Nucleic Acid Content Research Articles

Evaluation of Aspergillus aculeatus GC-09 for the biological control of citrus blue mold caused by Penicillium italicum

Blue mold caused by Penicillium italicum is a severe postharvest disease in citrus fruits. In this study, the fermentation product (FP-E) of Aspergillus aculeatus GC-09, an endophytic fungus isolated from a citrus plant, was found to exhibit antifungal activity against P. italicum with a MIC of 0.3125 mg/mL. The fungus A. aculeatus GC-09 was identified based on the studies of morphology and ITS nucleotide sequence. FP-E significantly inhibited the spore germination and mycelial growth of P. italicum. Scanning electron microscopy (SEM) results of P. italicum treated with FP-E showed shrunken, distorted and collapsed hyphae and conidiospores, indicative of the cell membrane damage, which was further confirmed by the propidium iodide (PI) fluorescent staining analysis. Consistent with the microscopy observation, FP-E led to the leakage of cellular constituents from P. italicum, which is evident from the increase in electrical conductivity and nucleic acid contents in the mycelial solution incubated with FP-E. In addition, FP-E treatment considerably increased the intracellular reactive oxygen species (ROS) content, and reduced the enzyme activities of both catalase (CAT) and peroxidase (POD) in P. italicum cells. Furthermore, orange fruits treated with FP-E showed fewer disease symptoms compared to the untreated fruits. These results suggested that the antifungal activity of FP-E might be associated with the disruption of cell membrane integrity, the accumulation of ROS level, and the reduction of the antioxidant enzymes activity of P. italicum. Therefore, A. aculeatus GC-09 might be a potential microbial resource for the biocontrol of citrus postharvest blue mold.

Antibacterial activity and mechanism of phillyrin against selected four foodborne pathogens

The antibacterial activity of phillyrin on Escherichia coli ATCC8739 (E. coli ATCC8739), Bacillus subtilis BS08, Staphylococcus aureus ATCC6538 (S. aureus ATCC6538) and Salmonella ATCC14028 was determined by filter paper method. The minimum inhibitory concentration (MIC) of the four tested bacteria was investigated by plate coating method to evaluate antibacterial ability. The antibacterial mechanism was further investigated by measuring growth curve, electric conductivity, nucleic acid content, Na+/K+-ATPase activity and polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that phillyrin had antibacterial effects and the MICs against Escherichia coli ATCC8739, Bacillus subtilis BS08, Staphylococcus aureus ATCC6538 and Salmonella ATCC14028 were 2.15, 3.16, 3.02 and 3.40 μg/mL respectively, and the antibacterial effect on Escherichia coli ATCC 8739 was more significant (p< 0.05) than that on other bacteria. Scanning electron microscopy (SEM) indicated that phillyrin destroyed the morphology of the cells and the cells ruptured. The leakage of intracellular substances led to an increase in nucleic acid content and an increase in electric conductivity in the bacterial suspension; SDS-PAGE analysis indicated that phillyrin could inhibit protein synthesis; in addition, phillyrin could reduce Na+/K+-ATPase activity. Therefore, phillyrin had obvious antibacterial ability and inhibited the expression of bacterial proteins by destroying the cell membrane structure, resulting in the death of the cells.

Механизм метаболической клеточной интеграции в симпатическом ганглии

Content of nucleic acids (NA) dynamics were studied in neuron and glial cells to assess the mechanisms of metabolic cellular integration using an experimental model “off function” nicotine cholinergic receptors (H-XP) in the cranial cervical sympathetic ganglion of rabbits. Content of NA was determined by cytophotometry after staining sections using the Einarson method. The article shows that there is a mechanism in the sympathetic ganglion coordinated metabolic changes neuron and gliocytes NA, an important component of which is the synaptic signal through H-XP.

Optimization of cationic polymer-mediated transfection for RNA interference.

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.

Limited-resource preparable chitosan magnetic particles for extracting amplification-ready nucleic acid from complex biofluids.

