Lysis Solution Research Articles

DETEKSI BAKTERI ENTEROHEMORRHAGIC ESCHERICHIA COLI PADA LAWAR MERAH BABI DENGAN MENGGUNAKAN TEKNIK POLYMERASE CHAIN REACTION (PCR) PADA WARUNG DI DENPASAR

Lawar merah babi is one of the typical Balinese dish made from pork, pork skin, some vegetables, coconut, various spices, and fresh blood as red colour. The processing of lawar still very traditional. In the process of processing and serving lawar merah babi, the sellers pay less attention to hygiene such as the materials that contaminated with bacteria, personal hygiene, and unclean cooking equipment. If it is not processed properly and hygienically, it is likely to be contaminated with Escherichia coli bacteria which if consumed will be pathogenic to cause diarrhea. This study aims to determine the presence of Escherichia coli bacterial contamination, especially the Enterohemorrhagic Escherichia coli pathotype on lawar merah babi that sold in stalls in Denpasar with the PCR technique. This research is a descriptive study with a cross sectional research design. The sampling technique used cluster purposive sampling technique with a total sample of 13 samples taken at stalls in Denpasar. All samples were examined at the Integrated Biomedical Laboratory, Department of Biochemistry, Faculty of Medicine, Udayana University. The research procedure starts from lawar merah babi was isolated for protein with a 0.6 probe and then DNA was isolated using a DNA isolation kit (cell lysis solution, nuclear lysis solution, protein precipitation solution, DNA rehydration solution). After that, EHEC was detected using stx-1 and stx-2 as target genes using PCR technique. Of the 13 samples studied, the results obtained did not detect any EHEC pathotype in all samples of the lawar merah babi.

Application of RapidHIT™ ID for cell authentication by fast and convenient STR profiling.

As cell therapies are injected directly into the body, cell authentication is essential. Short tandem repeat (STR) profiling is used for human identification in forensics as well as for cell authentication. The standard methodology (DNA extraction, quantification, polymerase chain reaction, and capillary electrophoresis) takes at least 6h and requires several instruments to obtain an STR profile. RapidHIT™ ID is a single automated instrument that provides an STR profile in 90 min. In this study, we aimed to propose a method to use RapidHIT™ ID for cell authentication. Four types of cells which are used for cell therapy or in the production process were used. The sensitivity of STR profiling was compared by the cell type and cell count using RapidHIT™ ID. Moreover, the effect of preservation solutions, pre-treatment with cell lysis solution, proteinase K, Flinders Technology Associates (FTA) cards, and dried or wet cotton swabs (with a single cell type or a mixture of two) were examined. The results were compared to those obtained by the standard methodology using genetic analyzer ThermoFisher SeqStudio. We accomplished a high sensitivity through our proposed method that can benefit cytology laboratories. Although the pre-treatment process affected the quality of the STR profile, other variables did not significantly affect STR profiling. As a result of the experiment, RapidHIT™ ID can be used as a faster and simpler instrument for cell authentication.

Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps.

In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus using the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such as S. aureus are harder to lyse than Gram-negative bacteria. Hence, the first step in the procedure for isolating gDNA from Gram-positive bacteria consists of a mechanical lysis step (e.g., using a bead beating grinder or homogenizer) or an enzymatic lysis step. For the method described here, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, the gDNA can be purified using procedures similar to those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the gDNA is subsequently used for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.

Peptidomic Analysis and Antimicrobial Activity of Serum Peptide from Hevea brasiliensis Clone BPM24.

