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.

Comparison of a novel antigen detection test with reverse transcription polymerase chain reaction assay for laboratory diagnosis of SARS-CoV-2 infection.

Molecular diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction (RT-PCR) in respiratory specimens is considered the gold standard method. This method is highly sensitive and specific but it has some limitations such as being expensive and requiring special laboratory equipment and skilled personnel. RapidFor™ Antigen Rapid Test Kit is a commercially available Ag-RDT which is produced in Turkey and designed to detect the nucleocapsid antigen of SARS-CoV-2 in nasopharyngeal swab samples. The aim of this study was to evaluate the performance of this novel SARS-CoV-2 antigen detection considering the RT-PCR method as the gold standard. Four hundred forty-four nasopharyngeal swab samples which were collected from the patients who met clinical criteria of COVID-19 from ten centers in Turkey between September 2020 and February 2021 were included in the study. All the nasopharyngeal swab samples were tested for SARS-CoV-2 RNA using commercial RT-PCR kits (Bioeksen and A1 Lifesciences, Istanbul, Turkey) according to the manufacturer's instructions. Viral loads were assessed according to the cycle threshold (Ct) values. RapidFor™ SARS-CoV-2 antigen test (Vitrosens Biotechnology, Istanbul, Turkey) was used to investigate the presence of SARS-CoV-2 antigen in all samples following the manufacturer's instructions. Out of 444 nasopharyngeal swab samples tested, 346 (77.9%) were positive and 98 (22.1%) were negative for SARS-CoV-2 RNA by RTPCR. Overall sensitivity of the RapidFor™. Antigen Rapid Test Kit was 80.3% whereas specificity was found to be 87.8%. Positivity rate of rapid antigen test in samples with Ct values over 25 and below 30 was 82.7%, while it increased to 95.7% in samples 20 ≤ Ct < 25 and reached 100% in samples with Ct values below 20. RapidFor™ SARS-CoV-2 Ag test might be a good choice in the screening of symptomatic and asymptomatic patients and their contacts for taking isolation measures early, with advantages over RT-PCR as being rapid, easy and being applicable in every laboratory and even at point of care.

Simple and low-cost antibiotic susceptibility testing for Mycobacterium tuberculosis using screen-printed electrodes.

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low- and middle-income countries, it is critical that low-cost and easy-to-use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low-cost, simple-to-use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow-growing M. tuberculosis (TB) pathogen in 96 h using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (108 -103  CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 103.82 and 1011.59  CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for the development of a comprehensive, low-cost, and simple-to-use AST system for prescribing in TB and multidrug-resistant tuberculosis.

Simple concentration method enables the use of gargle and mouthwash instead of nasopharyngeal swab sampling for the diagnosis of COVID-19 by PCR.

Since its emergence in December 2019, SARS-CoV-2 is causing one of the most devastating pandemics in human history. Currently, the most important method for definitive diagnosis of COVID-19 is identification of SARS-CoV-2 RNA in nasopharyngeal swab samples by RT-PCR. Nasopharyngeal swab sampling is a discomforting procedure sometimes with adverse effects, which also poses a risk for infection for the personnel performing the sampling. We have developed a new method for concentrating biological samples, which enabled us to use gargle and mouthwash samples to be used in RT-PCR, for the diagnosis of COVID-19, as an alternative to nasopharyngeal swab samples. We have analyzed nasopharyngeal and gargle and mouthwash samples, before and after concentration, of 363 patients by RT-PCR for the presence of SARS-CoV-2. Among 114 patients in which SARS-CoV-2 was identified in at least one of their samples, the virus was identified in 76 (66.7%), 67 (58.8%), and 101 (88.6%) of nasopharyngeal swab, gargle, and mouthwash samples before and after concentration, respectively. When concentrated by our new method, gargle and mouthwash samples can be used instead of nasopharyngeal samples in identification of SARS-CoV-2 by RT-PCR, with the same or better sensitivity. Eliminating the need for nasopharyngeal sampling will save the patients from an invasive and painful procedure and will lower the risk of infection for the healthcare personnel taking the sample. This easy sampling procedure may decrease the workload of hospitals, shorten the turnaround time of obtaining test results, and thus enable rapid isolation of infected patients.

Label-free molecular detection of antibiotic susceptibility for Mycobacterium smegmatis using a low cost electrode format.

Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.

