Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

Eu- and Tb-adsorbed Si3N4 and Ge3N4: tuning the colours with one luminescent host.

Phosphor-converted white light emitting diodes (pc-LEDs) are efficient light sources for applications in lighting and electronic devices. Nitrides, with their wide-ranging applicability due to their intriguing structural diversity, and their auspicious chemical and physical properties, represent an essential component in industrial and materials applications. Here, we present the successful adsorption of Eu and Tb at the grain boundaries of bulk ß-Si3N4 and ß-Ge3N4 by a successful combustion synthesis. The adsorption of europium and terbium, and the synergic combination of both, resulted in intriguing luminescence properties of all compounds (red, green, orange and yellow). In particular, the fact that one host can deliver different colours renders Eu,Tb-ß-M3N4 (M = Si, Ge) a prospective chief component for future light emitting diodes (LEDs). For the elucidation of the electronic properties and structure of ß-Si3N4 and ß-Ge3N4, Mott-Schottky (MS) measurements and density functional theory (DFT) computations were conducted for the bare and RE adsorbed samples.

Conservative management of pediatric temporomandibular disc displacement presenting as juvenile idiopathic arthritis: a case report.

There is limited research regarding management of temporomandibular disorders (TMD) in adolescents with imaging signs of juvenile idiopathic arthritis (JIA). An 11-year-old girl presented to a hospital-based chiropractor for evaluation of a 1.5-year history of unilateral temporomandibular joint (TMJ) pain and trismus. Previously, pediatric rheumatologists diagnosed JIA after contrast-enhanced magnetic resonance imaging revealed edema, effusion, and bilateral anterior disc displacement, and recommended methotrexate, corticosteroid injection, and arthrocentesis. The chiropractor questioned the JIA diagnosis, instead relating symptoms to a mechanical TMD/disc origin. Manual therapy, TMJ exercises, and acupuncture improved TMJ pain and opening. Invasive medical JIA interventions were avoided without long-term recurrence, further questioning the preceding JIA diagnosis. The success of this case suggests that stepped care, beginning with conservative treatment, has value for adolescents with TMD suspect for JIA. Integration of chiropractors and acupuncturists into healthcare institutions may facilitate this care model by affording nonpharmacologic interventions earlier in patient care.

Il existe peu de recherches sur la prise en charge des troubles temporo-mandibulaires (TTM) chez les adolescents présentant des signes d'imagerie de l'arthrite juvénile idiopathique (AJI). Une fillette de 11 ans s'est présentée chez un chiropraticien en milieu hospitalier pour l'évaluation d'un antécédent d'un an et demi de douleur unilatérale à l'articulation temporo-mandibulaire (ATM) et de trismus. Auparavant, les rhumatologues pédiatriques diagnostiquaient l'AJI une fois que l'imagerie par résonance magnétique avec contraste aurait révélé un œdème, un épanchement et un déplacement bilatéral du disque antérieur, et recommandaient le méthotrexate, l'injection de corticostéroïdes et l'arthrocentèse. Le chiropraticien a remis en question le diagnostic d'AJI, associant plutôt les symptômes à une origine mécanique du TTM/ disque. La thérapie manuelle, les exercices de l'ATM et l'acupuncture ont amélioré la douleur et l'ouverture de l'ATM. Les interventions médicales invasives d'AJI ont été évitées sans récidive à long terme, remettant davantage en question le diagnostic d'AJI précédent. Le succès de ce cas suggère que les soins par étapes, en commençant par un traitement conservateur, ont de la valeur pour les adolescents atteints de TTM supposés d'AJI. L'intégration des chiropraticiens et des acupuncteurs dans les établissements de santé peut faciliter ce modèle de soins en permettant des interventions non pharmacologiques plus tôt dans les soins aux patients.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

Senior Centers and LGBTQ Participants: Engaging older adults virtually in a pandemic.

