BioTechniques最新文献

Background: Adipose-derived stem cells (ADSCs) exhibit promising potential for the treatment of various diseases, including osteoarthritis. Spheroids derived from ADSCs are a viable treatment option with enhanced anti-inflammatory effects and tissue repair capabilities.

Objective: SphereRing^® is a rotating donut-shaped tube that efficiently produces large quantities of spheroids. However, accurately measuring spheroid size for spheroid quality assessment is challenging. This study aimed to develop an automated method for measuring spheroid size using deep learning through the ChatGPT Data Analyst for image recognition and processing.

Method: The area, perimeter, and circularity of spheroids generated with the SphereRing system were analyzed using ChatGPT Data Analyst and ImageJ. Measurement accuracy was validated using Bland-Altman analysis and scatter plot correlation coefficients.

Results: ChatGPT Data Analyst was consistent with ImageJ for all parameters. Bland-Altman plots demonstrated strong agreement; most data points were within the 95% limits.

Conclusion: The ChatGPT Data Analyst provides a reliable and efficient alternative for assessing spheroid quality. This method reduces human error and improves reproducibility to enhance spheroid quality control. Thus, this method has potential applications in regenerative medicine.

Neurological and psychiatric diseases and disorders affect more than half of the population. Many of these diseases are caused by the malfunctioning of protein synthesis, where too little or too much production of a protein harms a cell and its functions within the brain. We developed a drug screening platform to identify compounds that target the primary cause of these diseases, namely protein expression amounts. This cellular assay monitors protein expression of a target disease gene along with the protein expression of a control gene using the Protein Quantitation Ratioing (PQR) technique. PQR tracks protein concentration using fluorescence. We used human cells and CRISPR-Cas9 genome editing to insert the Protein Quantitation Reporter into target genes. These cells are used in high-throughput drug screening measuring the fluorescence as the assay. Drug hits can be validated using the same PQR technique or animal models of the disease.

Archival fixed tissues hold key insights into the evolutionary history of RNA viruses and the associated host immune response, yet access to the RNA sequence data is limited by a lack of robust methods for RNA extraction and sequence retrieval from these tissue types. Here we compared three commercial RNA extraction techniques (bead, column, and phase-based) on five fixed human brain tissues done in triplicate, that have been stored for up to 43 years. We found that for this sample set, bead-based extractions captured longer molecules and yielded a greater proportion of unique reads when aligned to the human genome, than did column and phase-based extraction methods. Via the incorporation of multiple extraction replicates, we quantified the variability in sequencing metrics resulting from tissue sample and extraction technique heterogeneity. Additionally, we compared pre- and post-sequencing metrics and found that the former poorly predicted post-sequencing on-target success. Our findings help inform future research on the recovery of RNA from archival fixed tissues.

Studies on simple DNA extraction methods from fish mucus, which is an ideal resource for noninvasive sampling, are scarce. In the aquaculture of sturgeons such as sterlets (Acipenser ruthenus), a high-throughput genetic sexing method is needed, as only females are reared to maturity for their roe. Here, DNA extraction methods using HotSHOT (hot sodium hydroxide and tris), a novel alkaline dip, alkaline glycol, and water were compared using the skin mucus of sterlets (n = 8) collected with a toothpick. High-throughput sexing of sterlets from six production batches (n = 3953) was also evaluated using HotSHOT or alkaline dip. An alkaline dip with 10-25 mM NaOH was an effective alternative to HotSHOT, eliminating the heating and neutralizing steps. Regarding high-throughput sexing, > 99% (3946) individuals were successfully genotyped on the first PCR trial, and genotype ratios of each batch were close to 1:1, showing applicability of the alkaline dip method for practical sterlet sexing.

Hospital-acquired infections (HAIs), also known as nosocomial infections, are illnesses contracted during treatment at a healthcare facility and can result in severe or life-threatening complications. HAIs are caused by microorganisms that exhibit resistance to standard antibiotics. HAIs can lead to severe complications, longer stays, and increased mortality, particularly in vulnerable patients. In our previous study, we demonstrated the ability of an engraved Petri dish, referred to as a "biosimulator," to induce adhesion of non-adherent cells and the microbiome. This paper explores the use of the biosimulator to elucidate the microbiome composition within intensive care units (ICUs) in a hospital setting. The biosimulator, with a nutrient-rich bacterial growth medium, was placed in ICUs for 24 h, then incubated for three days under aerobic and anaerobic conditions. Using 16S rRNA sequencing, we profiled the ICU microbiome from multiple samples. Our findings showed that ICU microbiomes closely mirrored those of patients, with microorganisms in the ICU exhibiting stronger interrelationships than in control conditions. The combined use of the biosimulator and profiling offers an effective approach for analyzing and understanding microbiome changes in healthcare settings, particularly in high-risk areas, such as ICUs.

Both bacterial and eukaryotic ribosomes can initiate protein synthesis with formylmethionine (fMet), but detecting fMet-bearing peptides and fMet-bearing proteins has been challenging due to the lack of effective anti-pan-fMet antibodies. Previously, we developed a polyclonal anti-fMet antibody using a fMet-Gly-Ser-Gly-Cys pentapeptide that detects those fMet-bearing peptides and fMet-bearing proteins regardless of their sequence context. In this study, we significantly improved the antibody's specificity and affinity by using a mixture of fMet-Xaa-Cys tripeptides (Xaa, any of the 20 amino acids) as the immunogen. This newly optimized anti-fMet antibody is a powerful, cost-effective tool for detecting fMet-bearing proteins across species. Furthermore, this approach provides a foundation for developing anti-pan-specific antibodies targeting other N-terminal modifications through acylation, alkylation, oxidation, arginylation, etc.

KinSNP^® v1.0, a software tool for human identification, has been widely used to measure IBD segment sharing between individuals using dense SNP data. Herein, the tool was validated using simulated pedigree data (up to 9^th degree relationships) from five diverse populations from the 1000 Genomes Project. Performance was further tested under conditions of simulated genotyping errors and allele or locus dropout. KinSNP data were benchmarked with IBIS, Ped-sim, and known ranges of centimorgan sharing. The calculated values from KinSNP aligned closely with IBIS and Ped-sim benchmarks, and accuracy was maintained with up to 75% simulated missing data. However, even slight increases in simulated sequence error rates negatively impacted performance. This study supports that KinSNP is a reliable solution for IBD-based analyses in forensic contexts.

Observation of plant root morphology in soil is of fundamental importance in plant research, but the lack of transparency of the soil hampers direct observation of roots. One of the approaches to overcome this technical limitation is the use of "transparent soil" (TS), hydrogel-based beads produced by spherification of gelling agents. However, the production of TS by natural dripping of gelling solution can be labor intensive, time consuming and difficult to maintain consistent product quality. Here we present a semi-automated system for TS production. A three-channel peristatic pump controls the critical parameters for spherification, such as drop height and ionic strength, allowing larger-scale TS production with less manual operation. This system improves the efficiency of experiments using TS and enables large-scale experiments requiring large amounts of TS.