Journal of biomolecular techniques : JBT最新文献

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; Telephone: (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.

Integrating advanced technologies into STEMM (Science, Technology, Engineering, Mathematics, and Medicine) education is essential for preparing a future-ready, diverse scientific workforce capable of addressing complex global challenges. This paper presents three interconnected programs-BRITE (Biotechnology Research Incubator for Teachers), ASPIRATION (AI-guided Scientist-Mentored Primary Literature Adaptation for STEMM Education), and C-REP (Cancer Research Education Program)-developed through collaborations with Advanced Technology Cores and multi-institutional participation, involving mentors ranging from core directors to graduate and medical students. While initially supported by internal funding, two of these initiatives have since secured NIH grant support. These initiatives provide immersive, hands-on training in genomics, proteomics, metabolomics, flow cytometry, and AI, reaching middle and high school teachers, high school students, and college students, respectively. Each program integrates mentorship, advanced seminars, and core facility tours to bridge the gap between research and education. Beyond enhancing STEMM literacy and technical skills, these initiatives also support core facilities through increased visibility, utilization, and staff development. Early outcomes indicate improved confidence and engagement among participants, while ongoing evaluations aim to assess long-term impact and scalability. Together, these programs represent a sustainable, collaborative model for integrating cutting-edge science into education.

Until recently, performing RNA-sequencing (RNA-seq) at low input often required specialized library preparation kits. The SMARTer® Stranded Total RNA-Seq Kit v3-Pico Input Mammalian workflow offered by Takara Bio is a popular option for generating high-quality libraries from low-input material. Recently, two RNA-seq library preparation kits, the Watchmaker RNA Library Prep Kit with Polaris Depletion by Watchmaker Genomics and the sparQ RNA-Seq HMR Kit by Quantabio, that offer a larger input range became available. These kits may offer a more accessible and affordable workflow for low-input RNA-seq; therefore, to determine their suitability for low-input applications, a comparative evaluation of the kits was performed using the market standard Takara Pico kit for benchmarking. To do this, two replicates of three total RNA samples of varied quality were prepared at three inputs (250 pg, 1 ng, and 10 ng) using each workflow. Paired-end 50 bp sequencing was performed on a NovaSeq 6000 to obtain 13 million reads per library. While both novel kits generated next-generation sequencing libraries at all inputs tested, neither workflow was specifically tailored for low inputs. We observed variability in library diversity, proportion of duplicate reads, types of transcripts detected, sensitivity of detection, and proportion of nuclear rRNA reads. This suggests that further optimization is required to obtain high-quality libraries from 250 pg to 10 ng input amounts using the novel workflows.

Introduction: Advanced technologies have transformed diagnostics and therapeutics, improving disease management. Secondary school educators play a vital role in fostering critical thinking, problem-solving, and inspiring students in science, technology, engineering, math, and medicine (STEMM) innovations. Comprehensive teacher training enhances education delivery and ensures long-term advancements in science education, empowering future generations.

Materials and methods: We launched the Biotechnology Research Incubator for Teachers (BRITE) pilot program to train secondary school teachers. This 3-week program immerses teachers in Advanced Technology Core facilities, providing hands-on experience with cutting-edge technologies, such as protein array technology, next-generation sequencing, and flow cytometry. It also offers collaborative opportunities with scientists, exposure to primary scientific literature, and support in developing STEMM-based lesson plans.

Results: Over 3 years, the pilot program trained 10 teachers, establishing a framework that secured a 5-year NIH Research Education Program (R25) Science Education Partnership Award to support 12-16 teachers annually. BRITE post-program surveys revealed that participants gained increased confidence and a deeper understanding of integrating STEMM concepts into their classrooms. By the end of summer, each teacher developed a lesson outline based on their experience. By the end of their first year after the training, 43% of the teachers had successfully created specialized teaching units for their classes. These findings highlight the program's success in enhancing teacher development.

Discussion: This program is a practical, scalable, and sustainable model for advancing STEMM education, adaptable for other institutions aiming to create similar teacher-focused programs. We believe this approach can extend to other fields beyond biomedical science.

Federal funding for academic research is currently under scrutiny. Significant changes are anticipated, and we are already observing large cuts across all government agencies. Bracing for possible reductions in federal funding, academic institutions are proactively reviewing operations to minimize the impact on institutional research. After 50 years of successfully enhancing academic research, shared research resources (SRRs) are a proven model of providing efficiency and cost-effectiveness. SRRs provide critical data that have led to a significant number of high-impact publications and a considerable increase in funding for investigators. SRRs are positioned to make further improvements to the institutional research ecosystem through targeted investments and proper strategic planning with institutional leadership. Improvements, such as those outlined in this study, can significantly drive academic biomedical research, increasing competitiveness for research funding, which can ease the impact of cuts to current federal funding models.

