Assessment of the potential occurrence of Cryptosporidium species in various water sources in Sharqia Governorate, Egypt.

Cryptosporidium species are enteric apicomplexan parasites associated with diarrhoeal disease in humans and animals globally. Waterborne outbreaks resulting from contamination with the infective oocysts are common worldwide. Updated reports on waterborne protozoal infections are needed to identify emerging pathogens and susceptible populations. Therefore, this study aimed to assess the current profile of Cryptosporidium contamination of various water sources in Sharqia Governorate, Northeastern Egypt. For this purpose, eighty samples were collected from five different water types (canal, tap, tank, filtered, and groundwater), distributed in four major cities (El-Hessenia, Fakous, Zagazig, and Belbies) in Sharqia Governorate. All water samples were examined using conventional microscopy, ELISA, and real-time PCR (RT-PCR) techniques. Based on microscopic analysis, the Cryptosporidium protozoan was identified in 25% of the tested water samples. The RT-PCR assay has allowed for the quantification of Cryptosporidium oocysts in different types of water. Canal water exhibited the highest Cryptosporidium contamination levels (mean = 85.15 oocysts/L), followed by water tanks (mean = 12.031 oocysts/L). The study also provided a comparative evaluation of ELISA and RT-PCR for the diagnosis of Cryptosporidium infection. RT-PCR performed better than ELISA in terms of analytical accuracy (97.50% vs. 86.25%) and specificity (100% vs. 83.33%). However, ELISA showed a higher sensitivity (95.00% vs. 90.00%) for Cryptosporidium recovery. Our findings could serve as a platform for further investigations into the potential risks associated with water contamination in Sharqia Governorate. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01675-1.

Ameliorative effects of androstenediol against acetic acid-induced colitis in male wistar rats via inhibiting TLR4-mediated PI3K/Akt and NF-κB pathways through estrogen receptor ß activation.

5-androstenediol (ADIOL) functions as a selective estrogen receptor ß (ERß) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERß as contributing mechanisms. METHODS: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-ß antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1ß), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERß and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. RESULTS: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and ß catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1ß, NGAL, MMP9, and PI3K while increased ERß and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERß antagonist, PHTPP, largely diminished these protective effects of ADIOL. CONCLUSION: ADIOL could be beneficial against AA-induced colitis mostly through activating ERß.

Vagal Stimulation Ameliorates Non-Alcoholic Fatty Liver Disease in Rats.

BACKGROUND: The harmful consequences of non-alcoholic fatty liver disease (NAFLD) are posing an increasing threat to public health as the incidence of diabetes and obesity increases globally. A non-invasive treatment with a range of autonomic and metabolic benefits is transcutaneous vagus nerve stimulation (tVNS). AIM OF THE STUDY: To investigate the possible preventive impacts of VNS against adult rats' NAFLD caused by a high-fat diet (HFD) and to clarify the underlying mechanisms. METHODS: A total of thirty-two adult male rats were split into two groups: the HFD-induced NAFLD group (n = 24) and the control normal group (n = 8). The obesogenic diet was maintained for 12 weeks to induce hepatic steatosis. The HFD-induced NAFLD group (n = 24) was separated into three groups: the group without treatment (n = 8), the group with sham stimulation (n = 8), and the group with VNS treatment (n = 8). VNS was delivered for 30 min per day for 6 weeks after the establishment of NAFLD using a digital TENS device. The subsequent assessments included hepatic triglyceride, cholesterol content, serum lipid profile, and liver function testing. In this context, inflammatory biomarkers (TNF-α, IL-6) and hepatic oxidative stress (MDA, SOD, and GPx) were also assessed. To clarify the possible mechanisms behind the protective benefits of VNS, additional histological inspection and immunohistochemistry analysis of TNF-α and Caspase-3 were performed. RESULTS: In the NAFLD-affected obese rats, VNS markedly decreased the rats' body mass index (BMI) and abdominal circumference (AC). Liver function markers (albumin, ALT, and AST) and the serum lipid profile-which included a notable decrease in the amounts of hepatic triglycerides and cholesterol-were both markedly improved. Additionally, oxidative stress and inflammatory indicators showed a considerable decline with VNS. Notably, the liver tissues examined by histopathologists revealed that there is evidence of the protective impact of VNS on the oxidative and inflammatory states linked to HFD-induced NAFLD while maintaining the architectural and functional condition of the liver. CONCLUSIONS: Our findings suggest that VNS may represent a promising therapeutic candidate for managing NAFLD induced by obesity. It can be considered to be an effective adjuvant physiological intervention for the obese population with NAFLD to spare the liver against obesity-induced deleterious injury.