Extraction and concentration of pure nucleic acid from complex biofluids are the prerequisite for nucleic acid amplification test (NAAT) applications in pathogen detection, biowarfare prevention, and genetic diseases. However, conventional spin-column mediated nucleic acid extraction is constricted by the requirement for costly power-intensive centralized lab infrastructure, making it unsuitable for limited-resource settings. Significant progress in lab-on-a-chip devices or cartridges (e.g., Cepheid GeneXpert®) that integrate nucleic acid extraction and amplification has been made, but these approaches either require additional equipment or are costly. Similarly, their complexities make them difficult to fabricate in low-resource settings by the end-user themselves. The application of magnetic particles such as silica-coated iron oxide beads for nucleic acid extraction is relatively instrument-free, rapid, user-friendly, and amenable to automation. But, they rely on hazardous chaotropic salt chemistry and ethanol desalting that could limit their efficacy for downstream NAATs. Recent advances in several types of novel material (e.g., polyamine) coated magnetic bead-based chaotropic salt-free extraction methods offer a possible solution to this problem. However, these materials also involve multistep synthesis impermissible in limited-resource settings. To offer a possible instrument-free magnetic particle-based nucleic acid extraction doable at limited-resource settings, we investigated the nucleic acid capture ability of two chitosan-coated magnetic particles that are preparable by minimally trained personnel using only a water bath and a magnetic stirrer within 6-8 h. We quantitatively probed the efficiency of the passive (without any electrical shaking or vortex-aided) DNA magnetocapture (i.e., binding to chitosan magnetic particles, physical separation from its sample of origin, and release from the particles) using UV260. To explore their suitability towards clinically relevant sensitive downstream NAATs, 100-1000 copies (i.e., in the order of zeptomole) of Escherichia coli (E. coli) or human genomic DNA from aqueous solution, crude cell lysate, and fetal bovine serum were extracted by them and then successfully detected using quantitative real-time loop-mediated isothermal amplification (LAMP) or real-time polymerase chain reaction (PCR). Alongside, their suitability with gel-based LAMP, colorimetric LAMP, and in situ (on beads) LAMP was also probed. The required optimization of the amplification methods has been discussed. Overall, the turnaround time for the magnetocapture combined with NAAT was 1.5-2 h and is thus expected to aid in rapid clinical decision making. With the ease of preparation, reproducibility, and compatibility with downstream NAATs, we anticipate that these magnetic particles would facilitate the expansion and decentralization of nucleic acid-based diagnosis for limited-resource settings.

Antibacterial Effect and Mechanism of the Ethyl Acetate Extract of Cichorium Intybus L. Against Staphylococcus Aureus

Objective: Cichorium intybus L. (CILEAE) has many functions, such as antibacterial, anti-inflammatory, hypoglycemic, lipid-lowering, liver-protection and anti-oxidation. The subject aims to investigate the antibacterial activity and mechanism of the ethyl acetate extract of CILEAE against Staphylococcus aureus (S. aureus). Methods: Firstly, the antibacterial activity of CLEAE against several kinds of frequently pathogenic bacteria was determined by microbroth dilution method, then the antibacterial mechanism was investigated by Scanning Electron Microscope (SEM), permeability of cell membrane, respiratory metabolic pathway, protein band change in SDS-PAGE of soluble protein, and DAPI fluorescence staining. Results: The results of the Minimum Inhibitory Concentration (MIC) showed that CILEAE could significantly inhibit S. aureus and Escherichia coli in the tested concentration range. SEM results showed that 24 h after the application of CILEAE to S. aureus, the surface areas of the phage showed signs of shrinkage, dryness, distortion and deformation, vesicular or irregularly shaped protrusions and contraction of other structures. Culture fluid conductivity results showed that the cell membrane permeability was changed by CILEAE; The results of respiratory metabolism inhibition experiments showed minimal superimposition rates of CILEAE; The results of protein band analysis showed that CILEAE could inhibit protein synthesis in S. aureus, and the results of DAPI fluorescence staining showed that CILEAE treatment significantly reduced the nucleic acid content in vivo. Conclusion: CILEAE has strong bacteriostatic activity against S. aureus. Its bacteriostatic mechanism may be through destroying the integrity of bacterial cell wall and cell membrane, inhibiting bacterial respiratory metabolism, and inhibiting the synthesis of bacterial protein.

