Treatment with the vascular endothelial growth factor-A antibody, bevacizumab, has sex-specific effects in a rat model of mild traumatic brain injury.

Mild traumatic brain injury (mTBI) involves damage to the cerebrovascular system. Vascular endothelial growth factor-A (VEGF-A) is an important modulator of vascular health and VEGF-A promotes the brain's ability to recover after more severe forms of brain injury; however, the role of VEGF-A in mTBI remains poorly understood. Bevacizumab (BEV) is a monoclonal antibody that binds to VEGF-A and neutralises its actions. To better understand the role of VEGF-A in mTBI recovery, this study examined how BEV treatment affected outcomes in rats given a mTBI. Adult Sprague-Dawley rats were assigned to sham-injury + vehicle treatment (VEH), sham-injury + BEV treatment, mTBI + VEH treatment, mTBI + BEV treatment groups. Treatment was administered intracerebroventricularly via a cannula beginning at the time of injury and continuing until the end of the study. Rats underwent behavioral testing after injury and were euthanized on day 11. In both females and males, BEV had a negative impact on cognitive function. mTBI and BEV treatment increased the expression of inflammatory markers in females. In males, BEV treatment altered markers related to hypoxia and vascular health. These novel findings of sex-specific responses to BEV and mTBI provide important insights into the role of VEGF-A in mTBI.

Bacterial clade-specific analysis identifies distinct epithelial responses in inflammatory bowel disease.

Abnormal immune responses to the resident gut microbiome can drive inflammatory bowel disease (IBD). Here, we combine high-resolution, culture-based shotgun metagenomic sequencing and analysis with matched host transcriptomics across three intestinal sites (terminal ileum, cecum, rectum) from pediatric IBD (PIBD) patients (n = 58) and matched controls (n = 42) to investigate this relationship. Combining our site-specific approach with bacterial culturing, we establish a cohort-specific bacterial culture collection, comprising 6,620 isolates (170 distinct species, 32 putative novel), cultured from 286 mucosal biopsies. Phylogeny-based, clade-specific metagenomic analysis identifies key, functionally distinct Enterococcus clades associated with either IBD or health. Strain-specific in vitro validation demonstrates differences in cell cytotoxicity and inflammatory signaling in intestinal epithelial cells, consistent with the colonic mucosa-specific response measured in patients with IBD. This demonstrates the importance of strain-specific phenotypes and consideration of anatomical sites in exploring the dysregulated host-bacterial interactions in IBD.

International medical tourism of US cancer patients for alternative cancer treatments: Financial, demographic, and clinical profiles of online crowdfunding campaigns.

BACKGROUND: Previous research has found that individuals may travel outside their home countries in pursuit of alternative cancer therapies (ACT). The goal of this study is to compare individuals in the United States who propose plans for travel abroad for ACT, compared with individuals who seek ACT domestically. METHODS: Clinical and treatment data were extracted from campaign descriptions of 615 GoFundMe® campaigns fundraising for individuals in the United States seeking ACT between 2011 and 2019. We examined treatment modalities, treatment location, fundraising metrics, and online engagement within campaign profiles. Clinical and demographic differences between those who proposed international travel and those who sought ACT domestically were examined using two-sided Fisher's exact tests. Differences in financial and social engagement data were examined using two-sided Mann-Whitney tests. RESULTS: Of the total 615 campaigns, 237 (38.5%) mentioned plans to travel internationally for ACT, with the majority (81.9%) pursuing travel to Mexico. Campaigns that proposed international treatment requested more money ($35,000 vs. $22,650, p < 0.001), raised more money ($7833 vs. $5035, p < 0.001), had more donors (57 vs. 45, p = 0.02), and were shared more times (377 vs. 290.5, p = 0.008) compared to campaigns that did not. The median financial shortfall was greater for campaigns pursuing treatments internationally (-$22,640 vs. -$13,436, p < 0.003). CONCLUSIONS: Campaigns proposing international travel for ACT requested and received more money, were shared more online, and had more donors. However, there was significantly more unmet financial need among this group, highlighting potential financial toxicity on patients and families.

The Clinical Significance of Maximum Tumor Diameter on MRI in Men Undergoing Radical Prostatectomy or Definitive Radiotherapy for Locoregional Prostate Cancer.