[Diversity of Leishmania Strains Isolated from Cutaneous Leishmaniasis Patients in Turkey and its Reflection to Clinics in Mice Model]. / Türkiye'de Kutanöz Leysmanyazis Hastalarindan Elde Edilen Leishmania Izolatlarindaki Farkliliklar ve Bunlarin Fare Modeline Klinik Yansimasi.

Although asexual reproduction has been attributed to Leishmania species, genetic exchange has recently been demonstrated, which helped emerging of hybrid isolates. Situated on the crossroads between three continents, Leishmania hybrids may be present in Turkey. In Turkey, visceral leishmaniasis caused by Leishmania infantum is less common, while cutaneous leishmaniasis (CL) caused by Leishmania tropica and L.infantum could reach 2500 reported cases a year. Our aim was to investigate genetic variability of local Leishmania species and presence of hybrid Leishmania strains in Turkey. Twenty CL patients from Sanliurfa and Hatay, where only L.tropica and both L.tropica and L.infantum cause CL, respectively, were registered equally. All isolates were assessed with real-time polymerase chain reaction (Rt-PCR), isoenzyme analysis, gene sequencing, two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF/TOFMS followed by in vivo analyses on mouse model. Identification of differentially expressed proteins was performed. These proteins were confirmed by sequence analysis. All isolates from Sanliurfa were found to be L.tropica which caused cutaneous infection in mice. However, one of 10 isolates from Hatay was found as Leishmania major which caused cutaneous infection. Five isolates were found as L.tropica with Rt-PCR and gene sequencing, one of which had one different protein from the reference L.tropica strain and caused cutaneous infection. Four of the five isolates had five different proteins compared to reference strain and caused both cutaneous and visceral infections. Remaining four isolates showed double melting curves in Rt-PCR, which were concordant with L.tropica and L.infantum. Their sequencing and isoenzyme analyses indicated them as L.infantum. They had six different proteins compared to reference L.infantum strain and caused cutaneous and visceral infections. It is concluded that the isolates with different proteins were hybrid Leishmania species. In the present study, outcomes of the proteomics, genomics, clinical manifestations and tissue tropism on animal models were evaluated together for the first time. In addition to L.tropica and L.infantum, L.major was identified as a causative agent for CL and hybrids of L.infantum/tropica were also shown to be present.

Oligomer based real-time detection of microorganisms producing nuclease enzymes.

In this study, we provide a method using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases in real time, while growing them in culture media. The detection of such microorganisms was possible in a short period of time, as short as 10 minutes up to a maximum of 8 hours, depending on the bacterial density. We also showed the suitability of this new method for determination of minimum inhibitory concentration (MIC) in culture media in a very short period of time, compared to conventional methods. We believe that it can make a significant contribution to gain new insights for analysis of complex materials such as clinical samples, food samples and environmental samples.

Simple Identification of Mycobacterial Species by Sequence-Specific Multiple Polymerase Chain Reactions.

Several species of mycobacteria cause infections in humans. Species identification of clinical isolates of mycobacteria is very important for the decision of treatment and in choosing the appropriate treatment regimen. We have developed a multiplex PCR method that can identify practically all known species of mycobacteria, by determination of single-nucleotide differences at a total of 13 different polymorphic regions in the genes of rRNA and hsp65, in four PCR mixes. To achieve this goal, single-nucleotide differences in these polymorphic regions were used to divide mycobacterial species into two groups, than four, eight, etc., in an algorithmic manner. It was sufficient to reach single species level by evaluating 13 polymorphic regions. Evaluation of the multiplex PCR patterns by observable real-time electrophoresis (ORTE) simplified species identification. This new method may enable easy, rapid, and cost-effective identification of all species of mycobacteria.

Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

OBJECTIVE: To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014. METHODS: Initial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L. major. Developing lesions were observed, and the thickening of footpads was measured weekly. RESULTS: Melting curve analyses of 18 isolates showed a peak concordant with L. major, and two of them were confirmed by isoenzyme analyses as L. major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3 weeks, which even progressed to extremity amputation. CONCLUSION: Cutaneous leishmaniasis-causing L. major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L. major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L. major infections in Turkey, contrary to the infections in neighbouring countries.

Design, Synthesis, and Molecular Docking Studies of a Conjugated Thiadiazole-Thiourea Scaffold as Antituberculosis Agents.

