The Performance of Minicircle DNA Versus Parental Plasmid in p53 Gene Delivery Into HPV-18-Infected Cervical Cancer Cells.

Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA-p53 and its precursor parental plasmid (PP)-p53 for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells. First, live cell imaging and fluorescence microscopy studies allowed to understand that mcDNA-p53 vector was able to enter cell nuclei more rapidly than PP-p53 vector, leading to a transfection efficiency of 68% against 34%, respectively. Then, p53 transcripts and protein expression assessment revealed that both vectors were able to induce transcription and the target protein expression. However, the mcDNA-p53 vector performance stood out, by demonstrating higher p53 expression levels (91.65 ± 2.82 U/mL vs. 74.75 ± 4.44 U/mL). After assuring the safety of both vectors by viability studies, such potential was confirmed by proliferation and apoptosis assays. These studies confirmed the mcDNA-p53 vector function toward cell cycle arrest and apoptosis in HPV-18-infected cervical cancer cells. Altogether, these results suggest that the mcDNA vector has a more promising and efficient role as a DNA vector than conventional pDNA, opening new investigation lines for cervical cancer treatment in the future.

Minicircle DNA: The Future for DNA-Based Vectors?

Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing.

Ligand screening to pre-miRNA 149 G-quadruplex investigated by molecular dynamics.

Using a molecular dynamics approach, the study of the interaction between six different known ligands and a predicted pre-miRNA 149 RNA G-quadruplex (rG4) structure is reported. The stabilization of rG4 structures formed within the pre-miRNA stem-loop regions using small ligands is an attractive anticancer strategy. Particularly, miRNA-149 is upregulated in a variety of cancers such as prostate cancer and is therefore a potential target for drug development. The results show that ligands C8 and PhenDC3 interact with the rG4 structure via stacking interactions with the end G-quartets. Ligands [16]phenN2, [32]phen2N4 and pyridostatin on the other hand bind the loops/groove interface of the rG4 being H-bonding and electrostatic interactions the driving force of the interaction. The C8 precursor, C8-NH2, emphasizes the structural nuances of the rG4 short loops as the lack of a large terminal aromatic moiety produced a mixed stacking-groove binding mode. Overall, this study may help the design of specific ligands for pre-miRNA rG4 towards anticancer therapeutics development.Communicated by Ramaswamy H. Sarma.

Smoothing membrane protein structure determination by initial upstream stage improvements.

Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands.

Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.

Blastic Plasmacytoid Dendritic Cell Neoplasm with Pulmonary Involvement and Atypical Skin Lesion.

BACKGROUND Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematodermic malignancy neoplasm with highly aggressive course and poor prognosis. This disease typically presents with cutaneous involvement as the first manifestation, with subsequent or simultaneous spread to bone marrow and peripheral blood.  CASE REPORT Here, we report the case of a 51-year-old woman who presented a violaceus skin lesion on the lateral region of the right thigh, weight loss, fever, and lymphadenopathies. Computed tomography (CT) displayed thoracic and abdominal lymph node and alveolar bleeding. Flow cytometry from circulating blastic cells was compatible with BPDCN (CD4+, CD56+ and CD123+). She underwent 5 cycles of hyper-CVAD alternating with high-dose methotrexate and cytarabine, but the patient died due to alveolar bleeding and sepsis. CONCLUSIONS We report a rare case of BPDCN characterized by an aggressive course, presence of atypical skin lesion, a finding suggestive of pulmonary infiltration, and nonresponse to induction chemotherapy, leading to late diagnosis and therapeutic management. Because of the late recognition of the skin lesion, neoplastic cells infiltrated the dermis and spread as the disease progressed rapidly to a fatal course.

Influenza Plasmid DNA Vaccines: Progress and Prospects.

Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

Analysis of Salvinorin A in urine using microextraction in packed syringe and GC-MS/MS.

BACKGROUND: The aim of this work was to develop and validate a method for the determination of Salvinorin A in human urine using microextraction by packed sorbent (MEPS) and GC-MS/MS. RESULTS: The technique uses a sample volume as low as 0.2 ml, and the analyte was extracted using a C18 sorbent. The method showed to be linear between 20 and 1000 ng/ml and presented a LOD of 5 ng/ml. Intra- and inter-day precision and accuracy were acceptable. Absolute recoveries ranged from 71 to 80%. CONCLUSION: GC-MS/MS with MEPS demonstrated to be a fast and simple procedure for the quantification of Salvinorin A in urine. This is the first time that GC-MS/MS with MEPS was used for the determination of this compound in biological fluids. Furthermore, the device could be reused for up to 80 extractions, which accounted for a lower cost of analysis.

Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification.

Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection.

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.

Role of microextraction sampling procedures in forensic toxicology.

The last two decades have provided analysts with more sensitive technology, enabling scientists from all analytical fields to see what they were not able to see just a few years ago. This increased sensitivity has allowed drug detection at very low concentrations and testing in unconventional samples (e.g., hair, oral fluid and sweat), where despite having low analyte concentrations has also led to a reduction in sample size. Along with this reduction, and as a result of the use of excessive amounts of potentially toxic organic solvents (with the subsequent environmental pollution and costs associated with their proper disposal), there has been a growing tendency to use miniaturized sampling techniques. Those sampling procedures allow reducing organic solvent consumption to a minimum and at the same time provide a rapid, simple and cost-effective approach. In addition, it is possible to get at least some degree of automation when using these techniques, which will enhance sample throughput. Those miniaturized sample preparation techniques may be roughly categorized in solid-phase and liquid-phase microextraction, depending on the nature of the analyte. This paper reviews recently published literature on the use of microextraction sampling procedures, with a special focus on the field of forensic toxicology.

Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA.

Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.

Development and validation of an analytical method for the determination of trans- and cis-resveratrol in wine: analysis of its contents in 186 Portuguese red wines.

A simple procedure based on solid-phase extraction and high performance liquid chromatography coupled to diode array detector has been developed and validated for the qualitative and quantitative analysis of cis- and trans-resveratrol in wines. The method was linear from 0.025 (lower limit of quantitation, LLOQ) to 15 µg/mL for trans-resveratrol and from 0.023 (LLOQ) to 0.92 µg/mL for cis-resveratrol, with correlation coefficients higher than 0.99 for both isomers. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in method validation, that is, CVs inferior to 15% and mean relative errors within a ±14% interval. The extraction presented mean efficiencies close to 100% for both analytes. The validated methodology was applied to 186 Portuguese red wines from different regions, grape varieties and vintage. The results obtained showed that the content of trans-resveratrol in red wines ranged from 0.05 to 10.9 µg/mL, while the concentrations of cis-resveratrol ranged from 0.04 to 8.71 µg/mL.

Hair: a complementary source of bioanalytical information in forensic toxicology.

Hair has been used for years in the assessment and documentation of human exposure to drugs, as it presents characteristics that make it extremely valuable for this purpose, namely the fact that sample collection is performed in a noninvasive manner, under close supervision, the possibility of collecting a specimen reflecting a similar timeline in the case of claims or suspicion of a leak in the chain of custody, and the increased window of detection for the drugs. For these reasons, testing for drugs in hair provides unique and useful information in several fields of toxicology, from which the most prominent is the possibility of studying individual drug use histories by means of segmental analysis. This paper will review the unique role of hair as a complementary sample in documenting human exposure to drugs in the fields of clinical and forensic toxicology and workplace drug testing.