The Role of Metagenomics and Next-Generation Sequencing in Infectious Disease Diagnosis.

Metagenomic next-generation sequencing (mNGS) for pathogen detection is becoming increasingly available as a method to identify pathogens in cases of suspected infection. mNGS analyzes the nucleic acid content of patient samples with high-throughput sequencing technologies to detect and characterize microorganism DNA and/or RNA. This unbiased approach to organism detection enables diagnosis of a broad spectrum of infection types and can identify more potential pathogens than any single conventional test. This can lead to improved ability to diagnose patients, although there remains concern regarding contamination and detection of nonclinically significant organisms. We describe the laboratory approach to mNGS testing and highlight multiple considerations that affect diagnostic performance. We also summarize recent literature investigating the diagnostic performance of mNGS assays for a variety of infection types and recommend further studies to evaluate the improvement in clinical outcomes and cost-effectiveness of mNGS testing. The majority of studies demonstrate that mNGS has sensitivity similar to specific PCR assays and will identify more potential pathogens than conventional methods. While many of these additional organism detections correlate with the expected pathogen spectrum based on patient presentations, there are relatively few formal studies demonstrating whether these are true-positive infections and benefits to clinical outcomes. Reduced specificity due to contamination and clinically nonsignificant organism detections remains a major concern, emphasizing the importance of careful interpretation of the organism pathogenicity and potential association with the clinical syndrome. Further research is needed to determine the possible improvement in clinical outcomes and cost-effectiveness of mNGS testing.

Gibberellic Acid and Jasmonic Acid Improve Salt Tolerance in Summer Squash by Modulating Some Physiological Parameters Symptomatic for Oxidative Stress and Mineral Nutrition.

Gibberellic acid (GA) and jasmonic acid (JA) are considered to be endogenous regulators that play a vital role in regulating plant responses to stress conditions. This study investigated the ameliorative role of GA, JA, and the GA + JA mixture in mitigating the detrimental effect of salinity on the summer squash plant. In order to explore the physiological mechanisms of salt stress alleviation carried out by exogenous GA and JA, seed priming with 1.5 mM GA, 0.005 mM JA, and their mixture was performed; then the germinated summer squash seedlings were exposed to 50 mM NaCl. The results showed that a 50 mM NaCl treatment significantly reduced shoot and root fresh and dry weight, water content (%), the concentration of carotenoid (Car), nucleic acids, K+, and Mg++, the K+/Na+ ratio, and the activity of catalase (CAT) and ascorbate peroxidase (APX), while it increased the concentration of proline, thiobarbituric acid reactive substances (TBARS), Na+, and Cl− in summer squash plants, when compared with the control. However, seed priming with GA, JA and the GA + JA mixture significantly improved summer squash salt tolerance by reducing the concentration of Na+ and Cl−, TBARS, and the Chl a/b ratio and by increasing the activity of superoxide dismutase, CAT, and APX, the quantities of K+ and Mg++, the K+/Na+ ratio, and the quantities of RNA, DNA, chlorophyll b, and Car, which, in turn, ameliorated the growth of salinized plants. These findings suggest that GA and JA are able to efficiently defend summer squash plants from salinity destruction by adjusting nutrient uptake and increasing the activity of antioxidant enzymes in order to decrease reactive oxygen species accumulation due to salinity stress; these findings offer a practical intervention for summer squash cultivation in salt-affected soils. Synergistic effects of the GA and JA combination were not clearly observed, and JA alleviated most of the studied traits associated with salinity stress induced in summer squash more efficiently than GA or the GA + JA mixture.