Upon the outbreak of Covid-19, recommendations to cease all non-essential in person services were mandated across the United States to prevent transmission to non-infected individuals. As a result, approximately 96% of all senior centers in the United States were closed to in-person programming. Senior centers have had a long history of engaging older adults, maintaining community connections, enhancing social support and reducing social isolation. SAGE, the first publicly funded senior center for LGBT older adults in the US, serves a traditionally under-served population with a vast array of services and programs. This exploratory, cross-sectional study utilized an online survey to evaluate the experiences of 113 SAGE members after the Coronavirus pandemic closed their senior center. Participants reported a relatively easy adaptation to technology, steady participation in programs and services, satisfaction with virtual senior center programming and a stable sense of engagement with their peers. Higher levels of engagement with senior center programs was associated with stronger feelings of social support. Additionally, stronger perceptions of social support and participation in exercise and fitness programming were associated with higher life satisfaction and lower depression and anxiety. Implications and recommendations for other gerontological service providers are offered.

Generation of a conditional Flpo/FRT mouse model expressing constitutively active TGFß in fibroblasts.

Transforming growth factor (TGFß) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFß concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFß in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSFTGFßCA] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSFTGFßCA allele consists in a transgene encoding a constitutively active mutant form of TGFß (TGFßCA) under the control of a Frt-STOP-Frt (FSF) cassette. The FSFTGFßCA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSFTGFßCA] animals do not present any obvious phenotype despite the correct expression of TGFßCA transgene in fibroblasts. This [Fsp1-Flpo; FSFTGFßCA] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFß concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases.

Generation of an Fsp1 (fibroblast-specific protein 1)-Flpo transgenic mouse strain.

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1-Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast-specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1-Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.

Glandular defects in the mouse uterus with sustained activation of TGF-beta signaling is associated with altered differentiation of endometrial stromal cells and formation of stromal compartment.

Uterine gland development, also known as adenogenesis, is a key uterine morphogenic process indispensable for normal uterine function and fertility. Our earlier studies have reported that overactivation of TGFB receptor 1 (TGFBR1) in the mouse uterus using progesterone receptor (Pgr)-Cre recombinase causes female infertility, defective decidualization, and reduced uterine gland formation, a developmental milestone of postnatal uterus. To understand mechanisms that underpin the disrupted uterine gland formation in mice with sustained activation of TGFBR1, we raised the question of whether early postnatal adenogenesis was compromised in these mice. Experiments were designed using mice with constitutive activation of TGFBR1 driven by Pgr-Cre to determine the timing of adenogenic defects and potential mechanisms associated with dysregulation of adenogenic genes, luminal epithelial cell proliferation and endometrial fibrotic changes. Uterine tissues from mice with constitutive activation of TGFBR1 were collected during the critical time window of adenogenesis and analyzed together with age-matched controls. Multiple approaches including immunohistochemistry, immunofluorescence, Trichrome staining, quantitative real-time PCR, western blot, conditional knockout and human endometrial cell culture were utilized. TGFBR1 activation in the mouse uterus suppressed adenogenesis during postnatal uterine development, concomitant with the aberrant differentiation of uterine stromal cells. Analysis of transcript expression of WNT pathway components revealed dysregulation of adenogenesis-associated genes. Notably, the adenogenic defects occurred in spite of the increased proliferation of uterine luminal epithelial cells, accompanied by increased expression of genes associated with fibrotic changes. Moreover, the adenogenic defects were alleviated in mice where TGFBR1 was activated in presumably half of the complement of uterine cells. Our results suggest that altered differentiation of endometrial stromal cells and formation of stromal compartment promote adenogenic defects.

Comparison of a sports-hydration drink containing high amylose starch with usual hydration practice in Australian rules footballers during intense summer training.

