Grb7 Ablation in Mice Improved Glycemic Control, Enhanced Insulin Signaling, and Increased Abdominal fat Mass in Females.

OBJECTIVES: Growth factor receptor bound protein 7 (GRB7) is a multidomain signaling adaptor. Members of the Grb7/10/14 family, specifically Gbrb10/14, have important roles in metabolism. We ablated the Grb7 gene in mice to examine its metabolic function. METHODS: Global ablation of Grb7 in FVB/NJ mice was generated. Growth, organ weight, food intake, and glucose homeostasis were measured. Insulin signaling was examined by Western blotting. Fat and lean body mass was measured by nuclear magnetic resonance, and body composition after fasting or high-fat diet was assessed. Energy expenditure was measured by indirect calorimetry. Expression of adiposity and lipid metabolism genes was measured by quantitative PCR. RESULTS: Grb7-null mice were viable, fertile, and without obvious phenotype. Grb7 ablation improved glycemic control and displayed sensitization to insulin signaling in the liver. Grb7-null females but not males had increased gonadal white adipose tissue mass. Following a 12-week high-fat diet, Grb7-null female mice gained fat body mass and developed relative insulin resistance. With fasting, there was less decrease in fat body mass in Grb7-null female mice. Female mice with Grb7 ablation had increased baseline food intake, less energy expenditure, and displayed a decrease in the expression of lipolysis and adipose browning genes in gonadal white adipose tissue by transcript and protein analysis. CONCLUSION: Our study suggests that Grb7 is a negative regulator of glycemic control. Our results reveal a role for Grb7 in female mice in the regulation of the visceral adipose tissue mass, a powerful predictor of metabolic dysfunction in obesity.

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male-sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knockout of three floral genes in both FT-expressing (early-flowering) and non-FT genotypes. We report male-sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3-like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3-like knockouts produced virtually no pollen. In addition to male-sterility, both erec8 and ehec3-like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3-like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild-type (non-early-flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single-leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss-of-function mutations in any of these three genes show promise as a means of inducing male- or complete sterility without impacting vegetative development.

Optical detection of total cholesterol based on a dye-displacement method.

In the present study, the dye methylene blue (MB) was entrapped in an agarose gel and used as a sensing probe for the detection of total cholesterol. When methylene blue-entrapped agarose cubes were added to the cholesterol solution, methylene blue was displaced by cholesterol and released into the solution. A calibration curve was prepared by plotting the rate of release of methylene blue at 664 nm against varying cholesterol concentrations. A linear response was observed in the concentration range of 1 to 5 mM (40 mg/dL to 200 mg/dL) which covers normal and elevated cholesterol levels in humans. Optical detection of cholesterol using this dye-replacement method is simple, economical, and non-toxic. Characterisation of the system was carried out by FT-IR spectroscopy and cyclic voltammetry. The optical method was validated to determine total cholesterol in serum samples with reasonable accuracy.

Evaluation of right ventricular function during liver transplantation with transesophageal echocardiography.

BACKGROUND: The pathophysiology of advanced liver cirrhosis may induce alterations in the circulatory system that may be challenging for the anesthesiologist to manage intraoperatively, and perioperative cardiovascular events are associated with worse outcomes in cirrhotic patients undergoing liver transplantation. It remains controversial whether right ventricular function is impaired during this procedure. Studies using transesophageal echocardiography for quantitative analysis of the right ventricle remain scarce in this setting, yielding conflicting results. The aim of this study was to perform a quantitative assessment of right ventricular function with two parameters derived from transesophageal echocardiography during liver transplantation. METHODS: Nineteen adult patients of both genders undergoing liver transplantation were evaluated in this observational study. The exclusion criteria were age under 18 or above 65 years old, fulminant hepatic failure, hepatopulmonary syndrome, portopulmonary hypertension, cardiopulmonary disease, and contraindications to the transesophageal echocardiogram. Right ventricular function was assessed at five stages during liver transplantation: baseline, hepatectomy, anhepatic, postreperfusion, and closure by measuring tricuspid annular plane systolic excursion and right ventricular fractional area change obtained with transesophageal echocardiography. RESULTS: Right ventricular function was found to be normal throughout the procedure. The tricuspid annular plane systolic excursion showed a trend toward a decrease in the anhepatic phase compared to baseline (2.0 ± 0.9 cm vs. 2.4 ± 0.7 cm; P = 0.24) but with full recovery after reperfusion. Right ventricular fractional area change remained nearly constant during all stages studied (minimum: 50% ± 10 at baseline and anhepatic phase; maximum: 56% ± 12 at postreperfusion; P = 0.24). CONCLUSIONS: Right ventricular function was preserved during liver transplantation at the time points evaluated by two quantitative parameters derived from transesophageal echocardiogram.