In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculosis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole in a single molecular backbone. Three compounds from our series were distinguished from the others by their promising activity profiles against Mycobacterium tuberculosis strain H37Rv. Compounds 11 and 19 were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and 11.48 µM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H37Rv with an MIC value of 17.81 µM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking studies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds 11, 15, and 19 were found to have good docking scores in the range of -7.12 to -7.83 kcal/mol.

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.

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.

Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.

Antimicrobial Peptides and Their Anti-Leishmanial Efficacies on Leishmania tropica Promastigotes In vitro.

OBJECTIVE: Antimicrobial resistance is a real threat to humanity. Pentavalent antimonials are reported non-effective in leishmaniasis treatment today, in countries like India. New treatment options have been assessed worldwide lately. Antimicrobial peptides (AMP) are the leading antibiotic candidates due to their large spectrum, fast efficacy, and low resistance risks. Cathelicidins are the AMP with well-documented antimicrobial activities against bacteria, fungi, and protozoa, over their positively charged membranes. Here, we aim to design cathelicidine-like helical peptides (CLHP), and compare their anti-Leishmanial efficacies in vitro, with meglumine antimoniate (MA) on Leishmania tropica. METHODS: A total of five study [TN-1-5] and two control (MA and non-drug) groups were formed. Cryopreserved L. tropica isolate was thawed and cultivated in Novy-MacNeal-Nicolle medium and then in RPMI. Five different CLHPs (TN1-5) were diluted in dimethyl sulphoxide. A total of 150 uL of CLHPs and MA were added into the first wells of the test plaques, followed by serial dilutions that revealed doses within 4 and 512 ug/mL. Then, 100 uL of cultures including 1x108/mL of L. tropica promastigotes were added into each well. Viability of promastigotes was checked with XTT, while the parasite count was assessed at 24th and 48th hours. RESULTS: TN3 was effective at 32 ug/mL. All tested CLHPs exhibited varying degrees of anti-Leishmanial activities, except TN5, even at its highest dose. CONCLUSION: TN3 showed a particular efficacy against L. tropica in vitro. Further studies including in vivo testing of the candidate's both efficacy and toxicity are essential.

Metagenomic analysis of atheroma plaques for identification of microorganisms indicates presence of Toxoplasma gondii as a possible etiological agent.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Vital organs like the heart are affected by the occlusion of blood vessels due to atherosclerotic plaque formation. However, the role of infectious agents has always been an essential subject of investigation. This study investigated the presence of microorganisms, including nanobacteria, in atherosclerotic plaques removed from human carotid arteries by microbiological and metagenomic examination. METHODS: Atheroma plaque samples were obtained from 20 patients with carotid artery stenosis who had atherectomy by surgery or percutaneous intervention. Nanobacteria were grown by culturing homogenates of the atheroma plaques. Whole genome sequencing was done for samples. Because of the high percentage of Toxoplasma gondii (T. gondii) DNA, PCR investigation was applied to detect T. gondii DNA in the samples. RESULTS: A molecular analysis of nanobacteria revealed them to be made of human proteins, supporting the theory that they are not living organisms. According to sequencing results, samples showed that more than 50 % of the metagenomic sequences belonged to Toxoplasma gondii. PCR investigation indicated that T. gondii DNA was positive in 8 (40 %) of 20 plaques. CONCLUSIONS: Further evidence regarding the role of T. gondii in the etiology of plaque formation may help determine the strategy for prevention and treatment of infections in preventing atheroma plaque formation in the future.

In vitro efficacy of different PEGylation designs on cathelicidin-like peptide with high antibacterial and antifungal activity.