Tenascin-C Activation of Lung Fibroblasts in a 3D Synthetic Lung Extracellular Matrix Mimic.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that match the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro. Here, a synthetic, bioactive hydrogel is synthesized that mimics the native lung modulus and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin-binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which enables quiescent culture of human lung fibroblasts (HLFs). Stimulation with transforming growth factor ß1 (TGF-ß1), metastatic breast cancer conditioned media (CM), or tenascin-C-derived integrin-binding peptide activated hydrogel-encapsulated HLFs demonstrates multiple environmental methods to activate HLFs in a lung ECM-mimicking hydrogel. This lung hydrogel platform is a tunable, synthetic approach to studying the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.

Tenascin-C activation of lung fibroblasts in a 3D synthetic lung extracellular matrix mimic.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that contain the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro . Here, we developed a synthetic, bioactive hydrogel that mimics the native lung modulus, and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which promotes quiescence of human lung fibroblasts (HLFs). Stimulation with transforming growth factor ß1 (TGF-ß1), metastatic breast cancer conditioned media (CM), or tenascin-C activated these hydrogel-encapsulated HLFs in a manner reflective of their native in vivo responses. We propose this lung hydrogel platform as a tunable, synthetic approach to study the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.

Anticancer evaluation and molecular docking of new pyridopyrazolo-triazine and pyridopyrazolo-triazole derivatives.

3-Amino-4,6-dimethylpyrazolopyridine was applied as a precursor for the synthesis of some new pyridopyrazolo-triazine and pyridopyrazolo-triazole derivatives through diazotization, followed by coupling with many 2-cyanoacetamide compounds, ethyl 3-(phenylamino)-3-thioxopropanoate, 3-oxo-N-phenylbutanethioamide, and α-bromo-ketone reagents [namely; 2-bromo-1-(4-fluorophenyl)ethan-1-one, 5-bromo-2-(bromoacetyl)thiophene, 3-(2-bromoacetyl)-2H-chromen-2-one and/or 3-chloroacetylacetone]. The prepared compounds were identified by spectroscopic analyses as IR, 1H NMR, and mass data. The anticancer activity of these pyrazolopyridine analogues was investigated in colon, hepatocellular, breast, and cervix carcinoma cell lines. The pyridopyrazolo-triazine compound 5a substituted with a carboxylate group gave a distinguished value of IC50 = 3.89 µM against the MCF-7 cell line compared to doxorubicin as a reference drug. Also, the pyridopyrazolo-triazine compound 6a substituted with the carbothioamide function gave good activity toward HCT-116 and MCF-7 cell lines with IC50 values of 12.58 and 11.71 µM, respectively. The discovered pyrazolopyridine derivatives were studied theoretically by molecular docking, and this study exhibited suitable binding between the active sides of pyrazolopyridine ligands and proteins (PDB ID: 5IVE). The pyridopyrazolo-triazine compound 6a showed the highest free binding energy (- 7.8182 kcal/mol) when docked inside the active site of selected proteins.

Synthesis, biological evaluation and molecular docking of new triphenylamine-linked pyridine, thiazole and pyrazole analogues as anticancer agents.

