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The human TMPRSS2 gene is pathogenetically implicated in both coronaviral lung infection and prostate cancer, suggesting its potential as a drug target in both contexts. SARS-COV-2 spike polypeptides are primed by the host transmembrane TMPRSS2 protease, triggering virus fusion with epithelial cell membranes followed by an endocytotic internalisation process that bypasses normal endosomal activation of cathepsin-mediated innate immunity; viral co-opting of TMPRSS2 thus favors microbial survivability by attenuating host inflammatory responses. In contrast, most early hormone-dependent prostate cancers express TMPRSS2:ERG fusion genes arising from deletions that eliminate the TMPRSS2 coding region while juxtaposing its androgen-inducible promoter and the open reading frame of ERG, upregulating pro-inflammatory ERG while functionally disabling TMPRSS2. Moreover, inflammatory oxidative DNA damage selects for TMPRSS2:ERG-fused cancers, whereas patients treated with antiinflammatory drugs develop fewer of these fusion-dependent tumors. These findings imply that TMPRSS2 protects the prostate by enabling endosomal bypass of pathogens which could otherwise trigger inflammation-induced DNA damage that predisposes to TMPRSS2:ERG fusions. Hence, the high oncogenic selectability of TMPRSS2:ERG fusions may reflect a unique pro-inflammatory synergy between androgenic ERG gain-of-function and fusogenic TMPRSS2 loss-of-function, cautioning against the use of TMPRSS2-inhibitory drugs to prevent or treat early prostate cancer.

While newborns and children with hearing loss are routinely offered genetic testing, adults are rarely clinically tested for a genetic etiology. One clinically actionable result from genetic testing in children is the discovery of variants in syndromic hearing loss genes. EYA4 is a known hearing loss gene which is also involved in important pathways in cardiac tissue. The pleiotropic effects of rare EYA4 variants are poorly understood and their prevalence in a large cohort has not been previously reported. We investigated cardio-auditory phenotypes in 11,451 individuals in a large biobank using a rare variant, genome-first approach to EYA4. We filtered 256 EYA4 variants carried by 6737 participants to 26 rare and predicted deleterious variants carried by 42 heterozygotes. We aggregated predicted deleterious EYA4 gene variants into a combined variable (i.e. “gene burden”) and performed association studies across phenotypes compared to wildtype controls. We validated findings with replication in three independent cohorts and human tissue expression data. EYA4 gene burden was significantly associated with audiometric-proven HL (p = $${5.43\times 10}^{-5})$$ , Mobitz Type II AV block (p = $${2.16\times 10}^{-5}$$ ) and the syndromic presentation of HL and primary cardiomyopathy (p = 0.0194). Analyses on audiogram, echocardiogram, and electrocardiogram data validated these associations. Prior reports have focused on identifying variants in families with severe or syndromic phenotypes. In contrast, we found, using a genotype-first approach, that gene burden in EYA4 is associated with more subtle cardio-auditory phenotypes in an adult medical biobank population, including cardiac conduction disorders which have not been previously reported. We show the value of using a focused approach to uncover human disease related to pleiotropic gene variants and suggest a role for genetic testing in adults presenting with hearing loss.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has significantly improved outcomes in the treatment of refractory or relapsed large B-cell lymphoma (LBCL). We evaluated the long-term course of hematologic recovery, immune reconstitution, and infectious complications in 41 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) at a single center. Grade 3+ cytopenias occurred in 97.6% of patients within the first 28 days postinfusion, with most resolved by 6 months. Overall, 63.4% of patients received a red blood cell transfusion, 34.1% of patients received a platelet transfusion, 36.6% of patients received IV immunoglobulin, and 51.2% of patients received growth factor (granulocyte colony-stimulating factor) injections beyond the first 28 days postinfusion. Only 40% of patients had recovered detectable CD19+ B cells by 1 year, and 50% of patients had a CD4+ T-cell count

Background Fungal prostatitis is exceedingly rare with mostly case reports. Methods Electronic medical records at three medical centers were searched for cases of fungal prostatitis due to endemic mycoses and Cryptococcus over the preceding 10 years. Results Seven cases were identified from 105 600 prostate biopsies within the Southern California Permanente Medical Group for an incidence of 0.0066%. An additional eight cases were identified from two other health care systems. Excluding four patients without available clinical data, 11 patients were reviewed, most of whom underwent biopsy due to elevated prostate-specific antigen. Four were asymptomatic and the remainder had nonspecific signs or symptoms. All biopsies revealed granulomatous inflammation and fungal organisms. Seven patients had coccidioidomycosis, three patients had cryptococcosis (confirmed in two cases and suspected by organism morphology in the other), and one patient had likely histoplasmosis based on organism morphology. Prolonged antifungal treatment was standard; outcomes were favorable. Conclusion Fungal prostatitis due to endemic mycoses and Cryptococcus is uncommon and associated with favorable outcomes but generally involves prolonged therapy.

The anterior pituitary gland drives highly conserved physiologic processes in mammalian species. These hormonally controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current cellular models of mammalian anteiror pituitary, largely built on candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex relationship between hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with complementary imaging-based single-cell analyses of mRNAs and proteins, to systematically map both cell-type diversity and functional state heterogeneity in adult male and female mouse pituitaries at single-cell resolution and in the context of major physiologic demands. These quantitative single-cell analyses reveal sex-specific cell-type composition under normal pituitary homeostasis, identify an array of cells associated with complex complements of hormone-enrichment, and undercover non-hormone producing interstitial and supporting cell-types. Interestingly, we also identified a Pou1f1-expressing cell population that is characterized by a unique multi-hormone gene expression profile. In response to two well-defined physiologic stresses, dynamic shifts in cellular diversity and transcriptome profiles were observed for major hormone producing and the putative multi-hormone cells. These studies reveal unanticipated cellular complexity and plasticity in adult pituitary, and provide a rich resource for further validating and expanding our molecular understanding of pituitary gene expression programs and hormone production. Electronic supplementary material The online version of this article (10.1007/s13238-020-00705-x) contains supplementary material, which is available to authorized users.

