Efficacy of extracts from cryopreserved placenta on third-degree burns in rats

Human placenta extracts have anti-inflammatory, antioxidant and wound-healing properties, so they are promising drugs for the treatment of wounds of various origins, including burns. Cryopreservation methods are widely used to preserve the biological activity of placental drugs for a long time. The aim of this work was to study the effect of low-temperature storage of the placenta on the regenerative properties of its extracts. Fragments of freshly obtained placentas were cooled by immersion in liquid nitrogen and stored at –196 °C for 6 months. The placenta was warmed in a water bath at 37 °C. The effect of low-temperature preservation of the placenta on the ability of its extracts to positively affect the wound healing process was studied in a model of thermal burn of III B degree in rats. The effectiveness of wound treatment with extracts from cryopreserved placenta was evaluated by planimetric and histological methods at 3, 7, 14, 21 and 28 days after the burn. The activity of antioxidant enzymes in the serum of animals was also determined. superoxide dismutase activity was assessed by inhibition of adrenaline autooxidation in carbonate buffer, catalase activity was assessed by the degree of inhibition of ammonium peroxide formation. It has been shown that the treatment of burns with extracts from cryopreserved placenta helped to accelerate the regeneration processes and the rate of wound healing. The formation of granulation tissue was detected on the 7th day of treatment with extracts, and on the 14th day in the control. The area of burn wounds during treatment with extracts probably differed from the control starting from 14 days after application of the burn. It was found that the dynamics of recovery of catalase activity after burns is probably higher on the 7th day of treatment with extracts. The obtained data testify to the high efficiency of application of placenta stored at low-temperature for the purpose of obtaining extracts from it with preservation of regenerative properties.

BLOOD BUFFER SYSTEMS (LECTURE)

This lecture is devoted to theoretical foundations of blood buffer systems functioning. Biochemical aspects and physiological activity of phosphate, hydrogen carbonate buffer and its combined activity with hemoglobin buffer, which ensures stability of blood pH, are presented. Chemical reactions to achieve the required blood pH are investigated. The combination of buffer properties, one of the components of which is CO2gas and autonomous self-regulation by intracellular hemoglobin ensures the blood plasma pH constancy. Stabilizing systems are considered -the respiratory apparatus and kidneys, which create the possibility of maintaining the stability of extracellular fluid pH. Respiratory acidosis, alkalosis, metabolic acidosis are considered on the biochemical level. This article presents information about hemoglobin structure: heme structure and globin subunits in different typesof hemoglobin. The following mechanismswhich provide maximumoxygen saturation of lungs and maximum oxygen emission in the tissues: heme-hemic interaction, Bohr effect and influence of 2,3-diphospho-glycerate connected with haemoglobin, are considered. The proteinbuffer system has been characterized in the in general. The capacity of the phosphate buffer system has been shown to be close to 1-2% of the whole buffer capacity of the blood and up to 50% of the buffer capacity of urine. The organic phosphates also exhibit buffering activity in the cell. Human and animal organisms can have intracellular pH from 4.5 to 8.5 depending on the type of cells, but the blood pH should be 7.4. This parameter is ensured by the hydrogen carbonate buffer system. Moreover,the blood pH depends not on the absolute concentrations of buffer components but on their ratio. The most powerful is hemoglobin buffer system that accounts for 75% of the whole blood buffer system. For stabilization of buffer capacity, the body uses two other stabilizing systems -the respiratory apparatus and kidneys. At the same time, the compensatory role of the respiratory system has shortcomings. Hyperventilation of lungs causes respiratory alkalosis. Hypoventilation has a counteracting effect by lowering the pH of the blood. Thus, the blood buffer system is ensured by a complex system that allows the organisms to adapt to changes in the fluid medium and regulate the pH under pathological conditions.Key words:homeostasis, hemoglobin, blood, acid-liquid equilibrium. У лекції розглядаються теоретичні основи механізмів дії буферних систем крові. Наводяться біохімічні аспекти та фізіологічна дія фосфатного, гідрогенкарбонатного буфера та його спільна дія з гемоглобіновим буфером, що забезпечує стабільність рН крові. Розглядаються хімічні реакції досягнення необхідного рівня рН крові. Поєднання властивостей буфера, одним з компонентів якого є газ СО2, та автономним саморегулюванням за рахунок внутрішньоклітинного гемоглобіну, забезпечує постійність рН плазми крові. Розглядаються стабілізуючі системи –дихальний апарат та нирки, які створюють можливості підтримання постійності рН позаклітинної рідини. На біохімічному рівні розглядаються дихальні ацидоз, алкалоз, метаболічний ацидоз. У статті представлені відомості про будову гемоглобіну: будову гему та субодиниць глобіну у різних видах гемоглобінів. Розглядаються механізми, що забезпечують максимальне насичення киснем легенів та максимальну віддачу кисню в тканинах: гем-гемова взаємодія, ефект Бора та вплив 2,3-дифосфо-гліцерату, зв’язаного з гемоглобіном. В загальних рисах охарактеризована білкова буферна система. Показано, що ємність фосфатної буферної системи становить близько 1-2% від всієї буферної ємності крові та до 50% буферної ємності сечі. При цьому органічні фосфати також виявляють буферну дію в клітині. В організмі людини і тварин значення внутрішньоклітинного рН може бути від 4,5 до 8,5 взалежності від типу клітин, проте рН крові має становити 7,4. Цей показник забезпечується гідрогенкарбонатною буферною системою. Причому, рН крові залежить не від абсолютних концентрацій компонентів буфера, а від їхнього співвідношення. Найбільш потужною є гемоглобінова буферна система, яка становить 75% від всієї буферної системи крові. Для стабілізації буферної ємності організм використовує ще дві стабілізуючі системи –дихальний апарат та нирки. Разом з тим, компенсаторна роль дихальної системи має недоліки. Гіпервентиляція легень спричиняє дихальний алкалоз. Гіповентиляція виявляє протилежну дію, знижуючи рН крові. Таким чином, буферна система крові забезпечується складною системою, що дозволяє організмові адаптуватися до змін оточуючого середовища та регулювати рН за патологічних умов.Ключові слова:гомеостаз, гемоглобін, кров, кислотно-лужна рівновага.

