Topical application of a CCL22-binding aptamer suppresses contact allergy.

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For the pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nt DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

Implementation of Emulsion PCR for Amplification of Click-Modified DNA During SELEX.

Biased amplification of enriched DNA libraries is a limitation in the SELEX process and reduces the chances for successful enrichment of target-binding sequences. Implementation of emulsion PCR into click-SELEX protocols for targeting proteins or cells prevents the formation of by-products and increases the probability of successful enrichment of binding sequences. Through compartmentalization even poorly amplifiable sequences can be enriched, and by-products formed by product-product or product-primer hybridization are reduced to a minimum. In this chapter, we describe a protocol for emulsion PCR and subsequent DNA recovery for implementation into click-SELEX protocols using click-modified DNA. Our emulsion PCR protocol is easily integrated into existing SELEX protocols, requires no special laboratory equipment, and can be performed with easily commercially available reagents.

Injuries as exposure events in providing medical services by nursing staff

INTRODUCTION: Among medical staff, nurses are particularly vulnerable to occupational exposure since they provide direct patient care and perform medical activities which often involve using sharps. AIM: The objective of the study was to examine the frequency of injuries and their causes in nursing. METHODS: A diagnostic survey was employed using an original questionnaire. The study was carried out from 3rd March to 2nd April 2017. The study group comprised part-time nursing students. 107 respondents participating in the study worked in out-patient (28%) and in-patient (72%) healthcare. Most of the respondents were aged 41­50 (34.6%). RESULTS: 61.7% of the respondents were injured at work. The injury reporting rate was: 19.7% always, 22.7% often, 30.3% rarely, and 27.3% never. The most commonly mentioned types of injuries included: prick (51.5%), cut (28.8%), scratch (10.6%), prick and cut (9.1%). The incidence of injuries varied. 48.5% of the studied people declared fewer than 5 incidents, 31.8% quoted 5­10 injuries, 6.1% recalled 11­20, 13.6% did not remember such a situation. For nurses with longer seniority, there is a significant increase in injuries (p=0.029). Sources of injuries were most often: injection needle (35.9%), ampoule with medicine (23.3%), pen (7.8%). CONCLUSIONS: Not all occupational exposure cases are reported by nurses. Seniority determines injury incidence among nurses. The longer the seniority, the more common the injuries. The needle causes injuries most frequently.

Systematic evaluation of cell-SELEX enriched aptamers binding to breast cancer cells.

The sensitive and specific detection of pathogenic cells is essential in clinical diagnostics. To achieve this, molecular tools are required that unequivocally recognise appropriate cell surface molecules, such as biomarkers that come along with disease onset and progression. Aptamers are short single-stranded oligonucleotides that interact with cognate target molecules with high affinity and specificity. Within the last years they have gained an increased attention as cell-recognition tools. Here, we report a systematic analysis of a cell-SELEX procedure, for the identification of aptamers that recognise breast cancer cells. Besides a comparison of conventional (Sanger) with high-throughput sequencing techniques (next-generation sequencing), three different screening techniques have been applied to characterise the binding properties of selected aptamer candidates. This method has been found to be beneficial in finding DNA aptamers, rarely enriched in the libraries. Finally, four DNA aptamers were identified that exhibit broad-spectrum interaction patterns to different cancer cell lines derived from solid tumours.

New validated HPLC methodology for the determination of (-)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase.

A new chromatographic method for the enantioseparation and the determination of (-)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157-164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (-)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

The new HPLC methodology for the chiral separation of tamsulosin enantiomers on amylose tris(3,5-dimethylphenylcarbamate) stationary phase.

New chromatographic method for the enantioseparation of (R,S)-tamsulosin and the determination of (R)- and (S)-tamsulosin was developed with the aid of amylose tris(3,5-dimethylphenylcarbamate) stationary phase. The method was compared to the known procedure for tamsulosin enantioseparation on cellulose tris(3,5-dimethylphenyl carbamate). Careful validation of both methods allowed to prove advantages of the new procedure: significantly better resolution as well as twice better sensitivity. The method was applied to quantification of (R)- and (S)-tamsulosin contents in prolonged release Apo-Tamis 0.4 mg hard capsules (Apotex Europe B.V) and Omnic Ocas 0.4 mg coated tablets (Astellas).

Structure-retention relationship in a series of chiral 1,4-disubstituted piperazine derivatives on carbohydrate chiral stationary phases.

New racemic 1,4-disubstituted piperazines chemically named ethyl 2-[(4-pyrimidin-2yl-piperazine-1yl)carbonyl]C3-C5-alkanoates 1-7 were synthesized. The compounds were resolved into enantiomers on cellulose tris(4-methylbenzoate) and amylose tris(3,5-dimethylphenylcarbamate) stationary phases using hexane/propan-2-ol mobile phases. The optimum separation conditions for the compounds were obtained on cellulose tris(4-methylbenzoate) with 5% of 2-propanol in hexane. The relationship between structural and chromatographic parameters is discussed.

Direct separation, identification and quantification of epimers 22R and 22S of budesonide by capillary gas chromatography on a short analytical column with Rtx-5 stationary phase.

The conditions for separation, identification and quantitative determination of epimers 22R and 22S of budesonide by capillary gas chromatography (GC) with FID detection and two various sample injection methods, namely split-splitless and cool on-column, were established. In analysis helium as carrier gas and Rtx-5 capillary column of 7 m in length along with stationary phase Crossbond 5% diphenyl-95% dimethyl polysiloxane were used. The individual epimers were identified under specified conditions by using standard samples of different declared concentration of each epimer under investigation: (1) 51.2% of epimer 22R and 47.3% of epimer 22S, and (2) 95.1% of 22R and 4.4% of 22S, as well as Pulmicort, a preparation containing micronized budesonide as an active substance. It seems that good parameters of preliminary validation achieved by the proposed methods can confirm its suitability for quantitative analysis purpose. The retention times obtained for epimers 22R and 22S, depending on injection technique are about 7.7 and. 8.3 min for split and, approx. 10.3 and 10.9 min for cool on-column. The limits of detection and quantitation are 5.7 and 6.2 ng, for 22R respectively, and 4.3 and 4.8 ng for 22S. The linearity is maintained for concentrations ranging from 0.01 to 0.20 mg/ml. The quantitative analysis features of repeatability, high precision and accuracy confirmed by the obtained results and its statistical evaluation.