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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cric searchable image database as a public platform for conventional pap smear cytology data

Amidst the current health crisis and social distancing, telemedicine has become an important part of mainstream of healthcare, and building and deploying computational tools to support screening more efficiently is an increasing medical priority. The early identification of cervical cancer precursor lesions by Pap smear test can identify candidates for subsequent treatment. However, one of the main challenges is the accuracy of the conventional method, often subject to high rates of false negative. While machine learning has been highlighted to reduce the limitations of the test, the absence of high-quality curated datasets has prevented strategies development to improve cervical cancer screening. The Center for Recognition and Inspection of Cells (CRIC) platform enables the creation of CRIC Cervix collection, currently with 400 images (1,376 × 1,020 pixels) curated from conventional Pap smears, with manual classification of 11,534 cells. This collection has the potential to advance current efforts in training and testing machine learning algorithms for the automation of tasks as part of the cytopathological analysis in the routine work of laboratories.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host–pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum–Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral ( x-y) pixel resolution of 10 nm and an axial ( z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum–Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Biochemistry & Molecular Biology↗
DOE OSTI2021

Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Kindlin-2 promoted the progression of keloids through the Smad pathway and Fas/FasL pathway

Keloids are benign skin tumors characterized by aggressive growth. To date, there is no exact treatment because little is known about its pathological mechanism. Therefore, it is important to investigate the mechanism of its occurrence and development to identify therapeutic targets. In this study, the expression of Kindlin-2 was higher in keloid fibroblasts (KFs) than in normal skin fibroblasts (NFs). In vitro experiments showed that knocking down Kindlin-2 in KFs could promote cell apoptosis and inhibit cell proliferation, cell migration and invasion, and contractile capability. Western blot results showed that the phosphorylation of Smad3 in KFs was inhibited after knocking down Kindlin-2, inhibiting the activation of the Smad pathway. Moreover, knocking down Kindlin-2 increased the expression of Fas and FasL in KFs, which demonstrated that knocking down Kindlin-2 promoted the activation of the exogenous apoptotic pathway of KFs and then facilitated apoptosis. The above results revealed that knocking down Kindlin-2 in KFs can inhibit the activation of the Smad pathway and promote the activation of the Fas/FasL exogenous apoptosis pathway, thereby altering the cytological function of KFs. Therefore, Kindlin-2 might play an important role in the occurrence and development of keloids and could become a new target to treat keloids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Novel Cell-Type-Specific Drought-Responsive Proteins in Root Tips of Field-Grown Perennial Switchgrass

The root-tip region of plants, including the root cap, forms the most basal terminal of the root and exhibits a high degree of cellular complexity in terms of morphology, cytological function, and interaction with environmental cues in the soil. Cells in this region follow a developmental trajectory, transitioning from stem cells to meristematic cells, and ultimately to fully differentiated cell types. However, our understanding of root-tip cell-type specific proteomic responses to abiotic stresses, such as drought, particularly under field conditions, remains limited. This study aimed to identify spatially resolved, cell type-specific proteomes in switchgrass (Panicum virgatum) root tips under drought stress. Root tips were collected from seven-year-old, field-grown switchgrass ‘Alamo’ plants excavated under both well-watered and long-term drought conditions. Cell type-specific proteins were identified using laser capture microdissection (LCM) coupled with nanoPOTS (Nanodroplet Processing in One Pot for Trace Samples) and nano-LC-MS proteomics analysis. Five distinct cell types were targeted: (1) cells in the quiescent center and stem cell niche (QuC), (2) protodermal epidermal cells (PEC) in the meristematic zone, (3) epidermal cells in the transition and elongation zones above the root cap (Epi), (4) peripheral root cap cells (PRC), forming 2–3 layers below the PEC and 1–2 layers above the root border cells, and (5) columella root cap cells (Col) comprising of the columella initials and a single underlying layer of cells undergoing active growth. Principal component analysis (PCA) revealed clear separation among the five targeted cell types, confirming distinct proteomic profiles. Proteins predominantly enriched in each cell type were linked to distinct cellular functions, with QuC cells showing involvement in chromosomal behavior, DNA replication, and mitosis—key processes for stem cell niche regulation. Drought stress resulted in alterations of proteostasis, as evidenced by significant decreases in ribosomal proteins and increases in protein synthesis inhibitors. Moreover, drought stress induced unique cell-type–specific proteins involved in phytohormone biosynthesis and signaling pathways, including auxin, cytokinin, and jasmonic acid. In particular, QuC cells were more highly enriched in proteins associated with DNA repair and mitotic processes. Metabolic pathways related to amino acids, carbohydrates, and lipids were differentially affected in a cell-type–dependent manner, whereas general stress-responsive proteins exhibited consistent changes across all five cell types. Overall, this study provides unique spatially resolved, cell-type-specific proteomic profiles in root tips, representing a significant advancement in our understanding of the cellular mechanisms underlying plant responses to drought stress in natural field conditions.

perennial grass↗
DOE OSTI2021

Significance of lysogeny for the metabolism of Desulfovibrio spp. strains isolated from aquatic environments of Georgia

Sulphate-reducing bacteria (SRB) are ecologically important group of anaerobic micro-organisms that can reduce sulphate to form hydrogen sulphide-a toxic gas causing iron corrosion on metal surfaces. In this work, SRB strains were isolated from aquatic environments in the country of Georgia to determine their lysogenicity and the role of temperate phages in host metabolism. SRB strains were isolated in samples from the Black Sea coast of Georgia. Based on their genetic, cytological and physiological properties of bacteria, 10 Georgian isolates were assigned to the genus Desulfovibrio. Temperate bacteriophages were induced from three out of ten strains by UV-exposure. Comparison of metal (Fe and Cr) reduction and utilization of various carbon sources by the wild-type (lysogenic) bacterial strains and their UV-irradiated counterparts was done. Temperate phage in the cells of SRB could alter significant functions of bacteria and may have a contribution in the acquisition of different traits by SRB. This article pointed to a significant role for temperate bacteriophages in the metabolism and metabolic potential of host strains of SRB, which were first isolated from the aquatic environment of Georgia.

temperate phage↗
DOE OSTI2023

A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Validation of a Proteomic Signature of Lung Cancer Risk from Bronchial Specimens of Risk-Stratified Individuals

A major challenge in lung cancer prevention and cure hinges on identifying the at-risk population that ultimately develops lung cancer. Previously, we reported proteomic alterations in the cytologically normal bronchial epithelial cells collected from the bronchial brushings of individuals at risk for lung cancer. The purpose of this study is to validate, in an independent cohort, a selected list of 55 candidate proteins associated with risk for lung cancer with sensitive targeted proteomics using selected reaction monitoring (SRM). Bronchial brushings collected from individuals at low and high risk for developing lung cancer as well as patients with lung cancer, from both a subset of the original cohort (batch 1: n = 10 per group) and an independent cohort of 149 individuals (batch 2: low risk (n = 32), high risk (n = 34), and lung cancer (n = 83)), were analyzed using multiplexed SRM assays. ALDH3A1 and AKR1B10 were found to be consistently overexpressed in the high-risk group in both batch 1 and batch 2 brushing specimens as well as in the biopsies of batch 1. Validation of highly discriminatory proteins and metabolic enzymes by SRM in a larger independent cohort supported their use to identify patients at high risk for developing lung cancer.

60 APPLIED LIFE SCIENCES↗