Activation Of Phospholipase Research Articles

Pre-clinical Efficacy and Immunogenicity of IgY Antibodies Directed Against Crotalus durissus cumanensis Venom

Background:: Crotalus durissus cumanensis (C.d.c.) is the most widely distributed snake in Venezuela, causing the majority of snakebite envenoming Objective:: The purpose of this study was to produce IgY antibodies against a C.d.c. venom pool from different Venezuelan regions and evaluate their neutralization capacity on various venom toxic activ-ities. Methods:: Anti-C.d.c. venom antibodies are purified from chicken egg yolks by precipitation with polyethylene glycol and further analyzed by Multiple Antigen Blot Assay, indirect ELISA, Western blot, and Inhibition assays. In addition, we evaluate the phospholipase, edematogenic, and hemor-rhagic activities. In addition, a new envenoming simulation study using anti-C.d.c. venom IgY in mice is presented. Results:: In this study, we show that anti-C.d.c. venom IgY is capable of neutralizing 4LD50 doses of the Cdc venom (i.e., 1.76 mg of IgY neutralized 14 μg of C.d.c. venom) and effectively neutralizing the phospholipase, edematogenic and hemorrhagic activities. Additionally, the anti C.d.c. venom IgY specifically recognizes polypeptide bands with apparent molecular masses of ~ 54.55, 30.39, 24.1, 14.02, and 9.44 kDa by western blot. The IgY specificity is demonstrated by a dose-dependent inhi-bition, in which antibodies pre-adsorbed with the C.d.c. venom does not recognize the proteins con-tained in the venom. Furthermore, in the simulation study of envenoming, the mice inoculated with IgY showed no response. Conclusion:: Our results support the use of anti-venom IgY as an alternative to traditional equine therapy in animals and, eventually, in human patients bitten by C.d.c snakes. conclusion: Our results support the use of anti-venom IgY as an alternative to traditional equine therapy in animals and, eventually, in human patients bitten by C.d.c snakes.

Study of the influence of ionizing radiation on the activity of the neurotoxin Vipoxin in a model monolayer system at the water/air interface

The main components of the cell membrane are phospholipids, and the enzymes that hydrolyze them for the purposes of many important physiological functions are the phospholipases whose properties strongly depend on the phospholipids’ structural organization. Here, we propose a proper experimental approach to study the effect of ionizing radiation on the catalytic action of the neurotoxin Vipoxin, a heterodimeric protein complex composed of an active basic secretory phospholipase A2 (PLA2) and an inactive acidic component, known as “inhibitor”, on the phospholipid substrate such as α-L-dilauroylphosphatidylcholine (DLPC) using as a model system Langmuir monolayer. First, the Vipoxin dissolved in buffer solution was irradiated in a controlled manner with radiation doses varying from 0.1 to 10 Gy, next, the Vipoxin PLA2 activity was followed in real-time, using a modified Langmuir trough, measuring the surface area change of the monolayer. This approach allowed us to obtain the dependence of the neurotoxin catalytic activity at a particular surface pressure and to determine the fundamental constants of the enzyme reaction. It was found that the activity of Vipoxin was influenced in a dose dependent manner. We also found that at higher irradiation doses the enzymatic activity increased, hypothetically due to dissociation of its dimeric Vipoxin complex.

Phosphatidylinositol-specific phospholipase C-associated phospholipid metabolism mediates DcGLRs channel to promote calcium influx under CaCl2 treatment in shredded carrots during storage