More than ten thousand peer-reviewed studies have assessed the role of fibroblast growth factors (FGFs) and their receptors (FGFRs) in cancer, but few patients have yet benefited from drugs targeting this molecular family. Strategizing how best to use FGFR-targeted drugs is complicated by multiple variables, including RNA splicing events that alter the affinity of ligands for FGFRs and hence change the outcomes of stromal-epithelial interactions. The effects of splicing are most relevant to FGFR2; expression of the FGFR2b splice isoform can restore apoptotic sensitivity to cancer cells, whereas switching to FGFR2c may drive tumor progression by triggering epithelial-mesenchymal transition. The differentiating and regulatory actions of wild-type FGFR2b contrast with the proliferative actions of FGFR1 and FGFR3, and may be converted to mitogenicity either by splice switching or by silencing of tumor suppressor genes such as CDH1 or PTEN. Exclusive use of small-molecule pan-FGFR inhibitors may thus cause nonselective blockade of FGFR2 isoforms with opposing actions, undermining the rationale of FGFR2 drug targeting. This splice-dependent ability of FGFR2 to switch between tumor-suppressing and -driving functions highlights an unmet oncologic need for isoform-specific drug targeting, e.g., by antibody inhibition of ligand-FGFR2c binding, as well as for more nuanced molecular pathology prediction of FGFR2 actions in different stromal-tumor contexts.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.

To evaluate terpinen-4-ol lid scrubs (Cliradex) with Microblepharoexfoliation (MBE) (BlephEx) in the treatment of Demodex blepharitis.An Institutional Review Board (IRB) approved the randomized prospective double-masked trial of 50 patients with Demodex positive blepharitis. All subjects were given an in-office MBE treatment and randomized to masked lid scrubs (terpinen-4-ol or sham) twice daily for 1 month. All subjects then returned for an additional MBE treatment and received open-label terpinen-4-ol scrubs twice daily for 1 month.Demodex levels, Ocular Surface Disease Index, tear osmolarity, MMP-9, Schirmer 1 and grading of the lid margin appearance, meibomian gland dropout, and meibomian gland secretions.Forty-six subjects (23 per group) were included for the analysis; 4 lost to follow up. Total Demodex folliculorum levels in the Cliradex group improved: 4.7 to 3.6 (first month interval P = 0.266) to 2.6 (second month interval P = 0.279), overall P0.05 over 2 months. Total levels in the sham group improved: 5.1 to 3.0 (first month interval P0.05) to 2.5 (second month interval P = 0.496), overall P0.05 over 2 months. No clear statistically meaningful improvements in other dry eye and blepharitis data metrics were observed.In-office MBE treatments combined with either Cliradex terpinen-4-ol medicated lid scrubs or sham scrubs showed a statistically significant reduction in D. folliculorum infestation levels; however, conclusions on clinical significance could not be made. In-office MBE with terpinen-4-ol lid scrubs showed no significant improvement over sham scrubs.

A 39-year-old man presented to the Bellevue Hospital emergency department in November with 1 week of fevers and cough. On the previous day, he had been assaulted, developing right buttock and thigh pain. His medical history was notable for alcohol use disorder treated with the opioid antagonist

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis, however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko) and gain (Shh-P1) of Shh signaling revealed a set of Shh responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provided novel insights into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh responsive genes. These datasets are useful entry points for deciphering Shh dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.SUMMARY STATEMENTAn integrated genomic approach identifies Shh responsive genes and associated regulatory sequences with known and previously uncharacterized roles in cochlear morphogenesis, including genes that prime the cochlea for sensory development.

Douglas Epstein and colleagues use de novo motif analysis to identify sequence motifs and cognate transcription factors for brain enhancers of Shh active in the zona limitans intrathalamica (zli). They find new zli enhancers in mice and a functional equivalent in hemichordates, indicating an ancient origin of these sequence elements. Genomic approaches have predicted hundreds of thousands of tissue-specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown1,2,3,4. Here we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen Sonic hedgehog (Shh)5,6. We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover new Shh zli enhancers in mice and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies and over extensive evolutionary distances.

Objective: Hypoglycemia due to insulin-like growth factor (IGF)-2 secretion by a tumor, referred to as non–islet cell tumor hypoglycemia, or IGF-2-oma, is a rare and serious paraneoplastic complication of malignancy. When surgical resection is not an option, studies have shown improvement of hypoglycemia with glucocorticoids. We describe the case of a patient with persistent hypoglycemia despite prolonged high-dose glucocorticoid therapy.Methods: A 22-year-old man with known metastatic hepatocellular carcinoma presented with dizziness and weakness. Initial blood glucose level was found to be 10 mg/dL. Multiple injections of 50% dextrose and continuous infusion with 25% dextrose were required to maintain normal blood glucose levels. Laboratory work-up revealed suppressed C-peptide and insulin levels with hypoglycemia and an elevated ratio of IGF-2 to IGF-1, consistent with IGF-2 secretion by the tumor.Results: Despite high-dose glucocorticoid therapy, continuous intravenous dextrose was necessary to prevent hypoglycemia. In addition, the patient's tumor progressed rapidly, and he was ultimately discharged to hospice on intravenous dextrose.Conclusion: Hypoglycemia due to IGF-2 secretion is a rare and challenging paraneoplastic complication. Cases of hypoglycemia that do not respond to glucocorticoid therapy may indicate rapid disease progression and imminent death.Abbreviation: IGF insulin-like growth factor

Eremothecium coryli is a dimorphic fungus of the Saccharomycetes class. While species within this class are known to cause human infection, Eremothecium species have previously only been known as phytopathogens and never been isolated from a human sample. Here, we report the first known case of human E. coryli infection.

The anterior pituitary gland drives a set of highly conserved physiologic processes in mammalian species. These hormonally-controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current models, largely built upon candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex models of hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with a set of orthogonal mRNA and protein imaging studies, to systematically map the cellular composition of adult male and female mouse pituitaries at single-cell resolution and in the setting of major physiologic demands. These analyses reveal sex-specific cellular diversity associated with normal pituitary homeostasis, and identify an array of cells with complex complements of hormone-enrichment as well as a series of non-hormone producing interstitial and supporting cell lineages. These scRNA-seq studies identify a major cell population that is characterized by a unique multi-hormone gene expression profile. The detection of dynamic shifts in cellular representations and transcriptome profiles in response to two well-defined physiologic stresses suggests corresponding roles of a number of these clusters in cellular plasticity within the adult pituitary. These studies point to an unanticipated complexity and plasticity in pituitary cellular composition that expands upon current models and concepts of pituitary gene expression and hormone production.