The Light Microscopy Research Group, a research group with the Association of Biomolecular Resource Facilities, organized a global study where participants were given artificially generated images of various specimens with various signal-to-noise and object proximity levels. Users were tasked with segmenting the images and providing measured metrics as part of the study. Rather than ranking algorithms, our goal was to study the sources of variability of the results across participants given the same task. This study highlights that substantial variability can exist between independent analyses of identical datasets, even when the analysis problem is relatively straightforward and the analysts are experienced. These findings further support the need for data and analysis methods that follow the findable, accessible, interoperable, reusable (FAIR) principles.

The morphology of a microglial cell determines its activation state and is indicative of various pathologies. Mouse brains are composed of 300,000-500,000 microglia, which makes the application of automated measurement an attractive method for activation status quantification. Additionally, automation can allow for the maximal amount of data per animal versus manual methods. Here we describe an annotated analysis pipeline that works with the commonly used analysis software package Nikon NIS-Elements to automate quantification of microglia cells in mounted and stained brain sections given user-specified parameters. The pipeline can be modified for use with other types of ramified cells, such as neurons. Our analysis pipeline includes pre-processing, thresholding, skeletonization, puncta detection, Sholl analysis, branch classification, and measurement. Once detection thresholds for the desired cell population are validated on representative fields of view, batch analysis can be run for as many image files as desired to generate tables of measurements for regions as large as entire microscope slides when run on a dedicated image processing workstation. The pipeline can also be customized for other purposes, unlike some other automated analysis tools. To our knowledge, a publicly available pipeline template does not exist that can process this amount of microglial morphology and yet is modular, utilizing the customizable NIS-Elements General Analysis 3. With our pipeline, analysis is fast and hands-off once initial thresholds are set for segmentation, introducing less human bias compared with manual measurements, generating more useful data per animal, and allowing less time to be spent on analysis and more time on experiments.

Light microscopy is a powerful tool that can detect and measure cellular and subcellular structural changes over time. This can be done quickly with transmitted light microscopy without perturbing the cells with stains or probes. For instance, cells undergoing apoptosis often shrink in size and have characteristic blebs in the plasma membrane. Many fluorescent probes/reporters are also used for different phases of apoptosis, such as tagged caspase 3. This paper will showcase the practical applications of several imaging modalities (transmitted light and fluorescence) for detecting apoptosis in endpoint and time-lapse images/sequences.

Preparing phage DNA in sufficient quantities for sequencing is often a challenging task, especially when a sensitive bacterial host is not available for phage propagation.¹ This limitation poses a significant obstacle in phage research as the availability of adequate phage DNA is often considered crucial for various analyses, including genome sequencing, functional studies, and therapeutic developments. Also, because DNA extraction from phage samples (e.g., from bacterial induction) can yield low amounts of genomic DNA, many studies utilize tagmentation for amplification-free quantitative sequencing. However, this technique has the drawback of losing phage genome ends (termini) and creating biases in genome coverage.²,³ Polymerase chain reaction (PCR)-free sequencing is often recommended or even necessary to obtain an unbiased characterization of phage genomes or communities. However, sequencing very low quantities of DNA without PCR amplification is challenging, and sequencing service providers, as well as library kit manufacturers, will only guarantee products and results with relatively high DNA inputs. In this study, we aimed to assess the feasibility of sequencing phage genomic DNA with very low DNA starting material and to determine the impact of decreasing DNA input on sequencing quality using both Illumina short-read and Nanopore long-read technologies. We analyzed the quantity and quality of output sequences (and their impact on genome assemblies) for different ranges of input DNA concentrations, starting at the recommended DNA inputs for each technology. We concluded that it is achievable to perform sequencing of high quality with DNA inputs that are lower (i.e., 1000-fold lower) than manufacturers' recommendations or requirements. In this study, we successfully sequenced phage genomic DNA (without PCR amplification) using as little as 1 ng of total input DNA (or 0.02 ng/uL in 50 uL eluted volume) for short-read sequencing with Illumina technology and 0.4 ng (or 0,036 ng/uL in 11 uL eluted volume) for long-read sequencing with Nanopore technology.