Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats.

Gene therapy for cystic fibrosis (CF) will require the safe transfer of CFTR cDNA to airway epithelia in vivo. We showed previously that a recombinant adenovirus, Ad2/CFTR-1, expresses CFTR in vitro. As adenovirus rarely integrates, treatment will require repeated vector administration. We applied Ad2/CFTR-1 to intrapulmonary airway epithelia of cotton rats and nasal epithelia of Rhesus monkeys. In both species we detected CFTR mRNA and protein after repeated administration and in monkeys, protein was detected six weeks after repeat administration. The vector did not replicate and was rapidly cleared. Despite an antibody response, there was no evidence of a local or systemic inflammatory response after repeat administration. These data indicate that repetitive administration of Ad2/CFTR-1 is both safe and efficacious.

Cross-sectional associations between short and mid-term blood pressure variability, cognition, and vascular stiffness in older adults.

Background: High blood pressure variability (BPV), particularly in older age, appears to be an independent risk factor for incident dementia. The current study aimed to investigate the association between different BPV measures (short- and mid-term BPV including circadian patterns) and cognitive functioning as well as vascular stiffness measures to better understand the role that BPV plays in cognitive impairment. Methods: 70 older adults (60-80-year-olds) without dementia completed a cognitive test battery and had their blood pressure (BP) assessed via a 24-hour ambulatory BP monitor (divided into sleep and wake for short-term BPV) and 4-day morning and evening home-based BP monitor (for day-to-day BPV). Arterial stiffness was evaluated via pulse wave analysis and pulse wave velocity (PWV) and cerebrovascular pulsatility was assessed via transcranial doppler sonography of the middle cerebral arteries. Results: High systolic as well as diastolic short- and mid-term BPV were associated with poorer cognitive functioning, independent of the mean BP. Higher short-term BPV was associated with poorer attention and psychomotor speed, whilst day-to-day BPV was negatively linked with executive functioning. Circadian BP patterns (dipping and morning BP surge) showed no significant relationships with cognition after adjusting for covariates. Higher systolic short-term BPV was associated with higher arterial stiffness (PWV) and higher diastolic day-to-day BPV was linked with lower arterial stiffness. No significant associations between BPV measures and cerebrovascular pulsatility were present. Conclusion: High BPV, independently of the mean BP, is associated with lower cognitive performance and increased arterial stiffness in older adults without clinically-relevant cognitive impairment. This highlights the role of systolic and diastolic BPV as a potential early clinical marker for cognitive impairment.

Exercise-induced oxidative stress: the effects of ß-alanine supplementation in women.

The purpose of this study was to evaluate the effects of ß-alanine supplementation on markers of oxidative stress. Twenty-four women (age: 21.7±2.1 years; VO2max: 2.6±0.3 l min(-1)) were randomly assigned, in a double-blind fashion, to a ß-alanine (BA, 2×800 mg tablets, 3× daily; CarnoSyn®; n=13) or placebo (PL, 2×800 mg maltodextrin tablets, 3× daily; n=11) group. A graded oxygen consumption test (VO2max) was performed to evaluate VO2max, time to exhaustion, ventilatory threshold and establish peak velocity (PV). A 40-min treadmill run was used to induce oxidative stress. Total antioxidant capacity, superoxide dismutase, 8-isoprostane (8ISO) and reduced glutathione were measured. Heart rate and ratings of perceived exertion were recorded during the 40 min run. Separate three- [4×2×2; acute (base vs. IP vs. 2 vs. 4 h)×chronic (pre- vs. post-)×treatment (BA vs. PL)] and two- [2×2; time (pre-supplement vs. post-supplement)×treatment (BA vs. PL)] way ANOVAs were used for analyses. There was a significant increase in VO2max (p=0.009), independent of treatment, with no significant changes in TTE (p=0.074) or VT (p=0.344). Ratings of perceived exertion values were significantly improved from pre- to post-supplementation for the BA group only at 40 min (p=0.02). The ANOVA model demonstrated no significant treatment effects on oxidative stress. The chronic effects of BA supplementation demonstrated little antioxidant potential, in women, and little influence on aerobic performance assessments.

Prostate cancer induced bone pain: pathobiology, current treatments and pain responses from recent clinical trials.

