Cell Lysis Step Research Articles

Using Repeated Lysis Steps Fractionates Between Heterotrophic and Cyanobacterial DNA Extracted from Xenic Cyanobacterial Cultures.

Extracting DNA from cyanobacteria can be a challenge because of their diverse morphologies, challenging cellular structure, and the heterotrophic microbiome often present within cyanobacterial cultures. As such, even when our DNA yields are sufficient for sequencing, the percentage of reads coming from the cyanobacterial host can be low, leading to incomplete genomes spread across several scaffolds. In this research, we optimized a DNA isolation protocol using three iterative cell lysis steps to enrich the portion of DNA isolated coming from the cyanobacterial host rather than the heterotrophic microbiome. In order to utilize in-house nanopore sequencing, we faced a challenge in that our lysis protocol led to DNA shearing and a lower molecular weight DNA extract than is suitable for this sequencing technology. As such we used two bead-based size selection steps to remove shorter molecules of DNA before nanopore sequencing. EPI2ME analysis of the processed reads from the iterative lysis steps showed that in the first lysis the heterotrophic microbiome could make up more than half of all reads, but with each lysis the proportion of reads coming from these other species decreased. Using our iterative lysis protocol, we were able to sequence the genomes of two cyanobacteria isolated from fresh water sources around northern Mississippi, namely Leptolyngbya sp. BL-A-14 and Limnothrix sp. BL-A-16. The genomes of these isolates were assembled as closed chromosomes of 7.2 and 4.5 Mb for Leptolyngbya sp. BL-A-14 and Limnothrix sp. BL-A-16, respectively. Because some cyanobacteria have symbioses with their heterotrophic microbiome it is not always possible to prepare axenic cultures of these organisms, we hope our approach will be useful for sequencing xenic cultures of cyanobacteria, but we can also imagine applications in studying this microbiome specifically by focusing sequencing efforts on the first fraction.

A fully automated Lab-on-a-Disc platform integrated a high-speed triggered siphon valve for PBMCs extraction

Human Peripheral Blood Mononuclear Cells (PBMCs) are isolated from peripheral blood and identified as any blood cell with a round nucleus that exhibits immune responses and undergoes immunophenotypic changes upon exposure to various pathophysiological stimuli. Obtaining high-recovery and clinical-grade PBMCs without decreasing cell viability and causing stress is crucial for disease diagnosis and successful immunotherapy. However, traditional manual PBMCs extraction methods rely on manual intervention with less recovery rate and reliability. In this study, we introduced a novel and efficient strategy for the fully automated extraction of PBMCs based on a Lab-on-a-Disk (LoaD) platform. The centrifugal chip used percoll as density gradient media (DGM) for separation and extraction on account of the density difference of cells in whole blood, without labeling and any additional extra cellular filtration or cell lysis steps. Above all, we proposed a high-speed triggered siphon valve, which was closed under the speed of cell sedimentation and subsequently opened by increasing speed to complete the extraction of PBMCs. It can avoid the problem that previous siphon valves rely on unstable hydrophilic surface treatment and prime under low/zero speed conditions. With valves and the clock channel integrated on the chip, users can achieve fully automated collection of PBMCs. Compared with the clinical laboratory results, the recovery rate of extracted PBMCs was 80 %. The experimental results prove that the high-speed triggered siphon valve improves the extraction efficiency of PBMCs. The robust chips, which are not only simple to manufacture and assemble but also stable and reliable to use, have great potential in biomedical and clinical applications.

FRI540 A Novel, Rapid, Sensitive Bioassay For Thyrotropin Receptor Stimulatory Antibodies Improves Diagnosis & Management Of Autoimmune Thyroid Disease