Recent reports on antibiotic resistance have highlighted the need to reduce the impact of this global health issue through urgent prevention and control. The World Health Organization currently considers antibiotic resistance as one of the most dangerous threats to global health. Therefore, Antimicrobial peptides (AMPs) are promising for the development of novel antibiotic molecules due to their high antimicrobial effects, non-inducing antimicrobial resistance (AMR) properties, and broad spectrum. Hence, in this study, we developed novel antimicrobial peptide/polymer conjugates to reduce the adverse effects of TN6 (RLLRLLLRLLR) peptide. We demonstrate how our constructs function in vitro in terms of antimicrobial activity, hemolytic activity, cytotoxicity, and protease resistance. Our findings show that our molecules are effective against different types of microorganisms such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, vancomycin-resistant Enteroccus faecium, and Candida albicans, which are known to be pathogenic and antibiotic-resistant. Our constructs generally showed low cytotoxicity relative to the peptide in HaCaT and 3T3 cells. Especially these structures are very successful in terms of hemotoxicity. In the bacteremia model with S. aureus, the naked peptide (TN6) was hemotoxic even at 1 µg/mL, while the hemotoxicity of the conjugates was considerably lower than the peptide. Remarkably in this model, the hemolytic activity of PepC-PEG-pepC conjugate decreased 15-fold from 2.36 to 31.12 µg/mL compared to the bacteria-free 60-min treatment. This is proof that in the case of bacteremia and sepsis, the conjugates specifically direct to bacterial cell membranes rather than red blood cells. In addition, the PepC-PEG-pepC conjugate is resistant to plasma proteases. Moreover, morphological and intracellular damage of the peptide/conjugates to Escherichia coli are demonstrated in SEM and TEM images. These results suggest our molecules can be considered potential next-generation broad-spectrum antibiotic molecule/drug candidates that might be used in clinical cases such as bacteremia and sepsis.

A Novel Peptide-Based Detection of SARS-CoV-2 Antibodies.

The need for rapidly developed diagnostic tests has gained significant attention after the recent pandemic. Production of neutralizing antibodies for vaccine development or antibodies to be used in diagnostic tests usually require the usage of recombinant proteins representing the infectious agent. However, peptides that can mimic these recombinant proteins may be rapidly utilized, especially in emergencies such as the recent outbreak. Here, we report two peptides that mimic the receptor binding domain of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and investigate their binding behavior against the corresponding human immunoglobulin G and immunoglobulin M (IgG and IgM) antibodies in a clinical sample using a quartz crystal microbalance (QCM) sensor. These peptides were immobilized on a QCM sensor surface, and their binding behavior was studied against a clinical serum sample that was previously determined to be IgG and IgM-positive. It was determined that designed peptides bind to SARS-CoV-2 antibodies in a clinical sample. These peptides might be useful for the detection of SARS-CoV-2 antibodies using different methods such as enzyme-linked immunosorbent assay (ELISA) or lateral flow assays. A similar platform might prove to be useful for the detection and development of antibodies in other infections.

Synthesis and antimycobacterial activity of some phthalimide derivatives.

Structurally modified phthalimide derivatives were prepared through condensation of phthalic and tetrafluorophthalic anhydride with selected sulfonamides with variable yields. All compounds were screened for their antimycobacterium activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) using a micro broth dilution technique. The fluorinated derivatives (compounds 2c, 2d, 2f and 2h) had antimycobacterium activity comparable with classical sulfonamide drugs. The minimum inhibitory concentration (MIC) of compounds 2c, 2d, 2f and 2h was greater than that of isoniazid (MIC<0.02 µg/mL) and in vitro activity was greater than that of pyrazinamide, another first line antimycobacterium drug (MIC 50-100 µg/mL). The new compounds could be considered new lead compounds in the treatment of multi-drug resistant tuberculosis.

Polymeric Approach to Adjuvant System in Antibody Production against Leishmaniasis Based on Hybridoma Technology.

Background: Leishmaniasis is a zoonotic disease, which is one of the serious public health problems in the world. Nowadays, antibody production using hybridoma technology may be a correct approach in terms of sensitivity in the diagnosis of diseases such as leishmaniasis. The aim of this study was investigation of the effectiveness of different adjuvants on polyclonal antibody production against L. tropica based on hybridoma technique. Methods: Accordingly, Freund's adjuvant (1956, M. tuberculosis), as a classic adjuvant in studies, was used comparatively with the non-toxic polymeric based Polyoxidonium adjuvant. All animal immunization procedures were conducted at Bezm-i Alem University Experimental Animal Research Center. The adjuvant response was tested both in the serum sample and in the antibodies produced by the hybridomas. The antibody titers were determined with ELISA. Results: Freund's and Polyoxidonium (PO) group blood titer's increased approximately 5.5 fold compared to control after the 6th and 8th immunization. Hybridomas produced from mice immunized with PO adjuvant induced only antigen-specific antibody response and did not develop an immune response against the adjuvant. Conclusion: Adjuvant selection is very important in terms of the specificity of antibody responses of cells produced in hybridoma technology. Therefore, PO is recommended as a new adjuvant system in this study.

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.