Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

COVID-19 laboratory preparedness and response in the Americas Region: Lessons learned

By the time the etiologic agent of the COVID-19 was identified as a novel coronavirus, no country in the Americas Region had laboratory capacity for detecting this new virus. A strategic multilevel approach with specific reagent purchase and delivery, regional trainings, in-country missions, and the provision of technical support was established for timely preparedness of national reference laboratories for SARS-CoV-2 detection. All countries should be prepared to timely detect any potential pandemic emerging agent. The rapid SARS-CoV-2 molecular detection implementation throughout the Americas showed the importance of an efficient and coordinated laboratory response for preparedness. Here we present how in 25 days the Americas Region went from no SARS-CoV-2 diagnostic capacity, to molecular detection fully implemented in 28 Member States, under the coordinated strategy of the Pan American Health Organization and collaborative work at regional and country level with national authorities and public health laboratories.

The thin ret(raction) line: biomedical journal responses to incorrect non-targeting nucleotide sequence reagents in human gene knockdown publications

The capacity of the scientific literature to self-correct is of vital importance, but few studies have compared post-publication journal responses to specific error types. We have compared journal responses to a specific reagent error in 31 human gene knockdown publications, namely a non-targeting or negative control nucleotide sequence that is instead predicted to target a human gene. The 31 papers published by 13 biomedical journals generated 26 published responses (14 retractions, 5 expressions of concern, 7 author corrections which included one resolved expression of concern) as well as 6 stated decisions to take no action. Variations in published responses were noted both between journals and by 4 journals that published different responses to at least 2 papers. A subset of published responses revealed conflicting explanations for the wrongly identified control reagent, despite 30/31 papers obtaining their gene knockdown reagents from the same external supplier. Viewed collectively, different journal responses to human gene knockdown publications with a common reagent error type suggest that editorial staff require more support to interpret post-publication notifications of incorrect nucleotide sequence reagents. We propose a draft template to facilitate the communication, interpretation and investigation of published errors, including errors affecting research reagents.

Spectrofluorometric determination of L-tryptophan in canary (Canarium indicum L.) seed protein hydrolysate

Canary (Canarium indicum L.) is an indigenous plant of Indonesia, which mainly grows in the eastern part of Indonesia, especially in the Maluku, North Sulawesi, and Seram islands. We believe that no scientific reports have been conducted about L-tryptophan content in Canarium indicum. Therefore, this study was conducted to determine the presence and quantitate the aromatic amino acid (L-tryptophan) in the canary protein hydrolysate by the spectrofluorometric method. The protein hydrolysate was prepared by two hydrolysis methods, enzymatic and alkaline hydrolysis. L-tryptophan can be differentiated from tyrosine directly without using any reagent by excitation of the sample at 295 nm in order to avoid tyrosine emission. The equation of calibration curve correlation using standard in the range 0.5-5 ppm was y = 6632.3x - 845.42 and correlation coefficient of 0.9997, while the coefficient of variance in linear regression was 1.29%. The detection limit and quantification limit obtained were 0.116 ppm and 0.35 ppm respectively. The recoveries of the accuracy test were obtained in the range of 95-96%. The relative standard deviation of intra-assay precision tests were obtained in the range of 0.5-1.8%, while the intermediate precision in the range of 2.18-3.74%. L-tryptophan was detected in all samples (papain, pepsin, and alkaline hydrolysate), with concentrations 5.6, 5 and 1.53 mg/100mg of protein respectively. The used fluorometric method complied with the validation requirements and can be used to analyze L-tryptophan in samples containing tyrosine without overlapping of spectra and without the use of any specific reagent.

Prospective Evaluation of Molecular Assays for Diagnosis of Vaginitis

ABSTRACT Molecular tests to diagnose conditions involving the disruption of normal microbiota are difficult to optimize. Using Nugent-scored Gram stain (NS) as the reference standard, we evaluated the performance of 3 molecular assays for the diagnosis of bacterial vaginosis (BV) and examined the impact of an incremental increase in bacterial targets. The BD Affirm assay includes a DNA probe for Gardnerella vaginalis, the Hologic transcription-mediated amplification (TMA) analyte-specific reagent (ASR) assay adds a second Lactobacillus sp. target, and the recently cleared in vitro diagnostic use (IVD) Aptima BV assay includes a third target (Atopobium vaginae). The diagnosis of vulvovaginal candidiasis (VVC) by the Affirm and Candida vaginitis Hologic TMA ASR assays was assessed using microscopy for yeast as the reference standard. From May to December 2018, 111 women with vaginitis symptoms prompting the clinician to order an Affirm test were enrolled with informed consent for the collection of additional specimens. Clinicians accurately predicted BV as the most likely diagnosis for 71% of the 45 patients with BV. Coinfection occurred in 13.5% of patients. For BV, the specificity of the Aptima IVD assay (86.3%) was higher than the Affirm assay (60.6%, P = 0.0002), but sensitivities were not significantly different. For VVC, the sensitivity of the ASR assay (100%) was higher than Affirm (75.9%; P = 0.023) and the specificity of the Affirm assay (98.8%) was higher than the ASR assay (86.6%; P = 0.004).