Abstract Disclosure: G.J. Kahaly: None. M.A. Lupo: None. A.T. Bossowski: None. L. Frommer: None. B. Hatun: None. A. George: None. J. Wolf: None. Background: Autoantibody mimicry of hormone action at the thyrotropin receptor (TSH-R) and aberrant signaling of TSH-R by autoantibodies (TSH-R-Ab) causes autoimmune thyroid disease (AITD), hyperthyroidism and hypothyroidism, both of which affect millions of patients worldwide. In this multicenter, single blind study, the specificity, sensitivity and performance of a novel bioassay for stimulatory TSH-R-Ab were tested. Methods: TSH-R-Ab were measured in a blinded manner with a TSH-R binding immunoassay (Cobas e411, Roche), and both stimulatory (TSAb) and blocking (TBAb) bioassays with luciferase release as readout (Thyretain®, Quidel) according to manufacturer’s instructions. TSH-R-Ab was also measured using the new TSAb bioassay (TurboTM). TurboTM utilizes a cAMP biosensor that is an engineered form of firefly luciferase in which the cAMP-binding domain of protein kinase A is fused between the N- and C-termini such that the luciferase is inactive until cAMP binds to the cAMP binding site. Using this biosensor, luciferase activity is proportional to intracellular cAMP levels. TurboTM detects TSAb in serum at room temperature, in a real-time homogeneous format, which delivers results in 60 minutes. It does not require cell culture, sample dilution, washing or cell lysis steps. Results: Eight-hundred forty-four samples of unselected, consecutive AITD patients and control subjects, devoid of autoimmune endocrine and thyroid disorders, were included. Mean age (SD) was 49 (14.7) years, range 1-89 years. Female: male sex ratio was 3.7:1. The four TSH-R-Ab assays were negative in all 174 controls. In contrast, the Turbo TSAb, Thyretain® TSAb and the binding assays detected TSAb in 557 of 670 (83%), 526/670 (78%) and 430/637 (67%) AITD samples, respectively (TurboTM bioassay versus binding immunoassay, chi-square test p<0.001). The results of the Turbo bioassay highly correlated with both thyroid function in Graves’ hyperthyroidism as well as with the clinical activity and severity of associated thyroid eye disease (p<0.001). Inter- (two users) and intra- (two lots) assay comparison for the Turbo bioassay showed coefficient of variations of only 4%, 3%, and 2%, respectively. The two TSAb bioassays, TurboTM and Thyretain®, correlated (Spearman’s r =0.7, p<0.001). Furthermore, the magnitude of serum levels in both TSAb bioassays correlated with TSH-R-Ab binding (both p<0.001). Finally, seventeen AITD samples showed dual TSH-R-Ab positivity in the TBAb and TSAb bioassays. Conclusions: The novel, rapid, easy-to-perform, sensitive and specific TurboTM bioassay performs better than the established TSH-R-Ab binding immunoassay and the Thyretain® bioassay. It provides clinically useful information for the accurate diagnosis and management of AITD patients. Presentation: Friday, June 16, 2023

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Abstract #1153940: Specific Detection of TSAb and TBAb in Serum of Suspected AITD Patients using Two Rapid Homogenous Bioassays

Two rapid homogenous bioassays, named as Turbo TSI and Turbo TBI, have been developed to either quantitatively detect Thyroid Stimulating Antibodies (TSAb) or qualitatively detect Thyroid Blocking Antibody (TBAb) in Autoimmune Thyroid Disease (ATID) patient sera. The two bioassays do not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time to 60 minutes. This study was designed to demonstrate the high specificity of the two bioassays in comparison with the TSHR Autoantibody (TRAb) ELISA Kit (Kronus).

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay

Background aimsThe current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. MethodsThe flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. ResultsOptimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. ConclusionsThe updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique

Cephalosporin C acylase (CCA) is an essential enzyme for the one-step conversion of cephalosporin C into 7-aminocephalosporanic acid (7-ACA), an intermediate compound used to synthesize various semi-synthetic cephalosporin antibiotics. The industrial process prefers to use enzymes in immobilized form rather than soluble. A crosslinked enzyme aggregate (CLEAs) is a potential matrix-less enzyme immobilization technique to produce stable immobilized enzymes with high activity and low production costs. This study aimed to optimize the CCA immobilization using the CLEAs technique with Chitosan as a co-aggregate. The CCA lysate was obtained from harvesting CCA fermentation broth using a mutant strain of Escherichia coli through cell separation and lysis steps. Partially purified CCA by ammonium sulfate addition was conducted to obtain an active fraction of 20-60% saturation, followed by co-aggregation with Chitosan to form physical CCA aggregates. The aggregates were then immobilized by a crosslinking technique using glutaraldehyde to form CLEAs-CCA. Optimization of the immobilization process was carried out by Response Surface Methodology in three steps, (i) screening of the influencing factors, (ii) determining the level of the significant factors, and (iii) optimizing the immobilization condition. The CLEAs-CCA activity was used as a response parameter. Under optimum conditions, CLEAs-CCA activity obtained was 85.91 Ug-1.

A rapid homogenous bioassay for detection of thyroid-stimulating antibodies based on a luminescent cyclic AMP biosensor

Graves' disease (GD) is an autoimmune disease caused by antibodies to the thyroid stimulating hormone receptor (TSHR). The FDA-cleared Thyretain™ TSI bioassay is a highly specific method to detect thyroid stimulating antibodies (TSAb/TSI) in the blood of patients with autoimmune thyroid disease (AITD), particularly GD. To simplify the workflow of this bioassay and to support a semi-quantitative result, we have generated a stable CHO-K1 cell line expressing both a chimeric TSH receptor (TSHR-Mc4) and a luciferase-based homogeneous cAMP biosensor (GS luciferase). Here, we describe a rapid, real-time, homogenous bioassay (Turbo™ TSI Bioassay) to directly assess the functional activity of TSI and produce results in International Units of IU/L. The Turbo™ TSI bioassay works by measuring changes in the intracellular cAMP level induced by a G-protein coupled receptor (G-PCR) signaling cascade which is triggered by the binding of TSI to the TSHR. Upon binding to cAMP, the GS luciferase reporter is activated through conformational changes and generates light that can be measured in intact cells with a luminometer. The LoD and LoQ of the assay were determined to be 0.016 IU/L and 0.03 IU/L, respectively and the preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain™ TSI bioassay results as reference. The analytical performance of the Turbo™ TSI bioassay is comparable to the Thyretain™ TSI bioassay as evidenced by similar EC50 values for a TSHR stimulating monoclonal antibody (M22). The specificity of the Turbo™ TSI bioassay was demonstrated by showing no response to a high concentration of a human monoclonal TSHR blocking antibody (K1–70). The precision of the assay was excellent with an overall within-laboratory precision <15% CV. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo™ TSI and the Thyretain™ TSI bioassays were 98.7% and 93.5%, respectively. While both bioassays yield equivalent analytical and clinical performances, the Turbo™ TSI bioassay is much simpler to perform. It does not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time from about 21 h to 60 min. In addition, the same cell line showed its capability of detecting thyroid blocking antibodies (TBAb/TBI) in a competitive format. The Turbo™ TSI bioassay is user-friendly and is a very promising advancement to aid the diagnosis of autoimmune thyroid disease (AITD).

MODIFIKASI METODE ISOLASI DNA CETYL TRIMETHYLAMMONIUM BROMIDE (CTAB) UNTUK SAMPEL EPITEL PIPI MANUSIA

Isolation of deoxyribonucleotide (DNA) is an important step in molecular analysis. In this process, DNA must be obtained in sufficient quantities and in good quality for any further analysis. The Cetyl Trimethylammonium Bromide (CTAB) method is commonly used in DNA isolation of plant or fungal. This method is an alternative in DNA isolation since it is easy and inexpensive. This study aims to modify the CTAB method for DNA isolation from human cheek epithelium for any molecular analysis. Epithelial cells were taken from the oral cavity of the researcher. The isolation protocol included cell lysis step with CTAB buffer and proteinase-K, purification step with the addition of chloroform:isoamylalcohol (24:1), precipitation step with isopropanol. The results of the ratio analysis of DNA spectrophotometer at wavelengths of 260 and 280 nm in the range of 1.73-1.85. The quality of DNA isolation was observed by agarose gel electrophoresis and a firm band was obtained after Ethidium Bromide staining. The DNA concentration in both methods ranged from 400-480 mg/mL. The time required for both methods ranges from 2.5-3 hours. The modified CTAB method DNA isolation protocol produces DNA that has good quality and quantity for molecular analysis processes, such as Polymerase Chain Reaction (PCR).

Quantification of the organophosphate flame retardant triphenylphosphate and its main metabolite in whole blood by liquid–liquid micro-extraction and liquid chromatography-tandem mass spectrometry

This work proposes a rapid, simple and accurate analytical method for quantification of the organophosphate flame retardant triphenylphosphate (TPhP) and the biomarker diphenylphosphate (DPhP) in whole blood by a simple and effective sample preparation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification. A liquid–liquid microextraction (µLLE) from 250 µL of mouse whole blood was developed and validated to quantify TPhP and DPhP. Nature of extraction solvent, grinding for cell lysis and purification step were investigated. The use of distilled solvents proved necessary to reduce and control the contamination of the ubiquitous target compounds throughout the analytical process and to maintain a stable and repeatable signal in the analytical controls.The validation was performed by matrix-matched calibration with a correction by labelled internal standards: the limit of quantification was 1 ng/mL for DPhP and 2 ng/mL for TPhP with good linearity over the range up to 48 ng/mL for each compound. Accuracy was between 92.5% and 106.7% with relative standard deviation < 20%. The µLLE-LC-MS/MS method was successfully tested during short exposures of mice to DPhP or TPhP via drinking water or by intravenous injection.

Next generation sequencing for pathogen detection in periprosthetic joint infections.

Periprosthetic joint infections (PJI) represent one of the most catastrophic complications following total joint arthroplasty (TJA). The lack of standardized diagnostic tests and protocols for PJI is a challenge for arthroplasty surgeons.Next generation sequencing (NGS) is an innovative diagnostic tool that can sequence microbial deoxyribonucleic acids (DNA) from a synovial fluid sample: all DNA present in a specimen is sequenced in parallel, generating millions of reads. It has been shown to be extremely useful in a culture-negative PJI setting.Metagenomic NGS (mNGS) allows for universal pathogen detection, regardless of microbe type, in a 24–48-hour timeframe: in its nanopore-base variation, mNGS also allows for antimicrobial resistance characterization.Cell-free DNA (cfDNA) NGS, characterized by lack of the cell lysis step, has a fast run-time (hours) and, together with a high sensitivity and specificity in microorganism isolation, may provide information on the presence of antimicrobial resistance genes.Metagenomics and cfDNA testing have reduced the time needed to detect infecting bacteria and represent very promising technologies for fast PJI diagnosis.NGS technologies are revolutionary methods that could disrupt the diagnostic paradigm of PJI, but a comprehensive collection of clinical evidence is still needed before they become widely used diagnostic tools.Cite this article: EFORT Open Rev 2021;6:236-244. DOI: 10.1302/2058-5241.6.200099

OPTIMIZATION OF REFOLDING PROCESS FOR PRODUCTION OF RECOMBINANT CRM197 PROTEIN FROM E.COLI

E. coli is most widely, well studied and highly characterized microorganism available for production of recombinant protein. rCRM197 (Recombinant Cross Reacting Material) is a genetically modified recombinant protein which is widely used as a carrier protein. When a recombinant protein is over expressed in heterologous expression system of E. coli, it gets misfolded and aggregated in the form of inclusion bodies. Recovery of active recombinant protein from the inclusion bodies is a cumbersome task. In the current study, rCRM197 was over expressed in E. coli based host system using optimized semi defined media. The purified inclusion bodies were isolated by optimization of Cell lysis step. Solubilization was performed using high concentration of Chaotropes at alkaline pH. A refolded and functional form of native CRM197 protein was recovered by dilution of solubilizing agent using Cystine/Cysteine redox agents in Refolding buffer. The optimized process resulted in generating high quality cost effective CRM197 with drastic reduction in the production time.

Conformational Differences Are Observed for the Active and Inactive Forms of Pinholin S21 Using DEER Spectroscopy.

Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S2168) and inactive antipinholin (S2168IRS) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron-electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S2168 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S2168IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

Structural Dynamics and Topology of the Inactive Form of S21 Holin in a Lipid Bilayer Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy.

The bacteriophage infection cycle plays a crucial role in recycling the world's biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S21 is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S2168. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S2168IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S2168IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N- and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S2168IRS were incorporated into the lipid bilayer where N- and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S2168IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N- and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

Overcoming the deceptively low viability of CD45+ cells in thawed cord blood unit segments.