Sonic hedgehog (Shh) plays well characterized roles in the development of several regions of the brain and spinal cord, but its functions in the hypothalamus have been more difficult to elucidate due to the complex neuroanatomy of this brain area. Here, we utilize fate-mapping and conditional deletion models in mice to define requirements for dynamic Shh activity at distinct stages of tuberal hypothalamic development, a brain region with important homeostatic functions. At early time points, Shh signaling regulates dorsoventral patterning, neurogenesis, and the size of the ventral midline. Fate mapping experiments demonstrate that Shh expressing and responsive progenitors contribute to distinct neuronal subtypes, accounting for some of the cellular heterogeneity in tuberal hypothalamic nuclei. Conditional deletion of the Hedgehog transducer Smoothened (Smo), after dorsoventral patterning has been established, reveals that Shh signaling is necessary to maintain proliferation and progenitor identity during peak periods of hypothalamic neurogenesis. We also find that mosaic disruption ofSmocauses a non-cell autonomous gain in Shh signaling activity in neighboring wild type cells, suggesting a mechanism for the pathogenesis of hypothalamic hamartomas, a benign tumor that forms during hypothalamic development.SUMMARY STATEMENTRequirements for dynamic Sonic hedgehog activity at distinct stages of tuberal hypothalamic development are defined using fate-mapping and conditional deletion models in mice.

Background While invasive fungal infections (IFIs) are common in patients with acute myeloid leukemia (AML) undergoing induction chemotherapy, little current data exist on the epidemiology of IFIs in this patient population given widespread use of antifungal prophylaxis. Because our institution does not administer antifungal prophylaxis, we are in a unique position to study the natural history of IFIs in these patients. Methods We evaluated the incidence of IFIs using established definitions in adults with AML undergoing induction chemotherapy at Stanford Health Care from 2012 to 2017. We also analyzed incidence of antifungal treatment, impact of IFI diagnosis on survival, and risk factors for IFI development. Patients were followed for up to 12 weeks after beginning induction chemotherapy. Results Of 488 patients analyzed, 243 were eligible for inclusion. The median age was 57 (interquartile range 45–65). Men composed 134 (55%) of the patients and 157 (65%) where white. Fifty-four (22%) had antecedent myelodysplastic syndrome; most received a “7 + 3” regimen involving cytarabine and an anthracycline. Thirty-one (13%) developed a proven or probable IFI; 104 (43%) developed a proven, probable, or possible IFI. Most IFIs were due to lower respiratory tract disease. Eighteen identified organisms were Candida, including six C. albicans. Eight organisms were mold, including four Aspergillus isolates (all but one A. fumigatus) and one isolate each of Fusarium solani, Rhizomucor, Rhizopus, and Scedosporium apiospermum/Pseudallescheria boydii. One hundred ninety patients (78%) received antifungals during their initial admission and 99 (46%) of patients surviving their initial admission were discharged on antifungals. Only 66.7% of patients with a proven or probable IFI survived through 12 weeks, compared with 92.2% of those without (P = 0.007). Baseline absolute neutrophil count ≤500 cells/μL and longer duration of neutropenia were significantly associated with development of proven or probable IFIs. Conclusion Among patients receiving induction chemotherapy for AML, IFIs due to Candida and mold remain frequent absent antifungal prophylaxis and are associated with worse survival. Our findings support the use of antifungal prophylaxis in this patient population. Disclosures All authors: No reported disclosures.

BACKGROUND Little is known about recent trends in surveillance among the more than 1 million US colorectal cancer (CRC) survivors. Moreover, for stage I disease, which accounts for more than 30% of survivors, the guidelines are limited, and the use of surveillance has not been well studied. Guidelines were changed in 2005 to include recommendations for computed tomography (CT) surveillance in select patients, but the impact of these changes has not been explored. METHODS A retrospective analysis of patients who were identified in the Survival, Epidemiology, and End Results–Medicare database and underwent resection of stage I to III CRC between 2001 and 2009 was performed. The receipt of guideline-determined sufficient surveillance, including office visits, colonoscopy, carcinoembryonic antigen (CEA) testing, and CT imaging, in the 3 years after resection was evaluated. RESULTS The study included 23,990 colon cancer patients and 5665 rectal cancer patients. Rates of office visits and colonoscopy were high and stable over the study period. Rates of CEA surveillance increased over the study period but remained low, even for stage III disease. Rates of CT imaging increased gradually during the study period, but the 2005 guideline change had no effect. Stage II patients, including high-risk patients, received surveillance at significantly lower rates than stage III patients despite similar recommendations. Conversely, up to 30% of stage I patients received nonrecommended CEA testing and CT imaging. CONCLUSIONS There continues to be substantial underuse of surveillance for CRC survivors and particularly for stage II patients, who constitute almost 40% of survivors. The 2005 guideline change had a negligible impact on CT surveillance. Conversely, although guidelines are limited, many stage I patients are receiving intensive surveillance. Cancer 2015;121:3435–43. © 2015 American Cancer Society.

Autoimmune thrombocytopenia is an uncommon but reported paraneoplastic manifestation of renal cell carcinoma (RCC). Treatment usually involves management of the underlying malignancy; however, steroids have shown a benefit in published case reports. Here, we describe a patient with profound thrombocytopenia secondary to metastatic RCC. It was refractory to steroid and intravenous immunoglobulin, but the platelet count improved markedly following initiation of everolimus. The possible explanation includes immunomodulation, tumour lysis or a combination of both effects. This is the first reported case of everolimus used in paraneoplastic thrombocytopenia from RCC. More studies are needed for further investigation of its potential use in secondary immune thrombocytopenia from RCC and perhaps other malignancies.

Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fate mapping experiments using two independent Wnt responsive driver strains (TopCreER and Gbx2(CreER)) that when crossed to inducible responder lines (Rosa(lacZ) or Rosa(zsGreen)) permanently labeled dorsomedial otic progenitors and their derivatives. Tamoxifen time course experiments revealed that most vestibular structures showed some degree of labeling when recombination was induced between E7.75 and E12.5, consistent with continuous Wnt signaling activity in this tissue. Remarkably, a population of Wnt responsive cells in the dorsal otocyst was also found to contribute to the sensory epithelium of the cochlear duct, including auditory hair and support cells. Similar results were observed with both TopCreER and Gbx2(CreER) strains. The ventral displacement of Wnt responsive cells followed a spatiotemporal sequence that initiated in the anterior otic cup at, or immediately prior to, the 17-somite stage (E9) and then spread progressively to the posterior pole of the otic vesicle by the 25-somite stage (E9.5). These lineage-tracing experiments identify the earliest known origin of auditory sensory progenitors within a population of Wnt responsive cells in the dorsomedial otic cup.

The completion of the human genome sequence sparked optimism about prospects for new anticancer drug development, but clinical progress over the last decade has proven slower than expected. Here it is proposed that unrealistically high expectations of first-generation discovery-based diagnostics have contributed to this problem. Hypothesis-based single-molecule tests (e.g., mutation screening of KRAS, EGFR, BRAF or KIT genes) continue to change clinical practice incrementally, whereas first-generation multiplex assays--such as gene expression profiling and proteomics--have identified few high-impact therapeutic targets despite numerous correlations with prognosis. To move forward, second-generation multiplex diagnostics should be based not on statistical patterns/associations alone, but on clinically interpretable ('high-signal-to-noise') data such as change-of-function mutations, gene amplifications, recurrent chromosomal anomalies, and abnormal phosphorylation profiles of ERK or mTOR signaling cascades.

Tbx2 and Tbx3 are two highly related members of the T-box transcription factor gene family that regulate patterning and differentiation of a number of tissue rudiments in the mouse. Both genes are partially co-expressed in the ventral diencephalon and the infundibulum; however, a functional requirement in murine pituitary development has not been reported. Here, we show by genetic lineage tracing that Tbx2+ cells constitute the precursor population of the neurohypophysis. However, Tbx2 is dispensable for neurohypophysis development as revealed by normal formation of this organ in Tbx2-deficient mice. By contrast, loss of Tbx3 from the ventral diencephalon results in a failure to establish the Tbx2+ domain in this region, and a lack of evagination of the infundibulum and formation of the neurohypophysis. Rathke's pouch is severely hypoplastic, exhibits defects in dorsoventral patterning, and degenerates after E12.5. In Tbx3-deficient embryos, the ventral diencephalon is hyperproliferative and displays an abnormal cellular architecture, probably resulting from a failure to repress transcription of Shh. We further show that Tbx3 and Tbx2 repress Shh by sequestering the SRY box-containing transcription factor Sox2 away from a Shh forebrain enhancer (SBE2), thus preventing its activation. These data suggest that Tbx3 is required in the ventral diencephalon to establish a Shh− domain to allow formation of the infundibulum.

Hemophilia patients use factor-clotting concentrates (factor VIII for hemophilia A and factor IX for hemophilia B) for improved blood clotting. These products are used to prevent or stop bleeding episodes. However, some hemophilia patients develop inhibitors (i.e., the patient's immune system develops antibodies against these factor concentrates). Hence, these patients do not respond well to the factor concentrates. A majority of hemophilia patients with inhibitors are managed on-demand with the following bypassing agents: recombinant factor VIIa (rFVIIa) and activated prothrombin complex concentrate (aPCC). The recently published U.S. registries Dosing Observational Study in Hemophilia (DOSE) and Hemostasis and Thrombosis Research Society (HTRS) reported higher rFVIIa on-demand use for bleed management than previously described.To estimate aPCC and rFVIIa prophylaxis costs relative to rFVIIa on-demand treatment cost based on rFVIIa doses reported in U.S. registries.A literature-based cost model was developed assuming a base case on-demand annual bleed rate (ABR) of 28.7 per inhibitor patient, which was taken from a randomized phase 3 clinical trial. The doses for rFVIIa on-demand were taken from the median dose per bleed reported by the DOSE and HTRS registries. Model inputs for aPCC and rFVIIa prophylaxis (i.e., dosing and efficacy) were derived from respective randomized clinical trials. Cost analysis was from the U.S. payer perspective, and only direct drug costs were considered. The drug cost was based on the Medicare Part B 2014 average sale price (ASP). Two-way sensitivity and threshold analyses were performed by simultaneously varying on-demand ABR, prophylaxis efficacy, and unit drug cost. In addition to studying relative costs associated with on-demand and prophylaxis treatments, relative cost per bleeding episode avoided were also calculated for aPCC and rFVIIa prophylaxis treatments. The prophylaxis efficacy reported in the trials were used to determine the number of bleeding episodes avoided.Based on the median on-demand dose of 695 mcg per kg per bleed, reported by the DOSE registry, the annual rFVIIa on-demand cost was $34,009 per kg of body weight. The annual rFVIIa on-demand cost was $22,020 per kg of body weight when the median dose of 450 mcg per kg per bleed reported by the HTRS registry was considered. The annual cost rose to $38,461 per kg of body weight when the rFVIIa on-demand dose of 786 mcg per kg per bleed among patients infusing an initial dose ≥ 250 mcg per kg was considered. The aPCC (85 units per kg per every other day) and rFVIIa (90 mcg per kg per every day) annual prophylaxis costs were $26,536 and $60,700, respectively. Also, aPCC and rFVIIa prophyaxis treatments were estimated to prevent a total of 20.8 and 12.9 annual bleeding episodes, respectively. When compared with the on-demand dose of 695 mcg per kg per bleed (DOSE registry), the annual aPCC and rFVIIa prophylaxis costs were 21.9% lower and 78.4% higher, respectively. Additionally, aPCC prophylaxis saved $360 per kg for each bleeding episode avoided. rFVIIa prophylaxis cost $2,066 per kg for each bleeding episode avoided. Compared with the on-demand dose of 450 mcg per kg per bleed (HTRS registry), aPCC and rFVIIa prophylaxis costs were 20.5% and 174.9% higher, respectively. In this case, aPCC and rFVIIa prophylaxis treatment costs were $217 per kg and $2,995 per kg, respectively, for each bleeding episode avoided. aPCC and rFVIIa prophylaxis costs were 31.0% lower and 57.8% higher, respectively, when compared with the rFVIIa on-demand dose of 786 mcg per kg per bleed, among patients infusing an initial dose ≥ 250 mcg per kg (HTRS registry). In this case, aPCC prophylaxis saved $573 per kg for each bleeding episode avoided, while rFVIIa prophylaxis costs $1,724 per kg for each bleeding episode avoided. Results of the 2-way sensitivity analyses were robust in the majority of the scenarios considered.aPCC prophylaxis may be cost saving for managing hemophilia patients with inhibitors who bleed frequently and infuse significant quantities of rFVIIa on-demand.