The rapid activation of phosphatidylinositol-specific phospholipase C (PI-PLC) occurs early after the stimulation of biotic and abiotic stress in plants, which directly associated with the calcium channel-induced calcium ion (Ca2+) influx. Exogenous calcium chloride (CaCl2) mediates the calcium signaling transduction to promote the γ-aminobutyric acid accumulation and nutritional quality in shredded carrots whereas the generation mechanism remains uncertain. Therefore, the involvement of PI-PLC-associated phospholipid metabolism was investigated in present study. Our result revealed that CaCl2 treatment promoted the expression and activity of PI-PLC and increased the inositol 1,4,5-trisphosphate and hexakisphosphate content in shredded carrots. The transcripts of multi-glutamate receptor-like channels (DcGLRs), the glutamate and γ-aminobutyric acid (GABA) content, and Ca2+ influx were induced by CaCl2 treatment in shredded carrots during storage. However, PI-PLC inhibitor (U73122) treatment inhibited the activation of PI-PLC, the increase of many DcGLRs family genes expression levels, and Ca2+ influx. Moreover, the identification of DcPI-PLC4/6 and DcGLRs proteins, along with the analysis of characteristic domains such as PLCXc, PLCYc, C2 domain, transmembranous regions, and ligand binding domain, suggests their involvement in phospholipid catalysis and calcium transport in carrots. Furthermore, DcPI-PLC4/6 overexpression in tobacco leaves induced the Ca2+ influx by activating the expressions of NtGLRs and the accumulation of glutamate and GABA. These findings collectively indicate that CaCl2 treatment-induced PI-PLC activation influences DcGLRs expression levels to mediate cytosolic Ca2+ influx, thus, highlighting the “PI-PLC-GLRs-Ca2+” pathway in calcium signaling generation and GABA biosynthesis in shredded carrots.

Abstract 3058: Cofilin 1 Mediates Fatty Acid-induced Foam Cell Formation And Inflammation In Mouse Bone Marrow-derived Macrophages

Background: Macrophages contribute to the development of atherosclerosis, in part through the accumulation of lipid droplets and subsequent transformation into highly inflammatory, lipid laden foam cells. Our previous work suggested that the RAGE/DIAPH1 axis plays a key role in regulating lipid metabolism, as deletion of Diaph1 in hepatocytes reduced nuclear translocation of SREBP1 through the actin cytoskeleton. Cofilin 1 (CFL1) is an actin polymerization molecule which has also been linked to the nuclear translocation of SRBP1, as well as triglyceride synthesis through activation of phospholipase D1. In the aforementioned study by our group, Diaph1 deletion was found to increase the Serine 3 phosphorylation (i.e., inactivation) of Cfl1 . Hypothesis: Accordingly, we hypothesized that CFL1 plays a role in lipid droplet accumulation in macrophages, potentially increasing the risk of atherosclerotic development. Methods: To investigate its role in macrophage lipid handling, we silenced Cfl1 in bone marrow derived macrophages (BMDMs) extracted from male C57BL/6J mice, using silencing RNAs and scramble controls. BMDMs were treated with a mixture of fatty acids palmitate and oleate (250 uM). The effects of Cfl1 knockdown on lipid accumulation were assessed using immunohistochemical staining (Bodipy). Quantitative (q)PCR was used to interrogate the mechanisms underlying these effects, and their impact on inflammation. Results: Cfl1- silenced (75% knockdown shown by qPCR) vs. scramble cells exhibited a significant decrease in the ratio of Bodipy to DAPI % area (p=0.0001). By qPCR, compared to scramble controls, BMDMs subjected to silencing of Cfl1 and incubated with fatty acids (palmitate-oleate) displayed significantly lower mRNA expression of Cd36 (p=0.0018) ; Ppargc1a (p=0.004) , Il6 (p<0.0001), and Tnfa (p=0.013). Conclusions: Our results indicate that silencing of Cfl1 leads to a decrease in lipid accumulation and markers of inflammation in BMDMs. Collectively, our data suggest that the effects of Cfl1 silencing on lipid handling result from decreased fatty acid uptake, which limits fatty acid oxidation on account of fatty acid availability in the cell. Studies are underway to probe the implications of these findings in atherosclerosis.

Melittin—The principal toxin of honeybee venom—Is also produced in the honeybee fat body

Melittin is a powerful toxin present in honeybee venom that is active in a wide range of animals, from insects to humans. Melittin exerts numerous biological, toxicological, and pharmacological effects, the most important of which is destruction of the cell membrane. The phospholipase activity of melittin and its ability to activate phospholipases in the venom contribute to these actions. Using analytical methods, we discovered that the honeybee Apis mellifera produces melittin not only in the venom gland but also in its fat body cells, which remain resistant to this toxin's effects. We suggest that melittin acts as an anti-bacterial agent, since its gene expression is significantly upregulated when honeybees are infected with Escherichia coli and Listeria monocytogenes bacteria; additionally, melittin effectively kills these bacteria in the disc diffusion test. We hypothesize that the chemical and physicochemical properties of the melittin molecule (hydrophilicity, lipophilicity, and capacity to form tetramers) in combination with reactive conditions (melittin concentration, salt concentration, pH, and temperature) are responsible for the targeted destruction of bacterial cells and apparent tolerance towards own tissue cells. Considering that melittin is an important current and, importantly, potential broad-spectrum medication, a thorough understanding of the observed phenomena may significantly increase its use in clinical practice.

