Clinical Implementation of MetaFusion for Accurate Cancer-Driving Fusion Detection from RNA Sequencing.

Oncogenic fusion genes may be identified from next-generation sequencing data, typically RNA-sequencing. However, in a clinical setting, identifying these alterations is challenging against a background of nonrelevant fusion calls that reduce workflow precision and specificity. Furthermore, although numerous algorithms have been developed to detect fusions in RNA-sequencing, there are variations in their individual sensitivities. Here this problem was addressed by introducing MetaFusion into clinical use. Its utility was illustrated when applied to both whole-transcriptome and targeted sequencing data sets. MetaFusion combines ensemble fusion calls from eight individual fusion-calling algorithms with practice-informed identification of gene fusions that are known to be clinically relevant. In doing so, it allows oncogenic fusions to be identified with near-perfect sensitivity and high precision and specificity, significantly outperforming the individual fusion callers it uses as well as existing clinical-grade software. MetaFusion enhances clinical yield over existing methods and is able to identify fusions that have patient relevance for the purposes of diagnosis, prognosis, and treatment.

The psychological impact on perioperative healthcare workers during Victoria's second COVID-19 wave: A prospective longitudinal thematic analysis.

The COVID-19 pandemic has had a profound psychological impact on our frontline healthcare workers. Throughout the entire second COVID-19 wave at one major tertiary hospital in Melbourne Australia, longitudinal qualitative data between perioperative staff members, and analyses of intrapersonal changes were reported. Inductive analysis of three open-ended questions generated four major themes: Organisational Response to the Pandemic, Psychological Impact, Changes in Feelings of Support Over Time and Suggestions for Changes. Understanding the challenges, perception and suggestions from this longitudinal study allows us to provide a range of support services and interventions to minimise the long-term negative psychological impact and be better prepared should another similar situation arises again.

Data on cecal and fecal microbiota and predicted metagenomes profiles of female mice receiving whole flaxseed or its oil and secoisolariciresinol diglucoside components.

Dietary flaxseed (FS) and its components including FS oil (FSO), secoisolariciresinol diglucoside (SDG) and fiber, are processed by the gut microbiota. These data are in support of the article entitled "Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components", Journal of Nutritional Biochemistry [1]. Here we describe data generated by 16S rRNA sequencing of DNA obtained from cecum contents and feces of C57BL/6 female mice fed either a basal diet (BD, AIN93G), or isocaloric diets containing 10% FS, or 10% FS-equivalent amounts of FSO or SDG for 21 days. These include bacterial community composition and inferred KEGG pathways; the raw data are publicly available at the NCBI SRA database (BioProject ID PRJNA683934). Furthermore, this work includes detailed experimentation procedures, total bacterial counts (qPCR) in the cecum content and feces, and correlation analysis between a selected bacterial genus, Bacteroides and a predicted metabolic pathway. FS is utilized worldwide, especially for the prevention and/or treatment of diseases including cardiovascular diseases, diabetes and cancer. These data will be valuable as a reference to study different FS cultivars and SDG- or FSO- enriched products on the gut microbiota, to study gut microbial responses to FS and its components in different mouse strains and mammalian hosts to elucidate individualized effects, and to understand the importance of the gut microbiota for FS benefits.

Discriminatory and cooperative effects within the mouse gut microbiota in response to flaxseed and its oil and lignan components.

Gut microbial processing of dietary flaxseed (FS) contributes to its health benefits, but the relative effects of its bioactive components (lignans, omega-3 fatty acids, fiber) on the microbiota are unclear. We investigated the gut microbial compositional and functional responses to whole FS and its isolated components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG) (precursor to microbial-derived enterolignans) to help understand their contribution to whole FS benefits. Cecum content and fecal samples were collected from C57BL/6 female mice fed a basal diet (AIN93G) or isocaloric diets containing 10% FS or 10% FS-equivalent amounts of FSO or SDG for 21 days. Cecal and fecal microbiota composition and predicted genomic functions, and their relationship with serum enterolignans were evaluated. Only FS modified the community structure. Shared- and diet-specific enriched taxa and functions were identified. Carbohydrate and protein processing functions were enriched in FS mice, and there was a positive correlation between select enriched taxa, encompassing fiber degraders and SDG metabolizers, and serum enterolignans. This was not observed in mice receiving isolated FSO and SDG, suggesting that FS fiber supports SDG microbial metabolism. In conclusion, the cooperative activities of a diverse microbiota are necessary to process FS components and, when administered at the amount present in FS, these components may act together to affect SDG-derived enterolignans production. This has implications for the use of FS, FSO and SDG in clinical practice.

Predicting need for orthognathic surgery in early permanent dentition patients with unilateral cleft lip and palate using receiver operating characteristic analysis.

INTRODUCTION: The purpose of this study was to predict the need for orthognathic surgery in patients with unilateral cleft lip and palate (UCLP) in the early permanent dentition. METHODS: In this retrospective cohort study, we included 61 patients with complete UCLP (36 male, 25 female; mean age, 18.47 years; range, 16.92-26.17 years). The subjects were grouped into an orthognathic surgery group and a nonsurgery group at the time of growth completion. Lateral cephalograms obtained at the age of 11 years were analyzed to compare the 2 groups. The receiver operating characteristic analysis was applied to predict the probability of the need for orthognathic surgery in early adulthood by using the measurements obtained at the age of 11 years. RESULTS: SNB, ANB, SN, overbite, overjet, maxillary length, mandibular body length, and L1-MP were found to be significantly different between the 2 groups. For a person with a score of 2 in the 3-variable-based criteria, the sensitivity and specificity for determining the need for surgical treatment were 90.0% and 83.9%, respectively (ANB, ≤-0.45°; overjet, ≤-2.00 mm; maxillary length, ≤47.25 mm). CONCLUSIONS: Three cephalometric variables, the minimum number of discriminators required to obtain the optimum discriminant effectiveness, predicted the future need for orthognathic surgery with an accuracy of 86.9% in patients with UCLP.

