Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

<p>Abstract</p> <p>Background</p> <p>Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts <it>in vivo</it>. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.</p> <p>Results</p> <p>The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.</p> <p>Conclusion</p> <p>The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.</p

The Pupils\u27 Opinion of the Examination and Grading Process in the Classroom

U članku se oslikava kompleksnost nastavnog provjeravanja i ocjenjivanja učenika kroz eksplikaciju značenja i zadataka ove etape nastavnog procesa te isticanje poteškoća koje se pojavljuju prilikom njene realizacije. Naznačeni su i zakonski okviri provedbe provjeravanja i ocjenjivanja učenika u osnovnim i srednjim školama.Praksa nastavnog provjeravanja i ocjenjivanja prikazana je kroz analizu i interpretaciju rezultata ispitivanja mišljenja skupine osnovnoškolskih i srednjoškolskih učenika (N=147) o navedenoj problematici. Utvrđeni su najčešći i preferirani oblici te mišljenja učenika o ne/korektnosti i transparentnosti provjeravanja i ocjenjivanja znanja i sposobnosti učenika u nastavnoj praksii prilikama za samoevaluaciju. Prikupljeni podaci upućuju na to da se u provođenju navedene etape nastavnog procesa ostvaruje nedovoljna raznovrsnost u oblicima njene realizacije, da učenicima nisu uvijek pružene adekvatne povratne informacije o rezultatima njihova rada te da je provjeravanje i ocjenjivanje dominantno nastavnikova aktivnost prilikom koje učenici najčešće nisu u prilici da samostalno ocijene svoj napredak u učenju i ostvarivanju zadataka nastave. Iz navedenog logično proizlazi često prisutan dojam učenika da su nekorektno ocijenjeni. Nastavno bi provjeravanje i ocjenjivanje trebalo uvažavati kompleksnost učenikove osobnosti i njen originalni doprinos u nastavi. Dijelom je to moguće ostvariti uporabom različitih oblika realizacije, pružanjem jasnih informacija o rezultatima učenja te većim stupnjem uključenosti učenika u ocjenjivanje svojeg napretka u učenju.The article portrays the complexity of classroom examination and grading of pupils via theexplication of the meaning and tasks of this stage of the teaching process and by stressingthe difficulties that arise during its realization. The legal framework for the conducting of theexamination and grading of pupils in elementary and high schools is also outlined.The practice of classroom examination and grading is shown through the analysis andinterpretation of the results of a survey of a group of elementary and highschool pupils (N=147)about the said process. The most common and the preferred types and the pupils\u27 opinion of thein/correctness and transparency of the testing and grading of pupils\u27 knowledge and abilities inclassroom practice were determined, as well as the opportunities for self-evaluation. The gathereddata indicate a lack of variety in the ways of conducting this stage of the teaching process, thepupils aren\u27t always given adequate feedback information about the results of their work andthe examination is predominantly a teachers activity during which the pupils most often aren\u27tgiven a chance to independently evaluate their progress in learning and carrying out classroomassignments. The above mentioned logically leads to the impression, commonly present amongpupils, that they were incorrectly graded.Classroom examination and grading should take into consideration the complexity of the pupil\u27spersonality and her original contribution in class. It is partially possible to achieve that by usingdifferent methods of realization, by giving clear information about the results of their studying,and by a greater degree of inclusion of the pupils in evaluating the progress of their studying.</p

Fungicidal activity of recombinant javanicin against Cryptococcus neoformans is associated with intracellular target(s) involved in carbohydrate and energy metabolic processes

The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents

Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR–Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log10 IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings

Direct Detection of Streptococcus suis from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future

Potentiality of melittin-loaded niosomal vesicles against vancomycin-intermediate Staphylococcus aureus and Staphylococcal skin infection

Background: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibiotic-resistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required. Methods: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection. Results: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120– 200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin. Conclusion: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery

Activity of Propolis Nanoparticles against HSV-2: Promising Approach to Inhibiting Infection and Replication

Herpes simplex type 2 (HSV-2) infection causes a significant life-long disease. Long-term side effects of antiviral drugs can lead to the emergence of drug resistance. Thus, propolis, a natural product derived from beehives, has been proposed to prevent or treat HSV-2 infections. Unfortunately, therapeutic applications of propolis are still limited due its poor solubility. To overcome this, a nanoparticle-based drug delivery system was employed. An ethanolic extract of propolis (EEP) was encapsulated in nanoparticles composed of poly(lactic-co-glycolic acid) and chitosan using a modified oil-in-water single emulsion by using the solvent evaporation method. The produced nanoparticles (EEP-NPs) had a spherical shape with a size of ~450 nm and presented satisfactory physicochemical properties, including positively charged surface (38.05 &plusmn; 7.65 mV), high entrapment efficiency (79.89 &plusmn; 13.92%), and sustained release profile. Moreover, EEP-NPs were less cytotoxic on Vero cells and exhibited anti-HSV-2 activity. EEP-NPs had a direct effect on the inactivation of viral particles, and also disrupted the virion entry and release from the host cells. A significant decrease in the expression levels of the HSV-2 replication-related genes (ICP4, ICP27, and gB) was also observed. Our study suggests that EEP-NPs provide a strong anti-HSV-2 activity and serve as a promising platform for the treatment of HSV-2 infections

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells-2

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells"</p><p>http://www.biomedcentral.com/1472-6750/8/5</p><p>BMC Biotechnology 2008;8():5-5.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2258298.</p><p></p>roxidase-conjugated mAb anti-HA (lane 1) and anti-His mAb (lane 2). The immunoreactive bands were visualized by ECL substrate detection system. The molecular weight is indicated. CD147-BCCP (open columns) or SVV-BCCP (black columns) was captured on the avidin-coated wells. Soluble scFv-M6-1B9 was subsequently added and the bound scFv was detected by peroxidase-conjugated mAb anti-HA. CD147-BCCP (lane 1) or SVV-BCCP (lane 2) proteins were separated on 12% SDS-PAGE, electroblotted onto a PVDF membrane, and then probed with soluble scFv-M6-1B9. The scFv was detected using peroxidase-conjugated mAb anti-HA. The positions of molecular mass markers are shown on the left. CD147 on U937 cells was stained with soluble scFv-M6-1B9 and then probed by mouse anti-HA-biotin. Subsequently, FITC-conjugated sheep anti-mouse immunoglobulins antibody was added. Monoclonal antibody M6-1B9 was used as a control system for detecting CD147 on U937 cells. The immunofluorescence on cells stained with soluble scFv-M6-1B9 (bold line) or mAb M6-1B9 (thin line) is shown. The dashed line represents background fluorescence of negative control mAb. The axis represents the number of events on a linear scale; the axis shows the fluorescence intensity on a logarithmic scale

Nano-Delivery System of Ethanolic Extract of Propolis Targeting <i>Mycobacterium tuberculosis</i> via Aptamer-Modified-Niosomes

Tuberculosis (TB) therapy requires long-course multidrug regimens leading to the emergence of drug-resistant TB and increased public health burden worldwide. As the treatment strategy is more challenging, seeking a potent non-antibiotic agent has been raised. Propolis serve as a natural source of bioactive molecules. It has been evidenced to eliminate various microbial pathogens including Mycobacterium tuberculosis (Mtb). In this study, we fabricated the niosome-based drug delivery platform for ethanolic extract of propolis (EEP) using thin film hydration method with Ag85A aptamer surface modification (Apt-PEGNio/EEP) to target Mtb. Physicochemical characterization of PEGNio/EEP indicated approximately −20 mV of zeta potential, 180 nm of spherical nanoparticles, 80% of entrapment efficiency, and the sustained release profile. The Apt-PEGNio/EEP and PEGNio/EEP showed no difference in these characteristics. The chemical composition in the nanostructure was confirmed by Fourier transform infrared spectrometry. Apt-PEGNio/EEP showed specific binding to Mycobacterium expressing Ag85 membrane-bound protein by confocal laser scanning microscope. It strongly inhibited Mtb in vitro and exhibited non-toxicity on alveolar macrophages. These findings indicate that the Apt-PEGNio/EEP acts as an antimycobacterial nanoparticle and might be a promising innovative targeted treatment. Further application of this smart nano-delivery system will lead to effective TB management