Unveiling the Significance of Peroxiredoxin 6 in Central Nervous System Disorders.

Peroxiredoxin 6 (Prdx6), a unique 1-Cys member of the peroxiredoxin family, exhibits peroxidase activity, phospholipase activity, and lysophosphatidylcholine acyltransferase (LPCAT) activity. Prdx6 has been known to be an important enzyme for the maintenance of lipid peroxidation repair, cellular metabolism, inflammatory signaling, and antioxidant damage. Growing research has demonstrated that the altered activity of this enzyme is linked with various pathological processes including central nervous system (CNS) disorders. This review discusses the distinctive structure, enzyme activity, and function of Prdx6 in different CNS disorders, as well as emphasizing the significance of Prdx6 in neurological disorders.

One-pot synthesis and biochemical characterization of the novel magnetic phospholipase D nanoflowers modified with polyphenols

Phospholipase D (PLD) can catalyze the hydrolytic cleavage of phosphodiester bonds and transphosphatidylation and has been widely employed in the chemical and pharmaceutical fields. To improve the stability and reusability of PLD, an immobilized enzyme system was developed using magnetic nanoparticles modified with polyphenols (dopamine and tannic acid) and metal hybrid nanoflowers to co-immobilize phospholipase D (PLD). This immobilization technique aimed to improve the enzymatic performance through increased surface area, reduced mass transfer limitations, and facilitated reuse. The hydrolytic and transphosphatidylation activities of PLD immobilized on polyphenol-modified magnetic nanoflowers (PDA-MNFs and TA-MNFs) were evaluated and compared to free PLD. PDA-MNFs and TA-MNFs showed significantly enhanced hydrolytic activity (19.9-fold and 21.7-fold increases, respectively) and transphosphatidylation activity (over 1.79-fold and 1.96-fold increases, respectively) compared to free PLD. Temperature and pH stability were also markedly improved for the immobilized PLD. After 10 reuse cycles, PDA-MNFs and TA-MNFs retained over 65 % of their initial transphosphatidylation activity. Therefore, this co-deposition and polymerization immobilization strategy using polyphenol-modified magnetic nanoflowers has the potential for efficiently increasing enzymatic performance and synthesizing industrially relevant compounds like phosphatidic acid and phosphatidylserine

Anti-Biofilm Activity of Assamsaponin A, Theasaponin E1, and Theasaponin E2 against Candida albicans.

Biofilm formation plays a crucial role in the pathogenesis of Candida albicans and is significantly associated with resistance to antifungal agents. Tea seed saponins, a class of non-ionic triterpenes, have been proven to have fungicidal effects on planktonic C. albicans. However, their anti-biofilm activity and mechanism of action against C. albicans remain unclear. In this study, the effects of three Camellia sinensis seed saponin monomers, namely, theasaponin E1 (TE1), theasaponin E2 (TE2), and assamsaponin A (ASA), on the metabolism, biofilm development, and expression of the virulence genes of C. albicans were evaluated. The results of the XTT reduction assay and crystal violet (CV) staining assay demonstrated that tea seed saponin monomers concentration-dependently suppressed the adhesion and biofilm formation of C. albicans and were able to eradicate mature biofilms. The compounds were in the following order in terms of their inhibitory effects: ASA > TE1 > TE2. The mechanisms were associated with reductions in multiple crucial virulence factors, including cell surface hydrophobicity (CSH), adhesion ability, hyphal morphology conversion, and phospholipase activity. It was further demonstrated through qRT-PCR analysis that the anti-biofilm activity of ASA and TE1 against C. albicans was attributed to the inhibition of RAS1 activation, which consequently suppressed the cAMP-PKA and MAPK signaling pathways. Conversely, TE2 appeared to regulate the morphological turnover and hyphal growth of C. albicans via a pathway that was independent of RAS1. These findings suggest that tea seed saponin monomers are promising innovative agents against C. albicans.

