Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.</p> <p>Results</p> <p>We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC₁D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC₁D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and <it>in vivo </it>showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. <it>In vivo </it>studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC₁D promoters.</p> <p>Conclusion</p> <p>This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.</p

Enhancement of cell-specific transgene expression from a Tet-Off regulatory system using a transcriptional amplification strategy in the rat brain

10.1002/jgm.1178Journal of Gene Medicine105583-592JGME

Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

<p>Abstract</p> <p>Background</p> <p>5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain.</p> <p>Results</p> <p>We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2) gene which selectively (97% co-localisation with TPH-2) target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca²⁺imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules.</p> <p>Conclusion</p> <p>For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this study open a host of new experimental opportunities and will greatly facilitate further studies of the central serotonergic system.</p

Development and validation of a chemotherapy tolerance prediction model for Chinese multiple myeloma patients: The TM frailty score

ObjectiveThe physical fitness of older individuals is heterogeneous, making it difficult to know their chemotherapy tolerance. The toxicities may offset the benefits of anti-myeloma therapy in frail patients. The accurate evaluation of frailty status before chemotherapy is essential. We aimed to explore the applicability of the IMWG GA and develop a new frailty screening tool more suitable for Chinese MM patients.Cases and methodsWe performed the IMWG GA and the full CGA in 167 MM patients and validated the applicability of the IMWG GA to chemotherapy and prognosis. The CGA domains were screened for their predictive value to improve IMWG GA and develop new frailty screening tools.ResultsThe results showed that the IMWG GA had limitations in distinguishing the risk of grade ≥3 adverse events (AEs) between fit and int-fit patients. Of the CGA domains, TUG and MNA-SF were independent prognostic factors for grade ≥3 AEs and OS and further stratified the risk of grade ≥3 AEs in the IMWG GA int-fit subgroup (P&lt; 0.05). We combined TUG and MNA-SF to construct the TM frailty score. The frail subgroup had a higher proportion of adverse outcomes, a higher hazard ratio (HR) in Cox regression and a higher Harrell’s C-index for distinguishing the risk of grade ≥3 AEs and OS than the IMWG GA frail subgroup.ConclusionThe TM frailty score is more suitable than the IMWG GA for evaluating chemotherapy tolerance and prognosis in the Chinese population

Astrocytes modulate brainstem respiratory rhythm-generating circuits and determine exercise capacity

Astrocytes are implicated in modulation of neuronal excitability and synaptic function, but it remains unknown if these glial cells can directly control activities of motor circuits to influence complex behaviors in vivo. This study focused on the vital respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) and determined how compromised function of local astrocytes affects breathing in conscious experimental animals (rats). Vesicular release mechanisms in astrocytes were disrupted by virally driven expression of either the dominant-negative SNARE protein or light chain of tetanus toxin. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity. These findings indicate that astrocytes modulate the activity of CNS circuits generating the respiratory rhythm, critically contribute to adaptive respiratory responses in conditions of increased metabolic demand and determine the exercise capacity

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating

Cross-species comparative studies are a powerful approach to understand the epithelial Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl- channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl- currents. Like its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl- channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. This article is protected by copyright. All rights reserved

STUDY ON SECONDARY CAROTENOIDS ACCUMULATION IN A GREEN ALGA, CHLOROCOCCUM SP

Ph.DDOCTOR OF PHILOSOPH