Label-free spectroscopic characterization of exosomes reveals cancer cell differentiation

Exosomes (EXOs) are considered an exceptionally promising source of cancer biomarkers for personalized medicine and liquid biopsy. Despite this potential, the EXOs translation process in diagnostics is still at its birth, and the development of reliable and reproducible methods for their characterization is highly demanded. Fourier Transform Infrared (FTIR) Spectroscopy is perfectly suited for this purpose, as it can provide a label-free biochemical profile of EXOs in terms of lipid, protein, and nucleic acid content. Here we evaluated the applicability of FTIR spectroscopy to the study of cancer-derived EXOs as a function of cell differentiation. For this purpose, we used N-acetyl-l-Cysteine (NAC) to induce a controlled differentiation in human colon carcinoma cells from a proliferative mesenchymal morphology to a less invasive epithelial phenotype, as measured with fluorescence and electron microscopy. EXOs derived from cells with different phenotypes showed significant variation in the relative intensity of the amide I-II and CH-stretching bands in the mid-IR range, indicating the spectroscopic lipid/protein ratio as an effective classification parameter. Additionally, we showed that different cell phenotypes are associated with a shape modification in these spectral bands that can be automatically detected by combining Principal Component Analysis (PCA) with Linear Discriminant Analysis (LDA).On the one hand, our study confirms that an FTIR analysis of EXOs allows scientists to precisely detect modifications occurring at the parental cell level; on the other hand, it unveils a set of effective spectral biomarkers able to monitoring cell changes from a mesenchymal to an epithelial phenotype, a clinically valuable piece of information considering that the epithelial-to-mesenchymal transition is a key step in the metastatic process.

Changes in Gut Bacterial Translation Occur before Symptom Onset and Dysbiosis in Dextran Sodium Sulfate-Induced Murine Colitis.

ABSTRACTLongitudinal studies on the gut microbiome that follow the effect of a perturbation are critical in understanding the microbiome’s response and succession to disease. Here, we use a dextran sodium sulfate (DSS) mouse model of colitis as a tractable perturbation to study how gut bacteria change their physiology over the course of a perturbation. Using single-cell methods such as flow cytometry, bioorthogonal noncanonical amino acid tagging (BONCAT), and population-based cell sorting combined with 16S rRNA sequencing, we determine the diversity of physiologically distinct fractions of the gut microbiota and how they respond to a controlled perturbation. The physiological markers of bacterial activity studied here include relative nucleic acid content, membrane damage, and protein production. There is a distinct and reproducible succession in bacterial physiology, with an increase in bacteria with membrane damage and diversity changes in the translationally active fraction, both, critically, occurring before symptom onset. Large increases in the relative abundance of Akkermansia were seen in all physiological fractions, most notably in the translationally active bacteria. Performing these analyses within a detailed, longitudinal framework determines which bacteria change their physiology early on, focusing therapeutic efforts in the future to predict or even mitigate relapse in diseases like inflammatory bowel diseases.IMPORTANCE Most studies on the gut microbiome focus on the composition of this community and how it changes in disease. However, how the community transitions from a healthy state to one associated with disease is currently unknown. Additionally, common diversity metrics do not provide functional information on bacterial activity. We begin to address these two unknowns by following bacterial activity over the course of disease progression, using a tractable mouse model of colitis. We find reproducible changes in gut bacterial physiology that occur before symptom onset, with increases in the proportion of bacteria with membrane damage, and changes in community composition of the translationally active bacteria. Our data provide a framework to identify possible windows of intervention and which bacteria to target in microbiome-based therapeutics.