On the stability of microwave-fabricated SERS substrates – chemical and morphological considerations

The stability of surface-enhanced Raman spectroscopy (SERS) substrates in different organic solvents and different buffer solutions was investigated. SERS substrates were fabricated by a microwave-assisted synthesis approach and the morphological as well as chemical changes of the SERS substrates were studied. It was demonstrated that the SERS substrates treated with methanol, ethanol, or N,N-dimethylformamide (DMF) were comparable and showed overall good stability and did not show severe morphological changes or a strong decrease in their Raman activity. Toluene treatment resulted in a strong decrease in the Raman activity whereas dimethyl sulfoxide (DMSO) treatment completely preserved or even slightly improved the Raman enhancement capabilities. SERS substrates immersed into phosphate-buffered saline (PBS) solutions were observed to be rather instable in low and neutral pH buffer solutions. Other buffer systems showed less severe influences on the SERS activity of the substrates and a carbonate buffer at pH 10 was found to even improve SERS performance. This study represents a guideline on the stability of microwave-fabricated SERS substrates or other SERS substrates consisting of non-stabilized silver nanoparticles for the application of different organic solvents and buffer solutions.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia

Background Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production. Results In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. Conclusions Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

Tau Protein Preferentially Associates With Synaptic Mitochondria in a Mouse Model of Tauopathy

BackgroundA consequence of an aging society is a continual and dramatic increase in the number of patients suffering from tauopathies, including Alzheimer’s disease (AD) and certain frontotemporal dementias. Accumulation of intracellular inclusions of abnormal fibrillar forms and hyperphosphorylated forms of microtubule-associated protein tau are hallmarks of AD and other tauopathies. Although tau pathology is associated with neuronal dysfunction the mechanism responsible remains obscure. In vitro, pathologically elevated expression of tau alters mitochondrial distribution by impairing cellular trafficking and thus may represent an important mediator of mitochondrial abnormalities contributing to neuronal dysfunction. We used the transgenic htau mouse model of tauopathy to investigate in vivo alterations in brain mitochondria in the presence of pathological forms of human tau.MethodsIn this study, we investigated alterations in bioenergetics and profiled the proteome of brain mitochondria from wild-type (WT) and htau mice at ages prior to and coinciding with pathologic tau deposition in htau mice. In addition, we characterized the expression of total and hyperphosphorylated forms of tau associated with synaptic mitochondria by biochemical fractionation and immunoblotting.ResultsSignificant tau pathology-dependent alterations in synaptic mitochondrial bioenergetics were observed at 8 mo nths, but not 5 months, of agein htau mice; however, non-synaptic mitochondrial function remained unaltered. Further, compared to control mice, proteins involved in microtubule-based movement were differentially expressed in htau mice at 8 months of age. In addition, significant accumulation of tau and its hyperphosphorylated forms was observed in synaptic mitochondria isolated from 8-month-old htau mice.ConclusionThese data suggest that tau preferentially associates with synaptic mitochondria as compared to non-synaptic mitochondria, and accumulation of pathologic forms of tau coincides with synaptic mitochondrial bioenergetic changes reminiscent of an aged synaptic mitochondrial phenotype reported in aging WT mice. Furthermore, the mitochondrially associated tau is soluble in carbonate buffer and more accessible to protease action suggesting it is not integrated into mitochochondrial membranes, but may rather be the result of protein-protein interactions.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia

Background: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production.Results: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99x107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37% and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. Conclusions: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

Establishment and Evaluation of an Indirect ELISA for Detection of Antibodies to Goat Klebsiella Pneumonia

Background: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are no systematic methods for detection of Klebsiella pneumoniae in goats currently. Results: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99x107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800. Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for other common bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37% and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. Conclusions: Taken together, our results suggested that the method that we established has promising applications in production, laying the foundation for epidemiology, serological investigation and clinical veterinary diagnosis of Klebsiella pneumoniae in goats.