological replicates per condition, 24 cDNA libraries were prepared over 11.5 h. Off-line Nanopore sequencing yielded 20.97 M classified reads with a Q-score > 9. Differential gene expression analysis revealed significant differences in transcriptomic profiles when comparing growth with glucose (exponential growth) to growth with pyruvate (stress conditions), allowing identification of 674 downregulated and 709 upregulated genes. Insignificant changes in gene expression patterns were measured when comparing growth with glucose and fructose, yielding only 64 differentially expressed genes. The expected differences in cellular responses identified in this study show a promising approach for transcriptomic profiling of bioreactor cultures, providing valuable insights on a molecular level at-line in a high-throughput fashion. Full article

This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes a differential extraction technique applied to sample pads from immunochromatographic tests, where the sample pads serve as the substrate. The method involves two sequential lysis steps to effectively separate non-sperm and sperm fractions, enabling the targeted isolation of distinct cell types for downstream DNA analysis. The efficiency of this procedure is demonstrated by the results within this paper, which highlights the successful recovery of both male autosomal and Y-STR profiles, even in mixed samples with a high female presence. Overall, this protocol demonstrates the effective recovery of DNA from sample pads, which is beneficial for forensic practitioners dealing with limited sample quantities, underscoring the value of using these pads in forensic analysis. Full article

Nowadays, the striking numbers of infertile couples that turn to assisted reproductive technologies (ART) drive the research toward a more comprehensive understanding of the underlying causes. Male factors contribute to the inability to conceive in half of the cases, and it has been suggested that sexually transmitted infections could have a role in the onset of spermatozoa impairments. Since the impact of HPV infection on sperm quality and sperm DNA integrity is debated, we wanted to analyze its impact on conventional seminal parameters and the sperm DNA fragmentation index (DFI). Therefore, 117 semen samples of patients undergoing IVF were evaluated for the following characteristics: HPV DNA detection and sperm DNA fragmentation, concentration, motility, and morphology. The results showed a higher rate of HPV-negative patients (59.8% vs. 40.2%) and no HPV-related effect on DFI, sperm concentration, total sperm number, and total motility. Only progressive motility and morphology were found as significantly influenced by HPV positivity. Moreover, we observed a statistically significant difference in DFI when comparing high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) genotypes. Our data suggest that the presence of any HPV type, whatever the exact localization of the virions, can impair some sperm parameters, while HR-HPVs specifically affect the integrity of spermatozoa DNA. Full article

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies. Full article