INTRODUCTION: Maximum tumor diameter (MTD) on pretreatment magnetic resonance imaging (MRI) has the potential to further risk stratify for men with prostate cancer (PCa) prior to definitive local therapy. We aim to evaluate the prognostic impact of radiographic maximum tumor diameter (MTD) in men with localized prostate cancer. PATIENTS AND METHODS: From a single-center retrospective cohort of men receiving definitive treatment for PCa (radical prostatectomy [RP] or radiotherapy [RT]) with available pretreatment MRI, we conducted univariable and multivariable Cox proportional-hazards models for progression using clinical variables including age, NCCN risk group, radiographic extracapsular extension (ECE), radiographic seminal vesical invasion (SVI), and MTD. RP and RT cohorts were analyzed separately. Covariates were used in a classification and regression tree (CART) analysis and progression-free survival was estimated with the Kaplan-Meier method and groups were compared using log-rank tests. RESULTS: The cohort included 631 patients (n = 428 RP, n = 203 RT). CART analysis identified 4 prognostic groups for patients treated with RP and 2 prognostic groups in those treated with RT. In the RP cohort, NCCN low/intermediate risk group patients with MTD>=15 mm had significantly worse PFS than those with MTD <= 14 mm, and NCCN high-risk patients with radiographic ECE had significantly worse PFS than those without ECE. In the RT cohort, PFS was significantly worse in the cohort with MTD >= 23 mm than those <= 22 mm. CONCLUSION: Radiographic MTD may be a useful prognostic factor for patients with locoregional prostate cancer. This is the first study to illustrate that the importance of pretreatment tumor size may vary based on treatment modality.

Timing and Motivations for Alternative Cancer Therapy With Insights From a Crowdfunding Platform: Cross-sectional Mixed Methods Study.

BACKGROUND: Alternative cancer therapy is associated with increased mortality, but little is known about those who pursue it. OBJECTIVE: We aimed to describe individuals' motivations for using alternative cancer therapies and determine whether motivations differ based on individuals' timing of seeking alternative therapies. METHODS: We used data from 649 campaigns posted on the website GoFundMe between 2011 and 2019 for beneficiaries with cancer pursuing alternative therapy. The data were analyzed using a mixed methods approach. Campaigns were categorized by timing of alternative therapy (either before or after experiencing conventional therapy). Qualitative analysis identified motivational themes. Chi-square tests of independence and Fisher tests (all 2-sided) determined significant differences in the presence of motivational themes between groups. RESULTS: The expression of concerns about the efficacy of conventional therapy was significantly more likely in campaigns for individuals who used conventional therapy first than in campaigns for individuals who started with alternative therapy (63.3% vs 41.7%; P<.001). Moreover, on comparing those who started with alternative therapy and those who switched from conventional to alternative therapy, those who started with alternative therapy more often expressed natural and holistic values (49.3% vs 27.0%; P<.001), expressed an unorthodox understanding of cancer (25.5% vs 16.4%; P=.004), referenced religious or spiritual beliefs (15.1% vs 8.9%; P=.01), perceived alternative treatment as efficacious (19.1% vs 10.2%; P=.001), and distrusted pharmaceutical companies (3.2% vs 0.5%; P=.04). CONCLUSIONS: Individuals sought treatments that reflected their values and beliefs, even if scientifically unfounded. Many individuals who reported prior conventional cancer therapy were motivated to pursue alternative treatments because they perceived the conventional treatments to be ineffective.

Integrative taxonomy resolves the cryptic and pseudo-cryptic Radula buccinifera complex (Porellales, Jungermanniopsida), including two reinstated and five new species.