PURPOSE: Metastatic spread of prostate cancer to the skeleton may result in debilitating bone pain. In this review, we address mechanisms underpinning the pathobiology of metastatic prostate cancer induced bone pain (PCIBP) that include sensitization and sprouting of primary afferent sensory nerve fibres in bone. We also review current treatments and pain responses evoked by various treatment modalities in clinical trials in this patient population. METHODS: We reviewed the literature using PubMed to identify research on the pathobiology of PCIBP. Additionally, we reviewed clinical trials of various treatment modalities in patients with PCIBP with pain response outcomes published in the past 7 years. RESULTS: Recent clinical trials show that radionuclides, given either alone or in combination with chemotherapy, evoked favourable pain responses in many patients and a single fraction of local external beam radiation therapy was as effective as multiple fractions. However, treatment with chemotherapy, small molecule inhibitors and/or immunotherapy agents, produced variable pain responses but pain response was the primary endpoint in only one of these trials. Additionally, there were no published trials of potentially novel analgesic agents in patients with PCIBP. CONCLUSION: There is a knowledge gap for clinical trials of chemotherapy, small molecule inhibitors and/or immunotherapy in patients with PCIBP where pain response is the primary endpoint. Also, there are no novel analgesic agents on the horizon for the relief of PCIBP and this is an area of large unmet medical need that warrants concerted research attention.

Ergolytic/ergogenic effects of creatine on aerobic power.

This study evaluated the effects of creatine (Cr) loading and sex differences on aerobic running performance. 27 men (mean±SD; age: 22.2±3.1 years, ht: 179.5±8.7 cm, wt: 78.0±9.8 kg) and 28 women (age: 21.2±2.1 years, ht: 166.0±5.8 cm, wt: 63.4±8.9 kg) were randomly assigned to either creatine (Cr, di-creatine citrate; n=27) or a placebo (PL; n=28) group, ingesting 1 packet 4 times daily (total of 20 g/day) for 5 days. Aerobic power (maximal oxygen consumption: VO2max) was assessed before and after supplementation using open circuit spirometry (Parvo-Medics) during graded exercise tests on a treadmill. 4 high-speed runs to exhaustion were conducted at 110, 105, 100, and 90% of peak velocity to determine critical velocity (CV). Distances achieved were plotted over times-to-exhaustion and linear regression was used to determine the slopes (critical velocity, CV) assessing aerobic performance. The results indicated that Cr loading did not positively or negatively influence VO2max, CV, time to exhaustion or body mass (p>0.05). These results suggest Cr supplementation may be used in aerobic running activities without detriments to performance.

Long-Chain Omega-3 Fatty Acid Intake Is Associated with Age but not Cognitive Performance in an Older Australian Sample.

BACKGROUND: Long-chain omega-3 polyunsaturated fatty acids (LCn-3 PUFA) are essential nutrients and may be capable of delaying age-related cognitive decline. However, previous studies indicate that Australians are not meeting recommendations for LCn-3 PUFA intake. The current study therefore examined LCn-3 PUFA intake in an older Australia sample, as well as associations between LCn-3 PUFA intake and cognitive function. METHODS: Cross-sectional data were collected from 90 adults aged 50 to 80 years. LCn-3 PUFA intake was assessed using a food frequency questionnaire and red blood cell fatty acid profiles were used to calculate the Omega-3 Index (RBC n-3 index). Cognitive function was measured using Addenbrooke's Cognitive Examination-III. RESULTS: Positive associations were observed between age and RBC n-3 index (b=0.06, 95% CI: 0.01 - 0.10, P=0.01), and age and LCn-3 PUFA intake from fish oil capsules (b=17.5, 95% CI: 2.4 - 32.5 mg/day, P=0.02). When adjusting for LCn-3 PUFA from fish oil capsules, the association between age and RBC n-3 index was no longer significant. No associations were observed between LCn-3 PUFA intake and cognitive function. CONCLUSION: LCn-3 PUFA and fish oil consumption increased with age in this sample of older Australians, particularly due to supplement intake. However, LCn-3 PUFA intake was not associated with cognitive function.

Intracellular protein trafficking defects in human disease.

Secretory proteins and integral membrane proteins travel through the secretory pathway to a variety of destinations. Their targets are often specified by signals in the amino acid sequence or signals added post-translationally. The KDEL sequence that retains soluble proteins in the endoplasmic reticulum and the mannose 6-phosphate group of lysosomal enzymes are well-characterized examples of targeting signals; other signals are less well understood. Given the complexity and importance of the intracellular trafficking pathways, it is perhaps not surprising that mutations that affect the trafficking of proteins are associated with some human genetic diseases.

