Cell Pellets Research Articles

Feasibility of using digital confocal microscopy for cytopathological examination in clinical practice

Optical imaging modalities are emerging as digital microscopy tools for tissue examination. The investigation of these techniques for potential applications in anatomic pathology practice has focused primarily on surgical pathology and has not included cytopathological specimens. We evaluated the feasibility of using digital confocal microscopy (CM) to examine cytopathological specimens. Smears and cell suspensions collected in RPMI solution were prepared from tissue scrapes obtained from surgical resections of breast, lung, liver, and kidney. Air-dried smears and cell pellets obtained from centrifugation of the cell suspensions were stained with 0.6 mM acridine orange and imaged with a CM platform. After completion of imaging, the smears were stained with Diff-Quik (DQ), and cell pellets were routinely processed, embedded in paraffin wax, cut, and stained with hematoxylin and eosin (H&E). We evaluated the mean time to acquire digital CM images; quality of images based on the extent of tissue recognition (0%, grade 0; 1–19%, grade 1; 20–50%, grade 2; >50%, grade 3); comparison of images with DQ- and H&E-stained specimens; and ability to make specific diagnoses. We imaged 91 smears and 52 cell pellets and acquired digital CM images within 2–3 min, with 92% and 88% of images, respectively, from smears and cell pellets showing grade 3 quality. On the basis of CM images, 8 smears (9%) and 7 cell pellets (14%) were categorized as benign, and 83 (91%) and 45 (88%), respectively, as malignant. Specific diagnoses were made by using digital CM images of smears and cell pellets that matched accurately with corresponding DQ- and H&E-stained preparations. The results of our first feasibility study clearly indicated the utility of CM as a next-generation digital microscopy tool for evaluating cytology specimens. Prospective clinical studies are warranted for validating our findings for potential incorporation into cytopathological clinical practice.

Internal pH and Acid Anion Accumulation in Listeria monocytogenes and Escherichia coli Exposed to Lactic or Acetic Acids at Mildly Acidic pH.

Organic acids are widely employed in the food industry to control growth of microbial pathogens such as Listeria monocytogenes and Escherichia coli. There is substantial evidence that intracellular accumulation of acid anions is a major inhibitor to cell viability, and that some bacteria are able to combat the toxic effects of anion accumulation via their ability to continue active metabolism at a lower intracellular pH (pHi). This study followed the accumulation of acid anion into the cell pellet and parallel changes in pHi in two human pathogenic strains of L. monocytogenes (N1-227 and R2-499) and in E. coli O157:H7 after exposure to sub-bacteriostatic levels of lactic and acetic acids at mildly acidic pH 6. The methodology employed in these studies included independent measures of pHi and intracellular anion accumulation. For the latter work, cells were pelleted through bromododecane to strip off extracellular water and solutes. Listeria strains accumulated 1.5-fold acetate or 2.5-fold lactate as compared to the external environment while mounting a defense against anion accumulation that included up to a 1-unit pHi drop from 7.5 to 6.5 for strain R2-499. E. coli accumulated 2.5-fold acetate but not lactate and apparently made use of combat mechanisms other than lowering pHi not explored in this study. Inulin was employed to estimate the fractional volume of cell pellet present as intracellular space. That intracellular fraction was 0.24 for E. coli, which infers that acid accumulation into the intercellular space was minimally 4 × that measured for the entire pellet. An intercellular fraction of pellet was not measurable for strains of L. monocytogenes. The data also bring into question the efficacy across bacterial species of the common, but confounding, practice of using intracellular anion accumulation as a measure of pHi, and vice versa.

A Single-Step Digestion for the Quantification and Characterization of Trace Particulate Silica Content in Biological Matrices Using Single Particle Inductively Coupled Plasma-Mass Spectrometry.