Hevea brasiliensis is severely affected by the fungal disease caused by Phytophthora spp. Significant loss of rubber yield is widespread and extensive use of chemical fungicides has resulted in health and environmental problems. This work aims to extract and identify the latex serum peptides from a disease tolerant clone of H. brasiliensis, and study the inhibitory efficacy against pathogenic bacteria and fungi. Serum peptides were extracted from H. brasiliensis BPM24 using mixed lysis solution. Low molecular weight peptides were screened and fractionated by solid-phase extraction and then identified by tandem mass spectrometry. Total and fractionated serum peptides were assayed for bacterial and fungal inhibition using broth microdilution and poisoned food methods. An inhibitory control study in the greenhouse was also performed using susceptible clones for pre and postinfection with Phytophthora spp. Forty-three serum peptide sequences were successfully identified. Thirty-four peptides matched with the proteins associated with plant defense response signaling, host resistance, and adverse environmental factors. The inhibitory study of total serum peptides demonstrated antibacterial and anti-fungal properties. The greenhouse study exhibited disease inhibitory efficacy of 60% for the treatment of Phytophthora spp. in post-infected plants and 80% for pre-treated samples. Latex serum peptides from disease tolerant H. brasiliensis revealed several proteins and peptides associated with plant defense and disease resistance. The peptides play a vital role for defense against bacteria and fungi pathogens, including Phytophthora spp. Enhanced disease protection can be obtained when the extracted peptides were applied to the susceptible plants before exposure to the fungi. These findings provided an insight and may pave the way for the development of biocontrol peptides from natural resources.

Separation and Purification of Recombinant β-Glucosidase with Hydrophobicity and Thermally Responsive Property from Cell Lysis Solution by Foam Separation and Further Purification.

The aim of this study was to separate and purify recombinant β-glucosidase (GLEGB) with elastin-like polypeptide (ELP) and graphene-binding peptide (GB) from cell lysis solution by foam separation and further purification. The study of foam property of GLEGB cell lysis solution indicated that it had excellent foaming property and foam stability, which was suitable for foam separation. This could be due to the GB tag with hydrophobicity, which made the recombinant β-glucosidase with GB preferentially adsorb on the surface of bubbles. At optimum operating conditions of foam separation, the enzyme activity recovery of GLEGB could reach 95.63 ± 1.0%. The foam solution of GLEGB was further purified based on the thermally responsive property of the ELP tag, and the purification fold of GLEGB could reach 29.6 ± 0.5 at the optimum operating conditions. The prominent purification effect indicates that this technique is a simple and efficient technique for the separation and purification of recombinant enzymes.

Rapid preparation of Candida genomic DNA: combined use of enzymatic digestion and thermal disruption

Nucleic acid based molecular technologies are the most promising tools for the early diagnosis of Candida infection. A simple and effective DNA preparation method is of critical for standardizing and applying molecular diagnostics in clinic laboratories. The goal of this study was to develop a Candida DNA preparation method that was quick to do, easy to perform, and bio-safe. Snailase and lyticase were screened and combined in this work to enhance the lysis of Candida cells. The lysis solution composition and metal bath were optimized to boost amplification efficiency and biosafety. A duplex real-time PCR was established to evaluate the sensitivity and specificity of the preparation method. Using the supernatant from the rapid preparation method as templates, the duplex PCR sensitivities for five common Candida species were determined to be as low as 100 CFUs. When compared to conventional preparation methods, the samples prepared by our method showed higher PCR detection sensitivity. PCR identification and ITS sequencing were 100% consistent, which was better than biochemical identification. This study demonstrates a rapid method for Candida DNA preparation that has the potential to be used in clinical laboratories. Meanwhile, the practical application of the method for clinical samples needs to be proven in future investigations.

CdSe-Co3O4@TiO2 nanoflower-based photoelectrochemical platform probing visible light-driven virus detection.

