Novel Use of Proton Magnetic Resonance Spectroscopy (1HMRS) to Non-Invasively Assess Placental Metabolism

Background

Placental insufficiency is a major cause of antepartum stillbirth and fetal growth restriction (FGR). In affected pregnancies, delivery is expedited when the risks of ongoing pregnancy outweigh those of prematurity. Current tests are unable to assess placental function and determine optimal timing for delivery. An accurate, non-invasive test that clearly defines the failing placenta would address a major unmet clinical need. Proton magnetic resonance spectroscopy (¹H MRS) can be used to assess the metabolic profile of tissue in-vivo. In FGR pregnancies, a reduction in N-acetylaspartate (NAA)/choline ratio and detection of lactate methyl are emerging as biomarkers of impaired neuronal metabolism and fetal hypoxia, respectively. However, fetal brain hypoxia is a late and sometimes fatal event in placental compromise, limiting clinical utility of brain ¹H MRS to prevent stillbirth. We hypothesised that abnormal placental ¹H MRS may be an earlier biomarker of intrauterine hypoxia, affording the opportunity to optimise timing of delivery in at-risk fetuses.

Methods and Findings

We recruited three women with severe placental insufficiency/FGR and three matched controls. Using a 3T MR system and a combination of phased-array coils, a 20×20×40 mm¹H MRS voxel was selected along the ‘long-axis’ of the placenta with saturation bands placed around the voxel to prevent contaminant signals. A significant choline peak (choline/lipid ratio 1.35–1.79) was detected in all healthy placentae. In contrast, in pregnancies complicated by FGR, the choline/lipid ratio was ≤0.02 in all placentae, despite preservation of the lipid peak (p<0.001).

Conclusions

This novel proof-of-concept study suggests that in severe placental insufficiency/FGR, the observed 60-fold reduction in the choline/lipid ratio by ¹H MRS may represent an early biomarker of critical placental insufficiency. Further studies will determine performance of this test and the potential role of 1H-MRS in the in-vivo assessment of placental function to inform timing of delivery.     

Natural selection on marine carnivores elaborated a diverse family of classical MHC class I genes exhibiting haplotypic gene content variation and allelic polymorphism

Pinnipeds, marine carnivores, diverged from terrestrial carnivores ~45 million years ago, before their adaptation to marine environments. This lifestyle change exposed pinnipeds to different microbiota and pathogens, with probable impact on their MHC class I genes. Investigating this question, genomic sequences were determined for 71 MHC class I variants: 27 from harbor seal and 44 from gray seal. These variants form three MHC class I gene lineages, one comprising a pseudogene. The second, a candidate nonclassical MHC class I gene, comprises a nonpolymorphic transcribed gene related to dog DLA-79 and giant panda Aime-1906. The third is the diversity lineage, which includes 62 of the 71 seal MHC class I variants. All are transcribed, and they minimally represent six harbor and 12 gray seal MHC class I genes. Besides species-specific differences in gene number, seal MHC class I haplotypes exhibit gene content variation and allelic polymorphism. Patterns of sequence variation, and of positions for positively selected sites, indicate the diversity lineage genes are the seals’ classical MHC class I genes. Evidence that expansion of diversity lineage genes began before gray and harbor seals diverged is the presence in both species of two distinctive sublineages of diversity lineage genes. Pointing to further expansion following the divergence are the presence of species-specific genes and greater MHC class I diversity in gray seals than harbor seals. The elaboration of a complex variable family of classical MHC class I genes in pinnipeds contrasts with the single, highly polymorphic classical MHC class I gene of dog and giant panda, terrestrial carnivores.     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Characterization of Salmonella type III secretion hyper-activity which results in biofilm-like cell aggregation

We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display.     

Effect of static magnetic fields on the growth,photosynthesis and ultrastructure of Chlorella kessleri microalgae

Microalgal biotechnology could generate substantial amounts of biofuels with minimal environmental impact if the economics can be improved by increasing the rate of biomass production. Chlorella kessleri was grown in a small‐scale raceway pond and in flask cultures with the entire volume, 1% (v/v) at any instant, periodically exposed to static magnetic fields to demonstrate increased biomass production and investigate physiological changes, respectively. The growth rate in flasks was maximal at a field strength of 10 mT, increasing from 0.39 ± 0.06 per day for the control to 0.88 ± 0.06 per day. In the raceway pond the 10 mT field increased the growth rate from 0.24 ± 0.03 to 0.45 ± 0.05 per day, final biomass from 0.88 ± 0.11 to 1.56 ± 0.18 g/L per day, and maximum biomass production from 0.11 ± 0.02 to 0.38 ± 0.04 g/L per day. Increased pigment, protein, Ca, and Zn content made the biomass produced with magnetic stimulation nutritionally superior. An increase in oxidative stress was measured indirectly as a decrease in antioxidant capacity from 26 ± 2 to 17 ± 1 µmol antioxidant/g biomass. Net photosynthetic capacity (NPC) and respiratory rate were increased by factors of 2.1 and 3.1, respectively. Loss of NPC enhancement after the removal of magnetic field fit a first‐order model well (R² = 0.99) with a half‐life of 3.3 days. Transmission electron microscopy showed enlarged chloroplasts and decreased thylakoid order with 10 mT treatment. By increasing daily biomass production about fourfold, 10 mT magnetic field exposure could make algal oil cost competitive with other biodiesel feedstocks. Bioelectromagnetics 33:298–308, 2012. © 2011 Wiley Periodicals, Inc.     

Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. ⁵⁴Mn²⁺ uptake occurred on stimulation but at a lower rate than ⁴⁵Ca²⁺ uptake. There was no delay of ⁵⁴Mn²⁺ uptake upon stimulation and ⁵⁴Mn²⁺ uptake was considerably prolonged compared to ⁴⁵Ca²⁺ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd²⁺>Mn²⁺>Ca²⁺>Sr²⁺.