The increased use of amorphous silica nanoparticles (SiNPs) in food products, materials science, cosmetics, and pharmaceuticals has raised questions about potential hazards in the environment and in human health. Although SiNPs are generally thought to be benign, recent studies have demonstrated toxicity in different cell and animal models. Despite their ubiquitous use, SiNPs are rarely analyzed quantitatively. Often, the methods used to analyze silicon and SiNPs are difficult, costly, require the use of dangerous reagents, and are prone to interferences. Additionally, characterization of SiNPs in complex matrices requires extensive sample preparation. To address this, we propose a single-step digestion method for the determination of trace SiNP content in biological matrices. For conventional inductively coupled plasma-mass spectrometry (ICP-MS) analysis, biological samples are often digested with concentrated HNO3. We found that with conventional ICP-MS, lower limits of detection (LLOD) of silicon are too high for trace analysis. However, we found that SiNPs are stable at a strong acidic pH; thus, concentrated HNO3 could be used to digest biological samples leaving SiNPs intact. Then, by analysis with single particle ICP-MS, we found that the smallest SiNP that could be read was 185nm in size. The concentration for the LLOD was found to be 0.032ppb with interday variability in sizing and concentration at 2.5% and 6.8% respectively. Utilizing this method, SiNPs were accurately sized and counted in cell pellets and media. Our proposed method can be used to accurately quantify and characterize SiNPs (or agglomerated SiNPs) larger than the derived LLOD in a variety of biological matrices and will assist in determining relationships between exposures of SiNPs and toxicity in humans and the environment.

Extracellular degradation of a polyurethane oligomer involving outer membrane vesicles and further insights on the degradation of 2,4-diaminotoluene in Pseudomonas capeferrum TDA1

The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine, a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

New information about the toxicological profile of Prorocentrum panamense (Prorocentrales, Dinophyceae) and its global distribution

SUMMARYDinoflagellates of the genera Prorocentrum and Dinophysis are known producers of toxic compounds belonging to the okadaic acid (OA) group. The ingestion of shellfish contaminated with these toxins causes a human disease named diarrheic shellfish poisoning (DSP). In this study, the first record of Prorocentrum panamense, a potential toxin‐producer species, was reported in the Canary Islands together with its toxicological characterization. Prorocentrum panamense cells were collected during April 2017 from natural pools located in the Northeastern part of Gran Canaria. This new record represents an expansion of P. panamense distribution area, previously restricted to the Pacific Ocean, Indian Ocean, Arabian Gulf and the Caribbean, and its introduction mechanisms are discussed. Laboratory cultures of P. panamense were settled and toxin production was assessed in both cell pellets and culture media at four different growth phases (latency, exponential, early stationary and late stationary) implementing LC‐MS/MS and neuro‐2a cell‐based assay (CBA). LC‐MS/MS allowed the identification of OA in the fraction corresponding to the late stationary phase, and tests performed on neuro‐2a cells showed, for most of the fractions, OA‐like activity observable by both cell morphology changes and cell mortality. This information is fundamental for a better understanding of the genus Prorocentrum global distribution, its ecology, and risks associated to toxic producing species.

Automated DNA, RNA, and Protein Extraction from Urine for Biobanking.

DNA, RNA, and proteins are unavoidable human biomarkers. Today, blood remains the commonly used source of biomarkers despite numerous limitations. Therefore, other sources of biomarkers such as urine could be more appropriate for research in the field of bladder cancer. The aim of this study was to set up a new automated procedure for urinary DNA, RNA, and protein extraction and to evaluate their quality and quantity. This study was conducted in the setting of the COBLAnCE cohort. Urinary DNA and RNA were extracted using the Maxwell 16 system, and urinary proteins were isolated by precipitation from the supernatant and the cell pellet. The concentration and purity of nucleic acids were determined by spectrophotometry. RNA integrity was determined by the Agilent Bioanalyzer. PCR assays were also used to ensure the quality of DNA and RNA samples. The quality of protein samples obtained was determined by Western blot analysis. PCR experiments performed highlighted that it is possible to use the DNA and RNA samples for amplification, gene expression, or genotyping. However, DNA and RNA recovery from urine was highly variable among patients, with a significant impact of the patient's gender. The samples were highly degraded. Finally, our protocol of protein isolation was effective in extracting urinary supernatant proteins as well as pellet proteins. Therefore, urine samples could constitute valuable resources for subsequent investigations in bladder cancer. These samples will allow identifying new easy-access biomarkers for the early detection of cancer, monitoring cancer progression, and assessing response to therapy.

A novel modified culture medium for enhancing redifferentiation of chondrocytes for cartilage tissue engineering applications

The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days. Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83-2.75, 5.00-7.50, and 1.00-1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.