Septo-optic dysplasia (SOD) is a congenital disorder characterized by optic nerve, pituitary and midline brain malformations. The clinical presentation of SOD is highly variable with a poorly understood etiology. The majority of SOD cases are sporadic, but in rare instances inherited mutations have been identified in a small number of transcription factors, some of which regulate the expression of Sonic hedgehog (Shh) during mouse forebrain development. SOD is also associated with young maternal age, suggesting that environmental factors, including alcohol consumption at early stages of pregnancy, might increase the risk of developing this condition. Here, we address the hypothesis that SOD is a multifactorial disorder stemming from interactions between mutations in Shh pathway genes and prenatal ethanol exposure. Mouse embryos with mutations in the Shh co-receptor, Cdon, were treated in utero with ethanol or saline at embryonic day 8 (E8.0) and evaluated for optic nerve hypoplasia (ONH), a prominent feature of SOD. We show that both Cdon−/− mutation and prenatal ethanol exposure independently cause ONH through a similar pathogenic mechanism that involves selective inhibition of Shh signaling in retinal progenitor cells, resulting in their premature cell-cycle arrest, precocious differentiation and failure to properly extend axons to the optic nerve. The ONH phenotype was not exacerbated in Cdon−/− embryos treated with ethanol, suggesting that an intact Shh signaling pathway is required for ethanol to exert its teratogenic effects. These results support a model whereby mutations in Cdon and prenatal ethanol exposure increase SOD risk through spatiotemporal perturbations in Shh signaling activity., Summary: In utero exposure to ethanol causes defects in optic nerve formation by interfering with Shh signaling activity in the growth and differentiation of retinal progenitor cells in the developing mouse eye.

SummarySepto-optic dysplasia (SOD) is a congenital brain anomaly that results in pituitary, optic nerve, and midline forebrain defects. The etiology of SOD is poorly understood, with the majority of cases being sporadic. In rare instances, SOD is caused by mutations in Sox2, Sox3, or Hesx1, but how this manifests in disease is not entirely certain. We demonstrate here that mouse embryos lacking Sonic hedgehog (Shh) in the prospective hypothalamus exhibit key features of SOD, including pituitary hypoplasia and absence of the optic disc. The hypothalamic source of Shh is required to maintain gene expression boundaries along the anteroposterior and mediolateral neural axes that are important for proper pituitary and eye development, respectively. We further reveal that Sox2 and Sox3 are dose-dependent regulators of Shh transcription that directly bind and activate a long-range Shh forebrain enhancer. These data indicate that reduced levels of Shh expression in the hypothalamus cause SOD.

In mouse embryos lacking sonic hedgehog (Shh), dorsoventral polarity within the otic vesicle is disrupted. Consequently, ventral otic derivatives, including the cochlear duct and saccule, fail to form, and dorsal otic derivatives, including the semicircular canals, endolymphatic duct and utricle, are malformed or absent. Since inner ear patterning and morphogenesis are heavily dependent on extracellular signals derived from tissues that are also compromised by the loss of Shh, the extent to which Shh signaling acts directly on the inner ear for its development is unclear. To address this question, we generated embryos in which smoothened (Smo), an essential transducer of Hedgehog (Hh) signaling, was conditionally inactivated in the otic epithelium (Smoecko). Ventral otic derivatives failed to form in Smoecko embryos, whereas vestibular structures developed properly. Consistent with these findings, we demonstrate that ventral, but not dorsal, otic identity is directly dependent on Hh. The role of Hh in cochlear-vestibular ganglion (cvg) formation is more complex, as both direct and indirect signaling mechanisms are implicated. Our data suggest that the loss of cvg neurons in Shh–/– animals is due, in part, to an increase in Wnt responsiveness in the otic vesicle, resulting in the ectopic expression of Tbx1 in the neurogenic domain and subsequent repression of Ngn1 transcription. A mitogenic role for Shh in cvg progenitor proliferation was also revealed in our analysis of Smoecko embryos. Taken together, these data contribute to a better understanding of the intrinsic and extrinsic signaling properties of Shh during inner ear development.

In caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

A novel optical biosensing platform utilizing the unique solubility and chemochromic properties of polyaniline is presented. A facile, ion-induced reprecipitation method leads to the entrapment of a chosen oxidoreductase enzyme, which, in the presence of its associated substrate, catalyzes a reversible redox change in the host polymer. This change is monitored via the UV–vis absorption and subsequently analyzed to fit a Michaelis–Menten model. Here, in vitro prototype devices demonstrate selective sensing of glucose, choline, and uric acid, and the potential to be adapted for use as part of real-time in vivo monitoring systems is discussed. © 2010 Elsevier B.V. All rights reserved.

Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6 fold increase in MnSOD activities leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation, and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase of the mitochondrial form of thioredoxin (thioredoxin 2), but not of several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.

