Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Corneal Dystrophy Mutations Drive Pathogenesis by Targeting TGFBIp Stability and Solubility in a Latent Amyloid-forming Domain

Numerous mutations in the corneal protein TGFBIp lead to opaque extracellular deposits and corneal dystrophies (CDs). Here we elucidate the molecular origins underlying TGFBIp's mutation-induced increase in aggregation propensity through comprehensive biophysical and bioinformatic analyses of mutations associated with every major subtype of TGFBIp-linked CDs including lattice corneal dystrophy (LCD) and three subtypes of granular corneal dystrophy (GCD 1–3). LCD mutations at buried positions in the C-terminal Fas1–4 domain lead to decreased stability. GCD variants show biophysical profiles distinct from those of LCD mutations. GCD 1 and 3 mutations reduce solubility rather than stability. Half of the 50 positions within Fas1–4 most sensitive to mutation are associated with at least one known disease-causing mutation, including 10 of the top 11 positions. Thus, TGFBIp aggregation is driven by mutations that despite their physico-chemical diversity target either the stability or solubility of Fas1–4 in predictable ways, suggesting straightforward general therapeutic strategies.     

Consensus based validation of membrane porins

Porins are outermembrane beta-barrel proteins. They have varied biological functionality ranging from phage receptors, immunogenicity, pathogenicity to apoptosis. However, only a small number has been structurally and functionally characterised. A validation mechanism and a database of porins would be useful for target selection in proteomics and structural genomics work. Here we report a validation mechanism developed for membrane porins. A database server for porins, PRNDS, has been created containing experimentally proven porins and likely putative porins. Each porin is validated and ranked using a weighted scoring system developed based on six, structure and sequence based criteria. The server also predicts possible porins.     

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.     

Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection

Background

Limited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.

Methods and Findings

We prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24.At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm³. HIV RNA was 5.5 log₁₀ copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/10⁶ PBMC) vs. Fiebig I (8 copy/10⁶ PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008).After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).

Conclusions

Gut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.Key Words: islets, stem-cells, development, epithelium, mesenchyme, pancreas, stomach, chick-quail, 3-dimensional, endocrine