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Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future. [ABSTRACT FROM AUTHOR]

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV). To evaluate the molecular mechanisms involved in the viral infection, in this study, we investigated the role of SARS-CoV Spike (S) protein in the regulation of cyclooxygenase-2 (COX-2). Expression of COX-2 stimulated by the S protein was verified by RT-PCR and western blot assay. To explore the relationship between S and COX-2, we constructed a series of plasmids containing truncated N-terminal fragments of the SARS-CoV S gene (designated from Sa to Si), which encoded truncated S proteins, and investigated whether these truncated proteins could induce effective expression of COX-2 in 293T cells. Our results showed that Sd that encoded a truncated S protein with 422 amino acid residues (from 1 to 422 aa), a part of 672 amino-acid S1 subunit is crucial for the induction of COX-2 expression. Immunofluorescence examinations also give the evidence that these N terminal 422 amino acids of the S protein were also required for the correct localization of the protein. We also compared S protein sequences of SARS-CoV isolated during the SARS break with that from palm civets, a possible source of SARS-CoV found in humans. S protein residues (344, 360), which mutated in the epitome from palm civet to human being were characterized in 3D modeling of 252–375 amino acid fragment. Collectively, these results indicate that S protein of SARS-CoV induces the expression of COX-2 and an N-terminal fragment of the Spike protein is crucial for the induction. Our finding may provide clue for the induction of inflammation by SARS-CoV and cast insight into the severity of the SARS epidemic. [ABSTRACT FROM AUTHOR]

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling. Myo-Inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) are important second messengers but their analysis remains challenging. Here, the authors develop a capillary electrophoresis-mass spectrometry method for the identification and quantitation of InsP and PP-InsP isomers in cells and tissues. [ABSTRACT FROM AUTHOR]