Noninvasive tracking of gene transcript and neuroprotection after gene therapy.

Gene therapy holds exceptional potential for translational medicine by improving the products of defective genes in diseases and/or providing necessary biologics from endogenous sources during recovery processes. However, validating methods for the delivery, distribution and expression of the exogenous genes from such therapy can generally not be applicable to monitor effects over the long term because they are invasive. We report here that human granulocyte colony-stimulating factor (hG-CSF) complimentary DNA (cDNA) encoded in self-complementary adeno-associated virus-type 2 adeno-associated virus, as delivered through eye drops at multiple time points after cerebral ischemia using bilateral carotid occlusion for 60 min (BCAO-60) led to significant reduction in mortality rates, cerebral atrophy and neurological deficits in C57black6 mice. Most importantly, we validated hG-CSF cDNA expression using translatable magnetic resonance imaging (MRI) in living brains. This noninvasive approach for monitoring exogenous gene expression in the brains has potential for great impact in the area of experimental gene therapy in animal models of heart attack, stroke, Alzheimer's dementia, Parkinson's disorder and amyotrophic lateral sclerosis, and the translation of such techniques to emergency medicine.

Altered Blood Flow in the Ophthalmic and Internal Carotid Arteries in Patients with Age-Related Macular Degeneration Measured Using Noncontrast MR Angiography at 7T.

BACKGROUND AND PURPOSE: Age-related macular degeneration is associated with reduced perfusion of the eye; however, the role of altered blood flow in the upstream ophthalmic or internal carotid arteries is unclear. We used ultra-high-field MR imaging to investigate whether the diameter of and blood flow in the ophthalmic artery and/or the ICA are altered in age-related macular degeneration and whether any blood flow changes are associated with disease progression. MATERIALS AND METHODS: Twenty-four patients with age-related macular degeneration and 13 similarly-aged healthy controls participated. TOF and high-resolution dynamic 2D phase-contrast MRA (0.26 × 0.26 × 2mm3, 100-ms effective sampling rate) was acquired at 7T. Vessel diameters were calculated from cross-sectional areas in phase-contrast acquisitions. Blood flow time-series were measured across the cardiac cycle. RESULTS: The ophthalmic artery vessel diameter was found to be significantly smaller in patients with age-related macular degeneration than in controls. Volumetric flow through the ophthalmic artery was significantly lower in patients with late age-related macular degeneration, with a significant trend of decreasing volumetric ophthalmic artery flow rates with increasing disease severity. The resistance index was significantly greater in patients with age-related macular degeneration than in controls in the ophthalmic artery. Flow velocity through the ophthalmic artery and ICA was significantly higher in patients with age-related macular degeneration. Ophthalmic artery blood flow as a percentage of ipsilateral ICA blood flow was nearly double in controls than in patients with age-related macular degeneration. CONCLUSIONS: These findings support the hypothesis that vascular changes upstream to the eye are associated with the severity of age-related macular degeneration. Additional investigation into the potential causality of this relationship and whether treatments that improve ocular circulation slow disease progression is warranted.

Discovery and Full Genome Characterization of SARS-CoV-2 in Stool Specimen from a Recovered Patient, China.

SARS-CoV-2 was found in a recovered patient's stool specimen by combining quantitative reverse transcription PCR (qRT-PCR) and genome sequencing. The patient was virus positive in stool specimens for at least an additional 15 days after he was recovered, whereas respiratory tract specimens were negative. The discovery of the complete genome of SARS-CoV-2 in the stool sample of the recovered patient demonstrates a cautionary warning that the potential mode of the virus transmission cannot be excluded through the fecal-oral route after viral clearance in the respiratory tract.

Candidate loci for insulin sensitivity and disposition index from a genome-wide association analysis of Hispanic participants in the Insulin Resistance Atherosclerosis (IRAS) Family Study.