The design and construction of a visible light-driven photoelectrochemical (PEC) device is describedbased on a CdSe-Co3O4@TiO2 nanoflower (NF). Moreover, an application to the ultrasensitive detection of viruses, such as hepatitis E virus (HEV), HEV-like particles (HEV-LPs), and SARS-CoV-2 spike protein in complicated lysate solution, is demonstrated. The photocurrent response output of a PEC device based on CdSe-Co3O4@TiO2 is enhanced compared with the individual components, TiO2 and CdSe-Co3O4. This can be attributed to the CdSe quantum dot (QD) sensitization effect and strong visible light absorption to improve overall system stability. A robust oxygen-evolving catalyst (Co3O4) coupled at the hole-trapping site (CdSe) extends the interfacial carrier lifetime, and the energy conversion efficiency was improved. The effective hybridization between the antibody and virus resulted in a linear relationship between the change in photocurrent density and the HEV-LP concentration ranging from 10fgmL-1 to 10ngmL-1, with a detection limit of 3.5fgmL-1. This CdSe-Co3O4@TiO2-based PEC device achieved considerable sensitivity, good specificity, and acceptable stability and demonstrated a significant ability to develop an upgraded device with affordable and portable biosensing capabilities.

Development of an Organic Dye‐Conjugated β‐Diketonate‐Europium Complex Luminescence Probe for Discrimination and Detection of Intracellular Biothiols

AbstractSmall molecular biothiols, cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play important roles in organisms, and their concentration levels are indicative of some human diseases. Herein we report an organic dye‐conjugated β‐diketonate‐Eu3+ complex, [Eu(NBD‐keto)3(DPBT)] (NBD‐keto: 7‐nitro‐2,1,3‐benzoxadiazole (NBD)‐conjugated to 1,1,1,2,2‐pentafluoro‐5‐phenyl‐3,5‐pentanedionate through a “O” ether bond; DPBT: 2‐(N,N‐diethylanilin‐4‐yl)‐4,6‐bis(3,5‐dimethylpyrazol‐1‐yl)‐1,3,5‐triazine), which acts as a unique luminescent probe for detecting and discriminating biothiols. [Eu(NBD‐keto)3(DPBT)] itself is not luminescent due to intramolecular interactions between NBD and β‐diketonate‐Eu3+ moieties. Upon reaction with biothiols, the β‐diketonate‐Eu3+ complex [Eu(keto)3(DPBT)] is generated, which emits long‐lived red emission at 610 nm. Meanwhile, three biothiol‐substituted NBD derivatives that exhibit different luminescence behaviors, green emissive (short‐lived) NBD‐NR (R=Cys or Hcy) at 540 nm and non‐luminescent NBD‐SR (R=GSH), are also generated. These luminescence response behaviors allow time‐gated and steady‐state luminescence modes to be combined for detecting total biothiols and discriminating GSH and Cys/Hcy. Using this probe, the quantitative detection and discrimination of GSH and Cys/Hcy in lysis solutions of HeLa cells were realized, which revealed the potential of the probe for biomedical applications.

Biomimetic one-pot preparation of surface biofunctionalized silica-coated magnetic composites for dual enzyme oriented immobilization without pre-purification

Surface functioned magnetic silica particles are efficient carriers to achieve facilitated separation and recycling of biocatalysts. However, traditional methods of modifying magnetic silica particles required time-costly sequential coating and surface modification steps and toxic solvents. Herein, a green and efficient routine was proposed to prepare the surface modified silica-coated magnetic microspheres (SCEs@SiO2 @Fe3O4) in one-pot. The elastin-like polypeptides (ELPs)-SpyCatcher chimera (SCEs) were purified by inverse transition cycling with high yield (275 mg/L) and incorporated into the magnetic silica spheres based on the biomimetic silicification capability of ELPs as proved by the EDS and SEM mapping. No SCEs leaked was observed within 48 h, indicating excellent stability in buffer. Then, the biofunctionalized carriers were used to purify and immobilize the target dual enzymes (xylanase-linker-SpyTag-linker-lichenase, bienzymes) directly from the crude cell lysis solution by the spontaneous isopeptide bond reaction between SpyCatcher and SpyTag. The immobilized bienzymes were sphere-like magnetic silica particles with uniform size, which had good magnetic responsiveness. The immobilization yield, immobilization efficiency and activity recovery for xylanase were 86%, 84 % and 72 %, while for lichenase was 92 %, 86 % and 79 %, respectively. Besides, the immobilized bienzymes showed good reusability (>60 %, 10 times for xylanase, >95 %, 8 times for lichenase). The SCEs modified silica-coated magnetic microspheres are expected to provide versatile platforms for single-step of purification and immobilization of multienzymes, offering great potentials in the field of biocatalysis.

