Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth.

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations.

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

Bcl6 is a subset-defining transcription factor of lymphoid tissue inducer-like ILC3.

Innate lymphoid cells (ILCs) are tissue-resident effector cells with roles in tissue homeostasis, protective immunity, and inflammatory disease. Group 3 ILCs (ILC3s) are classically defined by the master transcription factor RORγt. However, ILC3 can be further subdivided into subsets that share type 3 effector modules that exhibit significant ontological, transcriptional, phenotypic, and functional heterogeneity. Notably lymphoid tissue inducer (LTi)-like ILC3s mediate effector functions not typically associated with other RORγt-expressing lymphocytes, suggesting that additional transcription factors contribute to dictate ILC3 subset phenotypes. Here, we identify Bcl6 as a subset-defining transcription factor of LTi-like ILC3s in mice and humans. Deletion of Bcl6 results in dysregulation of the LTi-like ILC3 transcriptional program and markedly enhances expression of interleukin-17A (IL-17A) and IL-17F in LTi-like ILC3s in a manner in part dependent upon the commensal microbiota-and associated with worsened inflammation in a model of colitis. Together, these findings redefine our understanding of ILC3 subset biology.

The comparable tumour microenvironment in sporadic and NF2-related schwannomatosis vestibular schwannoma.

Bilateral vestibular schwannoma is the hallmark of NF2-related schwannomatosis, a rare tumour predisposition syndrome associated with a lifetime of surgical interventions, radiotherapy and off-label use of the anti-angiogenic drug bevacizumab. Unilateral vestibular schwannoma develops sporadically in non-NF2-related schwannomatosis patients for which there are no drug treatment options available. Tumour-infiltrating immune cells such as macrophages and T-cells correlate with increased vestibular schwannoma growth, which is suggested to be similar in sporadic and NF2-related schwannomatosis tumours. However, differences between NF2-related schwannomatosis and the more common sporadic disease include NF2-related schwannomatosis patients presenting an increased number of tumours, multiple tumour types and younger age at diagnosis. A comparison of the tumour microenvironment in sporadic and NF2-related schwannomatosis tumours is therefore required to underpin the development of immunotherapeutic targets, identify the possibility of extrapolating ex vivo data from sporadic vestibular schwannoma to NF2-related schwannomatosis and help inform clinical trial design with the feasibility of co-recruiting sporadic and NF2-related schwannomatosis patients. This study drew together bulk transcriptomic data from three published Affymetrix microarray datasets to compare the gene expression profiles of sporadic and NF2-related schwannomatosis vestibular schwannoma and subsequently deconvolved to predict the abundances of distinct tumour immune microenvironment populations. Data were validated using quantitative PCR and Hyperion imaging mass cytometry. Comparative bioinformatic analyses revealed close similarities in NF2-related schwannomatosis and sporadic vestibular schwannoma tumours across the three datasets. Significant inflammatory markers and signalling pathways were closely matched in NF2-related schwannomatosis and sporadic vestibular schwannoma, relating to the proliferation of macrophages, angiogenesis and inflammation. Bulk transcriptomic and imaging mass cytometry data identified macrophages as the most abundant immune population in vestibular schwannoma, comprising one-third of the cell mass in both NF2-related schwannomatosis and sporadic tumours. Importantly, there were no robust significant differences in signalling pathways, gene expression, cell type abundance or imaging mass cytometry staining between NF2-related schwannomatosis and sporadic vestibular schwannoma. These data indicate strong similarities in the tumour immune microenvironment of NF2-related schwannomatosis and sporadic vestibular schwannoma.

The genomic basis of temporal niche evolution in a diurnal rodent.

Patterns of diel activity-how animals allocate their activity throughout the 24-h daily cycle-play key roles in shaping the internal physiology of an animal and its relationship with the external environment.1,2,3,4,5 Although shifts in diel activity patterns have occurred numerous times over the course of vertebrate evolution,6 the genomic correlates of such transitions remain unknown. Here, we use the African striped mouse (Rhabdomys pumilio), a species that transitioned from the ancestrally nocturnal diel niche of its close relatives to a diurnal one,7,8,9,10,11 to define patterns of naturally occurring molecular variation in diel niche traits. First, to facilitate genomic analyses, we generate a chromosome-level genome assembly of the striped mouse. Next, using transcriptomics, we show that the switch to daytime activity in this species is associated with a realignment of daily rhythms in peripheral tissues with respect to the light:dark cycle and the central circadian clock. To uncover selection pressures associated with this temporal niche shift, we perform comparative genomic analyses with closely related rodent species and find evidence of relaxation of purifying selection on striped mouse genes in the rod phototransduction pathway. In agreement with this, electroretinogram measurements demonstrate that striped mice have functional differences in dim-light visual responses compared with nocturnal rodents. Taken together, our results show that striped mice have undergone a drastic change in circadian organization and provide evidence that the visual system has been a major target of selection as this species transitioned to a novel temporal niche.