Background:The metastatic propensity of invasive ductal carcinoma (IDC) of the breast correlates with axillary node involvement and with expression of the proliferation antigen Ki-67, whereas ductal carcinoma in situ (DCIS) is non-metastasising. To clarify whether concomitant DCIS affects IDC prognosis, we compared Ki-67 expression and node status of size-matched IDC subgroups either with DCIS (IDC-DCIS) or without DCIS (pure IDC).Methods:We analysed data from 1355 breast cancer patients. End points were defined by the association of IDC (with or without DCIS) with grade, nodal status, Ki-67, and ER/HER2.Results: Size-matched IDC-DCIS was more likely than pure IDC to be screen detected (P0.03), to occur in pre-menopausal women (P0.002), and to be either ER-positive (P0.002) or HER2-positive (P0.0005), but less likely to be treated with breast-conserving surgery (P0.004). Grade and Ki-67 were lower in IDC-DCIS than in pure IDC (P0.02), and declined as the DCIS enlarged (P0.01). Node involvement and lymphovascular invasion in IDC-DCIS increased with the size ratio of IDC to DCIS (P0.01). A 60-month cancer-specific survival favoured IDC-DCIS over size-matched pure IDC (97.4 vs 96.0%).Conclusion:IDC co-existing with DCIS is characterised by lower proliferation and metastatic potential than size-matched pure IDC, especially if the ratio of DCIS to IDC size is high. We submit that IDC-DCIS is biologically distinct from pure IDC, and propose an incremental molecular pathogenesis of IDC-DCIS evolution involving an intermediate DCIS precursor that remains dependent for replication on upstream mitogens. © 2010 Cancer Research UK All rights reserved., published_or_final_version

The renin-angiotensin system (RAS) is a prime target for cardiovascular drug therapy. Inhibition of the RAS lowers blood pressure and confers protection against cardiovascular and renal events. These latter benefits cannot be entirely attributed to blood pressure lowering. Angiotensin-converting enzyme (ACE)-inhibitors and angiotensin receptor blockers (ARBs) have been studied extensively and, while there is irrefutable evidence that these agents mitigate the risk for cardiovascular and renal events, their protection is incomplete. In outcomes studies that have employed ACE-inhibitors or ARBs there has been a relatively high residual event rate in the treatment arm and this has been ascribed, by some, to the fact that neither ACE-inhibitors nor ARBs completely repress RAS. For this reason, combined RAS blockade with an ACE-inhibitor and ARB has emerged as a therapeutic option. In hypertension, combined RAS blockade elicits only a marginal incremental drop in blood pressure and it does not further lower the risk for cardiovascular events. In chronic heart failure and proteinuric renal disease, combining these agents in carefully selected patients is associated with a reduction in clinical events. Irrespective of the setting, dual RAS blockade is associated with an increase in the risk for adverse events, primarily hyperkalemia and worsening renal function. The emergence of the direct renin inhibitor, aliskiren, has afforded clinicians a new strategy for RAS blockade. Renin system blockade with aliskiren plus another RAS agent is the subject of ongoing large-scale clinical trials and early studies suggest promise for this strategy. Currently, combined RAS blockade with an ACE-inhibitor and an ARB should not be routinely employed for hypertension; however, the combination of an ACE-inhibitor or ARB with aliskiren might be considered in some patients given the more formidable blood pressure-lowering profile of this regimen. In carefully selected patients with heart failure or kidney disease, combination therapy with two RAS inhibitors should be considered.

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

Down syndrome (DS) is the most common cause of mental retardation. Although structural and neurogenic abnormalities have been shown in the brains of DS patients, the molecular etiology is still unknown. To define it, we have performed structural and histological examinations of the brains of Ts1Cje and Ts2Cje, 2 mouse models for DS. These mice carry different length of trisomic segments of mouse chromosome 16 that are orthologous to human chromosome 21. At 3 months of age, ventricular enlargements were observed in both Ts1Cje and Ts2Cje brains at a similar degree. Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles (LVs) and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared with diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the genes that are responsible for these abnormal phenotypes and could be relevant to the mental retardation associated with DS.

Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4–6 months) and old (26–28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.

The hedgehog (Hh) signaling pathway is a key component of cross-talk during vertebrate gut development, involving endodermally secreted Sonic (Shh) and Indian hedgehog (Ihh) proteins that directly signal to adjacent mesoderm. Here we show that the closely linked mesenchymal forkhead transcription factors Foxf1 and Foxl1 are part of this signaling cascade. Analysis of conserved non-coding sequences surrounding Foxf1 and Foxl1 identified seven Gli binding sites, with two sites near Foxl1 being identical among mammalian, bird, fish, and amphibian species. In vitro experiments indicate that Gli2 binds to these Gli sites, several of which are critical for Gli2-mediated activation of a luciferase reporter in 293 cells. In addition, we demonstrate occupancy of one of these elements by Gli proteins in the intestine in vivo using chromatin immunoprecipitation. Furthermore, expression of both Foxf1 and Foxl1 is reduced in the Gli2/Gli3 mutant gut. These results provide compelling evidence that Foxf1 and Foxl1 are mediators of the Hh (endoderm) to mesoderm signaling pathway.

Alcohol consumption increases reactive oxygen species formation and lipid peroxidation, whose products can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. A possible role of manganese superoxide dismutase (MnSOD) on these effects has not been investigated. To test whether MnSOD overexpression modulates alcohol-induced mitochondrial alterations, we added ethanol to the drinking water of transgenic MnSOD-overexpressing (TgMnSOD) mice and their wild type (WT) littermates for 7 weeks. In TgMnSOD mice, alcohol administration further increased the activity of MnSOD, but decreased cytosolic glutathione as well as cytosolic glutathione peroxidase activity and peroxisomal catalase activity. Whereas ethanol increased cytochrome P-450 2E1 and mitochondrial ROS generation in both WT and TgMnSOD mice, hepatic iron, lipid peroxidation products and respiratory complex I protein carbonyls were only increased in ethanol-treated TgMnSOD mice but not in WT mice. In ethanol-fed TgMnSOD mice, but not ethanol-fed WT mice, mtDNA was depleted, and mtDNA lesions blocked the progress of polymerases. The iron chelator, DFO prevented hepatic iron accumulation, lipid peroxidation, protein carbonyl formation and mtDNA depletion in alcohol-treated TgMnSOD mice. Alcohol markedly decreased the activities of complexes I, IV and V of the respiratory chain in TgMnSOD, with absent or lesser effects in WT mice. There was no inflammation, apoptosis or necrosis, and steatosis was similar in ethanol-treated WT and TgMnSOD mice. In conclusion, prolonged alcohol administration selectively triggers iron accumulation, lipid peroxidation, respiratory complex I protein carbonylation, mtDNA lesions blocking the progress of polymerases, mtDNA depletion and respiratory complex dysfunction in TgMnSOD mice but not in WT mice.