Molecular data from three chloroplast markers resolve individuals attributable to Radula buccinifera in six lineages belonging to two subgenera, indicating the species is polyphyletic as currently circumscribed. All lineages are morphologically diagnosable, but one pair exhibits such morphological overlap that they can be considered cryptic. Molecular and morphological data justify the re-instatement of a broadly circumscribed ecologically variable R. strangulata, of R. mittenii, and the description of five new species. Two species Radula mittenii Steph. and R. notabilis sp. nov. are endemic to the Wet Tropics Bioregion of north-east Queensland, suggesting high diversity and high endemism might characterise the bryoflora of this relatively isolated wet-tropical region. Radula demissa sp. nov. is endemic to southern temperate Australasia, and like R. strangulata occurs on both sides of the Tasman Sea. Radula imposita sp. nov. is a twig and leaf epiphyte found in association with waterways in New South Wales and Queensland. Another species, R. pugioniformis sp. nov., has been confused with Radula buccinifera but was not included in the molecular phylogeny. Morphological data suggest it may belong to subg. Odontoradula. Radula buccinifera is endemic to Australia including Western Australia and Tasmania, and to date is known from south of the Clarence River on the north coast of New South Wales. Nested within R. buccinifera is a morphologically distinct plant from Norfolk Island described as R. anisotoma sp. nov. Radula australiana is resolved as monophyletic, sister to a species occurring in east coast Australian rainforests, and nesting among the R. buccinifera lineages with strong support. The molecular phylogeny suggests several long-distance dispersal events may have occurred. These include two east-west dispersal events from New Zealand to Tasmania and south-east Australia in R. strangulata, one east-west dispersal event from Tasmania to Western Australia in R. buccinifera, and at least one west-east dispersal from Australia to New Zealand in R. australiana. Another west-east dispersal event from Australia to Norfolk Island may have led to the budding speciation of R. anisotoma. In contrast, Radula demissa is phylogeographically subdivided into strongly supported clades either side of the Tasman Sea, suggesting long distance dispersal is infrequent in this species.

Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating.

The procedures of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are routinely used in modern medicine to overcome infertility and, in animal husbandry, to propagate lines with compromised fertility. However, there remains concern that manual selection and injection of whole sperm into oocytes could contribute to pre- and postnatal developmental defects. To address this, we have used gene expression profiling and immunophenotyping to characterize offspring generated by these procedures. We used gametes from glutathione peroxidase 1 knockout (Gpx1-/-) mice as a sensitized screen responsive to oxidative stress from artificial reproduction technologies (ART). There were no differences between IVF and ICSI derived offspring in gene expression patterns, and minor differences in hematopoietic parameters. Furthermore there were only minor differences between these IVF and ICSI pups and those derived from natural mating. These data demonstrate for the first time in that there is no significant phenotypic affects of ICSI when compared to IVF and we identified a relatively minor influence of the artificial fertilization methods on phenotype of offspring compared with natural mating. These observations would support the use of ICSI for derivation of mutant mouse lines and may be of some importance for the use of this technique in human ART.

Ultraviolet-induced apoptosis in embryonic stem cells in vitro.

Embryonic stem (ES) cells, and the inner cell mass from which they are derived, are hypersensitive to DNA damage and appear to have specific cellular defense systems for DNA repair and the elimination of damaged cells. These mechanisms differ from somatic cells and are vital to minimize developmental defects that would potentially result from the continued proliferation and differentiation of abnormal cells into adult cell lineages. Although the DNA damage-induced signaling cascades activated in these cells are known to include p38 and c-Jun N-terminal protein kinase mitogen-activated protein kinase pathways and activation of a variety of transcription factors, including p53, nuclear factor-kappaB, and activator protein-1, the nature of the specific mechanisms unique to these cells remains to be elucidated. Here, we describe the use of homozygous knockout ES cells to investigate the role of Ets1 in the response to DNA damage in these cells. These studies demonstrate that Ets1 is required for optimal p53 function in this response and further demonstrate the potential for knockout ES cells to elucidate the role of specific genes in early embryonic cell responses.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Ets1 as a marker of malignant potential in gastric carcinoma.

AIM: Ets1 proto-oncogene is a transcription factor involved in the activation of several genes of tumor invasion and metastasis. We aimed to determine the relationship between the extent and intensity of Ets1 expression and patients' clinicopathological factors in gastric carcinoma. METHODS: Immunohistochemical analysis was performed for gastric tumor paraffin-embedded sections, followed by image analysis. RESULTS: Ets1 was not expressed in the normal gastric epithelium and its surrounding cells. The percentage of Ets1 expressing cells detected increased significantly in both epithelial tumor and stromal cells from high T classification, lymph node metastasis positive, clinical advanced-stage groups (P<0.001). The level of Ets1 staining in epithelial tumor cells also reflected the degree of cell differentiation. The percentage of epithelial and stromal cells expressing Ets1 was significantly correlated with the presence of lymph node metastasis (P=0.014 and P<0.001 respectively). Ets1 expression was not observed in tissue samples from patients with benign gastric ulcers. CONCLUSION: Ets1 protein expression in epithelial tumor cells reflects the degree of differentiation, and the percentage of Ets1 positive tumor and stromal cells correlates with lymph node metastasis. Thus Ets1 is a valuable marker of malignant potential in terms of invasiveness and metastasis of gastric carcinoma. It is also possible that inhibition of Ets1 is a potential avenue for therapy in gastric cancer.