Early preclinical plasma protein biomarkers of brain trauma are influenced by early seizures and levetiracetam.

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Time-Dependent Changes in the Biofluid Levels of Neural Injury Markers in Severe Traumatic Brain Injury Patients-Cerebrospinal Fluid and Cerebral Microdialysates: A Longitudinal Prospective Pilot Study.

Monitoring protein biomarker levels in the cerebrospinal fluid (CSF) can help assess injury severity and outcome after traumatic brain injury (TBI). Determining injury-induced changes in the proteome of brain extracellular fluid (bECF) can more closely reflect changes in the brain parenchyma, but bECF is not routinely available. The aim of this pilot study was to compare time-dependent changes of S100 calcium-binding protein B (S100B), neuron-specific enolase (NSE), total Tau, and phosphorylated Tau (p-Tau) levels in matching CSF and bECF samples collected at 1, 3, and 5 days post-injury from severe TBI patients (n = 7; GCS 3-8) using microcapillary-based western analysis. We found that time-dependent changes in CSF and bECF levels were most pronounced for S100B and NSE, but there was substantial patient-to-patient variability. Importantly, the temporal pattern of biomarker changes in CSF and bECF samples showed similar trends. We also detected two different immunoreactive forms of S100B in both CSF and bECF samples, but the contribution of the different immunoreactive forms to total immunoreactivity varied from patient to patient and time point to time point. Our study is limited, but it illustrates the value of both quantitative and qualitative analysis of protein biomarkers and the importance of serial sampling for biofluid analysis after severe TBI.

Frizzled 7 modulates goblet and Paneth cell fate, and maintains homeostasis in mouse intestine.

Intestinal homeostasis depends on interactions between the intestinal epithelium, the immune system and the microbiota. Because of these complicated connections, there are many problems that need to be solved. Current research has indicated that genes targeted by Wnt signaling are responsible for controlling intestinal stem cell fate and for modulating intestinal homeostasis. Our data show that loss of frizzled 7 (Fzd7), an important element in Wnt signaling, interrupts the differentiation of mouse intestinal stem cells into absorptive progenitors instead of secretory progenitors (precursors of goblet and Paneth cells). The alteration in canonical Wnt and Notch signaling pathways interrupts epithelial homeostasis, resulting in a decrease in physical protection in the intestine. Several phenotypes in our Fzd7-deleted model were similar to the features of enterocolitis, such as shortened intestines, decreased numbers of goblet cells and Paneth cells, and severe inflammation. Additionally, loss of Fzd7 exacerbated the defects in a chemical-induced colitis model and could initiate tumorigenesis. These findings may provide important information for the discovery of efficient therapeutic methods to treat enterocolitis and related cancers in the intestines.

Blood-Based Biomarkers of Repetitive, Subconcussive Blast Overpressure Exposure in the Training Environment: A Pilot Study.

Because of their unknown long-term effects, repeated mild traumatic brain injuries (TBIs), including the low, subconcussive ones, represent a specific challenge to healthcare systems. It has been hypothesized that they can have a cumulative effect, and they may cause molecular changes that can lead to chronic degenerative processes. Military personnel are especially vulnerable to consequences of subconcussive TBIs because their training involves repeated exposures to mild explosive blasts. In this pilot study, we collected blood samples at baseline, 6 h, 24 h, 72 h, 2 weeks, and 3 months after heavy weapons training from students and instructors who were exposed to repeated subconcussive blasts. Samples were analyzed using the reverse and forward phase protein microarray platforms. We detected elevated serum levels of glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1 (UCH-L1), nicotinic alpha 7 subunit (CHRNA7), occludin (OCLN), claudin-5 (CLDN5), matrix metalloprotease 9 (MMP9), and intereukin-6 (IL-6). Importantly, serum levels of most of the tested protein biomarkers were the highest at 3 months after exposures. We also detected elevated autoantibody titers of proteins related to vascular and neuroglia-specific proteins at 3 months after exposures as compared to baseline levels. These findings suggest that repeated exposures to subconcussive blasts can induce molecular changes indicating not only neuron and glia damage, but also vascular changes and inflammation that are detectable for at least 3 months after exposures whereas elevated titers of autoantibodies against vascular and neuroglia-specific proteins can indicate an autoimmune process.