POS-804 Donor vascular endothelial growth factor gene polymorphism association with acute allograft rejection in live related renal transplant recipient patients

Renal allograft rejection risk associated with donor’s vascular endothelial growth factor (VEGF) gene polymorphism remain unelucidated till now. Although, studies have shown, an association of recipient’s VEGF polymorphism with the end-stage renal disease and early acute rejection. VEGF has pleiotropic function, which regulates vasculogenesis, endothelial cell survival signaling. Endothelial cell regulates tonicity, the permeability of blood vessels and egression of allo-stimulated inflammatory cell in intragraft compartments, thus regulate the events of rejection. In the current study, we aimed to investigate the distribution of VEGF -634C>G, -1154 G>A, -1190G>A, -1455T>C, -1499 C>T, -2578 C>A, -2549 18bp Insertion/Deletion, +405 C>G and +936 C>T SNPs among donors and recipients and to evaluate the VEGF mRNA and protein expression in intragraft tissue and in plasma. We analyzed VEGF gene polymorphism in 160 Kidney donor and 320 Kidney allograft recipients (n=160, rejecters and n=160, non-rejecters patients). One hours prior to patient admission in kidney transplant unit, a 5 ml of blood was collected in K2EDTA vials for plasma, DNA and RNA isolation from donor and recipients. After centrifugation at 1500RPM for 5 minutes, plasma supernatants were separated and 1ml cell pellet was used for DNA isolation and 1 ml pellet was dissolved in Trizole solution for RNA isolation. A core of allograft biopsy specimen was collected from 50 patients for allograft recipient for VEGF gene and protein expression in intragraft tissue by the RT-PCR and Immunohistochemistry techniques respectively and soluble plasma VEGF level was analyzed by the ELISA. Genotyping of VEGF SNPs were done through PCR- RFLP technique. Difference between donor and recipient group was calculated by the Fisher exact test. P value <0.05 was considered statistically significant. The odd ratio was calculated to measure the difference in association between genotypes. On comparison between donors and recipients genotypes of VEGF +936 C>T [CT (OR=7.16; 95% CI=4.33- 11.84; P=0.00) and TT (OR=49.30; 95% CI=11.84-205.29; P=0.00)], -1154 G>A [AG (OR=2.22; 95% CI=1.40-3.50; P=0.00)], -1190G>A [GG (OR=2.21; 95% CI=1.22-4.01; P=0.00)], -634C>G [GG (OR=2.34; 95% CI=1.34-4.10; P=0.00)], -2549 18bp Insertion/Deletion [ID (OR=1.58; 95% CI=1.01- 2.46; P=0.04) were significantly associated with risk of rejection. Genotypic frequencies of +936 C>T [TT (OR=2.43; 95% CI=1.33-4.44; P=0.004)], -1154 G>A [GG (OR=1.94; 95% CI=1.03-3.67; P=0.04)], -1190G>A [GA (OR=1.83; 95% CI=1.07-3.13; P=0.02)], -2549 18bp Insertion/Deletion [ID (OR=2.35; 95% CI=1.38- 3.99; P=0.002) and -1455T>C [TT (OR=3.13; 95% CI=1.07-9.10; P=0.03)] shown risk of allograft rejection whereas mutant genotypes of -2578 C>A [ CA (OR=0.45; 95% CI=0.26-0.79; P=0.005) and CC (OR=0.23; 95% CI=0.11-0.46; P=0.000)] and +405 C>G [GG (OR=0.43; 95% CI=0.20-0.91; P=0.02)] have shown protective association with rejection. The intragraft VEGF mRNA and protein expression and plasma soluble VEGF level was significantly higher in allograft recipients compared to healthy donor, which was further higher in rejecters compared to non-rejecters. Mutant genotypes of VEGF +936 C>T, -1190G>A, -2549 18bp Insertion/Deletion and -1455T>C SNPs was associated with increased risk for allograft rejection

