The mutational landscape of a US Midwestern breast cancer cohort reveals subtype-specific cancer drivers and prognostic markers.

BACKGROUND: Female breast cancer remains the second leading cause of cancer-related death in the USA. The heterogeneity in the tumor morphology across the cohort and within patients can lead to unpredictable therapy resistance, metastasis, and clinical outcome. Hence, supplementing classic pathological markers with intrinsic tumor molecular markers can help identify novel molecular subtypes and the discovery of actionable biomarkers. METHODS: We conducted a large multi-institutional genomic analysis of paired normal and tumor samples from breast cancer patients to profile the complex genomic architecture of breast tumors. Long-term patient follow-up, therapeutic regimens, and treatment response for this cohort are documented using the Breast Cancer Collaborative Registry. The majority of the patients in this study were at tumor stage 1 (51.4%) and stage 2 (36.3%) at the time of diagnosis. Whole-exome sequencing data from 554 patients were used for mutational profiling and identifying cancer drivers. RESULTS: We identified 54 tumors having at least 1000 mutations and 185 tumors with less than 100 mutations. Tumor mutational burden varied across the classified subtypes, and the top ten mutated genes include MUC4, MUC16, PIK3CA, TTN, TP53, NBPF10, NBPF1, CDC27, AHNAK2, and MUC2. Patients were classified based on seven biological and tumor-specific parameters, including grade, stage, hormone receptor status, histological subtype, Ki67 expression, lymph node status, race, and mutational profiles compared across different subtypes. Mutual exclusion of mutations in PIK3CA and TP53 was pronounced across different tumor grades. Cancer drivers specific to each subtype include TP53, PIK3CA, CDC27, CDH1, STK39, CBFB, MAP3K1, and GATA3, and mutations associated with patient survival were identified in our cohort. CONCLUSIONS: This extensive study has revealed tumor burden, driver genes, co-occurrence, mutual exclusivity, and survival effects of mutations on a US Midwestern breast cancer cohort, paving the way for developing personalized therapeutic strategies.

Transcriptome profile changes in the jejunum of nonhuman primates exposed to supralethal dose of total- or partial-body radiation.

The risk of exposure of the general public or military personnel to high levels of ionizing radiation from nuclear weapons or radiological accidents is a dire national security matter. The development of advanced molecular biodosimetry methods, those that measure biological response, such as transcriptomics, to screen large populations of radiation-exposed victims is key to improving survival outcomes during radiological mass casualty scenarios. In this study, nonhuman primates were exposed to either 12.0 Gy cobalt-60 gamma (total-body irradiation, TBI) or X-ray (partial-body irradiation, PBI) 24 h after administration of a potential radiation medical countermeasure, gamma-tocotrienol (GT3). Changes in the jejunal transcriptomic profiles in GT3-treated and irradiated animals were compared to healthy controls to assess the extent of radiation damage. No major effect of GT3 on radiation-induced transcriptome at this radiation dose was identified. About 80% of the pathways with a known activation or repression state were commonly observed between both exposures. Several common pathways activated due to irradiation include FAK signaling, CREB signaling in the neurons, phagosome formation, and G-protein coupled signaling pathway. Sex-specific differences associated with excessive mortality among irradiated females were identified in this study, including Estrogen receptor signaling. Differential pathway activation was also identified across PBI and TBI, pointing towards altered molecular response for different degrees of bone marrow sparing and radiation doses. This study provides insight into radiation-induced changes in jejunal transcriptional profiles, supporting the investigation for the identification of biomarkers for radiation injury and countermeasure efficacy.

A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1- null skeletal muscle.

KEY POINTS: Nrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses. ABSTRACT: Although Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2flox/flox and iMS-Keap1flox/flox , employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism.

Ecdysone signaling underlies the pea aphid transgenerational wing polyphenism.