The expression of H-Y antigen on preimplantation mouse embryos has been studied by complement dependent cytotoxicity. Embryos at the 8-cell stage were exposed to anti-H-Y and complement and then scored for cell death. Overall, half of the treated embryos were killed, retarded, or contained dead cells. Of those embryos which were not affected by anti-H-Y, 92% were female. In control experiments, neither normal serum plus complement nor complement alone had any specific effect on male embryos. It is concluded, therefore, that the H-Y antigen is present on preimplantation mouse embryos.

SummaryHoloprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPCmolecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Administration of methamphetamine (METH) to rats and nonhuman primates causes loss of terminals in the nigrostriatal dopaminergic system. The mechanism by which METH causes its neurotoxicity is not known. To evaluate further the role of oxyradicals in METH-induced neurotoxicity, we have tested its effects in CuZn superoxide dismutase (SOD) transgenic (Tg) mice, which express the human CuZnSOD gene. In non-Tg mice, acute METH administration causes significant decreases in levels of dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the striata and cortices of non-Tg mice. In contrast, there were no significant decreases in cortical or striatal DA in the SOD-Tg mice. The effects of METH on DOPAC were also attenuated in both structures of these SOD-Tg mice. Chronic METH administration caused decreases in levels of striatal DA and DOPAC in the non- Tg mice, whereas the SOD-Tg mice were not affected. These results suggest that METH-induced dopaminergic toxicity in mice may be secondary to increased production of reactive oxygen species such as the superoxide radical.

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase ( Sod1) and glutathione peroxidase-1 ( Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3–6 h in wild-type mice without any lethality. In contrast, treatment of Sod1−/−or Gpx1−/−mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1−/−mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1−/−, and Sod1−/−mice and in untreated Sod1−/−mice is an upregulation of p53 target genes ( p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

This report describes a family in which eight individuals in three generations had mental retardation in association with a characteristic pattern of clinical problems and physical abnormalities including short stature, eczema, hernias, delayed puberty, dysmorphic facies and digital anomalies. The family history was consistent with a chromosomal rearrangement with transmission through balanced carriers. Routine ASG banding studies showed extra chromosomal material on a chromosome 16 but failed to demonstrate any differences between the affected individuals and the presumed carriers. However, subsequent studies utilizing trypsin banding and microspectrophotometry of individual chromosomes demonstrated that the affected individuals were partially trisomic for the distal band of the long arm of chromosome 5 and that 0.273 units of a chromosome 5 were translocated to chromosome 16. This definitive cytogenetic diagnosis permitted accurate prenatal diagnosis to be carried out on the fetus of a balanced carrier female. The application of these techniques to previously obscure familial dysmorphic syndromes is recommended.

Tumor suppressor genes are classified by their somatic behavior either as caretakers (CTs) that maintain DNA integrity or as gatekeepers (GKs) that regulate cell survival, but the germ line role of these disease-related gene subgroups may differ. To test this hypothesis, we have used genomic data mining to compare the features of human CTs (n = 38), GKs (n = 36), DNA repair genes (n = 165), apoptosis genes (n = 622), and their orthologs. This analysis reveals that repair genes are numerically less common than apoptosis genes in the genomes of multicellular organisms (P < 0.01), whereas CT orthologs are commoner than GK orthologs in unicellular organisms (P < 0.05). Gene targeting data show that CTs are less essential than GKs for survival of multicellular organisms (P < 0.0005) and that CT knockouts often permit offspring viability at the cost of male sterility. Patterns of human familial oncogenic mutations confirm that isolated CT loss is commoner than is isolated GK loss (P < 0.00001). In sexually reproducing species, CTs appear subject to less efficient purifying selection (i.e., higher Ka/Ks) than GKs (P = 0.000003); the faster evolution of CTs seems likely to be mediated by gene methylation and reduced transcription-coupled repair, based on differences in dinucleotide patterns (P = 0.001). These data suggest that germ line CT/repair gene function is relatively dispensable for survival, and imply that milder (e.g., epimutational) male prezygotic repair defects could enhance sperm variation—and hence environmental adaptation and speciation—while sparing fertility. We submit that CTs and repair genes are general targets for epigenetically initiated adaptive evolution, and propose a model in which human cancers arise in part as an evolutionarily programmed side effect of age- and damage-inducible genetic instability affecting both somatic and germ line lineages.

DNA cytophotometry was used to resolve a cytogenetic ambiguity involving a possible deletion of part of the short arm of chromosome 18 in cells obtained by amniocentesis. Measurements of chromosomal DNA content were within normal limits. This is the first time that DNA cytophotometry has been used in cytogenetic diagnosis; it illustrates the potential of this new approach to cytogenetics.

Neural tube defects (NTDs) are very frequent congenital abnormalities in humans. Recently, we have documented independent association of Vangl1 and Vangl2 gene mutations with NTDs. In the Looptail mouse, homozygosity (but not heterozygosity) for loss-of-function alleles at Vangl2 causes the severe NTD craniorachischisis, whereas heterozygosity for mutant variants of VANGL1 is associated with NTDs in a human cohort of sporadic and familial cases. To understand the role of Vangl1 in normal development, we created a mouse mutant with an inactivating mutation at Vangl1 ( Vangl1 gt ). Vangl1 shows a dynamic pattern of expression in the developing neural tube and notochord at the time of neural tube closure. Vangl1 gt /+ heterozygotes and Vangl1 gt/gt homozygotes are viable and fertile, although Vangl1 gt/gt display subtle alterations in polarity of inner hair cells of the cochlea. Remarkably, and as opposed to healthy Vangl1 gt /+ and Vangl2 lp /+ heterozygotes, Vangl1 gt /+ ; Vangl2 lp /+ double heterozygotes show profound developmental defects that include severe craniorachischisis, inner ear defects (disorganization of the stereociliary bundles of hair cells of the organ of Corti), and cardiac abnormality (aberrant right subclavian artery). These results show that genetic interaction between Vangl1 and Vangl2 genes causes neural tube defects and raise the possibility that interaction between individual Vangl genes and other genetic loci and/or environmental factors may additionally contribute to the etiology of NTDs.