GENERATION OF ANTIGEN-SPECIFIC REGULATORY T CELLS BYLACTOBACILLUS SPECIES

Abstract INTRODUCTION There is a need for novel treatments for patients with inflammatory bowel disease (IBD) who do not respond to medical therapy. It is likely that IBD arises from dysfunctional interactions between the mucosal immune system and the gut microbiome, and manipulation of this interaction is therefore a promising therapeutic target. Signaling through the T cell receptor (TCR) may have a powerful role in controlling inflammation in IBD, as chimeric antigen receptor regulatory T cells (Tregs) which recognize colitis-associated antigens have been demonstrated to control inflammation in mouse models. We hypothesized that model antigen expression by two potential Treg-inducing organisms, Lactobacillus reuteri and L. plantarum, could stimulate antigen-specific Tregs which may be able to prevent colitis. Here, we demonstrate that expression of the ovalbumin-derived model peptides OT1 and OT2 by these species are recognized in vitro by transgenic CD8 and CD4 mouse T cells specific for OT1 and OT2, respectively. We also demonstrate that OT2 expression by L. reuteri can be recognized in vivo and generate antigen-specific Tregs. METHODS We generated expression of OT1 and OT2 peptides in L. reuteri and L. plantarum in three subcellular locations: secreted, cell membrane-associated, and cell wall-associated. For in vitro assays, mouse dendritic cells (DCs) were pulsed with heat-killed cell pellets from each of these constructs or culture supernatant from secreted constructs, then incubated with either OT1 CD8+ or OT2 CD4+ T cells pre-stained with CellTrace Violet (CTV) for 3.5 days. For in vivo analyses, mice were gavaged with L. reuteri transformed with either empty vector or wall-associated OT2, or given ovalbumin in drinking water, followed by adoptive transfer of CTV-stained OT2 cells. OT2 cells were recovered from mesenteric lymph nodes (mLN) 7 days after adoptive transfer and stained for Foxp3. RESULTS All OT2 constructs induced significant in vitro proliferation (decreased CTV staining) of OT2 cells compared to strains expressing empty vector, while secreted and membrane-associated OT1 constructs induced significant proliferation of OT1 cells. In vivo, L. reuteri expressing wall-associated OT2 induced significant OT2 proliferation in mLN compared to L. reuteri transformed with empty vector. A significantly higher proportion of these proliferated cells were Tregs (Foxp3+) compared to proliferated cells in mice treated with L. reuteri transformed with empty vector. CONCLUSION These findings suggest that Lactobacillus species can generate antigen-specific proliferation of T cells in vitro, and that L. reuteri may be capable of inducing antigen-specific Tregs in vivo. Expression of antigens by this species may represent a method of generating tolerance in the intestine, which could lead to novel therapies for controlling inflammation in IBD.

Microscopic and spectroscopic bioassociation study of uranium(VI) with an archaeal Halobacterium isolate.

The safe disposal of high-level radioactive waste in a deep geological repository is a huge social and technical challenge. So far, one of the less considered factors needed for a long-term risk assessment, is the impact of microorganisms occurring in the different host rocks. Even under the harsh conditions of salt formations different bacterial and archaeal species were found, e. g. Halobacterium sp. GP5 1–1, which has been isolated from a German rock salt sample. The interactions of this archaeon with uranium(VI), one of the radionuclides of major concern for the long-term storage of high-level radioactive waste, were investigated. Different spectroscopic techniques, as well as microscopy, were used to examine the occurring mechanisms on a molecular level leading to a more profound process understanding. Batch experiments with different uranium(VI) concentrations showed that the interaction is not only a simple, but a more complex combination of different processes. With the help of in situ attenuated total reflection Fourier-transform infrared spectroscopy the association of uranium(VI) onto carboxylate groups was verified. In addition, time-resolved laser-induced luminescence spectroscopy revealed the formation of phosphate and carboxylate species within the cell pellets as a function of the uranium(VI) concentration and incubation time. The association behavior differs from another very closely related halophilic archaeon, especially with regard to uranium(VI) concentrations. This clearly demonstrates the importance of studying the interactions of different, at first sight very similar, microorganisms with uranium(VI). This work provides new insights into the microbe-uranium(VI) interactions at highly saline conditions relevant to the long-term storage of radioactive waste in rock salt.

Diabetic nephropathy patients show hyper-responsiveness to N6-carboxymethyllysine.