Visible Light-Driven Photoelectrochemical Platform Based Biosensor

The characterization of the photoinduced carriers' production, separation, and interfacial reaction processes is critical in developing effective photoelectrochemical (PEC) detection. The design and fabrication of a visible-light-driven PEC device based on CdSe-Co3O4@TiO2 nanoflower are presented in this report. In addition, an application for the ultrasensitive detection of viruses, including hepatitis E virus (HEV), HEV-like particles (HEV-LPs), and SARS-CoV-2 spike protein in complex lysate solution. The photocurrent response output of a CdSe-Co3O4@TiO2 based PEC device is improved compared to the individual components, TiO2 and CdSe-Co3O4. This can be ascribed to forming a heterojunction structure obtained by the hybridization of TiO2 and CdSe-Co3O4. CdSe QDs sensitization effect and strong visible light absorption can extend interfacial carrier lifetime and enhance energy conversion efficiency coupling with a robust oxygen-evolving catalyst (Co3O4) at the hole trapping site (CdSe) to improve overall system stability. The developed PEC-based biosensing device has been effectively used for quantitative virus detection through effective hybridization among antibody and viral specimens. The effective capture of the virus resulted in a linear relationship of change in photocurrent output versus the HEV concentration ranging from 10 fg mL–1 to 100 ng mL–1 with a detection limit of 3.5 fg mL–1. This CdSe-Co3O4@TiO2-based PEC device shows a significant ability to develop an improved device with affordable and portable biosensing capabilities.

An insight into the value of circular RNA in psoriasis pathogenesis and its correlation with PASI score

Background Psoriasis is a chronic hyperproliferative inflammatory disease, in which genetic and environmental factors have an important role, but the exact cause is yet unknown. Circular RNAs (circRNAs) are regulatory noncoding RNAs. They play an important role in multiple physiological and pathological processes, such as cancer, cardiovascular diseases, and neuronal diseases. Some circRNAs are involved in regulating immunity, inflammation, and cell proliferation in many types of cells. Aim To detect the expression of circRNA (Circ0061012) in psoriatic skin of diseased patients as compared with normal control persons to investigate the possible role of circRNA in psoriasis pathogenesis and its correlation with disease severity. Patients and methods The present study included 30 psoriatic patients and 30 healthy controls. A 4-mm punch skin biopsy was taken from patients (psoriatic lesion) and controls, and it was kept in the lysis solution for the stability of the studied parameters and was kept frozen at -80°Celsius till analysis of circRNA by real-time polymerase chain reaction. Results The tissue level of circRNA (Circ0061012) was significantly higher among patients with psoriasis as compared with normal control individuals. Conclusion circRNA could contribute to psoriasis pathogenesis through its immunomodulatory role, so it might be a therapeutic target in the future.

Correlation between Sociodemographic Variables and OncoE6 Cervical Swab Test Positive Cases

