In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

Increased expression of axogenesis-related genes and mossy fibre length in dentate granule cells from adult HuD overexpressor mice

The neuronal RNA-binding protein HuD plays a critical role in the post-transcriptional regulation of short-lived mRNAs during the initial establishment and remodelling of neural connections. We have generated transgenic mice overexpressing this protein (HuD-Tg) in adult DGCs (dentate granule cells) and shown that their mossy fibres contain high levels of GAP-43 (growth-associated protein 43) and exhibit distinct morphological and electrophysiological properties. To investigate the basis for these changes and identify other molecular targets of HuD, DGCs from HuD-Tg and control mice were collected by LCM (laser capture microscopy) and RNAs analysed using DNA microarrays. Results show that 216 known mRNAs transcripts and 63 ESTs (expressed sequence tags) are significantly up-regulated in DGCs from these transgenic mice. Analyses of the 3′-UTRs (3′-untranslated regions) of these transcripts revealed an increased number of HuD-binding sites and the presence of several known instability-conferring sequences. Among these, the mRNA for TTR (transthyretin) shows the highest level of up-regulation, as confirmed by qRT–PCR (quantitative reverse transcription–PCR) and ISH (in situ hybridization). GO (gene ontology) analyses of up-regulated transcripts revealed a large over-representation of genes associated with neural development and axogenesis. In correlation with these gene expression changes, we found an increased length of the infrapyramidal mossy fibre bundle in HuD-Tg mice. These results support the notion that HuD stabilizes a number of developmentally regulated mRNAs in DGCs, resulting in increased axonal elongation

Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome.

BACKGROUND: Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital. RESULTS: Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters. CONCLUSIONS: Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages