Peptosome: A New Efficient Transfection Tool as an Alternative to Liposome.

Gene therapy is one of the most promising techniques for treating genetic diseases and cancer. The current most important problem in gene therapy is gene delivery. Viral and non-viral vectors like liposomes, used for gene delivery, have many limitations. We have developed new hybrid peptides by combining cell-penetrating peptides (CPPs) with the DNA-binding domain of the human histone H4 protein. These small peptides bind to DNA molecules through their histone domain, leaving the CPP part free and available for binding and penetration into cells, forming complexes that we named "peptosomes". We evaluated the transfection efficiency of several hybrid peptides by delivering a plasmid carrying the green fluorescent protein gene and following its expression by fluorescent microscopy. Among several hybrid peptides, TM3 achieved a gene delivery efficiency of 76%, compared to 52% for Lipofectamine 2000. TM3 peptosomes may become important gene delivery tools with several advantages over current gene delivery agents.

A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

Introduction: Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Methods: Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. Results: The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. Discussion: In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani population.

Colorectal cancer (CRC) is the third most prevalent cause of tumorigenesis and several pathogenic bacteria have been correlated with aggressive cases of cancer i.e., genotoxin (colibactin) producing Escherichia coli (E. coli). This study was designed to investigate the genetic diversity of clb+clb+ E. coli strains and their association with CRC. Pathogenic E. coli isolates from colorectal biopsies were characterized based on phylotypes, antibiotic resistance pattern, and (Enterobacterial Repetitive Intergenic Consensus Sequence-based Polymerase Chain Reaction) ERIC-PCR. Furthermore, isolates were screened for the presence of the Pks (polyketide synthase) Island specifically targeting colibactin genes A and Q. The selective clb+clb+ isolates were subjected to cytotoxicity assay using Human embryonic kidney (HEK) cell lines. We revealed that 43.47% of the cancer-associated E. coli isolates were from phylogroup B2 comparatively more pathogenic than rest while in the case of healthy controls no isolate was found from B2. Moreover, 90% were found positive for colibactin and pks (polyketide synthase) island, while none of the healthy controls were found positive for colibactin genes. All healthy and cancer-associated isolates were tested against 15 antibiotic agents, we observed that cancer-associated isolates showed a wide range of resistance from 96% against Nalidixic acid to 48% against Doxycycline. Moreover, E. coli isolates were further genotyped using ERIC-PCR, and selected clb+clb+ E. coli isolates were subjected to cytotoxicity assay. We recorded the significant cytotoxic activity of clb+clb+ E. coli phylogroup B2 isolates that might have contributed towards the progression of CRC or dysbiosis of healthy gut microbiota protecting against CRC pathogenesis. Our results revealed a significant p<0.023 association of dietary habits and hygiene p<0.001with CRC. This is the first study to report the prevalence of E. coli phylogroups and the role of colibactin most virulent phylogroup B2 among Pakistani individuals from low socioeconomic setup.

Feconomics®; a new and more convenient method, the routine diagnosis of intestinal parasitic infections.

Direct wet mount examination and concentration are the most commonly used methods for detecting intestinal parasites from fecal samples. Concentration methods are used when there are fewer protozoan cyst, coccidian oocyst, microsporidial spore, helminth egg, and larvae in the fecal samples. Early detection of the causative intestinal parasites plays a significant role in implementing timely and correct treatment, which relieves the patients' symptoms and also prevents recurrences. Formalin-ethyl acetate concentration (FEAC) is believed to be a gold standard method to detect most intestinal parasites. Thus, in this study, we evaluated the diagnostic value of Feconomics® [manufactured by Salubris Inc, Boston, USA. Patent application number (TR): 2010/07549] which is a simple, new, and rapid fecal concentration method for the detection of the intestinal parasites in human beings. We also compared the FEAC with Feconomics® and direct wet mount examination. A total of 918 fecal samples were collected from the patients suspected to have intestinal parasitic infection. Samples were examined with the direct wet mount, FEAC, and Feconomics® methods. Different parasite species 15.9% (146/918) with Feconomics®, 13.3% (122/918) with FEAC, and 9.8% (90/918) with direct wet mount examination, Feconomics® > FEAC > direct wet mount examinations were detected. They were statistically compared considering FEAC as the gold standard for parasitological diagnosis; the sensitivity and specificity of Feconomics® were calculated as 96 and 97%, respectively. Blastocystis hominis was found to be the most common parasite, followed by Giardia lamblia with direct wet mount examination, FEAC, and Feconomics® methods. Feconomics® proved to be better than not only FEAC in concentrating parasite egg and cyst forms as well as in maintaining characteristic morphology but it is also better in direct wet mount examination. Feconomics® eliminates the need for centrifugation by using absorbent beads that help the homogenization and concentration of the sample. Feconomics® in this study was considerably better than FEAC in detecting the trophozoites of Giardia lamblia. We suggest that Feconomics® be used for the routine diagnosis of intestinal parasitic infection in rural areas of developing countries due to the fact that a centrifuge is not required and it eliminates large stool particles.

