Journal of biomolecular techniques : JBT最新文献

Recent advances in massively parallel DNA sequencing have enabled researchers to study new areas of extreme environments. Of particular interest to many researchers are areas of the Arctic that have yet to be comprehensively examined using DNA techniques. These modern approaches to microbial profiling provide new critical data on systems biology not yet seen before from Arctic samples. The discovery of new microbes, microbial biochemical pathways, and biosynthetic gene clusters are critically important when characterizing the Arctic snow microbiome and can provide insights to discovering valuable biosynthetic gene clusters. In this study, 2 L of snow was collected from 15 sites 12 km east outside of Ilulissat, Greenland, using DNA-free sterile techniques. Snow was allowed to melt and immediately concentrated using the InnovaPrep CP sample concentrator. Whole genome DNA sequencing was performed on extracts using both Illumina and Nanopore sequencing as well as psychrophilic culturing. Individual cultures were also sequenced to determine whole genome content and species identity. The results showed a wide-ranging microbiome across the snow fields, including bacteria, yeast, and fungi, with Granulicella, Methylobabcterium, Nostoc, Sphingomonas, and Streptomyces being consistently detected at higher levels across the majority of sites and sequencing platforms, while Belnapia, Chlorogloea, Hymenobacter, Mesorhizobium, Narcardioides, Pseudomonas, Pseudonocardia, Roseomonas, and Solirubrobacter at comparatively lower abundances. The results of culture data for snow sites reveal Pseudomanas sp., Pseudomonas fluorescens Group, unknown Microbacteriaceae sp., Variovorax sp., Robbsia andropogonis, and low concentrations of Aureobasidium sp., Stylodothis sp., Sphingomonas sp., Hymenobacter sp., Caballeronia sordidicola, and two unknown species of yeast and one unknown species of bacteria.

Long-read DNA and RNA sequencing facilitate genome assembly, haplotyping, complex variant calling, and gene isoform identification. Structural variant calling is integral to the molecular characterization of tumors; thus, long-read nanopore DNA sequencing technology is becoming routinely used in cancer research. As a standard practice, high molecular weight (HMW) DNA is extracted from both tumor and matched normal samples from blood or buffy coat to redact germline variants from somatic mutations. However, we found that buffy coat DNA consistently underperformed compared to DNA extracted from tumor tissue. Furthermore, this observation was unique to DNA extracted from buffy coat cells collected in Streck, but not ethylenediaminetetraacetic acid (EDTA), tubes. We therefore investigated whether the released formaldehyde in Streck tubes resulted in DNA crosslinking, which would explain the low data throughput. Indeed, a decrosslinking step during Streck DNA extraction significantly improved data yield and fragment length without compromising data quality. We therefore recommend a tailored DNA extraction protocol of Streck- derived buffy coat samples for nanopore sequencing.

Nerve morphometry is a quantitative diagnostic laboratory technique used to analyze nerve structures, allowing clinicians to detect morphological differences in myelination and assess the progression of peripheral neuropathies. To quantify myelin and axon pathology, a nerve biopsy is prepared for observation using both light microscopy (LM) and transmission electron microscopy (TEM). Standardized morphometry enables the assessment of metrics, including myelinated fiber profile equivalent circle diameter and axon density. This process remains laborious and complex. This quality improvement study aimed to investigate a consolidated approach to conducting nerve morphometry using only TEM, or high-throughput whole-slide imaging (WSI) to replace standard LM imaging, reducing the complexity and time to conduct the procedure. Cases previously biopsied and analyzed for diagnosis by light microscopy were reimaged and assessed utilizing WSI and TEM. Data were then compared to those previously obtained for diagnosis by the standard LM procedure. While the WSI procedure resulted in good sensitivity and specificity (positive and negative predictive values, respectively) compared to the morphometric analyses originally obtained by the standard diagnostic LM, the results from the TEM analysis failed these statistical tests because of the inability to correctly characterize any of the normal cases. Therefore, we conclude that WSI may be an effective alternative to conducting standard LM myelinated fiber morphometry, reducing the time needed to complete the procedure, whereas the TEM morphometry protocol cannot be recommended at this time.