Synthesis of lipid during photosynthesis by phytoplankton of the southern ocean.

Assimilation of carbon-14 labeled bicarbonate into photosynthetic products was measured at four stations in the Southern Ocean. Phytoplankton populations incorporated as much as 80 percent of the fixed carbon into lipid under conditions of low temperatures (-0.2 degrees to -1.8 degrees C) and low light intensities. At higher temperatures (+0.3 degrees to +0.8 degrees C) and higher light intensities, incorporation into lipid accounted for less than 20 percent of the fixed carbon, synthesis of polysaccharide and protein being more prominent.

Dehydration and rehydration in a prebiological system.

A proteinoid microsphere suspension system was subjected to cyclic dehydration and rehydration. Particles having somewhat coacervate properties were observed, suggesting a relation between the coacervate and proteinoid origin of cells.

Synthesis of a sulfur-containing amino acid under simulated prebiotic conditions.

Ultraviolet irradiation of an aqueous solution of ammonium thiocyanate produces the sulfur-containing amino acid methionine. Synthesis of this class of biocompound fills another important gap in development of an overall picture of how prebiological chemistry may have evolved on primitive Earth.

Effect of deleting the R domain on CFTR-generated chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain.

Generation of cAMP-activated chloride currents by expression of CFTR.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (delta F508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR.

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

Acute effects of passive stretching vs vibration on the neuromuscular function of the plantar flexors.

This study examined the acute effects of passive stretching (PS) vs prolonged vibration (VIB) on voluntary peak torque (PT), percent voluntary activation (%VA), peak twitch torque (PTT), passive range of motion (PROM), musculotendinous stiffness (MTS), and surface electromyographic (EMG) and mechanomyographic (MMG) amplitude of the medial gastrocnemius (MG) and soleus (SOL) muscles during isometric maximal voluntary contractions (MVCs) of the plantar flexors. Fifteen healthy men performed the isometric MVCs and PROM assessments before and after 20 min of PS, VIB, and a control (CON) conditions. There were 10% and 5% decreases in voluntary PT, non-significant 3% and 2% decreases in %VA, 9-23% decreases in EMG amplitude of the MG and SOL after the PS and VIB, respectively, with no changes after the CON. PROM increased by 19% and MTS decreased by 38% after the PS, but neither changed after the VIB or CON conditions. Both PS and VIB elicited similar neural deficits (i.e., gamma loop impairment) that may have been responsible for the strength losses. However, mechanical factors related to PROM and MTS cannot be ruled out as contributors to the stretching-induced force deficit.

Efficacy of neutralizing buffered peptone water for recovery of Salmonella, Campylobacter, and Enterobacteriaceae from broiler carcasses at various points along a commercial immersion chilling process with peroxyacetic acid.

In 2016, USDA-Food Safety and Inspection Service began using a neutralizing buffered peptone water (nBPW) to rinse broiler carcasses for Salmonella and Campylobacter performance standard testing. The nBPW contains standard buffered peptone water (BPW) with compounds to neutralize residual antimicrobials that may be transferred from the carcass to the sample rinsate. However, a direct comparison of nBPW and BPW on carcasses commercially treated with antimicrobials has not been conducted. On 3 replicate days in a commercial broiler processing plant, an immersion chilling biomap using whole carcass rinse samples taken prior to any chilling treatment (30), after pre-chill treatment (30), after primary chill (30), after secondary chill (30), after post-chill treatment (50), and after post-chill treatment without the pre-chill treatment (49) were tested. Carcasses were rinsed with either BPW (without neutralizer) or nBPW. Rinsates were sampled for Salmonella and Campylobacter prevalence and both Enterobacteriaceae (EB) prevalence and counts. No significant differences were observed between sampling sites or rinse media for Salmonella due to an overall low prevalence (4 positive/219 samples). Campylobacter prevalence significantly decreased from prior to chilling (93%) to after all chilling steps (47%) as anticipated (P < 0.0001); however, overall significantly fewer Campylobacter positive carcasses were detected when nBPW was used (55%) in comparison to BPW (70%, P = 0.0258). Both EB prevalence and counts significantly decreased (both P < 0.0001) from prior to chilling (100%, 2.35 log10 CFU/mL) through after all chilling steps (52%, 0.47 log10 CFU/mL). The use of nBPW versus BPW did not impact EB prevalence; however, samples rinsed with nBPW had significantly higher overall counts (1.26 vs. 1.00 log10 CFU/mL, P = 0.0134). The results from this study indicate that the use of a PAA pre-chill treatment did not significantly impact bacteria recovery following all chilling steps. The use of nBPW was effective in neutralizing residual PAA in carcass rinsates when sampling for EB counts; however, nBPW may lessen the ability to detect Campylobacter in these same samples.