BACKGROUND: Fluid deficits exceeding 1.6% can lead to physical and cognitive impairment in athletes. Sport drinks used by athletes are often hyper-osmolar but this is known to be suboptimal for rehydration in medical settings and does not utilize colonic absorptive capacity. Colonic absorption can be enhanced by fermentative production of short chain fatty acids (SCFA) from substrates such as high amylose maize starch (HAMS). This study therefore compared, in elite Australian Football League (AFL) players at the height of outdoor summer training, a novel dual-action sports oral rehydration strategy that contained HAMS as well as glucose, to their usual rehydration practices (Control). The primary outcome markers of hydration were hematocrit and body weight. METHODS: A randomized single-blind crossover study was undertaken in thirty-one AFL players; twenty-seven completed the study which was conducted on four days (two days in the Intervention arm and two in Control arm). The Intervention arm was comprised a 50-100 g evening preload of an acetylated HAMS (Ingredion Pty Ltd) followed by consumption of a specially formulated sports oral rehydration solution (SpORS) drink during intense training and recovery. Players followed their usual hydration routine in the Control arm. Quantitative assessments of body weight, hematocrit and urine specific gravity were made at three time-points on each day of training: pre-training, post-training (90 min), and at end of recovery (30-60 min later). GPS tracking monitored player exertion. RESULTS: Across the three time-points, hematocrit was significantly lower and body weight significantly higher in Intervention compared to Control arms (p < 0.02 and p = 0.001 respectively, mixed effects model). Weights were significantly heavier at all three assessment points for Intervention compared to Control arms (Δ = 0.30 ± 0.13, p = 0.02 pre-training; Δ = 0.43 ± 0.14, p = 0.002 post training; and Δ = 0.68 ± 0.14, p < 0.001 for recovery). Between the pre-training and end-of-recovery assessments, the Control arm lost 0.80 kg overall compared with 0.12 kg in the Intervention arm, an 85% lower reduction of bodyweight across the assessment period. CONCLUSION: The combination of the significantly lower hematocrit and increased body weight in the Intervention arm represents better hydration not only at the end of training as well as following a recovery period but also at its commencement. The magnitude of the benefit seems sufficient to have an impact on performance and further studies to test this possibility are now indicated. TRIAL REGISTRATION: Trial is listed on the Australian New Zealand Clinical Trials Registry ( ACTRN 12613001373763 ).

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

A novel mouse model of testicular granulosa cell tumors.

STUDY QUESTION: What is the role of dysregulated transforming growth factor beta (TGFB) signaling in the development of sex cord-stromal tumors in the testis? SUMMARY ANSWER: Overactivation of TGFB signaling results in the development of testicular tumors resembling granulosa cell tumors (GrCTs). WHAT IS KNOWN ALREADY: In an earlier study, we demonstrated that constitutively active TGFB receptor 1 (TGFBR1) in ovarian somatic cells promotes the development of ovarian GrCTs. However, the consequence of dysregulation of TGFB signaling in the pathobiology of the testis, remains poorly defined. STUDY DESIGN, SIZE, DURATION: To identify the impact of dysregulation of TGFB signaling on the testis, we generated mice with constitutive activation of TGFBR1 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase. The effect of constitutively active TGFBR1 on testis development and the timeline of testicular tumor formation were examined. We further investigated the molecular features of testicular tumors and determined the expression of beta-catenin (CTNNB1) known to be involved in testicular GrCT development. PARTICIPANTS/MATERIALS, SETTING, METHODS: Male mice with constitutive activation of TGFBR1 were examined at various developmental stages (i.e. from 1 week up to 6 months) along with controls. Testis samples were collected and processed for histological and molecular analyses, including haematoxylin and eosin (H and E) staining, real-time PCR, immunohistochemistry, immunofluorescence and western blotting. Immunostaining/immunoblotting and real-time PCR experiments were performed using at least three animals per genotype. Data are presented as mean ± SEM. Statistical significance was determined using unpaired two-tail t-test and reported when P value is <0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Mice harboring constitutively active TGFBR1 in the testes developed tumors resembling testicular GrCTs, a rare type of tumors in the testis. The formation of testicular tumors led to altered cell proliferation, loss of germ cells and defective spermatogenesis. Immunohistochemically, these tumors were positive for inhibin alpha (INHA), forkhead box O1 (FOXO1), and more importantly, forkhead box L2 (FOXL2), a protein specifically expressed in the ovary and required for normal granulosa cell differentiation and function. Consistent with the immunohistochemical findings, FOXL2 proteins were only detectable in testes of TGFBR1-CAAcre mice but not those of controls by western blotting, suggesting potential alteration of Sertoli cell fate. To explore mechanisms underlying the tumor-promoting effect of TGFBR1 overactivation, we examined the expression of CTNNB1. The results revealed increased expression of CTNNB1 in testicular tumors in TGFBR1-CAAcre mice. Collectively, this study uncovered tumorigenic function of enhanced TGFB signaling in the testis. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed using mice, and the direct relevance of the experimental paradigm and findings to human testicular GrCTs awaits further investigation. Of note, constitutive activation of TGFBR1 was employed to enhance TGFB/SMAD signaling activity and may not be interpreted as the genetic cause of the disease. WIDER IMPLICATIONS OF THE FINDINGS: This mouse model may prove to be a useful addition to the mouse genetics toolkit for GrCT research. Our finding that dysregulation of TGFB signaling results in the development of testicular GrCTs supports a common origin between Sertoli cells and granulosa cells, and highlights the paramount importance of balanced TGFB signaling in reproduction and development. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Institutes of Health grant R03HD082416 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the New Faculty Start-up Funds from Texas A&M University awarded to Q.L. The authors declare no competing interest.

