Discovery of benzylisothioureas as potent divalent metal transporter 1 (DMT1) inhibitors

Inhibition of intestinal brush border DMT1 offers a novel therapeutic approach to the prevention and treatment of disorders of iron overload. Several series of diaryl and tricyclic benzylisothiourea compounds as novel and potent DMT1 inhibitors were discovered from the original hit compound 1. These compounds demonstrated in vitro potency against DMT1, desirable cell permeability properties and a dose-dependent inhibition of iron uptake in an acute rat model of iron hyperabsorption. Tricyclic compounds increased the in vitro potency by up to 16-fold versus the original hit. Diaryl compounds 6b and 14a demonstrated significant iron absorption inhibition in vivo with both 25 and 50 mg/kg doses. The diaryl and tricyclic compounds described in this report represent promising structural templates for further optimization.     

Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens

Tumor-associated carbohydrate antigens (TACAs) are being actively studied as targets for antitumor vaccine development. One serious challenge was the low immunogenecity of these antigens. Herein, we report the results of using the tobacco mosaic virus (TMV) capsid as a promising carrier of a weakly immunogenic TACA, the monomeric Tn antigen. The copper(I) catalyzed azide-alkyne cycloaddition reaction was highly efficient in covalently linking Tn onto the TMV capsid without resorting to a large excess of the Tn antigen. The location of Tn attachment turned out to be important. Tn introduced at the N terminus of TMV was immunosilent, while that attached to tyrosine 139 elicited strong immune responses. Both Tn specific IgG and IgM antibodies were generated as determined by enzyme-linked immunosorbent assay and a glycan microarray screening study. The production of high titers of IgG antibodies suggested that the TMV platform contained the requisite epitopes for helper T cells and was able to induce antibody isotype switching. The antibodies exhibited strong reactivities toward Tn antigen displayed in its native environment, i.e., cancer cell surface, thus highlighting the potential of TMV as a promising TACA carrier.     

An integrated computational and experimental study for overproducing fatty acids in Escherichia coli

Increasing demands for petroleum have stimulated sustainable ways to produce chemicals and biofuels. Specifically, fatty acids of varying chain lengths (C₆–C₁₆) naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids from plants and other microbial production hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways. In addition, precursors for fatty acids are used along other central metabolic pathways for the production of amino acids and biomass, which further complicates the engineering of microbial hosts for higher yields. Here, we demonstrate an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis techniques to meet this challenge. The OptForce procedure was used for suggesting and prioritizing genetic manipulations that overproduce fatty acids of different chain lengths from C₆ to C₁₆ starting with wild-type E. coli. We identified some common but mostly chain-specific genetic interventions alluding to the possibility of fine-tuning overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose (～39% maximum theoretical yield) of C_14–16 fatty acids in minimal M9 medium. These results highlight the benefit of using computational strain design and flux analysis tools in the design of recombinant strains of E. coli to produce free fatty acids.     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

Estimating the voltage-dependent free energy change of ion channels using the median voltage for activation

Voltage-gated ion channels are crucial for electrical activity and chemical signaling in a variety of cell types. Structure-activity studies involving electrophysiological characterization of mutants are widely used and allow us to quickly realize the energetic effects of a mutation by measuring macroscopic currents and fitting the observed voltage dependence of conductance to a Boltzmann equation. However, such an approach is somewhat limiting, principally because of the inherent assumption that the channel activation is a two-state process. In this analysis, we show that the area delineated by the gating charge displacement curve and its ordinate axis is related to the free energy of activation of a voltage-gated ion channel. We derive a parameter, the median voltage of charge transfer (V(m)), which is proportional to this area, and prove that the chemical component of free energy change of a system can be obtained from the knowledge of V(m) and the maximum number of charges transferred. Our method is not constrained by the number or connectivity of intermediate states and is applicable to instances in which the observed responses show a multiphasic behavior. We consider various models of ion channel gating with voltage-dependent steps, latent charge movement, inactivation, etc. and discuss the applicability of this approach in each case. Notably, our method estimates a net free energy change of approximately -14 kcal/mol associated with the full-scale activation of the Shaker potassium channel, in contrast to -2 to -3 kcal/mol estimated from a single Boltzmann fit. Our estimate of the net free energy change in the system is consistent with those derived from detailed kinetic models (Zagotta et al. 1994. J. Gen. Physiol. doi:10.1085/jgp.103.2.321). The median voltage method can reliably quantify the magnitude of free energy change associated with activation of a voltage-dependent system from macroscopic equilibrium measurements. This will be particularly useful in scanning mutagenesis experiments.     