There is no standard methodology for post-thaw sample preparation for viability analysis of umbilical cord blood units (CBU). A common challenge faced by CB bank is for their product to meet the post-thaw cell viability threshold for CD45+ cells set at 40% by NetCord-FACT. The objective of this work was to improve the post-thaw staining method to maximize CD45+ cell viability so that clinically valuable samples meet the NetCord-FACT threshold criteria for CD45+ and CD34+ cell viabilities. Samples of CBU buffy coats and CBU segments were thawed and taken for staining. Various parameters were evaluated on CD45+ and CD34+ cell viability as measured by 7-actinomycin D (7-AAD) staining. The results revealed that initiating the staining at 20min post-thaw instead of 30, shortening the red cell lysis treatment, or performing lysis on ice and removing this step all together, all improved the viability of CD45+ cells. Using CBU segments, it was shown that the most effective approach in increasing the viability of CD45+ cells was the complete omission of red cell lysis step. However, removal of the lysis step can create technical artefacts during flow cytometry acquisition that results in an underestimation of the viability of CD34+ cells. This can be avoided and CD34+ cell viability restored with additional thresholding on CD45 signal. CB CD45+ cells are sensitive to red cell lysis treatment post-thaw; omission of this step provides the best viability and ultimately better reflects the quality of cells used for transplantation.

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing.

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.

Accuracy of cell-free embryonic DNA testing for euploidy and aneuploidy in combined spent embryo culture medium and blastocoel fluid samples with or without using a cell lysis step before amplification

Accuracy of cell-free embryonic DNA testing for euploidy and aneuploidy in combined spent embryo culture medium and blastocoel fluid samples with or without using a cell lysis step before amplification

A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein

Protein purification is an indispensable step in diverse fields of biological research or production process. Conventional purification methods including the affinity purification or the usage of self-aggregating tags suffered from many drawbacks such as the complicated steps, high cost and low efficiency. Moreover, the fusion tag usually had negative effects on the activity of the target protein. To address the above issues, here we propose a novel protein purification method which needs simple operation steps, and this method is mediated by the combination of CipA protein and a mini-intein (Synechocystis sp. PCC6803 DnaB, Ssp DnaB), depending on the assembly function of CipA and the self-cleavage function of Ssp DnaB. To realize the purification, CipA-DnaB-eGFP protein was expressed and assembled into protein crystalline inclusions (PCIs) in E. coli. Then, only cell lysis, cleavage and centrifugation steps were required to purify eGFP. Purified eGFP was in the supernatant with a purity of over 90%. The cleavage efficiency and the yield of eGFP reached 51.96% and 13.99 ± 0.88 mg/L fermentation broth, respectively. Furthermore, to broaden the application of this approach, three other proteins which were maltose binding protein (MBP), ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhP) were purified with high cleavage efficiency. The purified Kivd and AdhP remained high specific activities. This work demonstrated an effective and convenient protein purification method.

Development and Validation of a Fast DNA Extraction Protocol for Fish Products

Fish species mislabeling is a typical form of food fraud widely reported around the world. The most applied technique in the detection of fish species mislabeling is based on DNA and the primary step of most DNA-based methods is to obtain sufficient amount of high-quality DNA quickly and efficiently. However, traditional DNA extraction method, for instance the salting-out method, can be greatly compromised by the fundamental step of cell lysis that generally costs hours or even overnight. Boiling protocol (BP), which can be achieved by an incubation for 10–15 min at 95–100 °C in a heat block or boiling water bath, is one of the simplest protocols for cell lysis. In this study, we applied BP method, together with a modified lysate, for DNA extraction from simulated fresh, frozen, and roasted fish products. Traditional proteinase K-based lysis method (TP) was used as the control and compared with BP method in DNA concentration, purity, conventional PCR, and real time PCR. Finally, BP method was validated on commercial fish products, followed by DNA barcoding to identify the labeling accuracy. The results highlighted a lower concentration and A260/A230 ratio for DNA extracted by BP method than TP method for most species, regardless of the simulation methods. While conventional PCR amplification of 650 bp COI barcode can be achieved for all species in all simulations. Moreover, no significant difference (p > 0.05) was observed with Ct value for the same species between BP method and TP method. Total DNA can be successfully extracted with BP method for all commercial fish products and poor labeling accuracy was revealed for commercial roasted fish fillet products, 72.7% of which were identified as containing multiple species. Therefore, in light of the lower demand for time and cost, BP method can be considered as a valid alternative of TP method.