The wing polyphenism of pea aphids is a compelling laboratory model with which to study the molecular mechanisms underlying phenotypic plasticity. In this polyphenism, environmental stressors such as high aphid density cause asexual, viviparous adult female aphids to alter the developmental fate of their embryos from wingless to winged morphs. This polyphenism is transgenerational, in that the pea aphid mother experiences the environmental signals, but it is her offspring that are affected. Previous research suggested that the steroid hormone ecdysone may play a role in this polyphenism. Here, we analyzed ecdysone-related gene expression patterns and found that they were consistent with a down-regulation of the ecdysone pathway being involved in the production of winged offspring. We therefore predicted that reduced ecdysone signaling would result in more winged offspring. Experimental injections of ecdysone or its analog resulted in a decreased production of winged offspring. Conversely, interfering with ecdysone signaling using an ecdysone receptor antagonist or knocking down the ecdysone receptor gene with RNAi resulted in an increased production of winged offspring. Our results are therefore consistent with the idea that ecdysone plays a causative role in the regulation of the proportion of winged offspring produced in response to crowding in this polyphenism. Our results also show that an environmentally regulated maternal hormone can mediate phenotype production in the next generation, as well as provide significant insight into the molecular mechanisms underlying the functioning of transgenerational phenotypic plasticity.

Quantitative Proteomics Identifies Novel Nrf2-Mediated Adaptative Signaling Pathways in Skeletal Muscle Following Exercise Training.

Exercise training (ExT) improves skeletal muscle health via multiple adaptative pathways. Nrf2 is a principal antioxidant transcription factor responsible for maintaining intracellular redox homeostasis. In this study, we hypothesized that Nrf2 is essential for adaptative responses to ExT and thus beneficial for muscle. Experiments were carried out on male wild type (WT) and iMS-Nrf2flox/flox inducible muscle-specific Nrf2 (KO) mice, which were randomly assigned to serve as sedentary controls (Sed) or underwent 3 weeks of treadmill ExT thus generating four groups: WT-Sed, WT-ExT, KO-Sed, and KO-ExT groups. Mice were examined for exercise performance and in situ tibialis anterior (TA) contractility, followed by mass spectrometry-based proteomics and bioinformatics to identify differentially expressed proteins and signaling pathways. We found that maximal running distance was significantly longer in the WT-ExT group compared to the WT-Sed group, whereas this capacity was impaired in KO-ExT mice. Force generation and fatigue tolerance of the TA were enhanced in WT-ExT, but reduced in KO-ExT, compared to Sed controls. Proteomic analysis further revealed that ExT upregulated 576 proteins in WT but downregulated 207 proteins in KO mice. These proteins represent pathways in redox homeostasis, mitochondrial respiration, and proteomic adaptation of muscle to ExT. In summary, our data suggest a critical role of Nrf2 in the beneficial effects of SkM and adaptation to ExT.

Fusion gene recurrence in non-small cell lung cancers and its association with cigarette smoke exposure.

Background: Lung cancer remains the leading cause of cancer-related deaths in the US despite novel treatment protocols, with about 235,000 new cases and 131,000 deaths expected from this cancer in 2021 alone. Lung adenocarcinoma and squamous cell carcinoma, which are both subtypes of non-small cell lung cancer, account for most lung cancer cases, and comparing the molecular signatures in these two cancers can identify novel mechanisms that contribute to non-small cell lung cancer oncogenesis. Methods: We, in this study, performed a comprehensive gene fusion profiling of these cancers, which is understudied in lung cancers. Using an alignment-free fusion detection tool, 'ChimeRScope', we screened for gene fusions in lung adenocarcinoma and squamous cell carcinoma datasets from The Cancer Gene Atlas database. Fusion profiles in these two cancer subtypes were essentially different with minimal overlap. Results: Our analysis revealed a positive association of smoking to fusion frequency in lung adenocarcinoma but not in squamous cell carcinoma and identified several fusion genes that could be explored as markers associated with cigarette smoke exposure. We also identified differentially regulated pathways linked to E2F, G2M checkpoint, and MTORC1 signaling upregulated and P53 pathway downregulated in samples containing high fusions in lung adenocarcinoma. Our results indicate that downregulation of the P53 pathway leads to higher gene fusion formation in lung adenocarcinoma. Conclusions: This manuscript provides a strong rationale for investigating the molecular mechanisms of cigarette smoke-induced gene fusion formation associated with lung cancer. Novel recurrent fusions associated with cigarette smoke were identified in our study, which could further be investigated for patient stratification, personalized therapy, and therapeutic monitoring.