Precipitating and relieving factors of migraine versus tension type headache

Background

The correlation between intracranial pressure (ICP) and intraocular pressure (IOP) is still controversial in literature and hence whether IOP can be used as a non-invasive surrogate of ICP remains unknown. The aim of the current study was to further clarify the potential correlation between ICP and IOP.

Methods

The IOP measured with Goldmann applanation tonometer was carried out on 130 patients whose ICP was determined via lumber puncture. The Pearson correlation coefficient between ICP and IOP was calculated, the fisher line discriminated analysis to evaluate the effectivity of using IOP to predict the ICP level.

Results

A significant correlation between ICP and IOP was found. ICP was correlated significantly with IOP of the right eyes (p?<?0.001) and IOP of the left eyes (p?=?0.001) and mean IOP of both eyes (p?<?0.001), respectively. However, using IOP as a measurement to predict ICP, the accuracy rate was found to be 65.4%.

Conclusion

Our data suggested that although a significant correlation exists between ICP and IOP, caution needs to be taken when using IOP readings by Goldmann applanation tonometer as a surrogate for direct cerebrospinal fluid pressure measurement of ICP.     

Probing alignment and phase behavior in intact wood cell walls using 2H NMR spectroscopy

This study presents the first application of (2)H NMR spectroscopy to quantify lignocellulose matrix orientation, and it demonstrates the ability to separately investigate oriented and unoriented amorphous domains in intact natural plant tissue. Matrix orientation is evaluated using NMR quadrupolar interactions in small deuterated probe molecules absorbed into bulk Liriodendron tulipifera sapwood. Ethylene glycol-d(4) deuterium spectra reveal two distinct amorphous domains, a highly oriented phase in the secondary wall S2 layer and an isotropic domain probably reflecting the compound middle lamella (CML). The oriented and isotropic signal areas exhibit thermally reversible changes, postulated to reflect probe redistribution between the S2 layer and the CML. Preliminary studies on a more powerful wood swelling agent, N,N-dimethylformamide-d(1), are also discussed. This (2)H NMR technique provides a new avenue for analysis and understanding of lignocellulose ultrastructure and promises to create new insights in correlating biomass processing with morphological change.     

Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks

The regulation of food intake in chickens (Gallus gallus domesticus) represents a complex homeostatic mechanism involving multiple levels of control, and regulation during high ambient temperatures (HT) is poorly understood. In this study, we examined hypothalamic mRNA expression of gonadotropin-inhibitory hormone (GnIH) to understand the effect of HT on an orexigenic neuropeptide. We examined the effects of HT (35 °C ambient temperature for 1, 24 or 48 h) on 14-day old chicks. HT significantly increased rectal temperature and suppressed food intake, and also influenced plasma metabolites. The expression of GnIH precursor mRNA in the diencephalon was significantly increased in chicks at 24-and 48 h of HT when food intake was suppressed significantly, whilst no change was observed for GnIH precursor mRNA and food intake at 1h of HT. In situ hybridization and immunocytochemistry further revealed the cellular localization of chicken GnIH precursor mRNA and its peptide in the paraventricular nucleus (PVN) in the chick hypothalamus. We examined plasma metabolites in chicks exposed to HT for 1 or 48 h and found that triacylglycerol concentration was significantly higher in HT than control chicks at 1h. Total protein, uric acid and calcium were significantly lower in HT chicks than control chicks at 48h. These results indicate that not only a reduction in food intake and alteration in plasma metabolites but also the PVN-specific expression of GnIH, an orexigenic agent, may be induced by HT. The reduced food intake at the same time as GnIH expression was increased during HT suggests that HT-induced GnIH expression may oppose HT-induced feeding suppression, rather than promote it. We suggest that the increased GnIH expression could be a consequence of the reduced food intake, and would not be a direct response to HT.     

Probabilistic RNA partitioning generates transient increases in the normalized variance of RNA numbers in synchronized populations of Escherichia coli

We explore the effects of probabilistic RNA partitioning during cell division on the normalized variance of RNA numbers across generations of bacterial populations. We first characterize these effects in model cell populations, where gene expression is modeled as a delayed stochastic process, as a function of the synchrony in cell division, the rate of division, and the RNA degradation rate. We further explore the additional variance that arises if the partitioning is biased. Next, in Escherichia coli cells expressing RNA tagged with MS2d-GFP, we measured the normalized variance of RNA numbers across several generations, with cell divisions synchronized by heat shock. We show that synchronized cell populations exhibit transient increases in normalized variance following cell divisions, as predicted by the model, which are not observed in unsynchronized populations. We conclude that errors in partitioning of RNA molecules generate diversity between the offspring of individual bacteria and thus constitute a form of reproductive bet-hedging.