Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series.

BACKGROUND: A microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO). METHODS: In total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer's test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system. RESULTS: From tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/µL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/µL (under a lower detection limit of 5.6 × 10-2). CONCLUSION: In conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

Two cases of dupilumab-associated conjunctivitis with high expression of IL-8 mRNA on the ocular surface: a case report.

BACKGROUND: Dupilumab-induced ocular surface disease (DIOSD) has been reported in patients with atopic dermatitis treated with dupilumab, and has been recognized as an adverse event of dupilumab. Our objective was to describe two cases of DIOSD with alterations in eotaxin-2 and interleukin (IL)-8 messenger ribonucleic acid (mRNA) expression on the ocular surface. CASE PRESENTATION: In the ocular surface test, specimens were collected from the patient's ocular surface, and eotaxin-2 and IL-8 mRNA levels in the specimens were measured using real-time polymerase chain reaction. The clinical score of ocular surface findings was quantified using a 5-5-5 exacerbation grading scale for allergic conjunctivitis. The first case was of a 27-year-old man who developed DIOSD 3 months after starting treatment with dupilumab injection for atopic dermatitis. After 5 weeks of topical instillation of tacrolimus ophthalmic suspension, the clinical score of ocular surface findings improved and IL-8 and eotaxin-2 mRNA expression levels gradually decreased. The second patient was a 55-year-old man who developed DIOSD 11 weeks after the start of treatment with dupilumab injection for atopic dermatitis. Four weeks after starting ophthalmological treatment with tacrolimus ophthalmic suspension, his clinical scores on ocular surface findings improved and IL-8 mRNA expression levels decreased. The ocular surface test in this case revealed increased expression levels of IL-8 mRNA on the ocular surface at the onset of DIOSD, which decreased with the improvement of objective findings. CONCLUSIONS: DIOSD, which has been successfully treated with tacrolimus ophthalmic suspension, may involve IL-8-related inflammation in addition to type 2 inflammation.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.

Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel ß-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.

On the similar spatial arrangement of active site residues in PAPS-dependent and phenolic sulfate-utilizing sulfotransferases.

Mammalian sulfotransferases (STs) utilize exclusively the sulfuryl group donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to catalyze the sulfurylation reactions based on a sequential transfer mechanism. In contrast, the commensal intestinal bacterial arylsulfate sulfotransferases (ASSTs) do not use PAPS as the sulfuryl group donor, but instead catalyze sulfuryl transfer from phenolic sulfate to a phenol via a Ping-Pong mechanism. Interestingly, structural comparison revealed a similar spatial arrangement of the active site residues as well as the cognate substrates in mouse ST (mSULT1D1) and Escherichia coli CFT073 ASST, despite that their overall structures bear no discernible relationship. These observations suggest that the active sites of PAPS-dependent SULT1D1 and phenolic sulfate-utilizing ASST represent an example of convergent evolution.

Conformational indeterminism in protein misfolding: chiral amplification on amyloidogenic pathway of insulin.

Unlike folding, protein aggregation is a multipathway, kinetically controlled process yielding different conformations of fibrils. The dynamics and determinism/indeterminism boundaries of misfolded conformations remain obscure. Here we show that, upon vortexing, insulin forms two distinct types of fibrils with opposite local chiral preferences, which manifest in the opposite twists of bound dye, thioflavin T. Occurrence of either type of fibrils in a test tube is only stochastically determined. By acting through an autocatalytic, "chiral amplification"-like mechanism, a random conformational fluctuation triggers conversion of the macroscopic amount of insulin into aggregates with uniformly biased chiral moieties, which bind and twist likewise the achiral dye. Although a convection-driven chiral amplification in achiral systems, which results in randomly distributed excesses of optically active forms, is known, observation of such a phenomenon in misfolded protein built of l-amino acids is unprecedented. The two optical variants of insulin fibrils show distinct morphologies and can propagate their chiral biases upon seeding to nonagitated insulin solutions. Our findings point to a new aspect of topological complexity of protein fibrils: a chiral feature of hierarchically assembled polypeptides, which is partly emancipated from the innate left-handedness of amino acids. Because altering chirality of a molecule changes dramatically its biological activity, the finding may have important ramifications in the context of the structural basis of "amyloid strains".

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism.

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.