Hepatic glucose metabolism during intraduodenal glucose infusion: impact of infection

We previously reported that infection decreases hepatic glucose uptake when glucose is given as a constant peripheral glucose infusion (8 mg. kg(-1) x min(-1)). This impairment persisted despite greater hyperinsulinemia in the infected group. In a normal setting, hepatic glucose uptake can be further enhanced if glucose is given gastrointestinally. Thus the aim of this study was to determine whether hepatic glucose uptake is impaired during an infection when glucose is given gastrointestinally. Thirty-six hours before study, a sham (SH, n = 7) or Escherichia coli-containing (2 x 10(9) organisms/kg; INF; n = 7) fibrin clot was placed in the peritoneal cavity of chronically catheterized dogs. After the 36 h, a glucose bolus (150 mg/kg) followed by a continuous infusion (8 mg. kg(-1). min(-1)) of glucose was given intraduodenally to conscious dogs for 240 min. Tracer ([3-(3)H]glucose and [U-(14)C]glucose) and arterial-venous difference techniques were used to assess hepatic and intestinal glucose metabolism. Infection increased hepatic blood flow (35 +/- 5 vs. 47+/-3 ml x g(-1) x min(-1); SH vs. INF) and basal glucose rate of appearance (2.1+/-0.2 vs. 3.3+/-0.1 mg x kg(-1) x min(-1)). Arterial insulin concentrations increased similarly in SH and INF during the last hour of glucose infusion (38+/-8 vs. 46+/-20 microU/ml), and arterial glucagon concentrations fell (62+/-14 to 30+/-3 vs. 624+/-191 to 208+/-97 pg/ml). Net intestinal glucose absorption was decreased in INF, attenuating the increase in blood glucose caused by the glucose load. Despite this, net hepatic glucose uptake (1.6+/-0.8 vs. 2.4+/- 0.9 mg x kg(-1) x min(-1); SH vs. INF) and consequently tracer-determined glycogen synthesis (1.3+/-0.3 vs. 1.0+/-0.3 mg. kg(-1) x min(-1)) were similar between groups. In summary, infection impairs net glucose absorption, but not net hepatic glucose uptake or glycogen deposition, when glucose is given intraduodenally.     

BioDry: An Inexpensive,Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry “field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.     

Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.     

Proteome-wide evidence for enhanced positive Darwinian selection within intrinsically disordered regions in proteins

Background  

Understanding the adaptive changes that alter the function of proteins during evolution is an important question for biology and medicine. The increasing number of completely sequenced genomes from closely related organisms, as well as individuals within species, facilitates systematic detection of recent selection events by means of comparative genomics.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

The flying buttress construct for posterior spinopelvic fixation: a technical note

Background

School screening for adolescent idiopathic scoliosis (AIS) is discussed. The aim of the present study was to describe the point prevalence of AIS and to evaluate the effectiveness of school screening in 12-year- old children.

Methods

Community nurses and physical therapists in the Southern Health region of Norway including about 12000 school children aged 12 years were invited to participate. All participating community nurses and physical therapists fulfilled an educational course to improve their knowledge about AIS and learn the screening procedure including the Adam Forward Bending Test and measurement of gibbus using a scoliometer.

Results

Sub-regions including 4000 school children participated. The prevalence of idiopathic scoliosis defined as a positive Adam Forward Bending Test, gibbus > 7° and primary major curve on radiographs > 10°, was 0.55%. Five children (0.13%) had a major curve > 20°. Bracing was not indicated in any child; all children were post menarche; four had Risser sign of 4, and one with Risser 1 did not have curve progression > 5° at later follow-up. In one of these 5 children however, the major curve progressed to 45° within 7 months after screening and the girl was operated.

Conclusion

The point prevalence of AIS in 12- year old children is in agreement or slightly lower than previous studies. The screening model employed demonstrates acceptable sensitivity and specificity and low referral rates. Screening at the age of 12 years only was not effective for detecting patients with indication for brace treatment.     