Lung transcriptome of nonhuman primates exposed to total- and partial-body irradiation.

The focus of radiation biodosimetry has changed recently, and a paradigm shift for using molecular technologies of omic platforms in addition to cytogenetic techniques has been observed. In our study, we have used a nonhuman primate model to investigate the impact of a supralethal dose of 12 Gy radiation on alterations in the lung transcriptome. We used 6 healthy and 32 irradiated animal samples to delineate radiation-induced changes. We also used a medical countermeasure, γ-tocotrienol (GT3), to observe any changes. We demonstrate significant radiation-induced changes in the lung transcriptome for total-body irradiation (TBI) and partial-body irradiation (PBI). However, no major influence of GT3 on radiation was noted in either comparison. Several common signaling pathways, including PI3K/AKT, GADD45, and p53, were upregulated in both exposures. TBI activated DNA-damage-related pathways in the lungs, whereas PTEN signaling was activated after PBI. Our study highlights the various transcriptional profiles associated with γ- and X-ray exposures, and the associated pathways include LXR/RXR activation in TBI, whereas pulmonary wound-healing and pulmonary fibrosis signaling was repressed in PBI. Our study provides important insights into the molecular pathways associated with irradiation that can be further investigated for biomarker discovery.

Cardiac Spinal Afferent Denervation Attenuates Renal Dysfunction in Rats With Cardiorenal Syndrome Type 2.

Cardiorenal syndrome type 2 (CRS2) is defined as a chronic cardiovascular disease, usually chronic heart failure (CHF), resulting in chronic kidney disease. We hypothesized that the cardiac spinal afferent reflex (CSAR) plays a critical role in the development of CRS2. Our data suggest that cardiac afferent ablation by resiniferatoxin not only improves cardiac function but also benefits the kidneys and increases long-term survival in the myocardial infarction model of CHF. We also found that renal denervation has a similar reno-protective effect in CHF rats. We believe this novel work contributes to the development of a unique neuromodulation therapy to treat CHF patients.

Fusion genes as biomarkers in pediatric cancers: A review of the current state and applicability in diagnostics and personalized therapy.

The incidence of pediatric cancers is rising steadily across the world, along with the challenges in understanding the molecular mechanisms and devising effective therapeutic strategies. Pediatric cancers are presented with diverse molecular characteristics and more distinct subtypes when compared to adult cancers. Recent studies on the genomic landscape of pediatric cancers using next-generation sequencing (NGS) approaches have redefined this field by providing better subtype characterization and novel actionable targets. Since early identification and personalized treatment strategies influence therapeutic outcomes, survival, and quality of life in pediatric cancer patients, the quest for actionable biomarkers is of great value in this field. Fusion genes that are prevalent and recurrent in several pediatric cancers are ideally suited in this context due to their disease-specific occurrence. In this review, we explore the current status of fusion genes in pediatric cancer subtypes and their use as biomarkers for diagnosis and personalized therapy. We discuss the technological advancements made in recent years in NGS sequencing and their impact on fusion detection algorithms that have revolutionized this field. Finally, we also discuss the advantages of pairing liquid biopsy protocols for fusion detection and their eventual use in diagnosis and treatment monitoring.

Identification of Fusion Transcripts from Unaligned RNA-Seq Reads Using ChimeRScope.

Fusion transcripts that are frequent in cancer can be exploited to understand the mechanisms of malignancy and can serve as diagnostic or prognostic markers. Several algorithms have been developed to predict fusion transcripts from DNA or RNA data. The majority of these algorithms align sequencing reads to the reference transcriptome for predicting fusions; however, this results in several undetected fusions due to the highly perturbed nature of cancer genomes. Here, we describe a novel method that uses a k-mer based algorithm to predict fusion transcripts accurately using the unaligned reads from the regular RNA-seq data analysis pipelines.

