Development of an Immortalized Porcine Fibroblast Cell Panel With Different Swine Leukocyte Antigen Genotypes

Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens.
We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase (hTERT) into primary fibroblast cells prepared from ear, fetal, and lung tissues of 10 pigs from five breeds and successfully cultured them for 30-45 passages.
The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar.
The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I (SLA-1, -2, and -3) and three class II genes (DQB1DRB1, and DQA), were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2, were identified.
Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes.
The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on a semiquantitative reverse transcription-polymerase chain reaction.
The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope-major histocompatibility complex interactions in pigs.

The core four – A panel of immunohistochemistry markers to diagnose and subtype testicular germ cell tumors.

 Immunohistochemistry (IHC) to differentiate germ cell tumors.
 The aim of the study is to differentiate seminomatous and nonseminomatous germ cell tumors (GCTs) with morphological overlap using a minimal and affordable panel of IHC markers.
This is a retrospective observational study.
 All testicular GCTs (TGCT) which were diagnosed on biopsies and/or resection specimens (prechemotherapy) between January 2014 and June 2019.
The demographic, clinical, and imaging findings were noted from the medical records. Hematoxylin and eosin (H and E)-stained sections were reviewed for morphology.
The IHC markers constituted Octamer-binding transcription factor (OCT) 3/4, glypican 3 (GPC3), CD117, CD30, placental-like alkaline phosphatase, Sal-like protein 4, and β-human chorionic gonadotropin (HCG).
IHC markers were performed in various combinations depending on the morphology, and a panel constituting OCT 3/4, CD117, GPC3, and CD30 was performed on cases with diagnostic dilemma and morphological overlaps.
Sensitivity, specificity, positive (PPV), and negative predictive value (NPV) were calculated for suggested panel of IHC OCT 3/4, CD117, GPC3, and CD30.
 The study included 36 patients with TGCT with a mean age of 27 (15-58) years. Nonseminomatous tumors were the most common (86%).
The concise panel was performed in 20/36 (56%) tumors to resolve the diagnosis. The sensitivity, specificity, PPV, and NPV for OCT3/4 were 80%, 55%, 31%, and 92% in seminomas and 65%, 100%, 100%, and 46% in embryonal carcinomas (EC), for CD117 was 89%, 82%, 73%, and 93% in seminomas and 60%, 77%, 60%, and 77% in yolk sac tumors (YST), for GPC3 was 95%, 90%, 95%, and 90% in YST, CD30 96%, 100%, 100%, and 91% in ECs, respectively.
Designing a novel concise and affordable IHC panel constituting OCT 3/4, CD117, GPC3, and CD30 has good sensitivity and specificity in differentiating seminomas, YST, and EC, respectively.
Additional markers, namely β-HCG, can be used in identifying the choriocarcinoma component.

Data-driven design of targeted gene panels for estimating immunotherapy biomarkers.

Tumour mutation burden and other exome-wide biomarkers are used to determine which patients will benefit from immunotherapy.
However, the cost of whole exome sequencing limits the widespread use of such biomarkers. Here, we introduce a data-driven framework for the design of targeted gene panels for estimating a broad class of biomarkers including tumour mutation burden and tumour indel burden.
Our first goal is to develop a generative model for the profile of mutation across the exome, which allows for gene- and variant type-dependent mutation rates.
Based on this model, we then propose a procedure for constructing biomarker estimators. Our approach allows the practitioner to select a targeted gene panel of prespecified size and construct an estimator that only depends on the selected genes.
Alternatively, our method may be applied to make predictions based on an existing gene panel, or to augment a gene panel to a given size.
We demonstrate the excellent performance of our proposal using data from three non small-cell lung cancer studies, as well as data from six other cancer types.

Panel of human cell lines with human/mouse artificial chromosomes.

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research.
Recently, microcell-mediated chromosome transfer (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalized mesenchymal stem cells (hiMSCs) and human-induced pluripotent stem cells (hiPSCs).
However, the conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via MMCT.
To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT.
Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors.
The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.

Glucose Metabolic Disorders Enhance Vascular Dysfunction Triggered by Particulate Air Pollution.

 Vascular dysfunction is a biological pathway whereby particulate matter (PM) exerts deleterious cardiovascular effects. The effects of ambient PM on vascular function in prediabetic individuals are unclear.
 A panel study recruited 112 Beijing residents with and without prediabetes. Multiple vascular function indices were measured up to 7×.
The associations between vascular function indices and short-term exposure to ambient PM, including fine PM (PM2.5), ultrafine particles, accumulation mode particles, and black carbon, and the modification of these associations by glucose metabolic status were examined using linear mixed-effects models.
Increases in brachial artery pulse pressure, central aortic pulse pressure, and ejection duration, and decreases in subendocardial viability ratio and reactive hyperemia index were significantly associated with at least one PM pollutant in all participants, indicating increased vascular dysfunction.
For example, for an interquartile range increment in 5-day moving average ultrafine particles, brachial artery pulse pressure, and central aortic pulse pressure increased 5.4% (0.8%-10.4%) and 6.2% (1.2%-11.5%), respectively.
Additionally, PM-associated changes in vascular function differed according to glucose metabolic status.

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Among participants with high fasting blood glucose levels (≥6.1 mmol/L), PM exposure was significantly associated with increased brachial artery systolic blood pressure, central aortic systolic blood pressure, brachial artery pulse pressure, central aortic pulse pressure, and augmentation pressure normalized to a heart rate of 75 bpm (AP75) and decreased subendocardial viability ratio and reactive hyperemia index. Weaker or null associations were observed in the low-fasting blood glucose group.
Glucose metabolic disorders may exacerbate vascular dysfunction associated with short-term ambient PM exposure.

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