The aim of this study was to evaluate the impact of N6-carboxymethyllysine (CML) on NF-κB gene expression and tumor necrosis factor (TNF) production in diabetic nephropathy. This was an observational study comprised of three groups: diabetic nephropathy (n=30), type II diabetes mellitus (n=28), and healthy volunteers (n=30). Blood samples collected from the study participants were cultured for 24 h in the presence of CML or an appropriate control. After incubation, the cultures were centrifuged to separate the cells from the conditioned media. cDNA was prepared from the cell pellet and used to quantify NF-κB gene expression by quantitative real-time polymerase chain reaction (PCR). The conditioned media were used to measure TNF production by enzyme-linked immunosorbent assay (ELISA). The CML-induced fold change in NF-κB gene expression was significantly different among the study groups (P=5.4×10-5). Also, the CML-induced fold change in TNF levels was significantly different among the three groups (P=4.3×10-8). These results imply that patients with diabetic nephropathy and type II diabetes mellitus showed an elevated response to CML.

Reduced sRAGE Production and ADAM10 Gene Expression in Peripheral Blood Samples of Diabetic Nephropathy Patients

Introduction: Excessive signalling via the Receptor for Advanced Glycation End products (RAGE) is implicated in inflammatory renal damage in Diabetic Nephropathy (DN). RAGE signaling is modulated by its soluble form (sRAGE) that arises due to ectodomain cleavage catalysed by A Disintegrin and Metalloproteinase 10 (ADAM10). The sRAGE functions as a decoy and competes with RAGE for binding to the cognate ligand. Aim: The aim of this study was to evaluate sRAGE and ADAM10 gene levels in peripheral blood samples of Diabetic Nephropathy patients. Materials and Methods: The present observational study was conducted in the Department of General Medicine, RL Jalappa Hospital and Research Centre, Kolar, Karnataka, India, between January 2019 to April 2020. Study comprised of three groups: group 1 of 30 DN patients; group 2 of 28 Type II Diabetis Mellitus (T2DM) patients without microvascular complications and group 3 comprised of 30 healthy volunteers. Blood samples obtained from the study participants were cultured for 24 hours along with insulin treatment or a suitable control. sRAGE levels were measured in the conditioned media by Enzyme Linked Immunosorbent Assay (ELISA) technique. Expression of the ADAM10 gene was measured in the cell pellet by using the quantitative real-time Polymerase Chain Reaction (PCR) technique. Results: A total of 88 subjects were included in the study, with 30 patients in group 1 (DN, mean age 55.34±7.76 years), 28 patients in group 2 (T2DM, mean age: 55.07±7.7 years) and 30 subjects in group 3 (healthy individuals, mean age: 55±8.05 years). sRAGE levels were significantly lower in nephropathy patients when compared to healthy volunteers (p=2.5×10-9). Likewise, ADAM10 expression levels were also significantly lower in nephropathy patients when compared to healthy volunteers (p=1.3×10-4). Insulin treatment led to significantly higher sRAGE production in healthy volunteers as compared to T2DM patients (p-value 3.3×10-11). Insulin treatment leads to significant upregulation of the ADAM10 gene expression in healthy volunteers as compared to DN and T2DM. Conclusion: Abnormal RAGE signaling in DN may arise due to diminished sRAGE production as a consequence of reduced ADAM10 expression.

Differentiation of CD117+ Amniotic Fluid Stem Cells towards Nephron Progenitors.