A cross-sectional, observational study was conducted to determine the relationship between sociodemographic variables and the development of cervical cancer through detection of detection of HPVE6 oncoprotein. A total of 47 endocervical swabs were collected from colposcopy clinic under the Department of Obstetrics & Gynaecology, Mymensingh Medical College, Mymensingh, Bangladesh, from January to December of 2015. Laboratory work was done in the Department of Microbiology of the same institution. E6 strip test is an immunochromatographic test based on detection of HPV-E6 oncoprotein in cervical swab samples. The swab specimen was treated with lysis solution and conditioning solution. Then the specimen solution was clarified by centrifugation. After that the sample solution was transferred into the detector mAb vial, wash solution vial, and finally, into developing solution vial. The test unit was then placed on a reading guide. Positive result was indicated by the appearance of purple colored test line. Out of 47 specimens, 21 (44.68%) were OncoE6 positive. Among those 21 positive cases, 19 (90.48%) were HPV-16 and 2 were (9.52%) HPV-18. Out of 11 histologically diagnosed CIN I cases, 1 (9.09%) case came out as positive. However, there were no positive cases by oncoE6 cervical swab test in CIN II and CINIII categories. Among histopathologically confirmed 22 cervical carcinoma cases, 20 (90.90%) came out as positive through this test. The results were compared with sociodemographic variables. OncoE6 is more common among ≥50 age group, and who got married before 18 years, and in multiparous women (P<0.05). Cervical cancer prevention services should be strengthen including health education, OncoE6 screening, and Anti-HPV vaccination.
 CBMJ 2022 July: vol. 11 no. 02 P: 152-157

Automating DUID methods using robotics for rapid high throughput sample preparation – Section 4 and Section 5A UK Road Traffic Act Compounds in Whole Blood

To fully automate a 96-well plate sample preparation technique using liquid handling robotics for the purpose of significantly increasing sample throughput for whole blood samples taken under the UK Road Traffic Act (RTA). Introduction: demand for analysis of whole blood samples taken under the Road Traffic Act 1988 has increased in recent years due to UK Policing and Government policy on tackling driving under the influence of drugs (DUID). Previous extraction techniques utilised by our laboratory include manual or semi-automated approaches that are lengthy and labour intensive. Both approaches require time consuming human input, to precipitate blood and prepare calibrators and quality controls. Recent developments have sped up chromatographic methods through use of techniques such as ultra-high performance liquid chromatography. However, there has been little progress with speeding up sample preparation and extraction processes, particularly for whole blood to compliment increased LC-MS/MS capacity. Use of 96-well plate formats for protein precipitation and extraction provide a platform that can be readily coupled to automated liquid handling (ALH) instruments. This provides a solution to the increase in demand by allowing for extraction of more samples, with minimal human input, in a much shorter amount of time. Here we present development, validation and implementation of an automated robotic sample preparation method for whole blood for analysis of drugs in RTA cases. Aliquots (200 μL) of blank blood and casework samples were dispensed into a 96-well plate using a robotic pipetting system before being loaded onto a Hamilton ALH. Casework samples and blank blood were spiked with internal standard. Additional blank blood samples were further spiked to produce calibrants and quality controls. The plate was mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate. A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a microelution SPE plate. Following extraction, the samples were reconstituted and the collection plate placed in the LC-MS/MS autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 30 mm, 1.7 μm, C18 column running a 4 minute gradient of water and methanol with 0.1% formic acid. Quantitation of 34 compounds (including common drugs of abuse and their metabolites) was successfully validated with CVs ranging from 3.0%–9.0% and accuracies of 85.8%–111.7%. Within batch precision had CVs of < 10.0%. The robotic platform was able to undertake sample pre-treatment and extraction of 40 case samples in 3.5 hours. The method was implemented into casework following UKAS accreditation and in 2021, 12,400 samples were analysed using the method. Introduction of this method to our laboratory has resulted in a significant increase in speed for a sample to be processed and results obtained. Each ALH is capable of extracting two batches per day; equating to 160 case sample versus 28 cases on semi-automated methods. This, along with a reduced sample volume requirement and reduced solvent consumption has had a very beneficial impact in terms of cost, turnaround times and ability to use low sample volume cases. Full automation of sample preparation of whole blood samples coupled with high speed chromatographic techniques can significantly increase throughput, decrease turnaround times and reduce solvent consumption.

