Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

Biofilm Formation and Multiplex PCR detection of icaABCD Operon in Staphylococcus capitis

Aims: The ability to form biofilm is a major virulence factor in the virulence of the Coagulase negative Staphylococcus (CoNS) group of bacteria. Being the most predominant member of CoNS, the ability of S. epidermidis in causing biofilm-associated infections has been well established. On the other hand, S. capitis and has always been regard as a non-pathogenic species although recently it was found to be responsible in a variety of infections. Hence, this study aimed to determine the biofilm formation capabilities and the presence of icaABCD genes in clinical isolates of S. capitis, which have emerged as an important opportunistic pathogen in clinical settings. Methodology: S. capitis was isolated and identified from 17 out of 200 clinical samples. Biofilm formation assay was performed quantitatively using a microtitre plate method. Mulitplex PCR primers for icaABCD genes were designed from DNA sequences coding for the icaA, B, C, and D structural genes of S capitis JF930147.1 which was compared together with five other species of Staphylococcus. Amplification of the icaABCD genes was performed using the designated primers. Results: From the 17 strains of S. capitis clinical isolates, 14 were identified as S. capitis subsp capitis while the remaining three were identified as S. capitis subsp ureolyticus. Except for two of the S. capitis subsp capitis isolates, the remaining strains were able to form biofilm, with majority of them were strong biofilm formers. Multiplex PCR was successful in amplifying the four icaABCD genes which was demonstrated in all the S. capitis isolates, including the two non-biofilm forming isolates. Conclusion: Majority of the S. capitis isolates were able to form biofilm phenotypically suggesting the possibility in causing opportunistic infections through indwelling medical devices. Multiplex PCR however was able to detect the presence of the icaABCD genes in all the S. capitis isolates. This suggests that the biofilm assessment on microtitre plate is not a definitive tool in determining the production of polysaccharide intercellular adhesion (PIA) but the production of the icaABCD genes could be a better assessment in determining biofilm production in Staphylococcus.

Factors affecting the accuracy and reliability of the measurement of anti-Müllerian hormone concentration in the clinic

Objective We aimed to identify the factors that influence serum anti-Müllerian hormone (AMH) concentration measurements. Methods We collected serum samples between May and September 2018 and compared the effect on AMH concentration measured by ELISA of conditions including venepuncture, storage time, storage temperature, locations of the reaction microplate, and the use of the oral contraceptive pill and gonadotrophin-releasing hormone (GnRH). Results AMH concentration was not affected by food intake but was affected by haemolysis. It was also much higher in samples on the edge of the ELISA microtitre plate. AMH concentration increased after incubation at room temperature for 1 day, 4°C for 3 days, −20°C for 1 month and −40°C for 4 months, but no change occurred during storage at −80°C for 9 months. AMH concentration was high in patients following GnRH agonist treatment but was not affected by oral contraceptives. Conclusions No fasting is required prior to AMH measurement. Placement of serum samples on the edge of microtitre plates affects the results of the AMH ELISA. If serum samples cannot be assayed immediately, it is best to store them at −80°C. Basal AMH concentration cannot be used as a measure of ovarian reserve after GnRH agonist treatment.

Protocol for evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative bacterial vector

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.

Protocol for evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative bacterial vector

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.

Inhibitory Effects of Antifungal Drugs on Biofilm Producing Aspergillus spp. Recovered from Drinking Water System