Objective:The study aimed at isolation of CD117+ stem cells from amniotic fluid samples followed by their invitro differentiation towards nephron progenitors that can be potentially used for regenerative medicine studies and to further understand pathways involved in renal pathogenesisMethods:This experimental study was conducted at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences, OJHA Campus Karachi from November 2019 to December 2020. After patient consent, a Pfannenstiel incision was performed by the gynecologist through abdominal and uterine muscles without cutting into Amniotic Membrane. Using a needle of 5CC syringe connected to sterile Redivac bottle, a blunt end insertion was passed through the membrane and the amniotic fluid was aseptically sucked into Redivac bottle, the ice bag was used for transporting amniotic fluid from hospital to the lab and samples were processed within 60 minutes after collection. Amniotic fluid was centrifuged at 4º C for 20 minutes at 1400xg. After centrifugation the cell pellet was treated for analysis of CD117+ cells using flowcytometry, once small percentage of CD117+ cells were identified the cells were prepared for differentiation and that was carried out using specific growth factors including BMP4, BMP7, FGF2, and retinoic acid, providing the niche to the stem cells for differentiation towards nephron progenitors which was confirmed by protein expression of Wilms Tumor-1 (WT1) using immunofluorescence analysis. The sample size for this invitro work was n=3.Results:We successfully isolated small percentage of CD117+ cells in amniotic fluid followed by in vitro expansion and differentiation towards nephron progenitor cells (NPCs) using well defined media and growth factors, initially differentiated cells were spindle shaped and showed fibroblastic appearance later at stage of nephron progenitors it attained the shape of rounded big clusters, differentiated cells stained positive for WT1 and negative for cluster of differentiation (CD117). Therefore, confirming the successful isolation and differentiation of amniotic fluid stem cells towards nephron progenitors.Conclusion:To the best of our knowledge this is the first study from the country on the use of Amniotic fluid stem cells and their differentiation towards nephron progenitors that can be used as substitution source of cell therapy for exploration of renal diseases at cellular and molecular level and potential regenerative medicine applications.

Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat β-cell lines INS-1 and INS-1832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Effect of the natural polymethoxylated flavone artemetin on lipid oxidation and its impact on cancer cell viability and lipids

The biochemical class of the polymethoxylated flavonoids represents uncommon phenolic compounds in plants presenting a more marked lipophilic behavior due to the alkylation of its hydroxylic groups. As a polymethoxylated flavone, which concerns a different bioavailability, artemetin (ART) has been examined in vitro against lipid oxidation and its impact on cancer cells has been explored. Despite this flavone only exerted a slight protection against in vitro fatty acid and cholesterol oxidative degradation, ART significantly reduced viability and modulated lipid profile in cancer Hela cells at the dose range 10–50 μM after 72 h of incubation. It induced marked changes in the monounsaturated/saturated phospholipid class, significant decreased the levels of palmitic, oleic and palmitoleic acids, maybe involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, ART compromised normal mitochondrial function, inducing a noteworthy mitochondrial membrane polarization in cancer cells. A dose-dependent absorption of ART was evidenced in HeLa cell pellets (15.2% of the applied amount at 50 μM), coupled to a marked increase in membrane fluidity, as indicate by the dose-dependent fluorescent Nile Red staining (red emissions). Our results validate the ART role as modulatory agent on cancer cell physiology, especially impacting viability, lipid metabolism, cell fluidity, and mitochondrial potential.

Reference Samples to Compare Next-Generation Sequencing Test Performance for Oncology Therapeutics and Diagnostics.

Diversity of laboratory-developed tests (LDTs) using next-generation sequencing (NGS) raises concerns about their accuracy for selection of targeted therapies. A working group developed a pilot study of traceable reference samples to measure NGS LDT performance among a cohort of clinical laboratories. Human cell lines were engineered via CRISPR/Cas9 and prepared as formalin-fixed, paraffin-embedded cell pellets ("wet" samples) to assess the entire NGS test cycle. In silico mutagenized NGS sequence files ("dry" samples) were used to assess the bioinformatics component of the NGS test cycle. Single and multinucleotide variants (n = 36) of KRAS and NRAS were tested at 5% or 15% variant allele fraction to determine eligibility for therapy with the EGFR inhibitor panitumumab in the setting of metastatic colorectal cancer. Twenty-one (21/21) laboratories tested wet samples; 19 of 21 analyzed dry samples. Of the laboratories that tested both the wet and dry samples, 7 (37%) of 19 laboratories correctly reported all variants, 3 (16%) of 19 had fewer than five errors, and 9 (47%) of 19 had five or more errors. Most errors were false negatives. Genetically engineered cell lines and mutagenized sequence files are complementary reference samples for evaluating NGS test performance among clinical laboratories using LDTs. Variable accuracy in detection of genetic variants among some LDTs may identify different patient populations for targeted therapy.

Development of an Autoinducible Plasmid for Recombinant Protein Production.

