Isolation Procedure Research Articles

Fungi Associated with Deterioration of Onion Variety (Colour)

The incidence of fungi associated with deterioration of three varieties of onion bulbs was investigated in Usmanu Danfodiyo University, Sokoto. The bulbs were purposively collected in sterile polyethene bags through random sampling from kasuwan daji in Sokoto and brought to Mycology Laboratory, Department of Biological Sciences for analysis. Samples were prepared using standard procedures for Isolation and Inoculation of fungal organisms. Isolates were identified based on Morphological and cultural identification using mycological atlas. Pathogenicity test was out carried out using standard procedures. The result obtained revealed the presence of Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus and Rhyzopus oryzae. R. oryzae had the highest percentage of occurrence (47.4%) in Red onion, A.niger and A. fumigatus had the highest occurrence (39.1%) in white onion, and in the purple onion A. niger and A. flavus had the highest percentage of occurrence with (42.8%). The pathogenicity test indicated that all the isolates were found to facilitate spoilage of the bulbs. R. oryzae was found to be more pronounced on white onion, A. niger was prominent in purple onion, A. flavus on red onion. Therefore, fungal spoilage of onion could be determined base on the type of fungal organisms and the type of onion variety. The study could help formers to detect the type of organisms that are responsible for spoilage on the different onion varieties thus, determine the appropriate type of pesticides to apply either in the field or on storage.

Dual-Purpose 3D-Silica Nanostructure Matrix for Rapid Epigenetic Reprogramming of Tumor Cell to Cancer Stem Cell Spheroid.

Cancer stem cells (CSCs), a rare subpopulation responsible for tumorigenesis and therapeutic resistance, are difficult to characterize and isolate. Conventional method of growing CSCs takes up to 2-8 weeks inhibiting the rate of research. Therefore, rapid reprogramming (RR) of tumor cells into CSCs is crucial to accelerate the stem cell oncology research. The current RR techniques cannot be utilized for CSC RR due to many limitations posed due to isolation requirements resulting in loss of vital data. Hence, a technique that can induce CSC RR without the need for isolation procedures is needed. Here, fabrication of a 3D-silica nanostructured extracellular matrix for RR and in situ monitoring is reported. The RR is tested using three preclinical cancer models. The 3D matrix and a zeta potential study confirm an intense material-cellular interaction resulting in the enhanced expressions of surface and epigenetic biomarkers. Cancer cells require only 3-day period to form CSC spheroids with 3D-silica extracellular matrix. Real-time single-cell monitoring of the methylene blue-induced photodynamic demonstrates the dual functionality. To the authors' knowledge, this is the first study to demonstrate a CSC epigenetic reprogramming using nanostructures. These findings may pave the path for accelerating the stem cell research in oncology.

Robust Data-Driven Design for Fault Diagnosis of Industrial Drives

Due to the presence of actuator disturbances and sensor noise, increased false alarm rate and decreased fault detection rate in fault diagnosis systems have become major concerns. Various performance indexes are proposed to deal with such problems with certain limitations. This paper proposes a robust performance-index based fault diagnosis methodology using input–output data. That data is used to construct robust parity space using the subspace identification method and proposed performance index. Generated residual shows enhanced sensitivity towards faults and robustness against unknown disturbances simultaneously. The threshold for residual is designed using the Gaussian likelihood ratio, and the wavelet transformation is used for post-processing. The proposed performance index is further used to develop a fault isolation procedure. To specify the location of the fault, a modified fault isolation scheme based on perfect unknown input decoupling is proposed that makes actuator and sensor residuals robust against disturbances and noise. The proposed detection and isolation scheme is implemented on the induction motor in the experimental setup. The results have shown the percentage fault detection of 98.88%, which is superior among recent research.

The Pivotal Role of Stem Cells in Veterinary Regenerative Medicine and Tissue Engineering.

The introduction of new regenerative therapeutic modalities in the veterinary practice has recently picked up a lot of interest. Stem cells are undifferentiated cells with a high capacity to self-renew and develop into tissue cells with specific roles. Hence, they are an effective therapeutic option to ameliorate the ability of the body to repair and engineer damaged tissues. Currently, based on their facile isolation and culture procedures and the absence of ethical concerns with their use, mesenchymal stem cells (MSCs) are the most promising stem cell type for therapeutic applications. They are becoming more and more well-known in veterinary medicine because of their exceptional immunomodulatory capabilities. However, their implementation on the clinical scale is still challenging. These limitations to their use in diverse affections in different animals drive the advancement of these therapies. In the present article, we discuss the ability of MSCs as a potent therapeutic modality for the engineering of different animals' tissues including the heart, skin, digestive system (mouth, teeth, gastrointestinal tract, and liver), musculoskeletal system (tendons, ligaments, joints, muscles, and nerves), kidneys, respiratory system, and eyes based on the existing knowledge. Moreover, we highlighted the promises of the implementation of MSCs in clinical use in veterinary practice.

Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome.

The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome. Cartilage tissues were transferred into a hypoxia chamber (4% O2) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.02%, 0.1%, 0.25%, 0.5%, 1%, and 2% was applied for 0.5, 1, 2, 4, and 18h to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4h. During 4-h digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis revealed that a 4-h digestion period with 1% or 2% collagenase II plus Actinomycin D optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis.

Initial experience of a novel method for electrical isolation of the superior vena cava using cryoballoon in patients with atrial fibrillation.

Damage to the sinus node (SN) has been described as a potential complication of superior vena cava (SVC) isolation. There have been reports of permanent SN injury requiring pacemaker implantation during isolation of the SVC. It is safe and effective to isolate SVC with the second-generation 28-mm cryoballoon by using a novel method. Forty-three patients (including six redo cases) with SVC-related atrial fibrillation (AF) from a consecutive series of 650 patients who underwent cryoballoon ablation were included. After pulmonary vein isolation was achieved, if the SVC trigger was identified, the SVC was electrically isolated using the cryoballoon. First, the cryoballoon was inflated in the right atrium (RA) and advanced towards the SVC-RA junction. After total occlusion was confirmed by dye injection with total retention of contrast in the SVC, the SVC-RA junction was determined. Next, the cryoballoon was deflated, advanced into SVC, then reinflated, and pulled back gently. The equatorial band of the cryoballoon was then set slightly (4.32 ± 0.71 mm) above the SVC-RA junction for isolation of the SVC. Real-time SVC potential was observed in all patients during ablation. The mean time to isolation was 24.5 ± 10.7 s. The SVC was successfully isolated in all patients. The mean number of freeze cycles was 2.5 ± 1.4 per patient, and the mean ablation time was 99.8 ± 22.7 s. A transient phrenic nerve (PN) injury occurred in one patient (2.33%). There were no SN injuries. Freedom from AF rates at 6 and 12 months was 97.7% and 93.0%, respectively. This novel method for SVC isolation using the cryoballoon is safe and feasible when the SVC driver during AF is determined and could avoid SN injury. PN function should still be carefully monitored during an SVC isolation procedure.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

A systematic study of the impact of the isoelectric precipitation process on the physical properties and protein composition of soy protein isolates

The functional properties of soy protein isolates (SPIs), which are crucial for their successful use in food applications, depend on their protein physical properties and composition. Although the production process of SPIs is well-known and established industrial practice, fundamental knowledge on how the different isolation steps and varying isolation conditions influence these properties is lacking. Here, these characteristics were systematically investigated by assessing the impacts of the various steps of a conventional isoelectric precipitation based SPI production protocol. Protein denaturation and colloidal state were evaluated with differential scanning calorimetry and dynamic light scattering combined with (ultra)centrifugation, respectively. The protein composition (on protein subunit level) was assessed via size-exclusion chromatography. Hexane defatting was found not to cause protein denaturation. Alkaline extraction at pH values between 7.0 and 9.0 resulted in no differences in protein physical properties or composition. Subsequent acid precipitation at pH 5.5 resulted in SPIs with a lower 7S/11S ratio and higher protein solubility at neutral pH than when produced at pH 4.5 and 3.5. SPIs obtained at all evaluated precipitation pH values contained a considerable amount of aggregated protein structures. Spray-drying of SPI did not result in a higher degree of protein denaturation or in a loss of protein solubility compared to freeze-drying, but a smaller amount of soluble aggregates was observed in spray-dried SPIs. Hence, alterations in the isolation procedure can result in SPIs with moderately different physical properties and protein composition, which might lead to different functional properties and thus applicabilities in certain food systems.

A simplified procedure for isolation of primary murine microglia.

There are various approaches in which one can isolate microglia from murine brains, such as immunomagnetic, density gradient, FACS and differential adhesive methods. In this procedure a modified flask-tapping approach was used due to its simplicity and reproducibility. Our protocol requires only a single step to isolate the microglia from the mixed cell population. Once the microglia were isolated, we characterized cell purity, microglial morphology and phagocytic activity. The single-step protocol, without the need for additional astrocyte or oligodendrocyte separation, allows microglial cells to be used immediately for experimental purposes. The protocol is low-cost and can be performed in any lab with standard cell-culture equipment.

Visualizable vs. standard, non-visualizable steerable sheath for pulmonary vein isolation procedures: Randomized, single-centre trial.

