Structure−Function Correlation of Chloroquine and Analogues as Transgene Expression Enhancers in Nonviral Gene Delivery

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N^4-(4-pyridinyl)-N^1,N^1-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N^4-(7-trifluoromethyl-4-quinolinyl)-N^1,N^1-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N^4-(4-quinolinyl)-N^1,N^1-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression:  (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid

Associations between body mass index across adult life and hip shapes at age 60 to 64:Evidence from the 1946 British birth cohort

Funding: The NSHD is funded by the UK Medical Research Council. SGM, RC, RJH and DK are supported by the UK Medical Research Council (Programme codes: MC_UU_12019/1, MC_UU_12019/2 and MC_UU_12019/4). This project was funded by the UK Medical Research Council (Grant MR/L010399/1) which supported SGM, AVP and FRS. The funders of the study had no role in study design, data collection, data analysis, data interpretation or writing of this manuscript. Acknowledgements: We thank Dr. Michael Machin for his valuable assistance obtaining the images and the University of Aberdeen Data Management Team for programming support for ‘Shape’. The authors are grateful to NSHD study members who took part in the clinic data collection for their continuing support. We thank members of the NSHD scientific and data collection teams at the following centres: MRC Unit for Lifelong Health and Ageing; Wellcome Trust (WT) Clinical Research Facility (CRF) Manchester; WTCRF and Medical Physics at the Western General Hospital in Edinburgh; WTCRF and Department of Nuclear Medicine at University Hospital Birmingham; WTCRF and the Department of Nuclear Medicine at University College London Hospital; CRF and the Department of Medical Physics at the University Hospital of Wales; CRF and Twin Research Unit at St Thomas' Hospital London. Data used in this publication are available to bona fide researchers upon request to the NSHD Data Sharing Committee via a standard application procedure. Further details can be found at: http://www.nshd.mrc.ac.uk/data; doi: http://dx.doi.org/10.5522/NSHD/Q102; doi: http://dx.doi.org/10.5522/NSHD/Q102A.Peer reviewedPublisher PD

A Multisite Study of Nurse-Reported Perceptions and Practice of ABCDEF Bundle Components

Objectives: ABCDEF bundle implementation in the Intensive Care Unit (ICU) is associated with dose dependent improvements in patient outcomes. The objective was to compare nurse attitudes about the ABCDEF bundle to self-reported adherence to bundle components. Research methodology/design: Cross-sectional study. Setting: Nurses providing direct patient care in 28 ICUs within 18 hospitals across the United States. Main outcome measures: 53-item survey of attitudes and practice of the ABCDEF bundle components was administered between November 2011 and August 2015 (n = 1661). Results: We did not find clinically significant correlations between nurse attitudes and adherence to Awakening trials, Breathing trials, and sedation protocol adherence (rs = 0.05-0.28) or sedation plan discussion during rounds and Awakening and Breathing trial Coordination (rs = 0.19). Delirium is more likely to be discussed during rounds when ICU physicians and nurse managers facilitate delirium reduction (rs = 0.27-0.36). Early mobilization is more likely to occur when ICU physicians, nurse managers, staffing, equipment, and the ICU environment facilitate early mobility (rs = 0.36-0.47). Physician leadership had the strongest correlation with reporting an ICU environment that facilitates ABCDEF bundle implementation (rs = 0.63-0.74). Conclusions: Nurse attitudes about bundle implementation did not predict bundle adherence. Nurse manager and physician leadership played a large role in creating a supportive ICU environment

GDNF Secreting Human Neural Progenitor Cells Protect Dying Motor Neurons, but Not Their Projection to Muscle, in a Rat Model of Familial ALS

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF) are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF.Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPC(GDNF)) and thus act as long-term 'mini pumps' in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1(G93A)). Following unilateral transplantation into the spinal cord of SOD1(G93A) rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no improvement in ipsilateral limb use.The potential to maintain dying motor neurons by delivering GDNF using neural progenitor cells represents a novel and powerful treatment strategy for ALS. While this approach represents a unique way to prevent motor neuron loss, our data also suggest that additional strategies may also be required for maintenance of neuromuscular connections and full functional recovery. However, simply maintaining motor neurons in patients would be the first step of a therapeutic advance for this devastating and incurable disease, while future strategies focus on the maintenance of the neuromuscular junction

An ALS-Linked Mutant SOD1 Produces a Locomotor Defect Associated with Aggregation and Synaptic Dysfunction When Expressed in Neurons of Caenorhabditis elegans

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking

The effect of intraluminal content on the bursting strength of vessels ligated with the harmonic ACE and LigaSure V.

PURPOSE: Energy-based surgical devices (ESDs) are critical for maintaining hemostasis during laparoscopy; however, there are no studies that have evaluated the function of ESDs under different physiologic conditions. We evaluated the effect of intraluminal vessel content on bursting pressure (BP) after ligation with two ESDs: the Harmonic ACE and the LigaSure V. MATERIALS AND METHODS: Bursting trials were performed on the vasculature of 24 pigs. Blood vessels were distended with blood of different hematocrit concentrations or an albumin solution of varying protein content. The vessel size and BP of each vessel was recorded after ligation with each ESD. RESULTS: In arteries 0 to 3 mm and veins 0 to 3 mm in size ligated with the Harmonic ACE or the LigaSure V, there were significantly elevated vessel BPs with supraphysiologic intraluminal hematocrits. In arteries and veins ligated with the Harmonic ACE, increasing albumin concentrations also led to increasing BPs, though these maximal BPs were lower than those obtained with supraphysiologic hematocrit levels. Increasing albumin concentrations did not increase the BP of the LigaSure V. Within the ranges tested, there was no decrease in vessel BP associated with anemia. CONCLUSION: In small vessels, a supraphysiologic hematocrit increased the BP of both arteries and veins when using the Harmonic ACE or the LigaSure V. With the devices tested, anemia did not seem to affect BP. While factors such as intraluminal protein concentration may play a role with ultrasonic energy devices, the mechanism of the increased BP remains unclear. Better understanding of ESDs will help in the design of future devices