Ribonucleotide reductase M2B in the myofibers modulates stem cell fate in skeletal muscle.

The balance among quiescence, differentiation, and self-renewal of skeletal muscle stem cells (MuSCs) is tightly regulated by their intrinsic and extrinsic properties from the niche. How the niche controls MuSC fate remains unclear. Ribonucleotide reductase M2B (Rrm2b) modulates MuSC quiescence/differentiation in muscle in response to injury. Rrm2b knockout in myofibers, but not in MuSCs, led to weakness of muscles, such as a loss of muscle mass and strength. After muscle injury, damaged myofibers were more efficiently repaired in the Rrm2b myofiber-specific knockout mice than the control mice, but these myofibers were thinner and showed weak functioning. Rrm2b-deleted myofibers released several myokines, which trigger MuSCs to differentiate but not re-enter the quiescent stage to replenish the stem cell pool. Overall, Rrm2b in the myofibers plays a critical role in modulating the MuSC fate by modifying the microenvironment, and it may lead to a possible strategy to treat muscle disorders.

Endurance exercise ameliorates Western diet-induced atherosclerosis through modulation of microbiota and its metabolites.

The World Health Organization determined cardiovascular disease to be the leading cause of death globally; atherosclerosis is the primary cause of the high morbidity and mortality rates. Regular physical activity is an effective strategy for maintaining endothelial health and function to prevent the development of atherosclerosis. Obesity is also a crucial risk factor for atherosclerotic progression in combination with various complications and systemic inflammation. Physiological homeostasis is modulated by the intestinal microbiota, but the mechanisms through which exercise attenuates atherosclerosis through the microbiota have not been elucidated. Therefore, we investigated the effects of endurance exercise on atherosclerosis induced by a Western diet (WD) and apolipoprotein E (ApoE) knockout in terms of microbiota parameters and metabolites. Genetically modified ApoE knockout mice (C57BL/6-Apoeem1Narl/Narl, ApoEKO) and wild-type mice (C57BL6/J) were divided into the following four groups (n = 6), namely, wild-type mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX), for a 12-week intervention. The WD significantly induced obesity and atherosclerotic syndrome in the ApoE WD group. Severe atherosclerotic lesions and arterial thickness were significantly elevated and accompanied by increases in VCAM-1, MCP-1, TNF-α, and IL-1ß for immune cell chemotaxis and inflammation during atherosclerotic pathogenesis in the ApoE WD group. In addition, dysbiosis in the ApoE WD group resulted in the lowest short-chain fatty acid (SCFA) production. Endurance exercise intervention (ApoE WD EX) significantly alleviated atherosclerotic syndrome by reducing obesity, significantly inhibiting VCAM-1, MCP-1, TNF-α, and IL-1ß expression, and increasing the production of SCFAs. Modulation of the microbiota associated with inflammation, such as Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge, and increased SCFA production, particularly through an abundance of Rikenellaceae and Dubosiella, were also observed after exercise intervention. Endurance exercise can alleviate WD-induced atherosclerosis through the amelioration of obesity, inflammation, and chemotaxis signaling, which are modulated by the microbiota and derived SCFAs.

The ADAM9/UBN2/AKR1C3 axis promotes resistance to androgen-deprivation in prostate cancer.

Metastatic and castration-resistant disease is a fatal manifestation of prostate cancer (PCa). The mechanism through which resistance to androgen deprivation in PCa is developed remains largely unknown. Our understanding of the tumor microenvironment (TME) and key signaling pathways between tumors and their TME is currently changing in light of the generation of new knowledge with regard to cancer progression. A disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) is a membranous bridge forming cell-cell and cell-matrix connections that regulate tumor aggressiveness and metastasis. However, it is not known whether ADAM9 expressed in the TME contributes to the CRPC phenotype. In this study, we aimed to investigate the expression patterns of ADAM9 in prostate cancer-associated fibroblasts (CAFs). We also intended to elucidate the effects of both stromal cell- and cancer cell-derived ADAM9 on the progression of CRPC and the implicated molecular pathways. By using both clinical specimens and cell lines, we herein showed that unlike the membrane anchored ADAM9 overexpressed by both PCa cells and prostate CAFs, the secreted isoform of ADAM9 (sADAM9) was strongly detected in CAFs, but rarely in tumor cells, and that could be a serum marker for PCa patients. We demonstrated that functionally sADAM9 are characterized as chemoattractant for the directed movement of androgen-independent PCa cells through integrin downstream FAK/AKT pathway, supporting that elevated sADAM9 by prostate CAFs could be responsible for the promotion of CRPC metastasis. Moreover, by stimulating PCa cells with sADAM9, we found that ubinuclein-2 (UBN2) expression was increased. A positive correlation of ADAM9 and UBN2 expression was observed in androgen receptor-expressing PCa cell lines and further confirmed in clinical PCa specimens. Using a genetic modification approach, we identified UBN2 as a downstream target gene of ADAM9 that is critical for the survival of androgen-dependent PCa cells in response to androgen deprivation, through the induction and effect of the aldo-keto reductase family 1 member C3 (AKR1C3). Collectively, our results reveal a novel action of ADAM9 on the transition of androgen-dependent PCa cells into an androgen-independent manner through the UBN2/AKR1C3 axis; the aforementioned action could contribute to the clinically-observed acquired androgen-deprivation therapy resistance.

Serum Protein Biomarkers of Inflammation, Oxidative Stress, and Cerebrovascular and Glial Injury in Concussed Australian Football Players.

Clinical decisions related to sports-related concussion (SRC) are challenging, because of the heterogenous nature of SRC symptoms coupled with the current reliance on subjective self-reported symptom measures. Sensitive and objective methods that can diagnose SRC and determine recovery would aid clinical management, and there is evidence that SRC induces changes in circulating protein biomarkers, indicative of neuroaxonal injury. However, potential blood biomarkers related to other pathobiological responses linked to SRC are still poorly understood. Therefore, here we analyzed blood samples from concussed (male = 30; female = 9) and non-concussed (male = 74; female = 27) amateur Australian rules football players collected during the pre-season (i.e., baseline), and at 2, 6, and 13 days post-SRC to determine time-dependent changes in serum levels of biomarkers related to glial (i.e., brain lipid-binding protein [BLBP]; phosphoprotein enriched in astrocytes 15) and cerebrovascular injury (i.e., von Willebrand factor, claudin-5), inflammation (i.e., fibrinogen, high mobility group box protein 1), and oxidative stress (i.e., 4-hydroxynoneal). In females, BLBP levels were significantly decreased at 2 days post-SRC compared with their pre-season baseline; however, area under the receiver operating characteristic curve (AUROC) analysis found that BLBP was unable to distinguish between SRC and controls. In males, AUROC analysis revealed a statistically significant change at 2 days post-SRC in the serum levels of 4-hydroxynoneal, however the associated AUROC value (0.6373) indicated little clinical utility for this biomarker in distinguishing SRC from controls. There were no other statistically significant findings. These results indicate that the serum biomarkers tested in this study hold little clinical value in the management of SRC at 2, 6, and 13 days post-injury.

WYSIWYG Design of Hypnotic Line Art.

Hypnotic line art is a modern form in which white narrow curved ribbons, with the width and direction varying along each path over a black background, provide a keen sense of 3D objects regarding surface shapes and topological contours. However, the procedure of manually creating such line art work can be quite tedious and time-consuming. In this article, we present an interactive system that offers a What-You-See-Is-What-You-Get (WYSIWYG) scheme for producing hypnotic line art images by integrating and placing evenly-spaced streamlines in tensor fields. With an input picture segmented, the user just needs to sketch a few illustrative strokes to guide the construction of a tensor field for each part of the objects therein. Specifically, we propose a new method which controls, with great precision, the aesthetic layout and artistic drawing of an array of streamlines in each tensor field to emulate the style of hypnotic line art. Given several parameters for streamlines such as density, thickness, and sharpness, our system is capable of generating professional-level hypnotic line art work. With great ease of use, it allows art designers to explore a wide variety of possibilities to obtain hypnotic line art results of their own preferences.

Distinct transcriptional programs stratify ovarian cancer cell lines into the five major histological subtypes.