Extracellular vesicle miRNA from cerebrospinal fluid as potential biomarkers for neurodegeneration

AbstractBackgroundExtracellular vesicles (EVs) are a diverse group of small membranous particles classified by protein and nucleic acid content, size, method of biogenesis, and cellular origin. The need for predictive biomarkers for neurodegenerative disease is critical for early detection and potential therapeutic intervention. We have previously identified multiple high ranking miRNAs in total cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD). To investigate whether these miRNAs are released from cells within EVs, we developed a pipeline to isolate and analyze EV miRNA.MethodEVs are isolated by size exclusion chromatography (SEC) from CSF collected from living donors. EV counts and sizes are determined by nanoparticle tracking analysis (NTA). Immunoblots are used to characterize EV composition and assess purity. Transmission electron microscopy (TEM) is used to assess size and EV structural characteristics. EV total RNA is isolated using commercial based methods. Quantitative PCR (qPCR) is performed using TaqMan Low Density Array (TLDA) miRNA cards.ResultThe CSF EV isolation protocol developed here is capable of isolating pure vesicles of the appropriate size and protein composition. Following miRNA qPCR, more than half of the high ranking miRNAs identified in total CSF from AD patients were observed in the isolated EV population.ConclusionThis pipeline is suitable for isolating EVs and assessing miRNA content in lumbar CSF. A large number of miRNAs isolated from EVs appear to correlate well with previously published data from total CSF, indicating a potential role for EV miRNA cargo as biomarkers for neurodegenerative diseases such as AD.

Viral Production in Seawater Filtered Through 0.2-μm Pore-Size Filters: A Hidden Biogeochemical Cycle in a Neglected Realm

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-μm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-μm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-μm filters and diluted with virus-free seawater. Viral production in the <0.2-μm filtrate was estimated from changes in viral abundance determined through flow cytometry. We found that viruses were produced in the <0.2-μm communities, which were strongly enriched with low nucleic acid content bacteria. Estimated viral production in the <0.2-μm filtrates accounted for up to 43% of total viral production and 10% of dissolved organic carbon production mediated by viral lysis of bacterial cells. By not considering viral production in these <0.2-μm communities, the virus reduction assay may underestimate viral production. Virus–bacteria interactions in <0.2-μm communities may represent a significant and overlooked role of viruses in marine food webs and carbon fluxes.

Zero-Emission of Palm Oil Mill Effluent Final Discharge Promoted Bacterial Biodiversity Rebound in the Receiving Water System

Zero-emission technology for palm oil mill effluent (POME) has led to a breakthrough in the palm oil industry in relation to the goal of sustainable development. However, there are limited resources on how this technology has affected the bacterial community in the receiving river that has previously been polluted with POME final discharge. Thus, the current study assessed the recoverability of the unexplored bacterial community in the receiving water of a constructed river water system post-zero emission of POME final discharge. An artificial river water system was constructed in this study, where the viability status and the composition of the bacterial community were assessed for 15 days using a flow cytometry-based assay and high-throughput sequencing by Illumina MiSeq, respectively. The zero-emission of POME final discharge reduced not only the physicochemical properties and nutrient contents of the receiving water, but also the bacterial cells’ viability from 40.3% to 24.5% and shifted the high nucleic acid (HNA) to low nucleic acid (LNA) content (38.7% to 34.5%). The proposed POME bacterial indicators, Alcaligenaceae and Chromatiaceae were not detectable in the rainwater (control) but were detected in the artificial river water system after the introduction of POME final discharge at the compositions of 1.0–1.3% and 2.2–5.1%, respectively. The implementation of a zero-emission system decreased the composition of Chromatiaceae from 2.2% on day 8 until it was undetectable on day 15, while Alcaligenaceae was continuously reduced from 1.2% to 0.9% within that similar time frame. As indicated by principal coordinate (PCO) analysis, the reductions in biological oxygen demand (BOD5) would further diminish the compositions of these bioindicators. The zero-emission of POME final discharge has demonstrated its efficacy, not only in reducing the polluting properties, but also in the bacterial biodiversity rebound in the affected water system.

Developing an Algorithm for Discriminating Oral Cancerous and Normal Tissues Using Raman Spectroscopy.