Intersection of DNA Repair Pathways and the Immune Landscape Identifies PD-L2 as a Prognostic Marker in Epithelial Ovarian Cancer.

BACKGROUND: Targeting DNA repair and immune checkpoint pathways has been the focus of multiple clinical trials. In this study, we explore the association between DNA repair proteins, immune response markers, and clinical outcome in women with EOC. METHODS: Immunohistochemical analysis of TMA with 181 EOC samples was used to determine expression levels for DNA repair proteins (PARP, PTEN, p53, H2Ax, FANCD2, and ATM) and immune-markers (CD4, CD8, CD68, PD-L2, PD-L1, and FOXP3). Biomarker expression was correlated to clinical data. Prognostic discriminatory ability was assessed per the combination of biomarkers. RESULTS: Tumor immunity biomarkers correlated with HRD biomarkers. High PD-L2 was significantly associated with high expression of CD8 (r = 0.18), CD68 (r = 0.17), and FOXp3 (r = 0.16) (all, p < 0.05). In a multivariate analysis, PD-L2 (hazard ratio (HR) 1.89), PARP (HR 1.75), and PTEN (HR 1.96) expressions were independently associated with decreased progression-free survival (PFS), whereas PD-L1 (HR 0.49) and CD4 (HR 0.67) were associated with improved PFS (all, p < 0.05). In 15 biomarker combinations, six combinations exhibited a discriminatory ability of >20% for the 4.5-year PFS rate, with four based on PD-L2 (PARP, PTEN, CD4, and PD-L1, 20.5-30.0%). CONCLUSIONS: Increased PD-L2 expression is a prognostic marker of decreased survival in EOC. Interaction between tumor DNA repair and microenvironment determines tumor progression and survival.

Targetable alterations in invasive pleomorphic lobular carcinoma of the breast.

BACKGROUND: Invasive pleomorphic lobular carcinoma (PLC) of the breast is a subtype of invasive lobular cancer which compromises approximately 1% of all epithelial breast malignancies and is characterized by higher nuclear pleomorphism and poorer prognosis than classic invasive lobular cancer (ILC). Since PLC is more aggressive than classical ILC, we examined the underlying molecular alterations in this subtype of breast cancer to understand the possible benefit from targeted therapies. METHODS: In this study, we investigate the clinical characteristics and molecular alterations in 16 PLC from our institution. Additionally, we examined the clinical and genomic features in 31 PLC from the Cancer Genome Atlas (TCGA). RESULTS: Overall, our analysis of PLC found that 28% had activating ERBB2 mutations, 21% had ERBB2 amplification, and 49% activating PIK3CA mutations. Among cases from our institution, we found 19% with activating ERBB2 mutations, 25% had ERBB2 amplification, and 38% with activating PIK3CA mutations. In data from TCGA, 32% had activating ERBB2 mutations, 19% had ERBB2 amplification, and 55% had activating PIK3CA mutations. While classic ILC in TCGA had similar percentages of PIK3CA alterations compared to PLC, activating ERBB2 alterations were exceedingly rare, with no activating ERBB2 mutations and only one case with ERBB2 amplification. Interestingly, in further examining TCGA data which included FGFR1 and PTEN, 94% of PLC had alterations in ERBB2, FGFR1, or the PI3K pathway. CONCLUSIONS: Our results show a high frequency of ERBB2 and PIK3CA alterations in PLC and suggest all PLC should be tested for potential therapeutic targeting.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

OBJECTIVE: Women with inherited mutations in BRCA1 gene have a high (40-70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1 mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations. MATERIALS AND METHODS: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1 mut and without BRCA1 mutations (BRCA wt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1 mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed. RESULTS: BRCA1 mut women had lower serum calcifediol levels compared to BRCA wt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCA wt patients, but was reduced in BRCA1 mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1 mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis. CONCLUSIONS: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1 mut women. VD treatment may limit BRCA1 mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1 mut carriers.

Social and legal implications of urine drug screen analysis in the neonate: A case of suspected specimen mishandling.