Automating DUID methods using robotics for rapid high throughput sample preparation – Analysis of Delta-9-Tetrahydrocannabinol and its major metabolite Delta-9-tetrahydrocannabinol acid in whole blood

To fully automate, using liquid-handling robotics, a liquid chromatography tandem mass spectrometry method for the analysis of delta-9-tetrahydrocannabinol (THC) and delta-9-tetrahydrocannabinol acid (THC-A) in whole blood. Uptake in usage of ultra-high performance liquid chromatography (UHPLC) has led to development of methods with very fast (typically < 5 minutes) chromatographic run times used in the forensic toxicology laboratory. Nevertheless, use of whole blood for quantitative analysis continues to present challenges for delivering high throughput methods with rapid analysis times. Whole blood requires the use of solid phase extraction (SPE) for sample pre-treatment and preparation, which is time consuming and labour intensive. Recent developments in SPE technologies have produced 96-well format platforms that are used to extract greater number of samples in parallel and are ideally suitable for automated liquid handlers. With recent sensitivity increases in mass spectrometers, smaller volumes (< 250 μL per aliquot of blood) can be used for extraction in 96-well plates. This facilitates the use of robotic platforms together with rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to give truly high throughput and fast analytical methods, increasing method efficiencies, reducing human intervention and errors. Here we present the development, validation and implementation of a fully automated robotic sample preparation method for whole blood for analysis of THC and THC-A in UK driving under the influence of drugs (DUID) cases. Aliquots (200 μL) of blank blood and casework samples previously dispensed into 96-well plates by a robotic system (BRAND) were prepared by the liquid handling system (Hamilton). Casework samples and blank blood were spiked with internal standard and blank blood samples were further spiked to produce calibrants and quality controls. The plate is then mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate (Waters). A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a C18 microelution SPE plate (Waters). Following extraction, the samples are reconstituted and the collection plate is placed in the autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 50 mm, 1.7 μm, C18 column running a 4 minute gradient of water and acetonitrile with 0.1% formic acid. The method was successfully validated, with LLOQs for THC and THC-A of 1 μg/L and 5 μg/L, respectively. Precision (%CV) for THC was < 8% and < 6% for THC-A. Accuracy was within 15% of the spiked concentration for THC and THC-A across all validation batches ( n = 11). The robotic platform was able to undertake sample pre-treatment and extraction in 3.5 hours. A batch of 40 casework samples can be completed in 13 hours including the LC-MS/MS run time. The introduction of this method to our laboratory has resulted in a significant increase in the speed for a sample to be processed and results obtained. We can prepare and extract double the amount of samples in less than half the time taken by our manual method. We have reduced the amount of sample used and decreased our turnaround times all for our customer's benefit. Full automation of the sample preparation of whole blood samples using liquid handling robots can significantly improve laboratory throughput and capacity and efficiently utilise high speed chromatography providing rapid analysis from start to finish.

All-in-One Luminescent Lanthanide Coordination Polymer Nanoprobe for Facile Detection of Protein Kinase Activity.

This study developed a novel luminescent assay for kinase activity using metal-organic coordination polymer nanoparticles (Tb/ATP-Zn) as the probe. Tb/ATP-Zn, self-assembled by adenosine triphosphate (ATP), Zn2+, and Tb3+, is non-luminescent. Protein kinase A (PKA) can catalyze the transformation of ATP within Tb/ATP-Zn nanoparticles to adenosine diphosphate (ADP), which in turn effectively sensitizes the luminescence of Tb3+. Based on this mechanism, Tb/ATP-Zn can realize the facile luminescent "turn-on" sensing of protein kinase activity without the use of external ATP and substrate peptide. Under optimized conditions, the fluorescence intensities of Tb/ATP-Zn at 550 nm are linear with the PKA activity within a range of 0.3-1.5 U·μL-1. The LOD (S/N = 3) of this method is down to 0.001 U·μL-1. The presented assay also features high selectivity, long-term stability, fast response, and convenient operation. Furthermore, Tb/ATP-Zn was successfully employed for monitoring PKA activity in cell lysis solutions. Probe Tb/ATP-Zn is thus expectable to be a powerful tool for the practical study of PKA in relevant biological events.