A new series of pyridine, thiazole, and pyrazole analogues were synthesized. The pyridone analogues 4a-e were synthesized by treating N-aryl-2-cyano-3-(4-(diphenylamino)phenyl)acrylamides 3a-e with malononitrile. Many 4-arylidene-thiazolidin-5-one analogues 6a-d were obtained by Knoevenagel reactions of 4-(diphenylamino)benzaldehyde (1) with their corresponding thiazolidin-5-one derivatives 5a-d. The structural elucidation of the products was proven by the collections of spectroscopic methods such as IR, 1H NMR, 13C NMR, and MS data. Their anti-cancer activity was examined against two cell lines, MDA-MB-231 (mammary carcinomas) and A-549 (lung cancer). Compared with cisplatin as a reference standard drug, 6-amino-4-(4-(diphenylamino)phenyl)-2-oxo-1-(p-tolyl)-1,2-dihydropyridine-3,5-dicarbonitrile (4b) and 6-amino-4-(4-(diphenylamino)phenyl)-1-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (4e) exhibited better efficiency against the A-549 cell line, with IC50 = 0.00803 and 0.0095 µM, respectively. Also, these compounds 4b and 4e showed the most potency among the examined compounds against MDA-MB-231 with IC50 = 0.0103 and 0.0147 µM, respectively. The newly synthesized compounds were docked inside the active sites of the selected proteins and were found to demonstrate proper binding. 2-Cyano-2-(4,4-(diphenylamino)benzylidene)-5-oxo-3-phenylthiazolidin-2-ylidene)-N-(p-tolyl)acetamide (6c) offered the highest binding affinity (- 8.1868 kcal/mol) when docked into (PDB ID:2ITO), in addition to 2-cyano-N-(4-(diethylamino)phenyl)-2-(4-(4-(diphenylamino)benzylidene)-5-oxo-3-phenylthiazolidin-2-ylidene)acetamide (6a) gave the highest energy score (- 9.3507 kcal/mol) with (PDB ID:2A4L).

Bioprospecting endophytic fungi for antifeedants and larvicides and their enhancement by gamma irradiation.

The search and discovery of new natural products with antifeedant and larvicidal potentials to mitigate harmful insects are scientific pressing issues in the modern agriculture. In this paper, the antifeedant and larvicidal potentials of 69 fungal isolates were screened against the Egyptian cotton leafworm Spodoptera littoralis. A total of 17 isolates showed the insecticidal potentials with three promising isolates. These strains were Aspergillus sydowii, Lasiodiplodia theobromae, and Aspergillus flavus isolated from Ricinus communis (bark), Terminalia arjuna (Bark), and Psidium guajava (twigs), respectively. The effect of gamma irradiation on the antifeedant and larvicidal activities of the three strains was investigated. Exposure of the fungal spores to 1000 Gy of gamma rays significantly intensified both the antifeedant and larvicidal potentials. To identify compounds responsible for these activities, extracts of the three strains were fractionated by thin layer chromatography. The nature of the separated compounds namely, Penitrem A, 1, 3, 5, 8- tetramethyl- 4, 6-diethyl- 7- [2- (methoxycarbonyl)ethyl] porphyrin (from A. sydowii), Penitrem A, 2, 7, 12, 17-Tetramethyl-3, 5:8, 10:13, 15:18, 20-tetrakis (2,2-dimethylpropano) porphyrin (from A. flavus), N,N-Diethyl-3-nitrobenzamide, and Diisooctyl-phthalate (from L. theobromae) were studied by GC-MS analysis. These findings recommend endophytic fungi as promising sources of novel natural compounds to mitigate harmful insects.

Cardiac autonomic neuropathy linked to left ventricular dysfunction in type 1 diabetic patients.