The aim of this study was to investigate the clinical potential of Raman spectroscopy (RS) in detecting oral squamous cell carcinoma (OSCC) in tumor and healthy tissues in surgical resection specimens during surgery. Raman experiments were performed on cryopreserved specimens from patients with OSCC. Univariate and multivariate analysis was performed based on the fingerprint region (700–1800 cm) of the Raman spectra. One hundred thirty-one ex-vivo Raman experiments were performed on 131 surgical resection specimens obtained from 67 patients. The principal component analysis (PCA) and partial least square (PLS) methods with linear discriminant analysis (LDA) were applied on an independent validation dataset. Both models were able to differentiate between the tissue types, but PLS–LDA showed 100% accuracy, sensitivity, and specificity. In this study, Raman measurements of fresh resection tissue specimens demonstrated that OSCC had significantly higher nucleic acid, protein, and several amino acid contents than adjacent healthy tissues. The specific spectral information obtained in this study can be used to develop an in vivo Raman spectroscopic method for the tumor-free resection boundary during surgery.

Picoplankton Diel Variability and Estimated Growth Rates in Epipelagic and Mesopelagic Waters of the Central Red Sea

The diel variability of the abundance and cell size of picoplanktonic groups in the central Red Sea was monitored every 2 h in situ on 4 occasions (once per season) from 2015 to 2016. We distinguished Prochlorococcus, low (LF-Syn) and high (HF-Syn) fluorescence Synechococcus, small (Speuk) and large (Lpeuk) picoeukaryotes and two groups of heterotrophic prokaryotes of low (LNA) and high (HNA) nucleic acid content. The diel variability in abundance was less marked than in cell size and more apparent in autotrophs than heterotrophs. Specific growth rates were estimated by an empirical relationship from measurements obtained in bottle incubations of surface and deep samples collected in the winter compared with in situ variations in cell size over 24 h. Autotrophic picoplankton groups generally grew faster (0.23–0.77 d–1) than heterotrophic prokaryotes (0.12–0.50 d–1). Surface to 100 m depth-weighted specific growth rates displayed a clear seasonal pattern for Prochlorococcus, with maxima in winter (0.77 ± 0.07 d–1) and minima in fall (0.52 ± 0.07 d–1). The two groups of Synechococcus peaked in spring, with slightly higher growth rates of LF-Syn (0.57 ± 0.04 d–1) than HF-Syn (0.43 ± 0.04 d–1). Speuk and Lpeuk showed different seasonal patterns, with lower values of the former (0.27 ± 0.02 and 0.37 ± 0.04 d–1, respectively). HNA consistently outgrew LNA heterotrophic prokaryotes, with a higher growth in the epipelagic (0–200 m, 0.36 ± 0.03 d–1) than in the mesopelagic (200–700 m, 0.26 ± 0.03 d–1), while no differences were found for LNA cells (0.19 ± 0.03 d–1 and 0.17 ± 0.02 d–1, respectively). With all data pooled, the mean diel abundances of autotrophic picoplankton in the upper epipelagic and of HNA cells in the epipelagic and mesopelagic layers were significantly correlated with the specific growth rates estimated from cell size variations. Our high-resolution sampling dataset suggests that changes in growth rates underlie the noticeable seasonality of picoplankton recently described in these tropical waters.

Absolute Lymphocyte Count Expansion Kinetics Can be Utilized to Assess Response to CAR T Cell Products