Prediction of matrilineal specific patatin-like protein governing in-vivo maternal haploid induction in maize using support vector machine and di-peptide composition

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6–7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity.

Purinergic signaling promotes premature senescence

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Up-regulation of CDR1 and MDR1 efflux pump genes and fluconazole resistance are involved in recurrence in Candida albicans-induced vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC) due to fluconazole resistance in Candida albicans isolates causes a wide range of complications. A number of 63 Candida albicans isolates obtained from vulvovaginal candidiasis (VVC) were identified by Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP). Antifungal susceptibility testing was performed by broth microdilution method according to the CLSI protocol. The role of CDR1 and MDR1 genes in progress of VVC to RVVC was examined and the activity of virulence-related enzymes was assessed. Candida albicans was diagnosed in 62.4 % cases, of which 22.2 % were confirmed as RVVC. Voriconazole was the most active drug among five tested antifungals. The mean expression level of CDR1 and MDR1 was higher in RVVC isolates compared to multidrug azole-resistant VVC isolates. Our results demonstrated that the expression of CDR1 and MDR1 and the level of phospholipase and proteinase activities could be quite important to induce fluconazole resistance in C. albicans and to progress of VVC to become RVVC in involved patients.

Obliteration of the Processus Vaginalis After Testicular Descent

The testis develops in the abdominal cavity and descends into the scrotum. Although numerous theories have been proposed, the mechanism of descent and the reason for its inhibition remain unknown. Furthermore, none of the explanations account for the other occurrences related to the descent, such as failed obliteration of the processus vaginalis, or the reasons for the decrease in fertility and increase in the risk of malignancy associated with an undescended testis. The gubernaculum is a primitive mesenchymal tissue that was first described in 1786. However, the role of the gubernaculum in the descent process remains obscure. The testis descends through the processus vaginalis. Although the processus vaginalis (PV) is usually defined as a simple peritoneal protrusion, it actively develops into the gubernaculum. The gubernaculum gives rise to the smooth muscles that surround the processus vaginalis. The striated cremaster muscle (CM) is also derived from the gubernaculum. Because the testis descends through the processus vaginalis, the muscles develop to propel the testis. After propelling the testis, the smooth muscle (SM) undergoes programmed cell death. The initiation of programmed cell death through the intrinsic pathway requires activation of phospholipase C. A transient shift in the autonomic balance via a decrease in the sympathetic tonus and an increase in the parasympathetic tonus is essential for initiating this programmed cell death. Programmed cell death in the SM is the physiological pathway for the obliteration of the processus vaginalis. Differences in the timing, intensity, or duration of this physiological pathway result in pathological conditions. A shift before testicular descent diminishes the SM content that is required to propel the testis, and thus inhibits descent. The early shift persists throughout childhood and results in the decrease in fertility and increase in the risk of malignancy because of the differences in signal transduction. Despite a successful descent, persistence of the shift alters the contractility of the CM by increasing the cytosolic calcium levels. Contracted CMs retracts or even ascends the testis. Inadequate intensity or duration of the shift of autonomic tonus causes failure of the programmed cell death. Persistence of the SM hinders the obliteration of PV and gives rise to hydroceles or inguinal hernias depending on the amount of residual smooth muscles. Similar findings from different countries support these explanations. Thus, our proposed mechanism satisfactorily explains the process of descent while considering all the factors related to the process of testicular descent.

First Report of the Emerging Pathogen Kodamaea ohmeri in Honduras.

Kodamaea ohmeri is an environmental yeast considered a rare emerging pathogen. In clinical settings, the correct identification of this yeast is relevant because some isolates are associated with resistance to antifungals. There is a lack of available data regarding the geographical distribution, virulence, and drug resistance profile of K. ohmeri. To contribute to the knowledge of this yeast, this study aimed to describe in depth three isolates of K. ohmeri associated with fungemia in Honduras. The identification of the isolates was carried out by sequencing the ribosomal ITS region. In addition, the susceptibility profile to antifungals was determined, and some properties associated with virulence were evaluated (exoenzyme production, biofilm formation, cell adhesion, and invasion). The isolates showed strong protease, phospholipase, and hemolysin activity, in addition to being biofilm producers. Adherence and invasion capacity were evident in the HeLa and Raw 264.7 cell lines, respectively. This study expands the understanding of the underlying biological traits associated with virulence in K. ohmeri, and it is the first report of the detection and identification of K. ohmeri in Honduras as a cause of human infection.