AIMS/HYPOTHESIS: The majority of type 2 diabetes genome-wide association studies (GWAS) to date have been performed in European-derived populations and have identified few variants that mediate their effect through insulin resistance. The aim of this study was to evaluate two quantitative, directly assessed measures of insulin resistance, namely insulin sensitivity index (S(I)) and insulin disposition index (DI), in Hispanic-American participants using an agnostic, high-density single nucleotide polymorphism (SNP) scan, and to validate these findings in additional samples. METHODS: A two-stage GWAS was performed in Hispanic-American samples from the Insulin Resistance Atherosclerosis Family Study. In Stage 1, 317,000 SNPs were assessed using 229 DNA samples. SNPs with evidence of association with glucose homeostasis and adiposity traits were then genotyped on the entire set of Hispanic-American samples (n = 1,190). This report focuses on the glucose homeostasis traits: S(I) and DI. RESULTS: Although evidence of association did not reach genome-wide significance (p = 5 x 10(-7)), in the combined analysis SNPs had admixture-adjusted p values of p (ADD) = 0.00010-0.0020 with 8 to 41% differences in genotypic means for S(I) and DI. CONCLUSIONS/INTERPRETATION: Several candidate loci were identified that are nominally associated with S(I) and/or DI in Hispanic-American participants. Replication of these findings in independent cohorts and additional focused analysis of these loci is warranted.

Prevalence of Cronobacter spp. and Salmonella in Milk Powder Manufacturing Facilities in the United States.

ABSTRACT: The U.S. Food and Drug Administration (FDA) conducted a sampling assignment in 2014 to ascertain the prevalence of Cronobacter spp. and Salmonella in the processing environment of facilities manufacturing milk powder. Cronobacter was detected in the environment of 38 (69%) of 55 facilities. The average prevalence of Cronobacter in 5,671 subsamples (i.e., swabs and sponges from different facility locations) was 4.4%. In the 38 facilities where Cronobacter was detected, the average prevalence of positive environmental subsamples was 6.25%. In 20 facilities where zone information of the sampling location was complete, Cronobacter was most frequently detected in zone 4, followed by zone 3, then zone 2, with zone 1 yielding the lowest percentage of positive samples. The prevalence of Cronobacter across the zones was statistically different (P < 0.05). There was no significant association between product type (i.e., lactose, whey products, buttermilk powder, and nonfat dried milk) and prevalence of Cronobacter in the facility. Salmonella was detected in the environment of three (5.5%) of the 55 facilities; all three facilities produced dried whey product. The overall prevalence of Salmonella in 5,714 subsamples was 0.16%. In facilities in which Salmonella was detected, the average prevalence was 2.5%. Salmonella was most frequently detected in zone 4, followed by zone 3. Salmonella was not detected in zone 1 or zone 2. The disparity between Salmonella and Cronobacter prevalence indicates that additional measures may be required to reduce or eliminate Cronobacter from the processing environment.

Insulin resistance, beta cell dysfunction and visceral adiposity as predictors of incident diabetes: the Insulin Resistance Atherosclerosis Study (IRAS) Family study.