The vascular endothelial growth factor (VEGF) family of polypeptide growth factors regulates a family of VEGF receptor (VEGFR) tyrosine kinases with pleiotropic downstream effects. Angiogenesis is the best known of these effects, but additional VEGF-dependent actions include increased vascular permeability, paracrine/autocrine growth factor release, enhancement of cell motility, and inhibition of apoptosis. In theory, therapeutic inhibition of angiogenesis should reduce tumor perfusion and thus increase tumor hypoxia and chemoresistance, but in clinical practice the VEGF antibody bevacizumab acts as a broad-spectrum chemosensitizer. Since VEGFR expression occurs in many tumor types, such chemosensitization is more readily explained by direct inhibition of tumor cell survival signals than by indirect stromal/vascular effects. The emerging model of anti-VEGF drug action being mediated primarily by tumoral (as distinct from endothelial) VEGFRs has clinically important implications for optimizing the anti-metastatic efficacy of this expanding drug class.

Background Antimicrobial resistance can be tracked by several methods. The optimal application of surveillance strategies is an integral part in curbing the resistance epidemic. Objectives The Antimicrobial Resistance Management (ARM) and Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) programs are ongoing antibiotic surveillance studies with different methodologies. Our aim was to compare susceptibility data generated by each program to determine if the two methods produce similar results. Methods ARM is a Web-based, antibiogram-driven surveillance system, whereas MYSTIC tests individual bacterial isolates in accordance with Clinical Laboratory Standards Institute methods. We compared national susceptibility rates during 1999 to 2004 from ARM and MYSTIC for Escherichia coli and Klebsiella pneumoniae. Results E. coli susceptibility was greater than 90% for all agents evaluated except ciprofloxacin and levofloxacin. Susceptibility rates in ARM and MYSTIC were similar (within 5%), with the exception of levofloxacin — for which susceptibility was reported as 92.3% in ARM and 83% in MYSTIC ( P < 0.0001). Amikacin, cefepime, ceftriaxone, imipenem, and meropenem were identified by both surveillance programs as the most active agents against E. coli and K. pneumoniae. Both systems detected increasing fluoroquinolone resistance ( P < 0.0001) and relatively stable activity among other agents tested. For K. pneumoniae, all agents displayed resistance rates less than 10%. Susceptibility rates in ARM and MYSTIC were remarkably similar. The largest difference was 2.9% for piperacillin/tazobactam (92.8% in ARM vs 95.7% in MYSTIC, P < 0.0001). Regionally, nonsusceptibility to E. coli and K. pneumoniae was similar, though for some comparisons MYSTIC reported higher rates of nonsusceptibility in the Northeast, Northwest, and South Central. Conclusion Surveillance of antibiotic activity patterns can be performed confidently with either method discussed above. Attempts should be made to integrate results from different surveillance programs to optimize surveillance.

The liver is distinguished from other tissues by (a) its detoxifying function, (b) its resistance to apoptosis, and (c) its regenerative response to damage. Hepatocellular carcinoma arises when chronic insults, such as hepatitis or iron overload, constitutively activate this regenerative program. Here, we propose that the proliferative response of the liver to damage underlies the resistance of hepatocellular carcinoma to cytotoxic therapy, and that hepatocellular carcinoma growth should therefore be more readily controlled by using a networked combination of noncytotoxic interventions to interrupt the damage-inducible regenerative pathway. To this end, hepatocellular carcinoma boasts a wealth of potential drug targets, including viral replication, the antiapoptotic immunosuppressant α-fetoprotein, hepatic iron overload, inflammatory signaling, extracellular proteases, and growth factors. By blocking these positive feedback loops in parallel, and so returning the host environment to a more normal state, epigenetic repression of tumor-suppressor gene function may be reversed and tumor dormancy restored. Noncytotoxic maneuvers that short circuit damage resistance loops may thus represent an indirect form of gene therapy meriting incorporation into hepatocellular carcinoma clinical trials.

Wild-type TP53 exons 5-8 contain CpG dinucleotides that are prone to methylation-dependent mutation during carcinogenesis, but the regulatory effects of methylation affecting these CpG sites are unclear. To clarify this, we first assessed site-specific TP53 CpG methylation in normal and transformed cells. Both DNA damage and cell ageing were associated with site-specific CpG demethylation in exon 5 accompanied by induction of a truncated TP53 isoform regulated by an adjacent intronic promoter (P2). We then synthesized novel synonymous TP53 alleles with divergent CpG content but stable encodement of the wild-type polypeptide. Expression of CpG-enriched TP53 constructs selectively reduced production of the full-length transcript (P1), consistent with a causal relationship between intragenic demethylation and transcription. 450K methylation comparison of normal (TP53-wildtype) and cancerous (TP53-mutant) human cells and tissues revealed focal cancer-associated declines in CpG methylation near the P1 transcription start site, accompanied by rises near the alternate exon 5 start site. These data confirm that site-specific changes of intragenic TP53 CpG methylation are extrinsically inducible, and suggest that human cancer progression is mediated in part by dysregulation of damage-inducible intragenic CpG demethylation that alters TP53 P1/P2 isoform expression. © 2015 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Genetic evolution depends in part upon a balance between negative selection and environmentally driven mutation. To explore whether this balance is affected by gene structure, we have used phylogenetic data mining to compare gene compositions across a range of species. Here we show that genomes of higher species exhibit a greater frequency of 5′ CpG islands and of CpG→TpG/CpA transitions. This latter mutational pattern exhibits a 5′-to-3′ trend in higher species, consistent with a length-dependent effect on methylation-dependent CpG suppression. Associated strand asymmetry (TpG>CpA) declines with gene length, implying attenuation of transcription-coupled repair 3′ to introns. A sharp 3′ rise in coding region single-nucleotide polymorphism frequency further supports a mechanistic role for intron length in promoting genetic variation by reducing repair and/or weakening negative selection. Consistent with this, the Ka/Ks ratio of 3′ exons exceeds that of centrally located exons in intron-containing, but not in intronless, genes (p