Aims: Biofilms formed in drinking water distribution systems serve as a continuous source of fungal infections. Biofilms are thick aggregates of adherent microorganisms including pathogenic species of fungi. Respiratory diseases and skin allergy reactions are caused by drinking water containing biofilm forming fungus and bacteria. One of the main causes of nosocomial infections and respiratory diseases in hospitals is due to the fungal biofilm formation in machines, catheters and other surgical instruments. There are some antifungal drugs which are used to control biofilm formation to minimize the infection rate. Methodology and results: The present study was conducted to isolate and identify Aspergillus species which are the main fungal spp. responsible for the biofilm formation in drinking water and to check their antifungal susceptibility against antifungal drugs. The isolated fungal samples from drinking water were cultivated on Potato dextrose agar for the isolation of Aspergillus species. Isolated Aspergillus species were identified on the basis of cultural, morphological and microscopic examination. Then in-vitro ability of biofilm produced by isolated Aspergillus species was estimated using microtitre plate method and quantification by crystal violet assay. Antifungal susceptibility testing against isolated fungal spp. was done by antifungal drug Amphotericin B. Results: From results, it is concluded that drinking water of labs, hospitals and common water chillers were more prevelant by Aspergillus species whereas water from reverse osmosis plants showed negative results. From microtitre plate method and crystal violet assay, it was concluded that Aspergillus spp. are Susceptible against Amphotericin B drug as compared to miconazole. Keywords: Aspergillus spp; Biofilm; Drinking water; Disk diffusion method; Amphotericin B

Inhibitory effects of antifungal drugs on biofilm producing Aspergillus spp. recovered from drinking water system

Aims: Biofilms formed in drinking water distribution systems serve as a continuous source of fungal infections. Biofilms are thick aggregates of adherent microorganisms including pathogenic species of fungi. Respiratory diseases and skin allergy reactions are caused by drinking water containing biofilm forming fungus and bacteria. One of the main causes of nosocomial infections and respiratory diseases in hospitals is due to the fungal biofilm formation in machines, catheters and other surgical instruments. There are some antifungal drugs which are used to control biofilm formation to minimize the infection rate. Methodology and results: The present study was conducted to isolate and identify Aspergillus species which are the main fungal spp. responsible for the biofilm formation in drinking water and to check their antifungal susceptibility against antifungal drugs. The isolated fungal samples from drinking water were cultivated on Potato dextrose agar for the isolation of Aspergillus species. Isolated Aspergillus species were identified on the basis of cultural, morphological and microscopic examination. Then in-vitro ability of biofilm produced by isolated Aspergillus species was estimated using microtitre plate method and quantification by crystal violet assay. Antifungal susceptibility testing against isolated fungal spp. was done by antifungal drug Amphotericin B.Results: From results, it is concluded that drinking water of labs, hospitals and common water chillers were more prevelant by Aspergillus species whereas water from reverse osmosis plants showed negative results. From microtitre plate method and crystal violet assay, it was concluded that Aspergillus spp. are Susceptible against Amphotericin B drug as compared to miconazole.

Comparison of Biofilm Producing and Non-Producing Escherichia coli Isolated from Urine Samples of Patients Visiting a Tertiary Care Hospital of Morang, Nepal

Objectives: The main objective of this study was to determine the prevalence of Escherichia coli among urinary tract infection (UTI) suspected patients visiting tertiary care hospital and to assess the biofilm producing ability of E. coli isolates. Methods: A prospective cross-sectional study was carried out in Biratnagar Metropolitan city, Eastern Nepal from December 2018 to May 2019. During the study 400 urine samples were collected from UTI suspected patients visiting a tertiary care hospital of Biratnagar. Urine samples were cultured by using semi-quantitative culture technique and identified. Antibiotic susceptibility testing was done by Kirby-Bauer Disk Diffusion method according to CLSI (2011) guidelines. Biofilm assays were performed by microtitre plate method. Results: This study reported 15% prevalence of E. coli out of 400 urine samples. 100% of E. coli isolates showed resistance to both Ampicillin and Amoxicillin while 100% were sensitive to Chloramphenicol. 70% (42/60) isolates were Multi Drug Resistance (MDR)E. coli. The maximum isolates (86.66%) were found to be biofilm producers by microtitre plate method. Resistance to other antibiotics such as Nalidixic acid (71.11% vs 46.66%), Norfloxacin (53.33% vs 46.66%), Cotrimoxazole (42.22% vs 26.66%) was comparatively higher among biofilm producers than non-biofilm producers. There was a significance of association between biofilm and MDR (p<0.05). Conclusion: There is relation between the ability of biofilm formation and drug resistance in the bacterium resulting to the failure of antibacterial drugs.