Optimized flow cytometry protocol for dihydrorhodamine 123-based detection of reactive oxygen species in leukocyte subpopulations in whole blood

Reactive oxygen species (ROS) and the ability of immune cells to mount an oxidative burst represent an important defense during microbial invasion, but is also recognized for playing a significant role in the progression of inflammatory disorders and disease. Although neutrophils produce the strongest ROS-response, other leukocytes and their cell subsets could play a significant role. Isolation of specific cells for determining their ROS-response can affect their functionality and is laborious or hard to replicate in different settings. We have therefore established a whole blood assay, that only requires 100 μL heparinized blood and utilizes the dihydrorhodamine (DHR) 123 ROS-probe combined with cell surface antibody staining for the specific detection of ROS in several subsets of cells simultaneously using flow cytometry. Although the flow markers chosen are interchangeable with other direct conjugated and cell specific antibodies depending on the research question, we focused on neutrophils (SSChighCD16brightHLA-DRneg/low), eosinophils (SSChighCD16lowHLA-DRlow/negCD193positiveCD125positive) and monocyte subsets (SSCintermediateHLA-DRhighCD14low-positiveCD16negative-positive). As a RBC-lysis reagent we compared BD FACS Lysis Solution to the in-house prepared ammonium-chloride‑potassium based ACK Lysis Buffer, that does not fix or permeabilize the immune cells. We find that ACK-lysis of stimulated and stained samples results in superior surface staining, decreased loss of cell subsets, and enhanced resolution of the DHR-signal. Compared to the other cells analyzed in healthy blood donors, neutrophils responded with the highest ROS-response to all tested stimuli (fMLP (low stimuli), E. coli, and PMA (high stimuli)), where eosinophils and the three monocyte subsets also showed an extensive ROS-response when stimulated with E. coli or PMA. Our assay provides the possibility for researchers to examine the ROS-response of specific cell subsets in specific patient groups ex vivo and could also allow the analysis of pharmacological intervention studies targeting ROS, which ultimately can advance the field of immunological research.

A Randomized Trial of Topical Fibrinogen-Depleted Human Platelet Lysate Treatment of Dry Eye Secondary to Chronic Graft-versus-Host Disease.

PurposeThe purpose of the study was to evaluate, as a pilot trial, safety and tolerability of CAM-101 10% and 30% topical ophthalmic fibrinogen-depleted human platelet lysate (FD hPL) solution in patients with dry eye disease (DED) secondary to graft-versus-host disease (GvHD) after 6 weeks of treatment.DesignA phase I/II, pilot, prospective, multicenter, randomized, double-masked clinical trial.ParticipantsPatients with DED secondary to GvHD.MethodsSixty-four adult patients were stratified by “symptom severity” (Ocular Surface Disease Index [OSDI], ocular discomfort Visual Analog Scale (VAS), ocular symptom frequency, and use of artificial tears) and then randomized 1:1:1 to CAM-101 (FD hPL) at 10% or 30% concentration or an electrolyte (Plasma-Lyte A) vehicle control, 1 drop in both eyes, 4 times daily, for 42 days. After 42 days, control patients were offered 42 days of open-label treatment with 30% FD hPL.Main Outcome MeasuresPrimary outcome safety measures were ocular and systemic adverse events and the number of patients in each group with clinically significant change from normal to abnormal in any ocular findings. Secondary outcomes were changes from baseline to day 42 in ocular discomfort, OSDI, fluorescein corneal staining, and lissamine green conjunctival staining relative to the vehicle control. The ocular symptom frequency was assessed on a 100-point VAS.ResultsFD hPL 10% and 30% were safe and well tolerated. Relative to the vehicle control, significant decreases from baseline to day 42 were seen in the FD hPL 30% group with regard to ocular discomfort (mean decrease = −18.04; P = 0.018), frequency of burning/stinging (−20.23; P = 0.022), eye discomfort (−32.97; P < 0.001), eye dryness (−21.61; P = 0.020), pain (−15.12; P = 0.044), photophobia (−24.33; P = 0.0125), and grittiness (−20.08; P = 0.0185). Decreases were also seen for itching and foreign body sensation, though not statistically significant. Improvements were seen in tear breakup time (mean increase = 1.30 seconds; P = 0.082) and the investigator’s global evaluation 4-point scale (mean decrease = −0.86; P = 0.026). Corneal fluorescein staining was not improved. The OSDI had a mean decrease of −8.88 compared to the vehicle, although not statistically significant.ConclusionsFibrinogen-depleted human platelet lysate appears to be well tolerated, with no significant toxicity at concentrations of 10% and 30%. These initial data suggest some efficacy, especially for subjective outcome measures relative to baseline assessments and treatment with the vehicle, but larger studies are needed to confirm these effects.