Combined spinal/caudal catheter anesthesia: extending the boundaries of regional anesthesia for complex pediatric urological surgery.

BACKGROUND: Spinal anesthesia (SA) is an established anesthetic technique for short outpatient pediatric urological cases. To avoid general anesthesia (GA) and expand regional anesthetics to longer and more complex pediatric surgeries, the authors began a program using a combined spinal/caudal catheter (SCC) technique. STUDY DESIGN: The authors retrospectively reviewed the charts of all patients scheduled for surgery under SCC between December 2016 and April 2018 and recorded age, gender, diagnosis, procedure, conversion to GA/airway intervention, operative time, neuraxial and intravenous medications administered, complications, and outcomes. The SCC technique typically involved an initial intrathecal injection of 0.5% isobaric bupivacaine followed by placement of a caudal epidural catheter. At the discretion of the anesthesiologist, patients received 0.5 mg per kilogram of oral midazolam approximately 30 min prior to entering the operating room. One hour after the intrathecal injection, 3% chloroprocaine was administered via the caudal catheter to prolong the duration of surgical block. Intra-operative management included either continuous infusion or bolus dosing of dexmedetomidine, as needed, for patient comfort and to optimize surgical conditions. Prior to removal of caudal catheter in the post-anesthesia care unit, a supplemental bolus dose of local anesthesia was given through the catheter to provide prolonged post-operative analgesia. RESULTS: Overall, 23 children underwent attempted SCC. SA was unsuccessful in three patients, and surgery was performed under GA. The remaining 20 children all had successful SCC placement. There were 11 girls and nine boys, with a mean age of 16.5 months (3.3-43.8). Surgeries performed under SCC included seven ureteral reimplantations, two ureterocele excisions/reimplantations, two megaureter repairs, four first-stage hypospadias repairs, one distal hypospadias repair, one second-stage hypospadias repair, two feminizing genitoplasties, and one open pyeloplasty. Average length of surgery was 109 min (range 63-172 min). Pre-operative midazolam was given in 13/20 (65%). All SCC patients were spontaneously breathing room air during the operation, and there were no airway interventions. Only one SCC patient received opioids intra-operatively. There were no intra-operative or perioperative complications. DISCUSSION: This pilot study shows that the technique of SCC allows one to do more complex urologic surgery under regional anesthesia than what would be possible under pure SA alone. The main limitations of the study include the relatively small number of patients and the small median length of the operative procedures. As a proof of concept, however, this does show that complex genital surgery bladder level procedures such as ureteral reimplantation can be performed under regional anesthesia. CONCLUSION: SCC allows for more complex surgeries to be performed exclusively under regional anesthesia, thus obviating the need for airway intervention, minimizing or eliminating the use of opioids, and thus avoiding known and potential risks associated with GA. The latter is of particular importance given current concerns regarding hypothetical neurocognitive effects of GA on children aged below 3 years.

Cognitive deficits associated with a high-fat diet and insulin resistance are potentiated by overexpression of ecto-nucleotide pyrophosphatase phosphodiesterase-1.

There is growing evidence that over consumption of high-fat foods and insulin resistance may alter hippocampal-dependent cognitive function. To study the individual contributions of diet and peripheral insulin resistance to learning and memory, we used a transgenic mouse line that overexpresses ecto-nucleotide pyrophosphatase phosphodiesterase-1 in adipocytes, which inhibits the insulin receptor. Here, we demonstrate that a model of peripheral insulin resistance exacerbates high-fat diet induced deficits in performance on the Morris Water Maze task. This finding was then reviewed in the context of the greater literature to explore potential mechanisms including triglyceride storage, adiponectin, lipid composition, insulin signaling, oxidative stress, and hippocampal signaling. Together, these findings further our understanding of the complex relationship among peripheral insulin resistance, diet and memory.

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.