Type 1 diabetes is a major cause of cardiovascular death; diabetic cardiomyopathy (DCM) is the most important cause of mortality among diabetic patients. There is an increasing body of evidence that the most important inducer of DCM is microvascular injury. The aim of this study is to establish a potential relationship between low frequency/high frequency (LF/HF) ratio and DCM and to set a possible predictive cutoff of LF:HF ratio for early detection of DCM. Methods: 75 type 1 diabetic patients together with 75 controls were assessed using tissue Doppler imaging for left ventricular (LV) and right ventricular (RV) diastolic function, and heart rate variability (HRV) indices including LF/HF ratio. Type 1 diabetic patients were also assessed for parameters of glycemic and lipid profile control. Results: Cases showed a statistically significant increase in LF/HF ratio compared to controls reflecting reduced HRV. Also, LV and RV diastolic function were reduced in cases compared to controls, there was a significant correlation between LV E/E' ratio (ratio of early transmitral velocity and average early mitral annular and basal septal velocities) and LF/HF ratio. LF/HF ratio was able to predict LV diastolic dysfunction as expressed by the LV E/E' ratio with a sensitivity of 96%. Conclusion: HRV indices notably LF/HF ratio seem to be an early and sensitive predictor of DCM, the latter finding not only underlines the role of microvascular injury in the induction of DCM but might help also for the early detection and reversal of it.

ATP hydrolysis tunes specificity of a AAA+ protease.

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is easy to alter. In vitro, wild-type ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that the rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states.

Synthesis of Novel Triphenylamine-Based Organic Dyes with Dual Anchors for Efficient Dye-Sensitized Solar Cells.

A new series of metal-free organic dyes (SM1-5) with dual anchors are synthesized for application in dye-sensitized solar cells (DSSC). Here, a simple triphenylamine (TPA) moiety serves as the electron donor, while di-cyanoacrylamide and di-thiazolidine-5-one units serve as the electron acceptors and anchoring groups. To understand the effect of dye structure on the photovoltaic characteristics of DSSCs, the photophysical and electrochemical properties, as well as molecular geometries calculated from density functional theory (DFT), are used for dyes SM1-5. The extinction coefficients of the organic dyes SM1-5 are high (5.36-9.54 104 M-1 cm-1), indicating a high aptitude for light harvesting. The photovoltaic studies indicated that using dye SM4 as a sensitizer showed a power conversion efficiency (PCE) of 6.09% (JSC = 14.13 mA cm-2, VOC = 0.624 V, FF = 68.89%). Interestingly, SM4 showed the highest values of VOC among all dyes, including N-719, due to its maximum dye coverage on the TiO2 surface, enhancing charge recombination resistance in the sensitized cell. The good agreement between the theoretically and experimentally obtained data indicates that the energy functional and basis set employed in this study can be successfully utilized to predict new photosensitizers' absorption spectra with great precision before synthesis. Also, these results show that bi-anchoring molecules have a lot of potentials to improve the overall performance of dye-sensitized solar cells.

Bioprospecting endophytic fungi for bioactive metabolites and use of irradiation to improve their bioactivities.

The search for new bioactive compounds with innovative modes of action and chemistry are desperately needed to tackle the increased emergence of drug-resistant microbes. With this view, this paper was conducted for the isolation, identification, and biological evaluation of fungal endophytes of eleven different plant species. A total of 69 endophytic strains were isolated and tested for the presence of bioactive metabolites with antifungal, antibacterial, anticancer, and antioxidant properties in their extracts. Upon screening, two promising strains were found to have all the before-mentioned activities. These strains were Aspergillus sydowii isolated from the bark of Ricinus communis and Aspergillus flavus isolated from the twigs of Psidium guajava. Major compounds present in extracts of the two strains were identified by GC-Mass analyses. Several well-known bioactive compounds as well as unreported ones were identified in the fungal extracts of the two strains. Furthermore, gamma irradiation (at 1000 Gy) of the fungal cultures resulted in improved bioactivities of extracts from the two strains. These findings recommend the two fungal strains as sources of antimicrobial, anticancer, and antioxidant compounds which may aid in the development of novel drugs. The presented research also explains the high-value of fungal endophytes as untapped sources of bioactive metabolites.

Prediction of In Vivo Pharmacokinetic Parameters and Time-Exposure Curves in Rats Using Machine Learning from the Chemical Structure.

