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Systematic ocular phenotyping of 8,707 knockout mouse lines identifies genes associated with abnormal corneal phenotypes.
Get PDFPURPOSE: Corneal dysmorphologies (CDs) are typically classified as either regressive degenerative corneal dystrophies (CDtrs) or defective growth and differentiation-driven corneal dysplasias (CDyps). Both eye disorders have multifactorial etiologies. While previous work has elucidated many aspects of CDs, such as presenting symptoms, epidemiology, and pathophysiology, the genetic mechanisms remain incompletely understood. The purpose of this study was to analyze phenotype data from 8,707 knockout mouse lines to identify new genes associated with the development of CDs in humans.
METHODS: 8,707 knockout mouse lines phenotyped by the International Mouse Phenotyping Consortium were queried for genes associated with statistically significant (P \u3c 0.0001) abnormal cornea morphology to identify candidate CD genes. Corneal abnormalities were investigated by histopathology. A literature search was used to determine the proportion of candidate genes previously associated with CDs in mice and humans. Phenotypes of human orthologues of mouse candidate genes were compared with known human CD genes to identify protein-protein interactions and molecular pathways using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Protein Analysis Through Evolutionary Relationships (PANTHER), and Kyoto Encyclopedia of Genes and Genomes.
RESULTS: Analysis of data from 8,707 knockout mouse lines identified 213 candidate CD genes. Of these, 37 (17%) genes were previously known to be associated with CD, including 14 in the mouse, 16 in humans, and 7 in both. The remaining 176 (83%) genes have not been previously implicated in CD. We also searched publicly available RNAseq data and found that 131 of the total 213 (61.5%) were expressed in adult human corneal tissue. STRING analysis showed several interactions within and between candidate and established CD proteins. All cellular pathways of the established genes were found in the PANTHER analysis of the candidate genes. Several of the candidate genes were implicated in corneal disease, such as TGF-ß signaling. We also identified other possible underappreciated mechanisms relevant to the human cornea.
CONCLUSIONS: We identified 213 mouse genes that resulted in statistically significant abnormal corneal phenotypes in knockout mice, many of which have not previously been implicated in corneal pathology. Bioinformatic analyses implicated candidate genes in several signaling pathways which are potential therapeutic targets
SINE retrotransposons import polyadenylation signals to 3\u27UTRs in dog (Canis familiaris).
Get PDFBACKGROUND: Messenger RNA 3\u27 untranslated regions (3\u27UTRs) control many aspects of gene expression and determine where the transcript will terminate. The polyadenylation signal (PAS) AAUAAA (AATAAA in DNA) is a key regulator of transcript termination and this hexamer, or a similar sequence, is very frequently found within 30 bp of 3\u27UTR ends. Short interspersed element (SINE) retrotransposons are found throughout genomes in high copy numbers. When inserted into genes they can disrupt expression, alter splicing, or cause nuclear retention of mRNAs. The genomes of the domestic dog and other carnivores carry hundreds of thousands of Can-SINEs, a tRNA-related SINE with transcription termination potential. Because of this we asked whether Can-SINEs may terminate transcript in some dog genes.
RESULTS: Each of the dog\u27s nine Can-SINE consensus sequences carry an average of three AATAAA PASs on their sense strands but zero on their antisense strands. Consistent with the idea that Can-SINEs can terminate transcripts, we find that sense-oriented Can-SINEs are approximately ten times more frequent at 3\u27 ends of 3\u27UTRs compared to further upstream within 3\u27UTRs. Furthermore, the count of AATAAA PASs on head-to-tail SINE sequences differs significantly between sense and antisense-oriented retrotransposons in transcripts. Can-SINEs near 3\u27UTR ends are likely to carry an AATAAA motif on the mRNA sense strand while those further upstream are not. We identified loci where Can-SINE insertion has truncated or altered a 3\u27UTR of the dog genome (dog 3\u27UTR) compared to the human ortholog. Dog 3\u27UTRs have peaks of AATAAA PAS frequency at 28, 32, and 36 bp from the end. The periodicity is partly explained by TAAA(n) repeats within Can-SINE AT-rich tails. We annotated all repeat-masked Can-SINE copies in the Boxer reference genome and found that the young SINEC_Cf type has a mode of 15 bp length for target site duplications (TSDs). All dog Can-SINE types favor integration at TSDs beginning with A(4).
CONCLUSION: Dog Can-SINE retrotransposition has imported AATAAA PASs into gene transcripts and led to alteration of 3\u27UTRs. AATAAA sequences are selectively removed from Can-SINEs in introns and upstream 3\u27UTR regions but are retained at the far downstream end of 3\u27UTRs, which we infer reflects their role as termination sequences for these transcripts
Targeting Ion Channels: Blockers Suppress Calcium Signals and Induce Cytotoxicity Across Medulloblastoma Cell Models.
Get PDFMedulloblastoma (MB) groups 3 and 4 lack targeted therapies despite their dismal prognoses. Ion channels and pumps have been implicated in promoting MB metastasis and growth; however, their roles remain poorly understood. In this study, we repurposed FDA-approved channel blockers and modulators to investigate their potential anti-tumor effects in MB cell lines (DAOY and D283) and primary cell cultures derived from a patient with MB. For the first time, we report spontaneous calcium signaling in MB cells. Spontaneous calcium signals were significantly reduced by mibefradil (calcium channel blocker), paxilline (calcium-activated potassium channel blocker), and thioridazine (potassium channel blocker). These drugs induced dose-dependent cytotoxicity in both the DAOY and D283 cell lines, as well as in primary cell cultures of a patient with group 3 or 4 MB. In contrast, digoxin and ouabain, inhibitors of the Na/K pump, reduced the calcium signaling by over 90% in DAOY cells and induced approximately 90% cell death in DAOY cells and 80% cell death in D283 cells. However, these effects were significantly diminished in the cells derived from a patient with MB, highlighting the variability in drug sensitivity among MB models. These findings demonstrate that calcium signaling is critical for MB cell survival and that the targeted inhibition of calcium pathways suppresses tumor cell growth across multiple MB models
ML-UrineQuant: A machine learning program for identifying and quantifying mouse urine on absorbent paper.