Comprehensive Characterization of Nanosized Extracellular Vesicles from Central and Peripheral Organs : Implications for Preclinical and Clinical Applications.

Extracellular vesicles (EV) are nano-sized vesicles that have been garnering a lot of attention for their valuable role as potential diagnostic markers and therapeutic vehicles for a plethora of pathologies. Whilst EV markers from biofluids such as plasma, serum, urine, cerebrospinal fluid and in vitro cell culture based platforms have been extensively studied, a significant knowledge gap that remains is the characterization of specific organ derived EVs (ODE). Here, we present a standardized protocol for isolation and characterization of purified EV isolated from brain, heart, lung, kidney and liver from rat and postmortem human tissue. Next, using quantitative mass spectrometry based proteomics, we characterized the respective tissue EV proteomes that identified synaptophysin (SYP), caveolin-3 (CAV3), solute carrier family 22 member 2 (SLC22A2), surfactant protein B (SP-B), and fatty acid-binding protein 1 (FABP1) as potential markers for the brain, heart, kidney, lung, and liver-EV, respectively. These respective tissue specific markers were further validated using both immunoblotting and a nanoplasmonic platform- single EV imaging analysis in the two species. To summarize, our study for the first time using traditional biochemical and high precision technology platforms provide a valuable proof of concept approach in defining specific ODE markers which further could be developed as potential therapeutic candidates for respective end-organ associated pathologies.

A large genomic insertion containing a duplicated follistatin gene is linked to the pea aphid male wing dimorphism.

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid (Acyrthosiphon pisum) male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated follistatin gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

Pan-Cancer Analysis Reveals the Diverse Landscape of Novel Sense and Antisense Fusion Transcripts.

Gene fusions that contribute to oncogenicity can be explored for identifying cancer biomarkers and potential drug targets. To investigate the nature and distribution of fusion transcripts in cancer, we examined the transcriptome data of about 9,000 primary tumors from 33 different cancers in TCGA (The Cancer Genome Atlas) along with cell line data from CCLE (Cancer Cell Line Encyclopedia) using ChimeRScope, a novel fusion detection algorithm. We identified several fusions with sense (canonical, 39%) or antisense (non-canonical, 61%) transcripts recurrent across cancers. The majority of the recurrent non-canonical fusions found in our study are novel, unexplored, and exhibited highly variable profiles across cancers, with breast cancer and glioblastoma having the highest and lowest rates, respectively. Overall, 4,344 recurrent fusions were identified from TCGA in this study, of which 70% were novel. Additional analysis of 802 tumor-derived cell line transcriptome data across 20 cancers revealed significant variability in recurrent fusion profiles between primary tumors and corresponding cell lines. A subset of canonical and non-canonical fusions was validated by examining the structural variation evidence in whole-genome sequencing (WGS) data or by Sanger sequencing of fusion junctions. Several recurrent fusion genes identified in our study show promise for drug repurposing in basket trials and present opportunities for mechanistic studies.

A citizen science initiative for open data and visualization of COVID-19 outbreak in Kerala, India.

OBJECTIVE: India reported its first coronavirus disease 2019 (COVID-19) case in the state of Kerala and an outbreak initiated subsequently. The Department of Health Services, Government of Kerala, initially released daily updates through daily textual bulletins for public awareness to control the spread of the disease. However, these unstructured data limit upstream applications, such as visualization, and analysis, thus demanding refinement to generate open and reusable datasets. MATERIALS AND METHODS: Through a citizen science initiative, we leveraged publicly available and crowd-verified data on COVID-19 outbreak in Kerala from the government bulletins and media outlets to generate reusable datasets. This was further visualized as a dashboard through a front-end Web application and a JSON (JavaScript Object Notation) repository, which serves as an application programming interface for the front end. RESULTS: From the sourced data, we provided real-time analysis, and daily updates of COVID-19 cases in Kerala, through a user-friendly bilingual dashboard (https://covid19kerala.info/) for nonspecialists. To ensure longevity and reusability, the dataset was deposited in an open-access public repository for future analysis. Finally, we provide outbreak trends and demographic characteristics of the individuals affected with COVID-19 in Kerala during the first 138 days of the outbreak. DISCUSSION: We anticipate that our dataset can form the basis for future studies, supplemented with clinical and epidemiological data from the individuals affected with COVID-19 in Kerala. CONCLUSIONS: We reported a citizen science initiative on the COVID-19 outbreak in Kerala to collect and deposit data in a structured format, which was utilized for visualizing the outbreak trend and describing demographic characteristics of affected individuals.