Route-dependent effect of nutritional support on liver glucose uptake

The liver is a major site of glucose disposal during chronic (5 day) total parenteral (TPN) and enteral (TEN) nutrition. Net hepatic glucose uptake (NHGU) is dependent on the route of delivery when only glucose is delivered acutely; however, the hepatic response to chronic TPN and TEN is very similar. We aimed to determine whether the route of nutrient delivery altered the acute (first 8 h) response of the liver and whether chronic enteral delivery of glucose alone could augment the adaptive response to TPN. Chronically catheterized conscious dogs received either TPN or TEN containing glucose, Intralipid, and Travasol for either 8 h or 5 days. Another group received TPN for 5 days, but approximately 50% of the glucose in the nutrition was given via the enteral route (TPN+EG). Hepatic metabolism was assessed with tracer and arteriovenous difference techniques. In the presence of similar arterial plasma glucose levels (approximately 6 mM), NHGU and net hepatic lactate release increased approximately twofold between 8 h and 5 days in TPN and TEN. NHGU (26 +/- 1 vs. 23 +/- 3 micromol.kg(-1).min(-1)) and net hepatic lactate release (44 +/- 1 vs. 34 +/- 6 micromol.kg(-1).min(-1)) in TPN+EG were similar to results for TPN, despite lower insulin levels (96 +/- 6 vs. 58 +/- 16 pM, TPN vs. TPN+EG). TEN does not acutely enhance NHGU or disposition above that seen with TPN. However, partial delivery of enteral glucose is effective in decreasing the insulin requirement during chronic TPN.     

Cardiac-specific blockade of NF-kappaB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo

The role of NF-kappaB in cardiac physiology and pathophysiology has been difficult to delineate due to the inability to specifically block NF-kappaB signaling in the heart. Cardiac-specific transgenic models have recently been developed that repress NF-kappaB activation by preventing phosphorylation at specific serine residues of the inhibitory kappaB (IkappaB) protein isoform IkappaBalpha. However, these models are unable to completely block NF-kappaB because of a second signaling pathway that regulates NF-kappaB function via Tyr42 phosphorylation of IkappaBalpha. We report the development of transgenic (3M) mouse lines that express the mutant IkappaBalpha(S32A,S36A,Y42F) in a cardiac-specific manner. NF-kappaB activation in cardiomyopathic TNF-1.6 mice is completely blocked by the 3M transgene but only partially blocked (70-80%) by the previously described double-mutant 2M [IkappaBalpha(S32A,S36A)] transgene, which demonstrates the action of two proximal pathways for NF-kappaB activation in TNF-alpha-induced cardiomyopathy. In contrast, after acute stimuli including administration of TNF-alpha and ischemia-reperfusion (I/R), NF-kappaB activation is blocked in both 2M and 3M transgenic mice. This result suggests that phosphorylation of the regulatory Ser32 and Ser36 predominantly mediates NF-kappaB activation in these situations. We show that infarct size after I/R is reduced by 70% in 3M transgenic mice, which conclusively demonstrates that NF-kappaB is involved in I/R injury. In summary, we have engineered novel transgenic mice that allow us to distinguish two major proximal pathways for NF-kappaB activation. Our results demonstrate that the serine and tyrosine phosphorylation pathways are differentially activated during different pathophysiological processes (cardiomyopathy and I/R injury) and that NF-kappaB contributes to infarct development after I/R.     

Impact of continuous and pulsatile insulin delivery on net hepatic glucose uptake

The pancreas releases insulin in a pulsatile manner; however, studies assessing the liver's response to insulin have used constant infusion rates. Our aims were to determine whether the secretion pattern of insulin [continuous (CON) vs. pulsatile] in the presence of hyperglycemia 1) influences net hepatic glucose uptake (NHGU) and 2) entrains NHGU. Chronically catheterized conscious dogs fasted for 42 h received infusions including peripheral somatostatin, portal insulin (0.25 mU x kg(-1) x min(-1)), peripheral glucagon (0.9 ng x kg(-1) x min(-1)), and peripheral glucose at a rate double the glucose load to the liver. After the basal period, insulin was infused for 210 min at either four times the basal rate (1 mU x kg(-1) x min(-1)) or an identical amount in pulses of 1 and 4 min duration, followed by intervals of 11 and 8 min (CON, 1/11, and 4/8, respectively) in which insulin was not infused. A variable peripheral glucose infusion containing [3H]glucose clamped glucose levels at twice the basal level ( approximately 200 mg/dl) throughout each study. Hepatic metabolism was assessed by combining tracer and arteriovenous difference techniques. Arterial plasma insulin (microU/ml) either increased from basal levels of 6 +/- 1 to a constant level of 22 +/- 4 in CON or oscillated from 5 +/- 1 to 416 +/- 79 and from 6 +/- 1 to 123 +/- 43 in 1/11 and 4/8, respectively. NHGU (-0.8 +/- 0.3, 0.4 +/- 0.2, and -0.9 +/- 0.4 mg x kg(-1) x min(-1)) and net hepatic fractional extraction of glucose (0.04 +/- 0.01, 0.04 +/- 0.01, and 0.05 +/- 0.01 mg x kg(-1) x min(-1)) were similar during the experimental period. Spectral analysis was performed to assess whether a correlation existed between the insulin secretion pattern and NHGU. NHGU was not augmented by pulsatile insulin delivery, and there is no evidence of entrainment in hepatic glucose metabolism. Thus the loss of insulin pulsatility per se likely has little or no impact on the effectiveness of insulin in regulating liver glucose uptake.