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis.

N-WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N-WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N-WASP in the initiation and progression of colorectal cancer. While deletion of N-wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche, and migration up the crypt-villus axis was enhanced. Loss of N-wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N-WASP in early intestinal carcinogenesis despite its later pro-invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre-neoplastic tissues. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pedicle screw placement accuracy using ultra-low radiation imaging with image enhancement versus conventional fluoroscopy in minimally invasive transforaminal lumbar interbody fusion: an internally randomized controlled trial.

OBJECTIVE A previous study found that ultra-low radiation imaging (ULRI) with image enhancement significantly decreases radiation exposure by roughly 75% for both the patient and operating room personnel during minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) (p < 0.001). However, no clinical data exist on whether this imaging modality negatively impacts patient outcomes. Thus, the goal of this randomized controlled trial was to assess pedicle screw placement accuracy with ULRI with image enhancement compared with conventional, standard-dose fluoroscopy for patients undergoing single-level MIS-TLIF. METHODS An institutional review board-approved, prospective internally randomized controlled trial was performed to compare breach rates for pedicle screw placement performed using ULRI with image enhancement versus conventional fluoroscopy. For cannulation and pedicle screw placement, surgery on 1 side (left vs right) was randomly assigned to be performed under ULRI. Screws on the opposite side were placed under conventional fluoroscopy, thereby allowing each patient to serve as his/her own control. In addition to standard intraoperative images to check screw placement, each patient underwent postoperative CT. Three experienced neurosurgeons independently analyzed the images and were blinded as to which imaging modality was used to assist with each screw placement. Screw placement was analyzed for pedicle breach (lateral vs medial and Grade 0 [< 2.0 mm], Grade 1 [2.0-4.0 mm], or Grade 2 [> 4.0 mm]), appropriate screw depth (50%-75% of the vertebral body's anteroposterior dimension), and appropriate screw angle (within 10° of the pedicle angle). The effective breach rate was calculated as the percentage of screws evaluated as breached > 2.0 mm medially or postoperatively symptomatic. RESULTS Twenty-three consecutive patients underwent single-level MIS-TLIF, and their sides were randomly assigned to receive ULRI. No patient had immediate postoperative complications (e.g., neurological decline, need for hardware repositioning). On CT confirmation, 4 screws that had K-wire placement and cannulation under ULRI and screw placement under conventional fluoroscopy showed deviations. There were 2 breaches that deviated medially but both were Grade 0 (< 2.0 mm). Similarly, 2 breaches occurred that were Grade 1 (> 2.0 mm) but both deviated laterally. Therefore, the effective breach rate (breach > 2.0 mm deviated medially) was unchanged in both imaging groups (0% using either ULRI or conventional fluoroscopy; p = 1.00). CONCLUSIONS ULRI with image enhancement does not compromise accuracy during pedicle screw placement compared with conventional fluoroscopy while it significantly decreases radiation exposure to both the patient and operating room personnel.