IntroductionCAR T-cell therapies are utilized to treat relapsed/refractory B-lymphoblastic leukemia (B-ALL), diffuse large B cell lymphoma, mantle cell lymphoma, and multiple myeloma. CAR T cell expansion kinetics after infusion impact response. However, these CAR T cell products vary in their target epitope and constituent molecules that makes it difficult to validate broad molecular or flow cytometric assays for use in the clinical setting. The lack of commercially available reagents also limits the ability of clinical laboratories to validate separate CAR T cell specific assays. We investigated the utility of common hematology laboratory parameters to measure CAR T cell expansion and response.MethodsClinical hematology laboratory parameters after infusion of autologous and allogeneic CAR T cell products directed were assessed. Concurrent CBC and cell population data (CPD) parameters from the Sysmex XN 3000 automated hematology analyzer were available in 82 patients. Absolute lymphocyte count (ALC) kinetics after infusion of autologous CD19, allogeneic CD19, CD22, CD33, allogeneic CD123-directed CAR T cell products were analyzed. Patients who received bone marrow transplant served as controls. CPD parameters included X (lateral light scatter-granularity), Y (fluorescence-nuclei acid content), Z (forward scatter-size), and their distribution widths WX, WY, WZ. Archived CellaVision cell morphology images from 118 patients who received CD19-directed CAR T cell products and 25 patients who received other CAR T cell products were analyzed. Response was determined from 1 month post-CAR bone marrow disease assessment.ResultsAbsolute Lymphocyte Counts, lymphocyte morphology and cell population data after infusion of CAR T cell productsCD19-CAR, UCAR19, and CD22-CAR all showed a distinct lymphocyte expansion phase post-infusion in responders (Figure 1A) that was absent in non-responders and controls(Figure 1B). ALC showed characteristic lag, expansion, and contraction phases in responders. CD19-CAR had a peak at day 8 while CD22-CAR and allogenic Universal(U)CAR19 had relatively delayed peak times occurring near day 15 (Figure 1B). The stem cell transplant control patients did not show ALC expansion in the first two weeks and instead showed normal lymphocyte regeneration that occurs in the third to fourth weeks. CD33-CAR and allogeneic UCAR123 non responders did not show ALC expansions.CAR T cell responders showed a distinct sequence of changes in lymphocyte morphology that was absent in non-responders and stem cell transplant controls (Figure 1C). This pattern was noted uniformly across various CAR T cell products (Figure 1D-1F). The morphological changes were categorized as: early, mid, and late. Early atypical cells were noted around days 4-8 after infusion and showed immunoblastic morphology that was present in 89% (n=105) of patients. Mid atypical cells were noted around days 5-14 after infusion and showed atypical large granular lymphocyte morphology that were seen in 95% (n=112) of patients. Late atypical cells showed the typical LGL morphology and was seen in 82% (n=97) of patients.WY fluorescence, which is a measure of nucleic acid content, was most useful in assessing changes in lymphocytes after CAR T cell infusion. WY was low (mean 402, n=57) at baseline pre-infusion timepoint and peaked (mean 1207) approximately 1 week after infusion (mean 7.9 days, n = 59). Peak WY was observed 3.7 days prior to peak ALC (mean day 11.6, n = 59).ConclusionWe demonstrate for the first time that common clinical laboratory parameters can be used to follow CAR T cell expansion after infusion in various autologous and allogeneic CAR T cell products. ALC expansion is a measure of CAR T cell expansion after infusion and correlates with response. Responders showed higher peak ALC compared to non-responders in all CAR T cell products. Timing of peak ALC expansion was determined by other factors such as expression of target antigen and CAR T-cell product used. Lymphocyte morphology followed ALC changes and showed a consistent sequence of changes that was seen across multiple CAR T cell products. Finally, CPD parameter WY which is a measure of nuclei acid content and activation, was an early harbinger of ALC expansion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative Raman spectroscopy of breast cancer malignancy utilizing higher-order principal components: A preliminary study