Toxic or medicinal? New approach to the pharmacological and toxicological properties of Echinodorus macrophyllus leaves

Echinodorus macrophyllus contains some phytochemicals that can induce toxicity but are also promising as ingredients in products to promote and maintain health, improve the immune system, and prevent diseases. In this study, the aqueous and ethanolic extracts of leaves of this species were characterized by high-performance liquid chromatography, investigated in vitro regarding their modulating action on snake venom enzymes, and evaluated in digestive enzyme inhibition assays. The extracts inhibited the phospholipase activity induced by Bothrops jararacussu and B. moojeni venoms. The hemolytic activity induced by Crotalus durissus terrificus venom was potentiated by 30-73% by the ethanolic extract at the ratios analyzed. The thrombolytic activity induced by B. moojeni venom was inhibited by 51% and 34% by the aqueous and ethanolic extracts at a ratio of 1:5 (w:w), respectively. The clotting time induced by B. moojeni and Lachesis muta muta venoms was prolonged after incubation with either extract. The extracts also exerted antigenotoxic action on DNA fragmentation induced by L. muta muta venom. Both extracts lowered the enzymatic activity of α-glucosidases, and the ethanolic extract inhibited α-amylases. The inhibition of digestive enzymes suggests the extracts could decrease calorie availability from carbohydrate intake. Results confirm the potential therapeutic use of E. macrophyllus in the prevention and treatment of hemostatic changes, since the proteins of venoms inhibited by it exhibit high homology with human proteins involved in various pathophysiological processes, such as inflammatory responses and blood coagulation.

Endothelial lipase variant T111I does not alter inhibition by angiopoietin-like proteins

High levels of HDL-C are correlated with a decreased risk of cardiovascular disease. HDL-C levels are modulated in part by the secreted phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL and decreases circulating HDL-C concentrations. A 584C/T polymorphism in LIPG, the gene which encodes EL, was first identified in individuals with increased HDL levels. This polymorphism results in a T111I point mutation the EL protein. The association between this variant, HDL levels, and the risk of coronary artery disease (CAD) in humans has been extensively studied, but the findings have been inconsistent. In this study, we took a biochemical approach, investigating how the T111I variant affected EL activity, structure, and stability. Moreover, we tested whether the T111I variant altered the inhibition of phospholipase activity by angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two known EL inhibitors. We found that neither the stability nor enzymatic activity of EL was altered by the T111I variant. Moreover, we found no difference between wild-type and T111I EL in their ability to be inhibited by ANGPTL proteins. These data suggest that any effect this variant may have on HDL-C levels or cardiovascular disease are not mediated through alterations in these functions.

Numerical Analysis of Compound Biochemical Calcium Oscillations Process in Hepatocyte Cells.

The hepatocyte cells regulate the wide range of liver function by moderating cellular activities such as lipid, protein metabolism, carbohydrate, and interact with other cells for proliferation and maintenance. In hepatocyte cells, the concentration of calcium uptake is quite extensive from various agonists such as active subunit, active phospholipase C, free calcium in the cytosol, and endoplasmic reticulum. The overproduction and degradation of calcium signals can cause homeostasis, liver inflammation, and liver diseases. The spatiotemporal behavior of calcium oscillation reveals the physiological role of these cellular entities in understanding the process of production and degradation. No computational attempt has been registered to date on the compound calcium regulation of these cellular entities including the memory of cells. Hence, the authors proposed a fractional order compartmental model that systematically simulates the exchange of calcium intake in cellular entities. The nonlinear equations of the rate of changes in the active subunit, active phospholipase C, free calcium in the cytosol, and endoplasmic reticulum are coupled to form a nonlinear fractional order initial value problem. The existence and uniqueness, stability analysis of the model is performed that validate the theoretical results and explore the dynamic behaviour of calcium oscillation in each compartment.

Obliteration of the Processus Vaginalis After Testicular Descent.

