Anaerobic Box Research Articles

Optimization of methods for sanitary-microbiological and sanitary-parasitological control of wastewater

Introduction. The main reason for the problems of surface reservoirs in many regions of Russia is the non-compliance with sanitary protection zones and the discharge of insufficiently treated wastewater into reservoirs. SanPiN 1.2.3685-21 introduced a list of the new controlled sanitary-microbiological and parasitological safety indicators for disinfected wastewater and surface water bodies. For some indicators, there are no methods of determination in the current methodological documents. In this work, studies were carried out using various methods, including accelerated ones, which make it possible to determine the normalized indicators in wastewater and surface waters. Goals and objectives - optimization of methods for the study of surface and wastewater in the implementation of sanitary-microbiological and sanitary-parasitological control. Materials and methods. Bacteriological, parasitological indicators, as well as coliphages were determined in samples of surface and wastewater. To detect and identify generalized coliform bacteria, E. coli, enterococci, P. aeruginosa and legionella, in addition to membrane filtration and direct seeding methods, test systems (IDEXX) were used: Colilert-18 and Colilert-24, Pseudolert, Enterolert, Legionelert. Obligate anaerobes of C. perfringens were cultured in an anaerobic box (Anaerobic Station A55, Don Whitley Scientific) in an atmosphere of a three-component gas mixture. Species identification of microorganisms was carried out by time-of-flight mass spectrometry (MALDI TOF MS) using a Microflex mass spectrometer with MALDI BioTyper software (Bruker, Germany). Isolation of RNA/DNA of viruses and bacteria was carried out using a set of reagents “AmpliSens. RIBOT-prep.” Results. As a result of the conducted studies, P. aeruginosa; E. coli, R. ornithinolytica, A. hydrophila, A. caviae, A. molluscorum; E. hirae, E. faecium, E.faecalis; C. perfringens; S. lutetiensis, S. suis were isolated and identified from wastewater at all stages of treatment, high fungal contamination was found, and also Group F adenovirus DNA (both in wastewater and surface water samples) and additionally rotovirus and norovirus RNA, Campylobacter DNA. SarsCov-2 virus RNA was not detected in any sample. Lamblia spp., Blastocystis spp. and Cryptosporidium parvum were identified; opportunistic Entamoeba spp.; helminth eggs - Toxocara spp., Hymenolepis diminuta, Hymenolepis nana, Asagis lumbricoides, egg oncospheres Tenia spp.; Nematodes spp. larvae. Limitations. Since clinically significant strains of bacteria, fungi, and parasitic protozoa were found in the studied water samples, it is important to expand knowledge about water sources as reservoirs and spreaders of these pathogens. Conclusion. Monitoring is now urgently needed to quantify pathogens circulating in surface water and wastewater.

Role of mitochondrial permeability transition pore in cholinergic anti-inflammatory pathway in cardiomyocytes

Objective To investigate the role of mitochondrial permeability transition pore (mPTP) in cholinergic anti-inflammatory pathway in cardiomyocytes. Methods After being cultured for 72 h, cardiomyocytes of neonatal Sprague-Dawley rats were randomly divided into 5 groups (n=36 each) using a random number table: control group (group C), anoxia/reoxygenation (A/R) group, specific α7 nicotinic acetylcholine receptor agonist PNU282987 group (PNU282987 group), mPTP opener atractyloside group(ATR group) and PNU282987+ ATR group (P+ A group). The cells were incubated in serum-free and low-glucose DMEM culture medium which was placed in an anaerobic box (O2 < 0.1%) lasting for 6 h, the culture medium was then replaced with high-glucose DMEM culture medium supplemented with 10% fetal bovine serum, and the cells were then re-incubated and exposed to 5% CO2 in normoxia in an incubator at 37 ℃ for 6 h to establish a model of A/R injury. In P and ATR groups, PNU282987 (final concentration 30 μmol/L) and atractyloside (final concentration 20 μmol/L) were added to the culture medium immediately after onset of reoxygenation, respectively, and in P+ A group, the combination of the two drugs previously mentioned was added instead.At 6 h of reoxygenation, lactate dehydrogenase (LDH) release rate, apoptosis, expression of NF-κBp65 and phosphorylated NF-κB p65 Ser536 (p-NF-κB p65 Ser536) in cardiomyocytes, concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant, and mitochondrial membrane potential (△Ψm) were detected. Results Compared to group C, the LDH release rate, early and late apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly increased, the expression of NF-κB p65 and p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in A/R, P, ATR and P+ A groups.Compared to A/R group, the LDH release rate, early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly decreased, the expression of p-NF-κB p65 Ser536 was down-regulated, and △Ψm was increased in group P, the LDH release rate, early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were increased, the expression of p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in ATR group, and the early apoptosis and concentrations of IL-6 and TNF-α in the supernatant were decreased, the expression of p-NF-κB p65 Ser536 was down-regulated, and there was no significant change in LDH release rate, late apoptosis and Δψ in P+ A group.Compared to group P, the LDH release rate, late apoptosis and concentrations of IL-6 and TNF-α in the supernatant were significantly increased, the expression of p-NF-κB p65 Ser536 was up-regulated, and △Ψm was decreased in P+ A group. Conclusion Cholinergic anti-inflammatory pathway exerts anti-inflammatory effect through inhibiting mPTP opening, thus reducing A/R injury to cardiomyocytes. Key words: Mitochondrial membrane transport protein; Acetylcholine; Vagus nerve; Myocardial reperfusion injury; Myocytes, cardiac

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.