Disruption of postnatal folliculogenesis and development of ovarian tumor in a mouse model with aberrant transforming growth factor beta signaling.

BACKGROUND: Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes. METHODS: A mouse model containing constitutively active TGFBR1 (TGFBR1CA) using Gdf9-iCre (termed TGFBR1-CAG9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CAG9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3'-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues. RESULTS: TGFBR1-CAG9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CAG9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues. CONCLUSIONS: These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.

The development of a monolith-based purification process for Orthopoxvirus vaccinia virus Lister strain.

The purification of large viruses remains an important field of research and development. The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8µm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1×109pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.

Corrigendum: Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

This corrects the article DOI: 10.1038/nature22056.

Acinar-to-Ductal Metaplasia Induced by Transforming Growth Factor Beta Facilitates KRASG12D-driven Pancreatic Tumorigenesis.

BACKGROUND & AIMS: Transforming growth factor beta (TGFß) acts either as a tumor suppressor or as an oncogene, depending on the cellular context and time of activation. TGFß activates the canonical SMAD pathway through its interaction with the serine/threonine kinase type I and II heterotetrameric receptors. Previous studies investigating TGFß-mediated signaling in the pancreas relied either on loss-of-function approaches or on ligand overexpression, and its effects on acinar cells have so far remained elusive. METHODS: We developed a transgenic mouse model allowing tamoxifen-inducible and Cre-mediated conditional activation of a constitutively active type I TGFß receptor (TßRICA) in the pancreatic acinar compartment. RESULTS: We observed that TßRICA expression induced acinar-to-ductal metaplasia (ADM) reprogramming, eventually facilitating the onset of KRASG12D-induced pre-cancerous pancreatic intraepithelial neoplasia. This phenotype was characterized by the cellular activation of apoptosis and dedifferentiation, two hallmarks of ADM, whereas at the molecular level, we evidenced a modulation in the expression of transcription factors such as Hnf1ß, Sox9, and Hes1. CONCLUSIONS: We demonstrate that TGFß pathway activation plays a crucial role in pancreatic tumor initiation through its capacity to induce ADM, providing a favorable environment for KRASG12D-dependent carcinogenesis. Such findings are highly relevant for the development of early detection markers and of potentially novel treatments for pancreatic cancer patients.

TGFß pathway limits dedifferentiation following WNT and MAPK pathway activation to suppress intestinal tumourigenesis.

Recent studies have suggested increased plasticity of differentiated cells within the intestine to act both as intestinal stem cells (ISCs) and tumour-initiating cells. However, little is known of the processes that regulate this plasticity. Our previous work has shown that activating mutations of Kras or the NF-κB pathway can drive dedifferentiation of intestinal cells lacking Apc. To investigate this process further, we profiled both cells undergoing dedifferentiation in vitro and tumours generated from these cells in vivo by gene expression analysis. Remarkably, no clear differences were observed in the tumours; however, during dedifferentiation in vitro we found a marked upregulation of TGFß signalling, a pathway commonly mutated in colorectal cancer (CRC). Genetic inactivation of TGFß type 1 receptor (Tgfbr1/Alk5) enhanced the ability of KrasG12D/+ mutation to drive dedifferentiation and markedly accelerated tumourigenesis. Mechanistically this is associated with a marked activation of MAPK signalling. Tumourigenesis from differentiated compartments is potently inhibited by MEK inhibition. Taken together, we show that tumours arising in differentiated compartments will be exposed to different suppressive signals, for example, TGFß and blockade of these makes tumourigenesis more efficient from this compartment.