Mycobacterial strains that stimulate the immune system most efficiently as candidates for the treatment of bladder cancer.

BACKGROUND: Intravesical bacillus Calmette-Guérin (BCG) application is widely used in the treatment of superficial bladder carcinoma. Despite being an effective therapy, the pathogenicity and lethal side effects of BCG limits its usage. Intensive research has been carried out to find less toxic and more potent therapeutic agents for the treatment of bladder cancer. Researchers have focused on Mycobacterium phlei as an alternative. The cell wall extract of M. phlei is sufficient for antitumoral activity. Our preliminary experiments indicate that the fractions rich in cell wall proteins cause activation of tumor necrosis factor (TNF)-α and interleukin (IL)-12. This study aims to identify powerful and less harmful mycobacteria among 88 strains in terms of how they stimulate the immune system. METHODS: Eighty-eight mycobacterial strains were grown in Middlebrook 7H9 medium. The bacterial cells were sonicated after heat treatment. The supernatants were incubated with the monocytic cell line THP-1, followed by measurement of TNF-α and IL-12 response. RESULTS AND CONCLUSION: In addition to M. phlei, the following 12 mycobacterial strains were selected as candidates for superficial bladder tumor treatment: M. agri, M. aichiense, M. aurum, M. brumae, M. chitae, M. chubuense, M. diernhoferi, M. gadium, M. murale, M. obuense, M. tokaiense and M. vaccae.

[A novel T-cloning vector system]. / Yeni bir T-klonlama vektör sistemi.

The use of cloning vectors has revolutionized molecular biology. Any vector with appropriate cloning sites can be used to clone a section of DNA and polymerase chain reaction (PCR) is a useful method for producing DNA fragments that are intended to be cloned. When Taq polymerase is used in PCR for polymerization, the enzyme adds an extra adenosyl (A) nucleotide to the 3' end of the extended strand, in a template independent manner. T-cloning vectors are created by adding a thymidine (T) residue to the ends of the cloning site. This aids ligation with PCR products possessing A-T. In this study, we prepared a T-cloning vector system, which guarantees insertion of open reading frames to the right position for expression. This method provides an easy way of cloning PCR products assuring at the same time in frame insertion.

Polymerase chain reaction in cutaneous tuberculosis: is it a reliable diagnostic method in paraffin-embedded tissues?

BACKGROUND: Most cutaneous tuberculosis lesions contain few bacilli, so identification of Mycobacterium tuberculosis in conventional laboratory tests is difficult. In vitro amplification of specific DNA sequences using polymerase chain reaction (PCR) has become a valuable tool in the rapid detection of slow-growing organisms like M. tuberculosis. AIM: To investigate the presence of M. tuberculosis DNA in cutaneous tuberculosis. METHODS: Twenty-two archival biopsy specimens diagnosed as cutaneous tuberculosis were investigated for the presence of M. tuberculosis DNA by PCR. Normal skin samples of 29 healthy patients were used as a control. RESULTS: Amplification of the M. tuberculosis DNA was observed in one of the cutaneous tuberculosis specimens and in a healthy control. CONCLUSION: Although PCR is a rapid diagnostic method, fixation procedures may decrease its sensitivity.