Animal pharmacokinetic (PK) data as well as human and animal in vitro systems are utilized in drug discovery to define the rate and route of drug elimination. Accurate prediction and mechanistic understanding of drug clearance and disposition in animals provide a degree of confidence for extrapolation to humans. In addition, prediction of in vivo properties can be used to improve design during drug discovery, help select compounds with better properties, and reduce the number of in vivo experiments. In this study, we generated machine learning models able to predict rat in vivo PK parameters and concentration-time PK profiles based on the molecular chemical structure and either measured or predicted in vitro parameters. The models were trained on internal in vivo rat PK data for over 3000 diverse compounds from multiple projects and therapeutic areas, and the predicted endpoints include clearance and oral bioavailability. We compared the performance of various traditional machine learning algorithms and deep learning approaches, including graph convolutional neural networks. The best models for PK parameters achieved R2 = 0.63 [root mean squared error (RMSE) = 0.26] for clearance and R2 = 0.55 (RMSE = 0.46) for bioavailability. The models provide a fast and cost-efficient way to guide the design of molecules with optimal PK profiles, to enable the prediction of virtual compounds at the point of design, and to drive prioritization of compounds for in vivo assays.

Repurposing Lovastatin Cytotoxicity against the Tongue Carcinoma HSC3 Cell Line Using a Eucalyptus Oil-Based Nanoemulgel Carrier.

Tongue cancer is one of the most common carcinomas of the head and neck region. The antitumor activities of statins, including lovastatin (LV), and the essential oil of eucalyptus (Eu oil), have been adequately reported. The aim of this study was to develop a nanoemulgel containing LV combined with Eu oil that could then be made into a nanoemulsion and assessed to determine its cytotoxicity against the cell line human chondrosarcoma-3 (HSC3) of carcinoma of the tongue. An I-optimal coordinate-exchange quadratic mixture design was adopted to optimize the investigated nanoemulsions. The droplet size and stability index of the developed formulations were measured to show characteristics of the nanoemulsions. The optimized LV loaded self-nanoemulsifying drug delivery system (LV-Eu-SNEDDS) was loaded into the gelling agent Carbopol 934 to develop the nanoemulgel and evaluated for its rheological properties. The cytotoxic efficiency of the optimized LV-Eu-SNEDDS loaded nanoemulgel was tested for cell viability, and the caspase-3 enzyme test was used against the HSC3 cell line of squamous carcinoma of the tongue. The optimized nanoemulsion had a droplet size of 85 nm and a stability index of 93%. The manufactured nanoemulgel loaded with the optimum LV-Eu-SNEDDS exhibited pseudoplastic flow with thixotropic behavior. The developed optimum LV-Eu-SNEDDS-loaded nanoemulgel had the best half-maximal inhibitory concentration (IC50) and caspase-3 enzyme values of the formulations developed for this study, and these features improved the ability of the nanoemulsion-loaded gel to deliver the drug to the investigated target cells. In addition, the in vitro cell viability studies revealed the synergistic effect between LV and Eu oil in the treatment of tongue cancer. These findings illustrated that the LV-Eu-SNEDDS-loaded gel formulation could be beneficial in the local treatment of tongue cancer.

Impact of cisplatin administration on cerebellar cortical structure and locomotor activity of infantile and juvenile albino rats: the role of oxidative stress.

The central neurotoxicity of cisplatin (CisPt) has always raised questions especially during development, but few studies are available. Hence, this work was designed to assess the CisPt's impacts on the postnatal rat cerebellum via evaluation of locomotor activity, histological and immunohistochemical studies, and to focus on cerebellar oxidative stress-related alterations. Eighty newborn pups were divided into 2 equal experimental groups: the control group was kept without any treatment and CisPt-treated group received a single subcutaneous injection of CisPt (5 µg /g b.w.) in their nape at PD10. Ten rats at PD11, PD17, and PD30 ages were weighed, then deeply anesthetized and sacrificed. For locomotor assessment, 20 pups were divided equally into control and CisPt-treated groups and tested at PD11-13, PD15-17, and PD28-30 ages. CisPt-treated rats suffered from decreased motor activity and showed decreased body and cerebellar weights, reduced levels of enzymatic antioxidants (SOD and CAT), and non-enzymatic antioxidant defense (GSH), and increase of lipid peroxidation marker (MDA). Histopathologically, CisPt sowed deleterious changes within cerebellar cortical layers in the form of vacuolations, decreased thickness, and hemorrhage (in PD17), while Purkinje cells exhibited profound degenerative changes in the form of swelling, disrupted arrangement, distortion, and nuclear shrinkage. In CisPt-treated rats, GFAP demonstrated upregulated, hypertrophied, and branched Bergmann glial fibers and reactive astrogliosis. Immuno-localization of Ki-67-positive cells revealed defective migration associated with decreased proliferation in early ages in addition to glial proliferation in PD30. In conclusion, CisPt causes oxidative stress-related deleterious effects on structure of developing cerebellar cortex and affects locomotor activity.