Oxygen tolerance in anaerobic pathogenic bacteria

A prerequisite for successful identification of anaerobic pathogenic bacteria from samples of clinical material is the method of cultivation. Currently, several methods of cultivation in anaerobic environment are used: cultivation in anaerobic box, anaerobic jar, and in nonrecurring cultivation system. Here, we determined the suitability of the above methods of cultivation using the estimation of the growth (diameters of colony size) of commonly isolated anaerobic pathogens (Bacteroides fragilis, Clostridium difficile, and Clostridium perfringens). The tested bacterial strains were exposed to atmospheric oxygen for various time periods and then they were cultivated using different anaerobic cultivation systems. Maximum growth differed, depending on the type of cultivation and the strain used. Thus, largest zone diameters, in the majority of measurements, were achieved in the anaerobic box. However, nonrecurring cultivation system seemed better in several cases; this applied to the cultivation of C. perfringens after 15, 30, and 60 min exposure to atmospheric oxygen as well as the cultivation of B. fragilis after 30 and 60 min of oxygen exposure. The cultivation in anaerobic box was the most convenient method for growth of C. difficile. In almost all cases, higher growth was observed in nonrecurring cultivation system than in the system of anaerobic jar. On the other hand, no significant differences were observed among these anaerobic cultivation systems which confirmed their applicability (taking into account some individual features concerning the optimization of cultivations) for identification of pathogenic anaerobes.

Changes in Aerobic and Anaerobic Capacity in CUSSA Junior Alpine Skiers

INTRODUCTION: US Ski and Snowboard Association requires serious athletes to participate in annual fitness evaluations. Of primary interest in this study is the pacer test (aerobic) and 40 cm box jump test (anaerobic). Although annual tests are required, questions still remain regarding the usefulness of such tests to improve an athletes physiological profile. PURPOSE: To determine if junior alpine ski racers are showing improvements in the aerobic 20 meter pacer test and anaerobic box test over the past 3 years. METHODS: Subjects (N= 171) were junior alpine skiers ages 15–19 and members of the Central United States Ski Association (CUSSA). Data were collected from 2004 to 2006 on skiers who where able to complete both the aerobic and anaerobic portions of the test. Subjects performed a 20 meter pacer test to determine VO2max. During 2004, and 2005 subjects completed a 40 second peak lactate box jump for anaerobic capacity, and in 2006 subjects completed a 60 second box jump. To enable to compare box jump scores over the 3 years, the number of jumps for the first 40 seconds was used for all 3 years. Changes in aerobic capacity and anaerobic capacity were then analyzed across the 3 years using an Analysis of Variance. RESULTS: No statistical difference in VO2max for all athletes from 2004 to 2006, respectively (43.8 ± 7.4, 43.18 ± 7.9, and 45.27 ± 6.7) was found (p< .05). Although improvements in females were not significant, they at least demonstrated a more consistent trend to improve VO2max over the 3 years. Box test scores where not different over the 3 years. CONCLUSIONS: There is no change in aerobic and anaerobic capacity for CUSSA junior alpine ski racers from 2004 to 2006. This suggests that annual fitness assessments have little value for motivating athletes to improve their physiological profile. This might also suggest coaches are not using this information effectively to improve individual weaknesses of their athletes.

Evaluation of methods of transport and cultivation of bacterial specimens from infected dental root canals

Five different methods of transport and cultivation of bacteria from dental root canal specimens were evaluated. Bacteria were recovered from 29 percent of the specimens when they were transported in VMG IV and subcultured in PRAS medium or on the surface of blood agar in an anaerobic box. When a PRAS-peptone-yeast extract broth was used for transport and the specimens were initially subcultured in the broth, bacteria were recovered from 49 percent of the specimens. When fluid thioglycollate medium USP (BBL) and Clausen medium (Oxoid) were used for transport as well as for initial subculture, bacteria were recovered from 58 percent and 47 percent of the specimens, respectively. The high recovery of bacteria by these media could to a significant degree be ascribed to the use of the media for initial subculture of the specimens. Recovery of bacteria by fluid thioglycollate medium USP was as good or even better as with the more elaborate PRAS medium. Fluid thioglycollate medium USP in screw-capped vials requires no special equipment in the dental clinic and is recommended for use in routine dental practice.