Steerable sheaths (SSs) are frequently used to improve catheter contact during pulmonary vein isolation (PVI) procedures. A new type of visualizable (by electroanatomical mapping system) SS has become available in clinical treatment. We aimed to compare procedural data of visualizable vs. non-visualizable steerable sheath assisted PVI procedures in patients with atrial fibrillation (AF). In this single-centre randomized study, we enrolled a total of 100 consecutive patients who underwent PVI due to AF. A total of 100 patients were randomized into 2 groups (visualizable SS group: 50; non-visualizable SS group: 50). Acute ablation success was 100% and the rate of the first pass isolation were similar (92% vs. 89%; p = 0.88). Using visualizable SS, left atrial (LA) procedure time (53.1 [41.3; 73.1] min vs. 59.5 [47.6; 74.1] min.; p = 0.04), LA fluoroscopy time (0 [0; 0] s vs. 17.5 [5.5; 69.25] s; p < 0.01) and LA fluoroscopy dose (0 [0; 0.27] mGy vs. 0.74 [0.16; 2.34] mGy; p < 0.01) was significantly less, however, there was no difference in the total procedural time (90 ± 35.2 min vs. 99.5 ± 31.8 min; p = 0.13), total fluoroscopy time (184 ± 89 s vs. 193 ± 44 s; p = 0.79), and total fluoroscopy dose (9.12 ± 1.98 mGy vs. 9.97 ± 2.27 mGy; p = 0.76). Compared to standard, non-visualizable SS group, the number of radiofrequency ablations was fewer (69 [58; 80] vs. 79 [73; 86); p < 0.01) as well as total ablation time was reduced (1049 sec. [853; 1175] vs. 1265 sec. [1085; 1441]; p < 0.01) in the visualizable SS cohort. No major complications occurred in either group. Compared to the standard, non-visualizable SS, visualizable SS significantly reduces the left atrial procedure time, RF delivery and fluoroscopy exposure without compromising its safety or effectiveness in patients undergoing PVI procedures for AF.

Microwave Assisted Esterification of Aryl/Alkyl Acids Catalyzed by N-Fluorobenzenesulfonimide

The susceptibility of the carbonyl group towards nucleophilic attack affords the construction of various organic compounds. Thus, investigations of carbonyl activation applying greener methodologies are highly important. In the present work, among the investigated N-halo compounds, N-fluorobenzenesulfonimide (NFSi) has been found as an efficient and selective catalyst in the reaction of direct esterification of aryl and alkyl carboxylic acids supported by microwave (MW) irradiation. The comprehensive esterification of different benzoic acids and mono-, di- and tri-carboxy alkyl derivatives was performed, whereby significant reaction time reductions were achieved. The presented method used NFSi as an easily manipulatable, non-metal, water- and air-tolerant catalyst, allowing simple synthetic and isolation procedures and energy saving, compared to conventional methodologies. Importantly, in contrast to esterification under thermal conditions, where N-halo compounds behave as pre-catalysts, in the MW-supported protocol, a distinct reaction mechanism has been proposed that assumes NFSi as a sustainable catalyst. Moreover, a scale-up of the industrially important derivative was performed.

An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.

DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

Selección de lo mejor del año 2022 en arritmias y estimulación cardiaca

From the numerous publications of the last year in the field of cardiac arrhythmias, we have made a selection of the most relevant. Hot topics such as opportunistic screening for atrial fibrillation, new developments in the indications and type of pulmonary vein isolation procedures are included. In the field of ventricular arrhythmias, some publications on pharmacological treatment are discussed and the message of ablation as the most effective strategy for the treatment of monomorphic ventricular tachycardias is reinforced. New developments in left bundle branch area pacing are being reported in the field of cardiac pacing, although the evidence is not yet strong in the form of large randomized studies.

The α-Fetoprotein Molecule has One or Two Rigid Domains Depending on the Protein Purification Procedure

Abstract: α-Fetoprotein samples isolated from human cord serum by different methods were compared using circular dichroism, scanning microcalorimetry and fluorescence spectroscopy. Their structural properties and conformational stability were appreciably discriminated. In particular, the protein molecule had either one or two relatively rigid domains which can be co-operatively disrupted by heating or urea treatment. This is due to the difference of the isolation procedures in their capabilities to strip the natural ligands from the protein molecule.

“Zero-Fluoro” Approach for the repeat pulmonary vein isolation procedures after initial cryoballoon ablation

“Zero-Fluoro” Approach for the repeat pulmonary vein isolation procedures after initial cryoballoon ablation

Prevalence and Antimicrobial Resistance Profile of Methicillin Resistant Staphylococcus aureus isolates from Wound Infections in Zaria, Nigeria.