Characterization of monoclonal antibodies specific to the transcription factor ETS-2 protein.

ETS-2 is a member of the ETS family of transcription factors. ETS-2 was initially characterized as a nuclear oncogene and has been shown to play a role in regulation of apoptosis and cell cycle progression. Members of the ETS family display high sequence homology, thus, there is considerable controversy concerning the specificity of existing ETS-2 polyclonal antibodies that have been used to define ETS-2 function. We therefore embarked on the production of ETS-2 specific monoclonal antibodies. In this report, we describe the production and characterization of six antibodies and the localization of their target epitopes to distinct domains of the ETS-2 protein. Four antibodies are ETS-2 specific and two antibodies cross-react with ETS-1, an ETS family member with the highest amino acid sequence homology to ETS-2. This report provides a comprehensive evaluation of ETS-2 specific monoclonal antibodies verified using ETS-2 null cells. These antibodies can be used for EMSA, Western blotting, immunoprecipitation and immunofluorescence staining experiments. Collectively, these reagents are invaluable molecular tools that should help better understand the biological function of ETS-2.

Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells.

Embryonic stem (ES) cells contain a p53-dependent apoptosis mechanism to avoid the continued proliferation and differentiation of damaged cells. We show that mouse ES cells lacking Ets1 are deficient in their ability to undergo UV-induced apoptosis, similar to p53 null ES cells. In Ets1(-/-) ES cells, UV induction of the p53 regulated genes mdm2, perp, cyclin G and bax was decreased both at mRNA and protein levels. While p53 protein levels were unaltered in Ets1(-/-) cells, its ability to transactivate genes such as mdm2 and cyclin G was reduced. Furthermore, electrophoretic mobility shift assays and immunoprecipitations demonstrated that the presence of Ets1 was necessary for a CBP/p53 complex to be formed. Chromatin immunoprecipitations demonstrated that Ets1 was required for the formation of a stable p53-DNA complex under physiological conditions and activation of histone acetyltransferase activity. These data demonstrate that Ets1 is an essential component of a UV-responsive p53 transcriptional activation complex in ES cells and suggests that Ets1 may contribute to the specificity of p53-dependent gene transactivation in distinct cellular compartments.

Inactivation of the transcription factor Elf3 in mice results in dysmorphogenesis and altered differentiation of intestinal epithelium.

BACKGROUND & AIMS: The mammalian small intestine is lined by a highly specialized epithelium that functions in the digestion and absorption of nutrients. The molecular mechanisms that direct intestinal epithelial cell morphogenesis and terminal differentiation are poorly understood. We have previously identified Elf3 (E74-like factor-3) as a member of the ETS transcription factor family strongly expressed in small intestinal epithelium. The aim of this study is to investigate the biological roles of Elf3 in vivo. METHODS: Mice with a null mutation of Elf3 were generated through targeted gene disruption. Characterization of intestinal development was performed by histologic and immunohistochemical techniques. RESULTS: Targeted disruption of Elf3 resulted in fetal lethality of about 30% at around embryonic day 11.5. Seventy percent of the Elf3-deficent progeny were born and displayed severe alterations of tissue architecture in the small intestine, manifested by poor villus formation and abnormal morphogenesis and terminal differentiation of absorptive enterocytes and mucus-secreting goblet cells. Crypt cell proliferation, however, appeared intact in Elf3-deficient mice.Elf3-deficient enterocytes express markedly reduced levels of the transforming growth factor beta type II receptor (TGF-beta RII), an inducer of intestinal epithelial differentiation. CONCLUSIONS: Elf3 is an important regulator of morphogenesis and terminal differentiation of epithelial cell lineages in the small intestine.