BACKGROUND: The legal implications associated with illicit drug use during pregnancy are significant, as providers are required to notify child protective services when a drug-exposed infant is identified. CASE REPORT: The case presented involves possible specimen mishandling in two infants at risk for in utero drug exposure and describes alternative methodologies available to confirm specimen identity. CONCLUSIONS: It is critical that institutions establish and adhere to stringent procedures when screening newborns.

Subcellular localization of FANCD2 is associated with survival in ovarian carcinoma.

OBJECTIVE: Ovarian cancer is a leading cause of death from gynecological cancers. Late diagnosis and resistance to therapy results in mortality and effective screening is required for early diagnosis and better treatments. Expression of the Fanconi Anemia complementation group D2 protein (FANCD2) is reduced in ovarian surface epithelial cells (OSE) in patients with ovarian cancer. FANCD2 has been studied for its role in DNA repair; however multiple studies have suggested that FANCD2 has a role outside the nucleus. We sought to determine whether subcellular localization of FANCD2 correlates with patient outcome in ovarian cancer. METHODS: We examined the subcellular localization of FANCD2 in primary OSE cells from consenting patients with ovarian cancer or a normal ovary. Ovarian tissue microarray was stained with anti-FANCD2 antibody by immunohistochemistry and the correlation of FANCD2 localization with patient outcomes was assessed. FANCD2 binding partners were identified by immunoprecipitation of cytoplasmic FANCD2. RESULTS: Nuclear and cytoplasmic localization of FANCD2 was observed in OSEs from both normal and ovarian cancer patients. Patients with cytoplasmic localization of FANCD2 (cFANCD2) experienced significantly longer median survival time (50 months), versus patients without cytoplasmic localization of FANCD2 (38 months; p < 0.05). Cytoplasmic FANCD2 was found to bind proteins involved in the innate immune system, cellular response to heat stress, amyloid fiber formation and estrogen mediated signaling. CONCLUSIONS: Our results suggest that the presence of cytoplasmic FANCD2 modulates FANCD2 activity resulting in better survival outcome in ovarian cancer patients.

14-3-3ζ loss leads to neonatal lethality by microRNA-126 downregulation-mediated developmental defects in lung vasculature.

BACKGROUND: The 14-3-3 family of proteins have been reported to play an important role in development in various mouse models, but the context specific developmental functions of 14-3-3ζ remain to be determined. In this study, we identified a context specific developmental function of 14-3-3ζ. RESULTS: Targeted deletion of 14-3-3ζ in the C57Bl/6J murine genetic background led to neonatal lethality due to respiratory distress and could be rescued by out-breeding to the CD-1 or backcrossing to the FVB/NJ congenic background. Histological analysis of lung sections from 18.5 days post coitum embryos (dpc) showed that 14-3-3ζ-/- lung development is arrested at the pseudoglandular stage and exhibits vascular defects. The expression of miR-126, an endothelial-specific miRNA known to regulate lung vascular integrity was down-regulated in the lungs of the 14-3-3ζ-/- embryos in the C57Bl/6J background as compared to their wild-type counterparts. Loss of 14-3-3ζ in endothelial cells inhibited the angiogenic capability of the endothelial cells as determined by both trans-well migration assays and tube formation assays and these defects could be rescued by re-expressing miR-126. Mechanistically, loss of 14-3-3ζ led to reduced Erk1/2 phosphorylation resulting in attenuated binding of the transcription factor Ets2 on the miR-126 promoter which ultimately reduced expression of miR-126. CONCLUSION: Our data demonstrates that miR-126 is an important angiogenesis regulator that functions downstream of 14-3-3ζ and downregulation of miR-126 plays a critical role in 14-3-3ζ-loss induced defects in lung vasculature in the C57Bl/6J genetic background.

14-3-3ζ loss impedes oncogene-induced mammary tumorigenesis and metastasis by attenuating oncogenic signaling.

The 14-3-3ζ protein belongs to the 14-3-3 family of regulatory eukaryotic proteins that modulate signaling by binding to wide variety of signaling molecules. 14-3-3ζ expression is amplified in over 40% breast cancer patients and is associated with a poor prognosis. Various in vitro and xenograft models have suggested that attenuating 14-3-3ζ expression may provide therapeutic benefits but there has been no study looking at tumor onset and metastasis in breast cancer mouse models with a targeted deletion of 14-3-3ζ. We generated a 14-3-3ζ knockout mouse model to characterize the role of 14-3-3ζ in breast cancer progression. Crossing 14-3-3ζ-/- mice with MMTV-PyMT and MMTV-Neu transgenic mice revealed that loss of 14-3-3ζ prolonged tumor latency and reduced lung metastasis as compared to MMTV-PyMT and MMTV-Neu mice. Mechanistically, loss of 14-3-3ζ suppressed tumor proliferation and angiogenesis and promoted apoptosis by suppressing the Akt and Erk pathway and upregulated the expression of the tumor suppressor p53. Our results provide evidence showing that attenuating 14-3-3ζ expression/activity in mammary tumors can provide a therapeutic benefit.