Get PDFThe void spot assay has gained popularity as a way of assessing functional bladder voiding parameters in mice, but analyzing the size and distribution of urine spot patterns on filter paper with software remains problematic due to inter-laboratory differences in image contrast and resolution quality and non-void artifacts. We have developed a machine learning algorithm based on Region-based Convolutional Neural Networks (Mask-RCNN) that was trained in object recognition to detect and quantitate urine spots across a broad range of sizes-ML-UrineQuant. The model proved extremely accurate at identifying urine spots in a wide variety of illumination and contrast settings. The overwhelming advantage it offers over current algorithms will be to allow individual labs to fine-tune the model on their specific images regardless of the image characteristics. This should be a valuable tool for anyone performing lower urinary tract research using mouse models
A ventilated perfused lung model platform to dissect the response of the lungs to viral infection.
No full textIn this study, we developed a 3D lung model that incorporated alveolar and vascular components, allowing for the investigation of lung physiology and responses to infection. We investigated the role of ventilation in formation of the alveolar epithelial layer and its response to viral infections. We subjected our perfused model to a continuous respiratory cycle at the air-liquid interface (ALI) for up to 10 days. The results revealed that ventilation increased tight-junction formation with better epithelial barrier function over time. Two viruses, influenza and respiratory syncytial virus (RSV), were tested, where ventilation enhanced infectivity with an increased progression of viral spread over time while sensitizing the epithelium for viral recognition. Ventilation also attenuated the production of key proinflammatory chemokines. Our findings represent a critical step forward in advancing our understanding of lung-specific viral responses and respiratory infections in response to ventilation, shedding light on vital aspects of pulmonary physiology and pathobiology
A call to action for deciphering genetic variants in human pluripotent stem cells for cell therapy.
No full textHuman pluripotent stem cell (hPSC)-based therapies offer promise but pose potential risks due to culture-acquired genetic variants, some of which have been linked with cancer. An international workshop addressed these concerns, highlighting the need for improved strategies to stratify variants and chart a path toward definitive guidelines in hPSC-based therapy
Alpha-smooth muscle actin-expressing dermal sheath cells are a major cellular contributor to heterotopic subcutaneous ossifications in a mouse model of Albright hereditary osteodystrophy.
Get PDFHeterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). Albright hereditary osteodystrophy is caused by heterozygous inactivation of GNAS, the gene that encodes the α-stimulatory subunit (Gαs ) of G proteins. Previously, we had shown using our laboratory’s AHO mouse model that SCOs develop around hair follicles. Here we show that SCO formation occurs due to inappropriate expansion and osteogenic differentiation of cells that express alpha-smooth muscle actin and that are located within the dermal sheath. We also show in AHO patients and mice that secreted frizzled related protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice leads to earlier development, greater severity, and acceleration of formation of SCOs. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies
Complete genome assemblies of two mouse subspecies reveal structural diversity of telomeres and centromeres.
No full textIt has been more than 20 years since the publication of the C57BL/6J mouse reference genome, which has been a key catalyst for understanding the biology of mammalian diseases. However, the mouse reference genome still lacks telomeres and centromeres, contains 281 chromosomal sequence gaps and only partially represents many biomedically relevant loci. Here we present the first telomere-to-telomere (T2T) mouse genomes for two key inbred strains, C57BL/6J and CAST/EiJ. These T2T genomes reveal substantial variability in telomere and centromere sizes and structural organization. We thus add an additional 213 Mb of new sequence to the reference genome, which contains 517 protein-coding genes. We also examined two important but incomplete loci in the mouse genome-the pseudoautosomal region (PAR) on the sex chromosomes and KRAB zinc-finger protein loci. We identified distant locations of the PAR boundary, different copy numbers and sizes of segmental duplications and a multitude of amino acid substitution mutations in PAR genes
Employee Perspectives on Genetic Testing in the Workplace: Results from a National Survey.
No full textINTRODUCTION: Genetic testing for health-related purposes is now offered in some workplace wellness programs, with notable ethical, legal, and social implications. However, little is known about employee perspectives on workplace genetic testing (wGT).
METHODS: We surveyed a large, diverse national sample of 2,000 employed adults (mean age = 43 years; 51% female). Survey measures assessed respondents\u27 wGT beliefs, perceptions of employer motivations for offering wGT, and privacy concerns.
RESULTS: Most respondents (57.4%) agreed that wGT would improve employees\u27 health, and 46.7% agreed it could aid recruitment and retention of employees. Many respondents attributed legally prohibited motivations to employers offering wGT, including charging employees higher insurance premiums based on wGT results (28.8% rated as very important to employers). Overall, 37% of respondents were not at all comfortable with their employer collecting their genetic information; in addition, most (83.6%) were somewhat or very concerned their employer would fail to protect the privacy of their genetic information or would share such information without permission (79.2%). Heightened privacy concerns were positively associated with employee characteristics, including age ≥55 years, self-identified Black or Asian race and ethnicity, and family history of common diseases, and inversely associated with prior genetic testing experience and employer trust.
CONCLUSIONS: Employees perceive potential health benefits of wGT but harbor substantial privacy concerns and show limited awareness of legal protections against employer misuse of wGT results. Findings suggest a need for robust employee education and informed consent in wGT, along with safeguarding of sensitive personal genetic information