A major challenge in analyses of molecular spectra from biological samples has been the detection of trace biomarkers, which are subtle biochemical alterations (in parts per million (ppm)) caused by disease, buried in pronounced background fluorescence. We report a quantitative chemometrics-assisted Raman study of subtle biochemical alterations associated with breast cancer malignancy using whole blood samples, based on a 785 nm laser excitation. To understand biochemical differences between healthy (control) and diseased samples, spectral analysis was undertaken in the 500–1800 cm−1 region using principal components analysis (PCA), linear discriminant analysis (LDA), and partial least squares discriminant analysis (PLS-DA). The subtle spectral markers at 589, 594, 630, 858, 868, 1005, 1160, 1250, 1347, 1358, 1626, 1630, and 1638 cm−1 differentiated controls from diseased patients and were assigned to proteins, lipids, and nucleic acids. Out of the above, six spectral regions were determined: 589, 594, 630, 1626, 1630 and 1638 cm−1, which can be regarded as new spectral markers for breast cancer. Various pure basic biochemical components were used to develop a partial least squares regression calibration model for quantitative analysis. The relative concentrations of biochemical alterations in healthy and diseased samples were estimated by applying the developed least squares fitting model to the determined trace spectral markers’ measured blood spectrum. The fitting model revealed that the relative concentrations of proteins, lipids, and nucleic acids increased with disease status (p < 0.05). Both PCA-LDA and PLS-DA models yielded sensitivities and specificities > 80%, and overall diagnostic accuracies between 90 and 100%. Considering the limited number of samples involved in this study, preliminary results from this approach are promising, encouraging further investigations.

AR-12 Has a Bactericidal Activity and a Synergistic Effect with Gentamicin against Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus (GAS) is an important human pathogen that can cause severe invasive infection, such as necrotizing fasciitis and streptococcal toxic shock syndrome. The mortality rate of streptococcal toxic shock syndrome ranges from 20% to 50% in spite of antibiotics administration. AR-12, a pyrazole derivative, has been reported to inhibit the infection of viruses, intracellular bacteria, and fungi. In this report, we evaluated the bactericidal activities and mechanisms of AR-12 on GAS infection. Our in vitro results showed that AR-12 dose-dependently reduced the GAS growth, and 2.5 μg/mL of AR-12 significantly killed GAS within 2 h. AR-12 caused a remarkable reduction in nucleic acid and protein content of GAS. The expression of heat shock protein DnaK and streptococcal exotoxins was also inhibited by AR-12. Surveys of the GAS architecture by scanning electron microscopy revealed that AR-12-treated GAS displayed incomplete septa and micro-spherical structures protruding out of cell walls. Moreover, the combination of AR-12 and gentamicin had a synergistic antibacterial activity against GAS replication for both in vitro and in vivo infection. Taken together, these novel findings obtained in this study may provide a new therapeutic strategy for invasive GAS infection.

Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p< .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p< .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p< .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

Evaluation of a downstream process for the recovery and concentration of a Cell-Culture-Derived rVSV-Spike COVID-19 vaccine candidate

rVSV-Spike (rVSV-S) is a recombinant viral vaccine candidate under development to control the COVID-19 pandemic and is currently in phase II clinical trials. rVSV-S induces neutralizing antibodies and protects against SARS-CoV-2 infection in animal models. Bringing rVSV-S to clinical trials required the development of a scalable downstream process for the production of rVSV-S that can meet regulatory guidelines. The objective of this study was the development of the first downstream unit operations for cell-culture-derived rVSV-S, namely, the removal of nucleic acid contamination, the clarification and concentration of viral harvested supernatant, and buffer exchange. Retaining the infectivity of the rVSV-S during the downstream process was challenged by the shear sensitivity of the enveloped rVSV-S and its membrane protruding spike protein. Through a series of screening experiments, we evaluated and established the required endonuclease treatment conditions, filter train composition, and hollow fiber-tangential flow filtration parameters to remove large particles, reduce the load of impurities, and concentrate and exchange the buffer while retaining rVSV-S infectivity. The combined effect of the first unit operations on viral recovery and the removal of critical impurities was examined during scale-up experiments. Overall, approximately 40% of viral recovery was obtained and the regulatory requirements of less than 10 ng host cell DNA per dose were met. However, while 86–97% of the host cell proteins were removed, the regulatory acceptable HCP levels were not achieved, requiring subsequent purification and polishing steps. The results we obtained during the scale-up experiments were similar to those obtained during the screening experiments, indicating the scalability of the process. The findings of this study set the foundation for the development of a complete downstream manufacturing process, requiring subsequent purification and polishing unit operations for clinical preparations of rVSV-S.