The role of nuclear factor kappaB in late-gestation liver development in the rat.

During the last 3 days of fetal development in the rodent, a burst of hepatocyte proliferation results in a tripling of liver size. Despite stimulation of mitogenesis via multiple signaling pathways, including some that are considered stress response pathways, little apoptosis accompanies this cell growth. Given the accepted role of nuclear factor kappaB (NF-kappaB) in preventing hepatocellular apoptosis during proliferation in mid-development, we predicted that NF-kappaB would be functional during the period of rapid growth during late gestation in the rat. NF-kappaB binding in electrophoretic mobility shift assays was low in embryonic day (E) 19 liver nuclear extracts relative to adult liver nuclear extracts. An additional band that was present in E19 liver was purified and identified as nucleolin. Tumor necrosis factor alpha (TNF-alpha) administration to E19 embryos in utero produced minimal induction of NF-kappaB p50 homodimers and p50/p65 heterodimers, yet baseline apoptosis was not affected. Although p65 was present in E19 hepatocyte cytoplasm in amounts comparable to adult liver, we observed little translocation of p65 to the liver nuclei following TNF-alpha administration. Additionally, expression of several NF-kappaB-responsive genes remained minimally induced in E19 liver following TNF-alpha treatment. In conclusion, although the NF-kappaB components are present in late-gestation fetal liver, NF-kappaB as a transcription factor is relatively inactive and unresponsive to TNF-alpha. Given this finding and the high level of proliferation in late-gestation fetal liver, we predict that alternative antiapoptotic mechanisms are active during this period of rapid hepatic growth.

Fas/FasL-mediated apoptosis in perinatal murine lungs.

Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the murine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index <0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression.

2,5-hexanedione-induced testicular injury.

Now in its third decade of mechanistic investigation, testicular injury caused by 2,5-hexanedione (2,5-HD) exposure is a well-studied model with a rich database. The development of this model reflects the larger changes that have moved biology from a branch of chemistry into the molecular age. Critically examined in this review is the proposed mechanism for 2,5-HD-induced testicular injury in which germ cell maturation is disrupted owing to alterations in Sertoli cell microtubule-mediated functions. The goal is to evaluate the technical and conceptual approaches used to assess 2,5-HD-induced testicular injury, to highlight unanswered questions, and to identify fruitful avenues of future research.

Absence of p53 and FasL has sexually dimorphic effects on both development and reproduction.

Reproduction and development are highly dependent on apoptosis to balance the proliferation that necessarily occurs during these processes. How the absence of two apoptotic factors in mice would affect reproduction and development was examined. Given previous reports of increased neural tube defects in p53-/- female fetuses, decreased fertility in gld female mice, and altered spermatogenesis in both p53 and gld male mice, the possibility that these phenotypes might be enhanced by the elimination of a second apoptotic factor was investigated. The reproductive vigor and the health of offspring were monitored during the production of the new double-deficient strain (FasL-/-p53-/-) for any changes from the reported phenotypes. Thus, any unusual phenotypes that could lead to new models for studying mechanisms of health and disease would be identified. Double-deficient male offspring appeared healthy and occurred at expected frequencies. Additionally, spermatogenesis and male fertility were unaffected by the gene deficiencies. On the other hand, FasL+/+p53-/- and FasL-/-p53-/- female mice were susceptible to increased malformations and post-natal death. These abnormalities were consistent with previous reports of neural tube defects in p53-/- female mice. Fertility rates were also significantly decreased in p53-/- female mice that lived to be adults, an observation not previously reported. Finally, the absence of both FasL and p53 led to dystocia in pregnant female mice, suggesting that the two genes play complementary roles in parturition. Therefore, although male mouse development and reproduction remained unaffected by p53 and FasL deficiencies, female mouse development was adversely affected by the absence of p53, and no live litters were born to female mice with the combined absence of both FasL and p53. In this report, we suggest a potential mechanism involving corpora luteal regression to explain this defect in parturition in FasL-/-p53-/- female mice.

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis.

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.

FasL deficiency enhances the development of tumors in p53+/- mice.

In the multistep process of tumor development, several events occur to transform cells from normal to malignant. Although p53 is one of the most commonly mutated genes in a wide variety of tumors, how other genes interact with p53 to transform cells is only just beginning to be understood. To study the effects of the interaction of the Fas system with p53 in tumor progression and development, mice with a targeted disruption of the p53 tumor supressor gene and a mutation in Fas ligand were bred. Organ weights, life expectancy, and tumor and tissue histology were assessed. Although spleen weights were drastically increased in FasL -/- p53 -/- mice, the FasL deficiency had no effect on life expectancy or the tumor spectrum of homozygous p53-deficient mice. The FasL deficiency reduced the median time to death from 12.1 months in FasL +/+ p53 +/- mice to 9.6 months in FasL -/- p53 +/- mice, and led to a shift in tumor spectrum from predominantly sarcomas (63%) when FasL was present to a large number of lymphomas (76%) in FasL -/- p53 +/- mice. Given the reduced life span and increased incidence of lymphoma in FasL -/- p53 +/- mice, these mice could be useful in carcinogenicity testing, particularly for understanding mechanisms of compounds that are nongenotoxic.