Mnk kinase pathway: Cellular functions and biological outcomes.

The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs (p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E (eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4E. The role of Mnk kinases in inflammation and inflammation-induced malignancies is also discussed.

Essential role for the Mnk pathway in the inhibitory effects of type I interferons on myeloproliferative neoplasm (MPN) precursors.

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.

Sprouty proteins are negative regulators of interferon (IFN) signaling and IFN-inducible biological responses.

Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses.

Mnk Kinases in Cytokine Signaling and Regulation of Cytokine Responses.

The kinases Mnk1 and Mnk2 are activated downstream of the p38 MAPK and MEK/ERK signaling pathways. Extensive work over the years has shown that these kinases control phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and regulate engagement of other effector elements, including hnRNPA1 and PSF. Mnk kinases are ubiquitously expressed and play critical roles in signaling for various cytokine receptors, while there is emerging evidence that they have important functions as mediators of pro-inflammatory cytokine production. In this review the mechanisms of activation of MNK pathways by cytokine receptors are addressed and their roles in diverse cytokine-dependent biological processes are reviewed. The clinical-translational implications of such work and the relevance of future development of specific MNK inhibitors for the treatment of malignancies and auto-immune disorders are discussed.

Recognition of an incidental abscess and a hematoma during ultrasound-guided femoral nerve block.

Ultrasound guidance for femoral nerve blockade allows visualization of normal and abnormal anatomy. Two cases of femoral nerve blockade under ultrasound guidance are presented where a major perineural pathologic lesion was incidentally revealed. These pathologic lesions, an abscess and a hematoma, resulted in significant adaptations in clinical care and have not been reported previously. We review and discuss incidental pathologic lesions that can be found in the femoral region. Identification of these 2 pathologic lesions facilitated our perioperative management strategies in two separate cases.

Diglyceride prodrug strategy for enhancing the bioavailability of norfloxacin.

Prodrug approach using diglyceride as a promoiety is a promising strategy to improve bioavailability of poorly absorbed drugs and the same was explored in the present work to improve oral bioavailability of norfloxacin; a second generation fluoroquinolone antibacterial. The prodrug was synthesized by standard procedures using dipalmitine as a carrier and the structure was confirmed by spectral analysis. Higher LogP indicated improved lipophilicity. The ester linkage between norfloxacin and dipalmitine would be susceptible to hydrolysis by lipases to release the parent drug and carrier in the body. In vivo kinetic studies in rats indicated 53% release of norfloxacin in plasma at the end of 8h. The prodrug exhibited improved pharmacological profile than the parent compound at equimolar dose that indirectly indicated improved bioavailability.

Essential role for Mnk kinases in type II interferon (IFNgamma) signaling and its suppressive effects on normal hematopoiesis.

IFNγ exhibits potent antitumor effects and plays important roles in the innate immunity against cancer. However, the mechanisms accounting for the antiproliferative effects of IFNγ still remain to be elucidated. We examined the role of Mnk1 (MAPK-interacting protein kinase 1) in IFNγ signaling. Our data demonstrate that IFNγ treatment of sensitive cells results in engagement of Mnk1, activation of its kinase domain, and downstream phosphorylation of the cap-binding protein eIF4E on Ser-209. Such engagement of Mnk1 plays an important role in IFNγ-induced IRF-1 (IFN regulatory factor 1) gene mRNA translation/protein expression and is essential for generation of antiproliferative responses. In studies aimed to determine the role of Mnk1 in the induction of the suppressive effects of IFNs on primitive hematopoietic progenitors, we found that siRNA-mediated Mnk1/2 knockdown results in partial reversal of the suppressive effects of IFNγ on human CD34+-derived myeloid (CFU-GM) and erythroid (BFU-E) progenitors. These findings establish a key role for the Mnk/eIF4E pathway in the regulatory effects of IFNγ on normal hematopoiesis and identify Mnk kinases as important elements in the control of IFNγ-inducible ISG mRNA translation.