Cell-Autonomous Requirement for Rx Function in the Mammalian Retina and Posterior Pituitary

Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx−/− cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx−/− and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.     

Acute down-regulation of sodium-dependent phosphate transporter NPT2a involves predominantly the cAMP/PKA pathway as revealed by signaling-selective parathyroid hormone analogs

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) in cells of the renal proximal tubule mediates the reduction in membrane expression of the sodium-dependent P(i) co-transporters, NPT2a and NPT2c, and thus suppresses the re-uptake of P(i) from the filtrate. In most cell types, the liganded PTHR1 activates Gα(S)/adenylyl cyclase/cAMP/PKA (cAMP/PKA) and Gα(q/11)/phospholipase C/phosphatidylinositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/PKC (IP(3)/PKC) signaling pathways, but the relative roles of each pathway in mediating renal regulation P(i) transport remain uncertain. We therefore explored the signaling mechanisms involved in PTH-dependent regulation of NPT2a function using potent, long-acting PTH analogs, M-PTH(1-28) (where M = Ala(1,12), Aib(3), Gln(10), Har(11), Trp(14), and Arg(19)) and its position 1-modified variant, Trp(1)-M-PTH(1-28), designed to be phospholipase C-deficient. In cell-based assays, both M-PTH(1-28) and Trp(1)-M-PTH(1-28) exhibited potent and prolonged cAMP responses, whereas only M-PTH(1-28) was effective in inducing IP(3) and intracellular calcium responses. In opossum kidney cells, a clonal cell line in which the PTHR1 and NPT2a are endogenously expressed, M-PTH(1-28) and Trp(1)-M-PTH(1-28) each induced reductions in (32)P uptake, and these responses persisted for more than 24 h after ligand wash-out, whereas that of PTH(1-34) was terminated by 4 h. When injected into wild-type mice, both M-modified PTH analogs induced prolonged reductions in blood P(i) levels and commensurate reductions in NPT2a expression in the renal brush border membrane. Our findings suggest that the acute down-regulation of NPT2a expression by PTH ligands involves mainly the cAMP/PKA signaling pathway and are thus consistent with the elevated blood P(i) levels seen in pseudohypoparathyroid patients, in whom Gα(s)-mediated signaling in renal proximal tubule cells is defective.     

Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity

The simian virus 40 large T antigen (SVLT) induces replication of plasmids bearing the SV40 origin of replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ~10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the β-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Subclinical inflammation on MRI of hand and foot of anticitrullinated peptide antibody–negative arthralgia patients at risk for rheumatoid arthritis

Introduction

It is known that anticitrullinated peptide antibody (ACPA)–positive rheumatoid arthritis (RA) has a preclinical phase. Whether this phase is also present in ACPA-negative RA is unknown. To determine this, we studied ACPA-negative arthralgia patients who were considered prone to progress to RA for local subclinical inflammation observed on hand and foot magnetic resonance imaging (MRI) scans.

Methods

We studied a total of 64 ACPA-negative patients without clinically detectable arthritis and with arthralgia of the small joints within the previous 1 year. Because of the character of the patients’ symptoms, the rheumatologists considered these patients to be prone to progress to RA. For comparisons, we evaluated 19 healthy, symptom-free controls and 20 ACPA-negative RA patients, who were identified according to the 1987 American Rheumatism Association criteria. All participants underwent MRI of unilateral wrist, metacarpophalangeal and metatarsophalangeal joints. Synovitis and bone marrow oedema (BME) were scored according to the OMERACT rheumatoid arthritis magnetic resonance imaging scoring system, and the scores were summed to yield the ‘MRI inflammation score’. Scores were compared between groups. Among the ACPA-negative arthralgia patients, MRI inflammation scores were related to C-reactive protein (CRP) levels and the tenderness of scanned joints.

Results

MRI inflammation scores increased progressively among the groups of controls and ACPA-negative arthralgia and RA patients (median scores = 0, 1 and 10, respectively; P < 0.001). The MRI inflammation scores of ACPA-negative arthralgia patients were significantly higher than those of controls (P = 0.018). In particular, the synovitis scores were higher in ACPA-negative arthralgia patients (P = 0.046). Among the ACPA-negative arthralgia patients, inflammation was observed predominantly in the wrist (53%). The synovitis scores were associated with CRP levels (P = 0.007) and joint tenderness (P = 0.026). Despite the limited follow-up duration, five patients developed clinically detectable arthritis. These five patients had higher scores for MRI inflammation (P = 0.001), synovitis (P = 0.002) and BME (P = 0.003) compared to the other patients.

Conclusion

Subclinical synovitis was observed in the small joints of ACPA-negative arthralgia patients, and especially in patients whose conditions progressed to clinically detectable arthritis. This finding suggests the presence of a preclinical phase in ACPA-negative RA. Further longitudinal studies of these lesions and patients are required to confirm this hypothesis.     

Genetic engineering of trees: progress and new horizons

Genetic engineering of trees to improve productivity, wood quality, and resistance to biotic and abiotic stresses has been the primary goal of the forest biotechnology community for decades. We review the extensive progress in these areas and their current status with respect to commercial applications. Examples include novel methods for lignin modification, solutions for long-standing problems related to pathogen resistance, modifications to flowering onset and fertility, and drought and freeze tolerance. There have been numerous successful greenhouse and field demonstrations of genetically engineered trees, but commercial application has been severely limited by social and technical considerations. Key social factors are costly and uncertain regulatory hurdles and sweeping market barriers in the form of forest certification systems that disallow genetically modified trees. These factors limit and, in many cases, preclude field research and commercial adoption. Another challenge is the high cost and uncertainty in transformation efficiency that is needed to apply genetic engineering and gene editing methods to most species and genotypes of commercial importance. Recent advances in developmental gene-based transformation systems and gene editing, if combined with regulatory and certification system reform, could provide the foundation for genetic engineering to become a significant tool for coping with the increasing environmental and biological stresses on planted and wild forests.