The testis develops in the abdominal cavity and descends into the scrotum. Although numerous theories have been proposed, the mechanism of descent and the reason for its inhibition remain unknown. Furthermore, none of the explanations account for the other occurrences related to the descent, such as failed obliteration of the processus vaginalis, or the reasons for the decrease in fertility and increase in the risk of malignancy associated with an undescended testis. The gubernaculum is a primitive mesenchymal tissue that was first described in 1786. However, the role of the gubernaculum in the descent process remains obscure. The testis descends through the processus vaginalis. Although the processus vaginalis (PV) is usually defined as a simple peritoneal protrusion, it actively develops into the gubernaculum. The gubernaculum gives rise to the smooth muscles that surround the processus vaginalis. The striated cremaster muscle (CM) is also derived from the gubernaculum. Because the testis descends through the processus vaginalis, the muscles develop to propel the testis. After propelling the testis, the smooth muscle (SM) undergoes programmed cell death. The initiation of programmed cell death through the intrinsic pathway requires activation of phospholipase C. A transient shift in the autonomic balance via a decrease in the sympathetic tonus and an increase in the parasympathetic tonus is essential for initiating this programmed cell death. Programmed cell death in the SM is the physiological pathway for the obliteration of the processus vaginalis. Differences in the timing, intensity, or duration of this physiological pathway result in pathological conditions. A shift before testicular descent diminishes the SM content that is required to propel the testis, and thus inhibits descent. The early shift persists throughout childhood and results in the decrease in fertility and increase in the risk of malignancy because of the differences in signal transduction. Despite a successful descent, persistence of the shift alters the contractility of the CM by increasing the cytosolic calcium levels. Contracted CMs retracts or even ascends the testis. Inadequate intensity or duration of the shift of autonomic tonus causes failure of the programmed cell death. Persistence of the SM hinders the obliteration of PV and gives rise to hydroceles or inguinal hernias depending on the amount of residual smooth muscles. Similar findings from different countries support these explanations. Thus, our proposed mechanism satisfactorily explains the process of descent while considering all the factors related to the process of testicular descent.

Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation.

Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).

Catabolite control protein C contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes.

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

Renal distress syndrome in surgery and uronephrology: concept, pathogenesis, basics of prevention and correction

Based on objective criteria of the structural and functional state of the kidneys in various urgent surgical and uronephrological pathologies (peritonitis, pancreonecrosis, intestinal obstruction, urinary peritonitis, acute purulent secondary pyelonephritis) to identify and prove the general pattern of development of renal changes, their influence on the pathogenesis of homeostasis disorders at the organizational level and to form the evidence base of a new symptom complex - renal distress syndrome in surgery and uronephrology; to establish the effectiveness of Remaxol in its correction. Experimentally on 60 mongrel dogs with acute peritonitis, pancreatitis, intestinal obstruction of varying severity, the state of renal function, including detoxification, was assessed based on the assessment of the inflow-outflow difference in the level of toxins and in the parenchyma of organs - the composition of lipids, the activity of peroxidation of membrane lipids and phospholipases. Clinical and laboratory studies included patients with acute peritonitis (48), acute pancreatitis (18), intestinal obstruction (21), acute purulent secondary pyelonephritis (19). Patients with peritonitis (20) underwent standardized therapy (the first subgroup) or with the inclusion of Remaxol (28) (the second subgroup). In the experiment and in the clinic, in acute surgical and some urological diseases with a different nature of the inflammatory process, there is a significant decrease in renal function. The recorded acute renal injury is combined with a pronounced membrane-stabilizing process in the tissue structures of the kidneys, the degree of which is associated with the severity of the disease. In order to correct and stabilize the indicated changes, Remaxol was used. The drug increased kidney tolerance to trigger pathogenetic agents, which improved the functional status of the kidneys. These data confirm the significance of the studied molecular mechanisms in kidney damage in urgent pathology. Experimental and clinical evidence has been obtained for the formation of a new concept - a symptom complex in acute surgical and uronephrological diseases of the abdominal cavity and retroperitoneal space called renal distress syndrome. Renal distress syndrome in surgery and uronephrology is a set of pathological processes of the body, the most important manifestation of which is the progression of endogenous intoxication due to a violation of the functional status of the kidneys as a result of membrane-stabilizing phenomena of organ cells due to oxidative stress and phospholipase activity.