Tissue and serum lipoxin A4 levels in psoriasis patients and their relation with disease severity

Background Psoriasis disease is a chronic hyperproliferative inflammatory disease, in which genetic and environmental factors have an important role, but the exact cause is yet unknown. Lipoxin A4 plays a role in improvement of psoriasis cases, it blocks the production of the proinflammatory cytokine tumor necrosis factor with increasing the production of the anti-inflammatory cytokine, c–c chemochine receptor-5 by T lymphocytes, and it reduces the perception of pain due to inflammation. Aim Detection of the expression of lipoxin A4 in the tissue and serum of psoriatic patients as compared with normal-control persons to investigate the possible role of lipoxin A4 in the psoriasis pathogenesis and its correlation with disease severity. Patients and methods The present study included 25 psoriatic patients and 25 age, sex-matched healthy controls. Four-mm punch-skin biopsy was taken from patients (psoriatic lesion) and control persons and it was kept in lysis solution for the stability of the studied parameters and it was kept frozen at –80°C till analysis of lipoxin A4 by PCR. Peripheral blood samples for detection of serum lipoxin A4 were taken. Blood samples were centrifuged and the serum was stored at –80°C and used for enzyme-linked immunosorbent assay. Results Psoriatic patients had significantly lower tissue and serum lipoxin A4 levels as compared with normal-control persons. Conclusions Lipoxin A4 could contribute to psoriasis pathogenesis through its immunomodulatory role.

Switchable Multiplex Photoelectrochemical Immunoassay of Aβ42 and Aβ40 Based on a pH-Responsive i-Motif Probe and Pyrene-Based MOF Photocathode.

In accurately diagnosing Alzheimer's disease (AD) and distinguishing AD from other dementia, the concentration ratio of amyloid-beta 42 (Aβ42) to Aβ40 is more reliable than the concentration of Aβ42 alone. For the multiplex PEC assay, generating an independent photocurrent of multiple targets on a single interface is a great challenge. Herein, an i-motif-based switchable sensing approach is proposed to construct a pH-regulated multiplex PEC immunosensor for Aβ42 and Aβ40 by using Bi-TBAPy as an efficient photoactive cathode material. An independent photocurrent signal of Aβ42 and Aβ40 is produced through the regulation of the electron-transfer tunneling distance by a pH-dependent configuration transition of the i-motif DNA. In a 96-well plate, immunological recognition of Aβ42 (or Aβ40) coupled with an enzymatic catalytic reaction produces an acidic (or alkaline) lysis solution, which triggers the formation and unravelment of the i-motif structure. The above configuration transition regulates the distance between Au NPs labeled SH-DNA and Bi-TBAPy, leading to PEC signal switching. Smart integration of the pH-responsive switchable DNA probe with a high-efficiency photocathode enables the precise monitoring of Aβ42 and Aβ40 at a single interface in a wide detection range (10 fg/mL ∼ 1 μg/mL and 1 pg/mL ∼ 1 μg/mL) with detection limit of 4.5 fg/mL and 0.52 pg/mL, respectively. The proposed i-motif-based switchable sensing strategy paves a new avenue for a multiplex PEC assay on a single interface, showing great prospects in bioanalysis and early disease diagnosis.