Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum : a new approach

Reported homocysteine (HCY) concentrations in human serum show poor concordance amongst laboratories due to endogenous HCY in the matrices used for assay calibrators and QCs. Hence, we have developed a fully validated LC–MS/MS method for measurement of HCY concentrations in human serum samples that addresses this issue by minimising matrix effects. We used small volumes (20 μL) of 2% Bovine Serum Albumin (BSA) as surrogate matrix for making calibrators and QCs with concentrations adjusted for the endogenous HCY concentration in the surrogate matrix using the method of standard additions. To aliquots (20 μL) of human serum samples, calibrators or QCs, were added HCY-d4 (internal standard) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) as reducing agent. After protein precipitation, diluted supernatants were injected into the LC–MS/MS. Calibration curves were linear; QCs were accurate (5.6% deviation from nominal), precise (CV% ≤ 9.6%), stable for four freeze–thaw cycles, and when stored at room temperature for 5 h or at −80 °C (27 days). Recoveries from QCs in surrogate matrix or pooled human serum were 91.9 and 95.9%, respectively. There was no matrix effect using 6 different individual serum samples including one that was haemolysed. Our LC–MS/MS method has satisfied all of the validation criteria of the 2012 EMA guideline.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF‑2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF‑2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β‑glycerophosphate). FGF‑2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF‑2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT‑qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF‑2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF‑2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.