BACKGROUND: Epithelial ovarian cancer (OC) is a heterogenous disease consisting of five major histologically distinct subtypes: high-grade serous (HGSOC), low-grade serous (LGSOC), endometrioid (ENOC), clear cell (CCOC) and mucinous (MOC). Although HGSOC is the most prevalent subtype, representing 70-80% of cases, a 2013 landmark study by Domcke et al. found that the most frequently used OC cell lines are not molecularly representative of this subtype. This raises the question, if not HGSOC, from which subtype do these cell lines derive? Indeed, non-HGSOC subtypes often respond poorly to chemotherapy; therefore, representative models are imperative for developing new targeted therapeutics. METHODS: Non-negative matrix factorisation (NMF) was applied to transcriptomic data from 44 OC cell lines in the Cancer Cell Line Encyclopedia, assessing the quality of clustering into 2-10 groups. Epithelial OC subtypes were assigned to cell lines optimally clustered into five transcriptionally distinct classes, confirmed by integration with subtype-specific mutations. A transcriptional subtype classifier was then developed by trialling three machine learning algorithms using subtype-specific metagenes defined by NMF. The ability of classifiers to predict subtype was tested using RNA sequencing of a living biobank of patient-derived OC models. RESULTS: Application of NMF optimally clustered the 44 cell lines into five transcriptionally distinct groups. Close inspection of orthogonal datasets revealed this five-cluster delineation corresponds to the five major OC subtypes. This NMF-based classification validates the Domcke et al. analysis, in identifying lines most representative of HGSOC, and additionally identifies models representing the four other subtypes. However, NMF of the cell lines into two clusters did not align with the dualistic model of OC and suggests this classification is an oversimplification. Subtype designation of patient-derived models by a random forest transcriptional classifier aligned with prior diagnosis in 76% of unambiguous cases. In cases where there was disagreement, this often indicated potential alternative diagnosis, supported by a review of histological, molecular and clinical features. CONCLUSIONS: This robust classification informs the selection of the most appropriate models for all five histotypes. Following further refinement on larger training cohorts, the transcriptional classification may represent a useful tool to support the classification of new model systems of OC subtypes.

Prenatal Bisphenol a Exposure, DNA Methylation, and Low Birth Weight: A Pilot Study in Taiwan.

Prenatal exposure to bisphenol A (BPA) may increase the risk of abnormal birth outcomes, and DNA methylation might mediate these adverse effects. This study aimed to investigate the effects of maternal BPA exposure on maternal and fetal DNA methylation levels and explore whether epigenetic changes are related to the associations between BPA and low birth weight. We collected urine and blood samples originating from 162 mother-infant pairs in a Taiwanese cohort study. We measured DNA methylation using the Illumina Infinium HumanMethylation 450 BeadChip in 34 maternal blood samples with high and low BPA levels based on the 75th percentile level (9.5 µg/g creatinine). Eighty-seven CpGs with the most differentially methylated probes possibly interacting with BPA exposure or birth weight were selected using two multiple regression models. Ingenuity pathway analysis (IPA) was utilized to narrow down 18 candidate CpGs related to disease categories, including developmental disorders, skeletal and muscular disorders, skeletal and muscular system development, metabolic diseases, and lipid metabolism. We then validated these genes by pyrosequencing, and 8 CpGs met the primer design score requirements in 82 cord blood samples. The associations among low birth weight, BPA exposure, and DNA methylation were analyzed. Exposure to BPA was associated with low birth weight. Analysis of the epigenome-wide findings did not show significant associations between BPA and DNA methylation in cord blood of the 8 CpGs. However, the adjusted odds ratio for the dehydrogenase/reductase member 9 (DHRS9) gene, at the 2nd CG site, in the hypermethylated group was significantly associated with low birth weight. These results support a role of BPA, and possibly DHRS9 methylation, in fetal growth. However, additional studies with larger sample sizes are warranted.

Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Aged Skeletal Muscle Retains the Ability to Remodel Extracellular Matrix for Degradation of Collagen Deposition after Muscle Injury.

Aging causes a decline in skeletal muscle function, resulting in a progressive loss of muscle mass, quality, and strength. A weak regenerative capacity is one of the critical causes of dysfunctional skeletal muscle in elderly individuals. The extracellular matrix (ECM) maintains the tissue framework structure in skeletal muscle. As shown by previous reports and our data, the gene expression of ECM components decreases with age, but the accumulation of collagen substantially increases in skeletal muscle. We examined the structural changes in ECM in aged skeletal muscle and found restricted ECM degradation. In aged skeletal muscles, several genes that maintain ECM structure, such as transforming growth factor ß (TGF-ß), tissue inhibitors of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and cathepsins, were downregulated. Muscle injury can induce muscle repair and regeneration in young and adult skeletal muscles. Surprisingly, muscle injury could not only efficiently induce regeneration in aged skeletal muscle, but it could also activate ECM remodeling and the clearance of ECM deposition. These results will help elucidate the mechanisms of muscle fibrosis with age and develop innovative antifibrotic therapies to decrease excessive collagen deposition in aged muscle.