AIMS/HYPOTHESIS: Central obesity, insulin resistance and beta cell dysfunction are independent risk factors for incident type 2 diabetes, although few studies have used detailed measures of these disorders. Our objective was to study the association of directly measured visceral and subcutaneous adipose tissue (VAT, SAT), insulin sensitivity (S (I)) and the acute insulin response (AIR) with incident type 2 diabetes. METHODS: Participants were 1,230 Hispanic-Americans and African-Americans in the Insulin Resistance Atherosclerosis Study (IRAS) Family Study who were free of type 2 diabetes at baseline (2000-2002). S (I) and AIR were determined from frequently sampled IVGTTs with minimal model analysis. VAT and SAT were determined by computed tomography. Impaired fasting glucose and type 2 diabetes were defined according to American Diabetes Association criteria. RESULTS: Incident type 2 diabetes was diagnosed in 90 participants after 5 years. After adjustment for age, sex, ethnicity, centre, impaired fasting glucose, triacylglycerol, HDL-cholesterol and systolic BP, both S(I) and AIR were inversely associated with type 2 diabetes (S (I), OR 0.53, 95% CI 0.39-0.73; AIR, OR 0.22, 95% CI 0.14-0.34 per SD; both p < 0.001), while both VAT and SAT were positively associated with type 2 diabetes (VAT, OR 1.68, 95% CI 1.22-2.33; SAT, OR 1.49, 95% CI 1.13-1.99; both p < 0.01). In a model including all four factors, S (I) and AIR (S (I), OR 0.55, 95% CI 0.37-0.80; AIR, OR 0.21, 95% CI 0.13-0.33; both p < 0.01) were significant predictors of type 2 diabetes, although associations with VAT and SAT were no longer significant. A significant sex x VAT interaction indicated a stronger association of VAT with type 2 diabetes in women than in men. CONCLUSIONS/INTERPRETATION: Insulin resistance, beta cell dysfunction and VAT predicted incident type 2 diabetes, with evidence of a stronger association of VAT with type 2 diabetes among women.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), a candidate gene for polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogenic, complex common genetic disease. Multiple pathways are involved in its pathogenesis, including the androgen signaling pathway and insulin signaling pathway. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) is a putative member of the androgen receptor-chaperone-co-chaperone complex, and may play a role in androgen signaling as a co-chaperone. Polymorphisms in the SGTA gene have not been evaluated for a role in PCOS. METHODS: Women with and without PCOS (287 cases, 187 controls) were genotyped for three single nucleotide polymorphisms (SNPs) in SGTA. SNPs and haplotypes were determined and tested for association with PCOS and component traits of PCOS. RESULTS: For SNP rs1640262, homozygotes for the minor allele were protected against PCOS (P = 0.009). Haplotype 1 (G-A-T) was associated with increased risk of PCOS (P = 0.015). In women with PCOS, haplotype 2 (A-G-C) was associated with increased insulin resistance (P = 0.013), consequently resulting in increased insulin secretion (P = 0.014). CONCLUSIONS: This study presents genetic evidence suggesting a potential role of SGTA in the pathogenesis of PCOS. SGTA may provide a connection between multiple pathways in PCOS.

Prevalence and Level of Listeria monocytogenes in Ice Cream Linked to a Listeriosis Outbreak in the United States.

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

Postprandial triglyceride and retinyl ester responses to oral fat: effects of fructose.

It has been shown that addition of fructose to an oral fat load results in higher postprandial concentrations of triglyceride. The present study, performed in 11 healthy volunteers, was initiated to see whether the effect of fructose on fat-induced lipemia also involved changes in postprandial concentrations of triglyceride-rich lipoproteins of intestinal origin. Vitamin A was used to label intestinal lipoproteins, and the retinyl palmitate concentrations were determined in plasma and in the Sf > 400 and Sf 20-400 lipoprotein fractions (Sf denotes the Svedberg flotation index). Addition of fructose (50 g) to a standard (40-g oral) fat load resulted in higher postprandial concentrations of triglyceride and retinyl palmitate in plasma and the Sf > 400 lipoprotein fraction (P < 0.001, analysis of variance), and the higher the fasting plasma triglyceride concentration, the greater the magnitude of the fructose effect (r = 0.83, P < 0.002). These data show that triglyceride-rich lipoproteins of intestinal origin play a role in the fructose-induced accentuation of postprandial lipemia.

Striatal volume loss in HD as measured by MRI and the influence of CAG repeat.

BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative disease that results from the expansion of a trinucleotide (CAG) repeat on chromosome 4. Progressive degeneration of the striatum is the pathologic hallmark of the disease. Little is known about the regional selectivity of the neurodegeneration and its relationship to the genetic expansion. METHODS: The authors used high-resolution MRI to determine the relationship between the genetic expansion and the degree of striatal degeneration. Morphometric analyses of the striatum from high-resolution MR images from 27 subjects with HD were compared with those of 24 healthy control subjects. RESULTS AND CONCLUSIONS: Striatal volumes were reduced in subjects with HD as compared with control subjects, in agreement with previously published reports. Left-sided volumes were smaller than right-sided volumes in subjects with HD; in healthy subjects, right-sided volumes were smaller. Finally, volume loss was significantly correlated with CAG repeat number. These results have potential implications for the design and assessment of therapeutic agents in the future.

Insulin secretion and action in noninsulin-dependent diabetes mellitus. Is insulin resistance secondary to hypoinsulinemia?

The insulin-stimulated glucose metabolic clearance rate, assessed by the insulin clamp technique, was compared in 40 normal subjects and 40 age- and weight-matched patients with noninsulin-dependent diabetes mellitus. These studies were conducted at steady-state plasma insulin levels of approximately 100 microU/ml, and the mean (+/- standard error of the mean) glucose metabolic clearance rate of patients with noninsulin-dependent diabetes mellitus was 81 +/- 9 ml/m2 per minute, as compared with a value of 235 +/- 14 ml/m2 per minute for normal subjects. This difference was highly statistically significant (p less than 0.001) and documents the extreme resistance to insulin-stimulated glucose utilization seen in noninsulin-dependent diabetes mellitus. Patients with noninsulin-dependent diabetes mellitus were also shown to have a lower than normal plasma insulin response to an oral glucose challenge. In contrast, ambient plasma insulin concentrations of normal subjects and patients with noninsulin-dependent diabetes mellitus were found to be quite comparable when measured throughout the day in response to the ingestion of conventional mixed meals. Consequently, absolute hypoinsulinemia is not characteristic of patients with noninsulin-dependent diabetes mellitus under conditions of daily living. Finally, the ability of intensive insulin treatment to improve insulin resistance was studied after one and six weeks of therapy. These results indicated that successful control of hyperglycemia led to a significant improvement in insulin action as early as one week after the initiation of insulin therapy, with no further changes noted after prolonged insulin administration. The degree to which insulin action approached normal values was greater when studies were carried out at circulating insulin levels of approximately 2,000 microU/ml as compared with insulin levels of approximately 100 microU/ml, but in both instances insulin-treated diabetic patients remained insulin-resistant as compared with normal subjects. These results have corroborated the fact that abnormalities of both insulin action and secretion can be documented in patients with noninsulin-dependent diabetes mellitus. However, patients with noninsulin-dependent diabetes mellitus were not found to be absolutely hypoinsulinemic in their daily existence, and control of their hyperglycemia with exogenous insulin did not restore insulin-stimulated glucose utilization to normal. Consequently, these data are not consistent with the view that the insulin resistance in noninsulin-dependent diabetes mellitus is entirely a secondary consequence of the hypoinsulinemia presumed to be present in these patients.

Resistance to insulin-mediated glucose uptake and hyperinsulinemia in women who had preeclampsia during pregnancy.

Plasma glucose and insulin responses to a 75-g oral glucose load, and the steady-state plasma insulin (SSPI) and glucose (SSPG) concentrations after an infusion of somatostatin, insulin, and glucose, were determined 2 months after delivery in 26 women; 13 who had a normal pregnancy and 13 who developed preeclampsia. The plasma glucose response to oral glucose was not different in the two groups, but the plasma insulin response was significantly greater (P < .02) in those who had been preeclamptic. Although the mean (+/- SE) SSPI concentrations during the infusion study were similar in the two groups (51 +/- 2 v 56 +/- 2 microU/mL), the SSPG concentrations were significantly higher (P < .02) in those who developed preeclampsia (160 +/- 17 v 119 +/- 17 mg/dL). Thus, when studied 2 months after delivery, women who developed preeclampsia were relatively insulin resistant and hyperinsulinemic when compared to those who had an uncomplicated pregnancy.

Lack of evidence for an association between alpha-adducin and blood pressure regulation in Asian populations.