As climate change continues to disrupt the polar regions of our planet, a comprehensive understanding of both phenotypic and genotypic characteristics of naturally occurring psychrophilic microorganisms is needed, not only from a microbial profiling and taxonomic aspect but also from an industrial potential standpoint. Knowing and understanding the organisms that have the genetic potential to break down environmental contaminants, such as microplastics, is of great interest. In this research, the primary focus was to isolate and characterize the psychrophilic microorganisms from a snow field near Ilulissat, Greenland and use a multi-omics approach to identify and characterize the biodegradation potential against certain biodegradable plastics. Bacterial stains isolated from Greenland were inoculated into small individual bioreactor tubes containing a minimal salts media combined with either polylactic acid or the proprietary Novamont material used in compostable bags. After 4 weeks of incubations at 6°C, turbidity (growth) was measured, and DNA and RNA were extracted and sequenced to identify putative plastic-degrading genes and biosynthetic gene clusters and determine if they are actively expressed in culture conditions. Cultured bacteria comprise 3 genera of bacteria: Pseudomonas, Duganella, and Massilia. Culture tubes comprised Pseudomonas or Duganella isolates alone or Pseudomonas in combination with either Duganella or Massilia isolates. Genomes assembled from cultures contained genes implicated in plastic degradation, and several contained the complete pathway for octane oxidation. Cultures contained active transcripts for most of the identified genes. Several biosynthetic gene clusters were also identified, which may play a role in biofilm formation or adaptation to psychrophilic growth. These data are believed to be the first laboratory culture experiments of psychrophilic microbial degradation of microplastics by organisms isolated from polar regions.

The past decade has seen widespread advances in quality control (QC) materials and software tools focused specifically on mass spectrometry-based proteomics, yet the rate of adoption is inconsistent. Despite the fundamental importance of QC, it typically falls behind learning new techniques, instruments, or software. Considering how important QC is in a core setting where data is generated for non-mass spectrometry experts and confidence in delivered results is paramount, we have created this quick-start guide focusing on off-the-shelf QC materials and relatively easy-to-use QC software. We hope that by providing a background on the different levels of QC, different materials and their uses, describing QC design options, and highlighting some current QC software, implementing QC in a core setting will be easier than ever. There continues to be development in each of these areas (such as new materials and software), and the current generation of QC for mass spectrometry-based proteomics is more than capable of conveying confidence in results as well as minimizing laboratory downtime by guiding experimental, technical, and analytical troubleshooting from sample to results.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

The cold-shock expression system in Escherichia coli was developed on a manual induction approach using optical density at 600 nm (OD₆₀₀) measurements and isopropyl β-D-1-thiogalactopyranoside (IPTG) addition. In this study, we show that cold-shock expression performs equally well using an autoinduction approach wherein OD₆₀₀ measurements and IPTG addition may be eliminated. We further demonstrate that cold-shock expression with autoinduction can better facilitate high-throughput experiments.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, AU-UGA Medical Partnership, 1425 Prince Avenue, Athens GA 30606. Tel; (706) 713-2216: Fax; (706) 713-2221: Email; cslaught@uga.edu or to any member of the editorial board. Article summaries reflect the reviewer's opinions and not necessarily those of the Association.

Introduction/objective: Academic institutions often struggle to meet the unique professional development needs of shared resource personnel, who require business skills, project and people management expertise, and an active, collaborative network of shared resource colleagues.

Materials and methods: We launched the Vanderbilt Core Exchange professional development and networking program in 2020. The program was intentionally designed with core personnel input and supports faculty and staff from more than 80 shared resources across Vanderbilt University and Vanderbilt University Medical Center. Resources offered include a quarterly seminar series with both virtual and in-person events, a website for accessing professional development materials and session recordings, and a dedicated online discussion group for core personnel networking.

Results: There have been 11 Vanderbilt Core Exchange events to date: 2 in person and 9 virtual. In-person events averaged 35 attendees, and virtual events averaged 45 attendees. Topics included equipment grant writing, marketing, handling difficult conversations, managing different workplace work styles, communication and project management tools, and the importance of self-care. Survey responses collected after each event were highly positive and informed areas of improvement and future event topics.

Discussion: This model of local shared resource professional development serves as a template for institutions who desire to create opportunities for collaboration and community building. With a small coordinating committee of dedicated individuals, an institution-wide professional development and networking program can be successfully established even with limited resources.