EFEKTIFITAS PEMANFAATAN POTENSI SENYAWA FENOLIK KUBIS UNGU (Brassica Oleraceae var.carpitata. L) SECARA INSTRUMEN UV-VIS

Cabbage Purple (Brassica oleracea var. Capitata L.) is one of the genus Brasica genus that is widely available in Indonesia. Purple cabbage has many benefits because it has many content, among others, vitamins A, B, C and E, mineral potassium, calcium, phosphorus, sodium and iron, sulforafan and contain anthocyanin. The purpose of this study was to perform and determine the total phenolic content of purple cabbage (Brassica oleracea var, capitata L.). The method used in qualitative analysis is through color reaction by using specific reagent, while for quantitative analysis done by UV-Vis spectrofotometry method. For antioxidant activity test through absorbance analysis of acid error and sample absorbance from observation result on UV-Vis spectrophotometer. The results of this study contain phenolic compounds, as for the total phenolic content of methanol extracts that the purple cabbage (Brassica oleraceavar, capitata L.) Amounted to 1.9873 mgGAE/g extract.

Synthesis and Spectro-Kinetic Study of the Complexes of Pd+2 Ion with 2-(5-Bromo-2- pyridylazo)5diethylaminophenol Reagent in Ethanol – Water Solutions

The complexes of Pd+2 ion with 2-(5-bromo-2-pyridylazo)-5-dieıhyl aminophenol (BPADAP) were studied kinetically and spectrophotometrically in aqueous ethanolic solutions. The reagent forms 1:1, 2:1 square planer and 1:1 bridged shape binuclear complexes with Pd+2 ion. All these complexes (violet colour) absorb light in the same region at 540, 575 and 618nm. The band at 618 nm seems to be specific for complexes of Pd+2ion with BPADAP. The rate constants of the growth in 93% H2O + 7% ethanol of 1:1 and 2:1 complexes at 575 and 618 nm were followed the first order kinetics and are quite of the same values , 0.495 and 0.463 min- 1 respectively. The rate constants of the decay of 2:1 complex at 618 and 575 nm were followed the first order kinetics; their values are 0.075 and 0.065 min-1 respectively which are slower than the rate of the decay of 1:1 complex. Whereas, the rate constants of the growth and decay of 1:1 bridged binuclear complex also followed the first order kinetics but they are much slower than those of 1:1 and 2:1 complexes: the values of the growth are 025min-1at 575 nm , and 0.16 min-1 at 618 nm. And the values of the decay are 0.0476 min-1 at 575 nm and 0.008min-1 at 61 8 nm. It was found that BPADAP can identify and detect in ethanol at 575 and 618 nm less than one microgram up to more than seven micrograms of Pd+2 ion . The interference of UO2+2, Th+4, Pb+2 , Cd+2,Bi+3, Hg+2 and Zn+ 2 in the detection of Pd+2 ion at 575 nm ranges between (25.3 - 67)%. Whereas, the interference of these ions with the detection at 618 nm is only between (2.3 -3.9) %, so it is reasonable to suggest that BPADAP is a specific reagent for Pd+2 ion at 618 nm

Validasi Metode Spektrofotometri Visible Untuk Penentuan Kadar Formaldehida Pada Pembalut Wanita Yang Beredar Di Pasaran

ABSTRAKPembalut menjadi kebutuhan wanita yang sangat penting karena digunakan untuk menyerap cairan darah ketika mengalami menstruasi. Pada pembuatan pembalut wanita dimungkinkan adanya pemakaian formaldehida. Oleh karena itu, pembalut wanita termasuk salah satu alat kesehatan yang kandungan dan bahan penyusunnya diatur oleh pemerintah. Pada penelitian ini menggunakan metode spektrofotometri visibel untuk penentuan kadar formaldehida dalam pembalut wanita sekali pakai. Sebelum digunakan, maka metode spektrofotometri visibel ini harus divalidasi terlebih dahulu untuk memastikan bahwa metode spektrofotometri yang digunakan dapat memberikan hasil yang akurat. Tujuan penelitian ini adalah melakukan validasi metode spektrofotometri visibel untuk penetapan kadar formaldehida dalam pembalut wanita sekali pakai menggunakan pereaksi nash sebagai reagen spesifik. Metode penelitian yang digunakan adalah pembuatan dan pembakuan larutan baku formaldehida, menentukan panjang gelombang maksimal, pembuatan kurva kalibrasi, melakukan uji linieritas, uji LOD dan LOQ, serta uji kesesuaian dan kecermatan, dan menentukan kadar formaldehida pada pembalut wanita. Hasil dari penelitian ini adalah bahwa metode spektrofotometri visibel memiliki selektifitas, linieritas,batas deteksi dan kuantitasi, presisi dan akurasi yang baik. Kadar rata-rata formaldehida pada ke lima sampel pembalut sebesar 2,88 mg/kg - 4,05 mg/kg.Kata kunci: pembalut, formaldehida, validasi, spektrofotometri visibelABSTRACTSanitary napkins are a very important woman’s need to absorb blood fluids when menstruating. In the manufacture of sanitary napkins may contain formaldehyde additives. Therefore, sanitary napkins are one of the medical devices whose composition is regulated by the government. In this study to identify the use of formaldehyde in sanitary napkins was carried out by visible spectrophotometry using nash reagent. This method should be validated in advance to ensure that the method used can provide accurate data. The aim of this research is to validate visible spectrophotometry method for determination of formaldehyde content indisposable sanitary napkins using nash reagent as specific reagent. Validation of UV – Vis spectrophotometry method for determination of formaldehyde showed that Nash reagent was suitable to determine formaldehyde. This method is linear with correlation coefficient (r2) of 0,99967. The validation characteristics include accuracy and precision, linearity, limit of detection, and limit of quantitation. The acceptance validation criteria were found in all case. Qualitative determination in five sanitary napkins samples showed positive results and the quantitative analysis confirmed that the average content of formaldehyde in five sanitary napkins samples was 2,88 mg/kg – 4,05 mg/kg.Keywords: sanitary napkins, formaldehyde, validation, visible spectrophotometry