Recent studies have found the tryptophan allele of a glycine to tryptophan polymorphism at position 460 (G460W) of the alpha-adducin protein to be associated with essential hypertension in European populations. We examined whether the tryptophan allele is associated with hypertension in a different population, comprised of subjects of Chinese origin from Taiwan, and Chinese and Japanese origin from the San Francisco Bay area and Hawaii. We adapted the 5' allelic discrimination assay or TaqMan to type individuals for the G460W polymorphism, and using this method we typed more than 1000 individuals. The frequency of the W allele was slightly increased in the treated subjects in the Chinese population (0.458 v 0.423) but not the Japanese population (0.549 v 0.558). We considered dominant, recessive, and additive models in our analysis. There was a significant result for a recessive model for systolic blood pressure in the Chinese population (chi2 6.84, df = 2, P < .05), but only suggestive evidence for diastolic blood pressure (chi2 3.30). In contrast, in the Japanese population, there was no evidence for a positive association under any model. For the combined Chinese and Japanese samples, the evidence for association with alpha-adducin was not significant.

An integrated strategy for evaluation of metabolic and oxidative defects in neurodegenerative illness using magnetic resonance techniques.

The number of physiologic and metabolic phenomena amenable to analysis using magnetic resonance (MR) techniques is increasing every year. MR techniques can now evaluate tissue parameters relevant to TCA cyclemetabolism, anerobic glycolysis, ATP levels, blood-brain barrier permeability, macrophage infiltration, cytotoxic edema, spreading depression, cerebral blood flow and volume, and neurotransmitter function. The paramagnetic nature of certain oxidation states of iron leads to the ability to map out brain function using deoxyhemoglobin as an endogenous contrast agent, and also allows for mapping of local tissue iron concentrations. In addition to these metabolic parameters, the number of ways to generate anatomic contrast using MR is also expanding; and in addition to conventional anatomic scans, mapping of axonal fiber tracts can also be performed using the anisotropy of water diffusion. A strategy for integration of these multifarious parameters in a comprehensive neurofunctional exam in neurodegenerative illness is outlined in this paper. The goals of the integrated exam, as applied to a given neurodegenerative illness, can be subdivided into three categories: etiology, natural history, and therapeutic end points. The consequences of oxidative stress and/or mitochondrial dysfunction are explored in the context of the various parameters that can be measured using the integrated MR exam.

Ability of exercise to inhibit carbohydrate-induced hypertriglyceridemia in rats.

The ability of spontaneous running to prevent carbohydrate-induced hypertriglyceridemia was studied in young, nonobese rats. Exercise-trained and sedentary rats were fed a diet consisting of (as percent total calories) 12% fat, 22% protein, and 66% carbohydrate. The source of the carbohydrate was varied, and experiments were carried out with sucrose and glucose as the sole dietary carbohydrate. Plasma triglyceride (TG) levels rose in response to both forms of dietary carbohydrate in both sedentary and exercise-trained rats, but the magnitude of the elevation was greatly attenuated in the exercise-trained group. Plasma insulin concentrations were also significantly lower in exercise-trained rats. Measurements of hepatic very low density lipoprotein (VLDL)-TG secretion rate and adipose tissue lipoprotein lipase (LPL) activity were made in an effort to determine how exercise-training prevented the development of carbohydrate-induced hypertriglyceridemia. The results of these studies indicated that perfused livers of exercise-trained rats secreted significantly less VLDL-TG, whereas adipose tissue LPL activity of the two groups was similar. On the basis of these results, it is postulated that the ability of exercise-training to inhibit carbohydrate-induced hypertriglyceridemia is due to an increase in insulin sensitivity resulting from chronic exercise. As a result, the postprandial insulin responses to high carbohydrate diets would be relatively reduced in exercise-trained rats, leading to decreased hepatic VLD-TG secretion, and lower plasma triglyceride concentrations.

