Effect of peak pressure and pressure gradient on subsurface shear stresses in the neuropathic foot

The pressure distribution on the plantar surface of the foot may provide insights into the stresses within the subsurface tissues of patients with diabetes mellitus and peripheral neuropathy (PN) who are at risk for skin breakdown. The purposes of this study were to (1) estimate the stress distribution in the subsurface soft tissue from a measured surface pressure distribution and determine any differences between values in the forefoot and rearfoot, and (2) determine the relationship between maximum shear stress (MSS) (magnitude and depth) and characteristics of the pressure distribution. The measured in-shoe pressure distributions during walking characterized by the peak plantar pressure and maximum pressure gradient on the plantar surface of the feet for 20 subjects with diabetes, PN and history of a mid foot or forefoot plantar ulcer were analyzed. The effects of peak pressure and maximum pressure gradient at the peak pressure location on the stress components in the subsurface soft tissue were studied using a potential function method to estimate the subsurface tissue stress. The calculated MSSs are larger in magnitude and located closer to the surface in the forefoot, where most skin breakdown occurs, compared to the rearfoot. In addition, the MSS (magnitude and depth) is highly correlated with the pressure gradient (r=-0.77 & 0.61) and the peak pressure (r=-0.61 & 0.91). The peak pressure and the maximum pressure gradient obtained from the surface pressure distribution appear to be important variables to identify where MSSs are located in the subsurface tissues on the plantar foot that may lead to skin break down.     

Effects of an oral glucose tolerance test on the myogenic response in healthy individuals

The myogenic response, the inherent ability of blood vessels to rapidly respond to changes in transmural pressure, is involved in local blood flow autoregulation. Animal studies suggest that both acute hyperglycemia and hyperinsulinemia may impair myogenic vasoconstriction. The purpose of this study was to examine the effects of an oral glucose load on brachial mean blood velocity (MBV) during increases in forearm transmural pressure in humans. Eight healthy men and women (38 +/- 5 yr) underwent an oral glucose tolerance test (OGTT). MBV (in cm/s; Doppler ultrasound) responses to a rise in forearm transmural pressure (arm tank suction, -50 mmHg) were studied before and every 30 min for 120 min during the OGTT. Before the start of the OGTT, MBV was lower than baseline values 30 and 60 s after the application of negative pressure. This suggests that myogenic constriction was present. During the OGTT, blood glucose rose from 88 +/- 2 to 120 +/- 6 mg/dl (P < 0.05) and insulin rose from 14 +/- 1 to 101 +/- 32 microU/ml (P < 0.05). Glucose loading attenuated the reduction in MBV with arm suction (Delta-0.73 +/- 0.14 vs. Delta-1.67 +/- 0.43 cm/s and Delta-1.07 +/- 0.14 vs. Delta-2.38 +/- 0.54 cm/s, respectively, during 30 and 60 s of suction postglucose compared with preglucose values; all P < 0.05). We observed no such time effect for myogenic responses during a sham OGTT. In an additional 5 subjects, glucose loading had no effect on brachial diameters with the application of negative pressure. Oral glucose loading leads to attenuated myogenic vasoconstriction in healthy individuals. The role that this diminished postglucose reactivity plays in mediating postprandial hypotension and/or orthostasis needs to be further explored.     

The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3′-5′ turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.     

Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis

MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.     

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

Background

Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.

Methods

Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2^d) prior to transplanting into C57BL/6 mice (H-2^b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).

Results

Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.

Conclusion

Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.     

Development of a MLST-biased SNP microarray for Candida albicans

We have developed a single nucleotide polymorphism (SNP) detection microarray for the human pathogenic yeast, Candida albicans, consisting of synthetic oligonucleotides bound to microscope slides. The array consists of multiple replicates of 79 SNPs, derived from 19 discrete loci located on all eight chromosomes. These loci include seven genes consisting of 57 SNPs that comprise a multi-locus sequence typing (MLST) consensus scheme for the species. The remaining 22 SNPs are from 12 additional loci located at intervals on the remaining chromosomes. In order to include highly informative polymorphisms from the MLST set on the array we performed a linkage analysis of major genotypes between the two pairs of MLST-linked genes. In addition, we performed a matched-set analysis for each SNP located within individual MLST loci. This analysis resulted in the reduction of informatively redundant mutations in the array for a large percentage of strains. We believe that a SNP array will be helpful in extending our knowledge of the epidemiology and genetics of C. albicans as a supplement to MLST typing.     

Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.

PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum. We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme. When expressed intracellularly, the enzyme was produced in a largely insoluble state. However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E. coli periplasm. Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates.     

Common BMI and diabetes-related genetic variants: A pilot study among indigenous people in the Brazilian Amazon

This study was carried out to investigate the frequency of genetic variants related to body mass index (BMI) and type 2 diabetes (T2D) and evaluating the potential impact of risk alleles on susceptibility to these disorders in six indigenous peoples from Brazilian Amazon region. The majority of Fst values for pairwise population comparisons among the indigenous groups are low or moderate. The indigenous people show high values of differentiation with Africans, Europeans and Southeast Asians and moderate values with East Asian and American populations, as expected. The allelic frequencies among indigenous indicate that the majority of associations observed with T2D in continental populations can be replicated in native Amazonians. The genetic risk scores calculated for T2D in indigenous are high and similar to those calculated for Americans and East Asians, while the estimates obtained for obesity are low, probably due to the low frequencies of the risk allele of the FTO gene found in our samples. ADRB3-rs4994 and ABCC8-rs1799854 genes showed a significant association with BMI and waist circumference, and the KCNJ11-rs5219 gene with hyperglycemia. These results emphasize the importance of knowing the genetic variability underlying complex genetic diseases in indigenous peoples and the search for particular or rare variants. Keywords: T2D, BMI, SNP, Amerindians, Brazilian     

The Mitochondrial Genome and Human Mitochondrial Diseases

To date, more than 100 point mutations and several hundreds of structural rearrangements of mitochondrial DNA (mtDNA) are known too be connected with characteristic neuromuscular and other mitochondrial syndromes varying form those causing death at the neonatal stage to diseases with late ages of onset. The immediate cause of mitochondrial disorders is a defective oxidative phosphorylation. Wide phenotypic variation and the heteroplasmy phenomenon, which some authors include in mutation load, are characteristic of human mitochondrial diseases. As the numbers of cases identified and pedigrees described increase, data on the genotype–phenotype interaction and the structure and frequency of pathogenic and conditionally pathogenic mtDNA mutations in human populations are rapidly accumulated. The data on the genetics and epidemiology of mitochondrial diseases are not only important for differential diagnosis and genetic counseling. Since both neutral and mildly pathogenic mutations of mtDNA are progressively accumulated in maternal phyletic lines, molecular analysis of these mutations permits not only reconstruction of the genealogical tree of modern humans, but also estimation of the role that these mutations play in natural selection.     

Interior Least Tern (Sternula antillarum) breeding distribution and ecology: implications for population‐level studies and the evaluation of alternative management strategies on large,regulated rivers

Interior Least Terns (Sternula antillarum) (ILT) are colonial, fish‐eating birds that breed within active channels of large sand bed rivers of the Great Plains and in the Lower Mississippi Valley. Multipurpose dams, irrigation structures, and engineered navigation systems have been present on these rivers for many decades. Despite severe alteration of channels and flow regimes, regulation era floods have remained effective at maintaining bare sandbar nesting habitat on many river segments and ILT populations have been stable or expanding since they were listed as endangered in 1985. We used ILT breeding colony locations from 2002 to 2012 and dispersal information to identify 16 populations and 48 subpopulations. More than 90% of ILT and >83% of river km with suitable nesting habitat occur within the two largest populations. However, replicate populations remain throughout the entire historical, geophysical, and ecological range of ILT. Rapid colonization of anthropogenic habitats in areas that were not historically occupied suggests metapopulation dynamics. The highest likelihood of demographic connectivity among ILT populations occurs across the Southern Plains and the Lower Mississippi River, which may be demographically connected with Least Tern populations on the Gulf Coast. Paired ecological and bird population models are needed to test whether previously articulated threats limit ILT population growth and to determine if management intervention is necessary and where. Given current knowledge, the largest sources of model uncertainty will be: (1) uncertainty in relationships between high flow events and subsequent sandbar characteristics and (2) uncertainty regarding the frequency of dispersal among population subunits. We recommend research strategies to reduce these uncertainties.