The production of recombinant proteins in E. coli involves such factors as host strains, expression vectors, culture media, and induction methods. The typical procedure to produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable vector to construct an overexpression plasmid, (2) transformation of a strain specialized for protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium and induction of the protein production at a right moment, and (4) further growth to get the maximum yield. There are hurdles involved in each of these steps, and researchers have developed many materials or methods, which often require special recipes or procedures. To eliminate the special requirements for recombinant protein production by using readily available materials. Also to save time and effort in the routine protein production work. We started with a vector capable of producing a target protein fused to the C-terminus of the maltose-binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase) was replaced with sacB. We chose 3 stationary phase promoters and made hybrids of them by mixing halves from each one. The T5 promoter was replaced with these stationary phase promoters or their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST genes were inserted in the place of sacB, and their production yields were compared with the original plasmid in the conventional way of expression. We constructed an expression plasmid with an autoinducible promoter working in a host that was not specially designed for protein production and in a TB medium that did not contain any secret ingredient, nor was it difficult to prepare unlike Studier's defined medium. This plasmid also contains a color indicator that turns red when protein production is successful. We tested our system with the maltose-binding protein (MBP) and the glutathione S-transferase (GST), and showed that both proteins were produced to a level comparable to what the commercial medium and/or the specialized strain yielded. We developed a plasmid equipped with an autoinducible promoter, a hybrid of the two promoters which were activated at the stationary phase. This plasmid does not need a special E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color, which can be used as an indicator of a successful production of the target protein and as a predictive measure of the amount of the produced target protein. We speculate that this plasmid will have its greatest advantage when growing cells at low temperatures, which would inevitably take a long time.

Accumulation of radio-iron and plutonium, alone and in combination, in Pseudomonas putida grown in liquid cultures

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose–response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this work Pseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10–20 mGy d−1) of 239Pu and 55Fe, both alone and in combination, for a period of 20 days, and the accumulation of 239Pu and 55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with 239Pu and stable Fe (primarily 56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that 239Pu inhibited the uptake of 55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by 239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

Characterization and cytotoxicity assessment of biosurfactant derived from Lactobacillus pentosus NCIM 2912.

Noteworthy properties of biosurfactant (BS) are fascinating scientific fraternity to explore them for food, medicinal, cosmetic, or pharmaceutical etc. applications. Newer products intended for pharmaceutical purposes are mandatory to go through pragmatic evaluation protocols. BS, being less cytotoxic, offers an ideal candidature for widespread applications in the healthcare sector. The goal of the current study was the isolation, physico-chemical characterization, and safety assessment of cell-associated biosurfactant (CABS) from Lactobacillus pentosus NCIM 2912. The culture was grown in a 3-L fermentor to produce CABS from the cell pellets through procedures like centrifugation, filtration, dialysis, column chromatography, and freeze-drying. Further, physical properties like surface tension (ST), critical micelle concentration (CMC), contact angle (CA), emulsification activity, stability of emulsion (height of emulsion, the extent of coalescence, and appearance), and ionic character of CABS were evaluated. Analytical characterization through TLC, FTIR, NMR, and GC-MS was carried out. The physico-chemical characterization revealed CABS as an anionic, multicomponent glycolipopeptide having a hydrophobic chain comprising butanoic acid (C4), decanoic acid (C10), undecanoic acid (C11), tridecanoic acid (C13), hexadecenoic acid (C16), and octadecanoic acid (C18). The oil-in-water (O/W) emulsions formed by CABS with various oils (olive, sesame, soybean, coconut) were stabilized up to the 7th day of storage and were analogous with polysorbate 80 (emulsifier/defoamer used in food industries). The O/W emulsions are quite stable at room temperature with no evidence of coalescence of droplets around 1week. The cytotoxicity of CABS was evaluated through MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cytotoxicity study performed on the human embryonic kidney (HEK 293), mouse fibroblast ATCC L929 and human epithelial type (HEP-2) cell lines recorded viability of 90.3 ± 0.1%, 99.2 ± 0.43, and 94.3 ± 0.2% respectively. The toxicity of the BS was comparable to that of the commercially used rhamnolipid sample. Thus, CABS derived from L. pentosus NCIM 2912 pose promising applications in the pharmaceutical, food industries acquiescently. The multifunctional potential of the incredibly versatile microbial product like BS from lactic acid bacteria (LAB) certainly contributes to wider avenues for varied industries.