Detection of dopaminergic cell loss and neural transplantation using pharmacological MRI, PET and behavioral assessment.

We demonstrate the use of magnetic resonance imaging (MRI) for detection of neurotransmitter stimulation using the dopamine transporter ligands amphetamine and CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) as pharmacological challenges. We demonstrate that the unilateral loss of a hemodynamic response to either amphetamine or CFT challenge by unilateral 6-hydroxydopamine lesioning is restored by transplantation of fetal dopamine neurons in the striatum. The time course for the hemodynamic changes parallels the time courses for dopamine release, measured by prior microdialysis studies, and also for the rotational behavior in the unilaterally lesioned animals. Transplantation of the fetal cells results in hemodynamic time courses after CFT or amphetamine challenges at the graft site that are identical to those induced both before transplantation and on the intact contralateral side. The transplantation also results in complete behavioral recovery. The spatial extent of the dopaminergic recovery in the lesioned striatum is the same when measured using either PET of tracer levels of [11C]CFT binding or MRI. These results show great promise for the application of pharmacological MRI for application to studies of dopamine cell loss and potential recovery in Parkinson's disease.

Laterality, somatotopy and reproducibility of the basal ganglia and motor cortex during motor tasks.

We investigated the basal ganglia, motor cortex area 4, and supplementary motor area (SMA) using functional magnetic resonance imaging (fMRI) and five motor tasks: switching between finger and toe movements, writing, finger tapping, pronation/supination, and saccadic eye movements. We found reliable activation in the caudate nucleus and putamen in single subjects without the need for inter-subject averaging. Percent signal changes in basal ganglia were smaller by a factor of three than those in SMA or motor cortex (1% vs. 2.5-3%). There was a definite foot-dorsal, hand-ventral basal ganglia somatotopy, similar to prior data from primates. Saccadic eye movements activated the caudate nucleus significantly more than the other tasks did. Unilateral movements produced bilateral activation in the striatum even when motor cortex activation was unilateral. Surprisingly, bilateral performance of the tasks led, on average, to consistently smaller basal ganglia activation than did unilateral performance (P<0.001), suggesting less inhibition of contralateral movements during bilateral tasks. Moreover, there was a striking dominance pattern in basal ganglia motor activation: the left basal ganglia were more active than the right for right handers, regardless of the hand used. This lateralization appears much stronger than that previously reported for motor cortex. Comparisons of inter-subject and intra-subject reproducibility indicated a much larger variability in basal ganglia and SMA compared to motor cortex, in spite of similar percent signal changes in the latter two structures.

Molecular markers for identification of the hyperparasitoids Dendrocerus carpenteri and Alloxysta xanthopsis in Lysiphlebus testaceipes parasitizing cereal aphids.

Polymerase chain reaction (PCR)-based molecular markers have been developed to detect the presence of primary parasitoids in cereal aphids and used to estimate primary parasitism rates. However, the presence of secondary parasitoids (hyperparasitoids) may lead to underestimates of primary parasitism rates based on PCR markers. This is because even though they kill the primary parasitoid, it's DNA can still be amplified, leading to an erroneous interpretation of a positive result. Another issue with secondary parasitoids is that adults are extremely difficult to identify using morphological characters. Therefore, we developed species-specific molecular markers to detect hyperparasitoids. A 16S ribosomal RNA mitochondrial gene fragment was amplified by PCR and sequenced from two secondary parasitoid species, Dendrocerus carpenteri (Curtis) (Hymenoptera: Megaspilidae) and Alloxysta xanthopsis (Ashmead) (Hymenoptera: Charipidae), four geographic isolates of the primary parasitoid, Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae), and six aphid species common to cereal crops. Species-specific PCR primers were designed for each insect on the basis of these 16S rRNA gene sequences. Amplification of template DNA, followed by agarose gel electrophoresis, successfully distinguished D. carpenteri and A. xanthopsis from all four isolates of L. testaceipes and all six cereal aphid species in this laboratory test.