Diurnal and developmental differences in gene expression between adult dispersing and flightless morphs of the wing polymorphic cricket, Gryllus firmus: Implications for life-history evolution.

The functional basis of life history adaptation is a key topic of research in life history evolution. Studies of wing-polymorphism in the cricket Gryllus firmus have played a prominent role in this field. However, prior in-depth investigations of morph specialization have primarily focused on a single hormone, juvenile hormone, and a single aspect of intermediary metabolism, the fatty-acid biosynthetic component of lipid metabolism. Moreover, the role of diurnal variation in life history adaptation in G. firmus has been understudied, as is the case for organisms in general. Here, we identify genes whose expression differs consistently between the morphs independent of time-of-day during early adulthood, as well as genes that exhibit a strong pattern of morph-specific diurnal expression. We find strong, consistent, morph-specific differences in the expression of genes involved in endocrine regulation, carbohydrate and lipid metabolism, and immunity - in particular, in the expression of an insulin-like-peptide precursor gene and genes involved in triglyceride production. We also find that the flight-capable morph exhibited a substantially greater number of genes exhibiting diurnal change in gene expression compared with the flightless morph, correlated with the greater circadian change in the hemolymph juvenile titer in the dispersing morph. In fact, diurnal differences in expression within the dispersing morph at different times of the day were significantly greater in magnitude than differences between dispersing and flightless morphs at the same time-of-day. These results provide important baseline information regarding the potential role of variable gene expression on life history specialization in morphs of G. firmus, and the first information on genetically-variable, diurnal change in gene expression, associated with a key life history polymorphism. These results also suggest the existence of prominent morph-specific circadian differences in gene expression in G. firmus, possibly caused by the morph-specific circadian rhythm in the juvenile hormone titer.

Cry1F resistance among lepidopteran pests: a model for improved resistance management?

The Cry1Fa protein from the bacterium Bacillus thuringiensis (Bt) is known for its potential to control lepidopteran pests, especially through transgenic expression in maize and cotton. The maize event TC1507 expressing the cry1Fa toxin gene became commercially available in the United States in 2003 for the management of key lepidopteran pests including the European corn borer, Ostrinia nubilalis, and the fall armyworm, Spodoptera frugiperda. A high-dose/refuge strategy has been widely adopted to delay evolution of resistance to event TC1507 and other transgenic Bt crops. Efficacy of this strategy depends on the crops expressing a high dose of the Bt toxin to targeted pests and adjacent refuges of non-Bt host plants serving as a source of abundant susceptible insects. While this strategy has proved effective in delaying O. nubilalis resistance, field-evolved resistance to event TC1507 has been reported in S. frugiperda populations in Puerto Rico, Brazil, and the southeastern United States. This paper examines available information on resistance to Cry1Fa in O. nubilalis and S. frugiperda and discusses how this information identifies opportunities to refine resistance management recommendations for Bt maize.

Failure to find association between febrile seizures and SCN1A rs3812718 polymorphism in south Indian patients with mesial temporal lobe epilepsy and hippocampal sclerosis.

We compared the allele and genotype frequencies of SCN1A SNP rs3812718 between patients with MTLE-HS of south Indian ancestry with and without febrile seizures (FS) and with ethnically matched controls. While we observed no significant difference in allele and genotype frequencies of rs3812718 between MTLE-HS patients with and without FS, A allele and AA genotype were overrepresented in MTLE-HS patients when compared to controls. We conclude that in the population studied, although rs3812718 polymorphism increases the susceptibility to MTLE-HS, this is not by increasing the susceptibility to FS.