Methicillin Resistant Staphylococcus aureus (MRSA) has been a major culprit in many problematic infections especially in wound infections due to its failure to respond to antibiotics and a leading bacterial agent in community acquired infections and infections acquired in hospitals. This study investigated the prevalence and antibiotic resistant profile of MRSA in patients with wound infections in Zaria. Two hundred and seventy-two wound swabs were collected from three hospitals in Zaria, Kaduna State. These were analyzed using standard microbiological and biochemical procedures for staphylococcal isolation. Confirmed S. aureus isolates were evaluated for methicillin resistance using cefoxitin disc and multidrug resistance status. Of the 272 suspected Staphylococcal isolates analyzed, 73 (27%) were confirmed as S. aureus isolates, of which 63 (86 %) were MRSA. The infection rate of MRSA was found to be higher in males than in females. MRSA strains were 100 % resistant to the β- lactam antibiotics, with 57.1 % being multidrug resistant, but were susceptible to Gentamicin, Ciprofloxacin, Trimethoprim-sulphamethoxazole and Tetracycline. We recorded a high proportion of MRSA and Multidrug resistant-MRSA isolates from wound infections in Zaria, thus, much emphasis on infection control measures should be prioritized. There is the need to increase the effectiveness of prevention and infection control measures as well as treatment of MRSA infections.

Generating robust human valvular interstitial cell cultures: Protocol and considerations

Research in heart valve biology is a growing field that has yet to elucidate the fundamentals of valve disease. Human valvular interstitial cells (hVICs) are the best option for studying the cellular mechanisms behind valvular pathologies. However, there is a wide range of isolation procedures for these cells published in the literature. To what extent various isolation methods, patient pathologies, and seeding densities influence the behaviour of hVICs remains unclear. Here, we present an optimised method of hVIC isolation from diseased human valves donated at the time of surgery. We show that two rounds of 1000 U/mL collagenase digestion for not >2 h results in a phenotypically stable cell culture with a near complete absence of endothelial cell contamination. We also suggest that cells should be seeded at 10,000 cells/cm2 for experimentation. We found that patient pathology does not affect the success of the isolation procedure, and that instead, successful cultures are predicted by ensuring >500 mg valve tissue as starting material.

HDL Isolated by Immunoaffinity, Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.

Is there an association between left atrial outpouching structures and recurrence of atrial fibrillation after catheter ablation?

ObjectiveThe aim of this study was to investigate the impact of left atrial diverticula (LADs), left sided septal pouches (LSSPs) and middle right pulmonary veins (MRPVs) on recurrent atrial fibrillation (rAF) in patients undergoing laser pulmonary vein isolation procedure (PVI).Material and methodsThis retrospective study enrolled 139 patients with pre-procedural multiple detector computed tomography (MDCT) imaging and 12 months follow-up examination. LADs, LSSPs and MRPV were identified by two radiologists on a dedicated workstation using multiplanar reconstructions and volume rendering technique. Univariate and bivariate regression analyses with patient demographics and cardiovascular risk factors as covariates were performed to reveal independent factors associated with rAF.ResultsLADs were recorded in 41 patients (29%), LSSPs in 20 (14%) and MRPVs in 15 (11%). The right anterosuperior wall of the left atrium was the most prevalent location of LADs (68%). rAF occured in 20 patients, thereof, 15 exhibited an outpouching structure of the left atrium (LAD: 9, LSSP: 2 and MRPV: 3). Presence of an LAD (HR: 2.7, 95%CI: 1.0–8.4, p = 0.04) and permanent AF (HR: 4.8, 95%CI: 1.5–16.3, p = 0.01) were independently associated with rAF.ConclusionsLAD, LSSP and MRPV were common findings on pre-procedural cardiac computed tomography. LADs were revealed as potential independent risk factor of rAF, which might be considered for treatment planning and post-treatment observation.

Extracellular vesicle approach to major psychiatric disorders.

Over the last few years, extracellular vesicles (EVs) have received increasing attention as potential non-invasive diagnostic and therapeutic biomarkers for various diseases. The interest in EVs is related to their structure and content, as well as to their changing cargo in response to different stimuli. One of the potential areas of use of EVs as biomarkers is the central nervous system (CNS), in particular the brain, because EVs can cross the blood-brain barrier, exist also in peripheral tissues and have a diverse cargo. Thus, they may represent "liquid biopsies" of the CNS that can reflect brain pathophysiology without the need for invasive surgical procedures. Overall, few studies to date have examined EVs in neuropsychiatric disorders, and the present evidence appears to lack reproducibility. This situation might be due to a variety of technical obstacles related to working with EVs, such as the use of different isolation strategies, which results in non-uniform vesicular and molecular outputs. Multi-omics approaches and improvements in the standardization of isolation procedures will allow highly pure EV fractions to be obtained in which the molecular cargo, particularly microRNAs and proteins, can be identified and accurately quantified. Eventually, these advances will enable researchers to decipher disease-relevant molecular signatures of the brain-derived EVs involved in synaptic plasticity, neuronal development, neuro-immune communication, and other related pathways. This narrative review summarizes the findings of studies on EVs in major psychiatric disorders, particularly in the field of biomarkers, and discusses the respective therapeutic potential of EVs.