Imputation of sensory properties using deep learning.

Predicting the sensory properties of compounds is challenging due to the subjective nature of the experimental measurements. This testing relies on a panel of human participants and is therefore also expensive and time-consuming. We describe the application of a state-of-the-art deep learning method, Alchemite™, to the imputation of sparse physicochemical and sensory data and compare the results with conventional quantitative structure-activity relationship methods and a multi-target graph convolutional neural network. The imputation model achieved a substantially higher accuracy of prediction, with improvements in R2 between 0.26 and 0.45 over the next best method for each sensory property. We also demonstrate that robust uncertainty estimates generated by the imputation model enable the most accurate predictions to be identified and that imputation also more accurately predicts activity cliffs, where small changes in compound structure result in large changes in sensory properties. In combination, these results demonstrate that the use of imputation, based on data from less expensive, early experiments, enables better selection of compounds for more costly studies, saving experimental time and resources.

Molecular detection of brucellosis in dromedary camels of Qatar by real-time PCR technique.

BACKGROUND AND METHODOLOGY: Brucellosis in livestock causes huge economic loses of developing countries and demonstrates a serious health risks to the patrons of infected dairy goods and meat. Prevention of brucellosis in cattle relies on the trustworthiness of the techniques utilized to detect the causative pathogen. In the current investigation, we described the exploration of the well-known real-time PCR technique based on the Brucella-specific IS711 with different tissue samples of dromedary camels in Qatar. RESULTS: The findings of the real-time PCR unveiled the occurrence of Brucella spp in 60 % of lung tissues, 71.42 % of liver tissues, 72 % of spleen tissues, 25 % of kidney tissues, and 42.42 % of placental fluid samples. Among them, the liver tissues and spleen tissues possessed the highest number of positive results, whereas kidney tissues displayed the lowest number of positive results for Brucella spp. CONCLUSION: The findings of this investigation discloses that PCR technique is a sensitive and specific technique for the identification of Brucella spp. in dromedary camels. Most of the tissues samples showed the presence of Brucella spp. and also we concluded that there is a need for further studies to be conducted in order to identify specific species of Brucella.

Degradation of Lon in Caulobacter crescentus.

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae.

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Understanding Conditional Associations between ToxCast in Vitro Readouts and the Hepatotoxicity of Compounds Using Rule-Based Methods.

Current in vitro models for hepatotoxicity commonly suffer from low detection rates due to incomplete coverage of bioactivity space. Additionally, in vivo exposure measures such as Cmax are used for hepatotoxicity screening and are unavailable early on. Here we propose a novel rule-based framework to extract interpretable and biologically meaningful multiconditional associations to prioritize in vitro end points for hepatotoxicity and understand the associated physicochemical conditions. The data used in this study were derived for 673 compounds from 361 ToxCast bioactivity measurements and 29 calculated physicochemical properties against two lowest effective levels (LEL) of rodent hepatotoxicity from ToxRefDB, namely 15 mg/kg/day and 500 mg/kg/day. To achieve 80% coverage of toxic compounds, 35 rules with accuracies ranging from 96% to 73% using 39 unique ToxCast assays are needed at a threshold level of 500 mg/kg/day, whereas to describe the same coverage at a threshold of 15 mg/kg/day, 20 rules with accuracies of between 98% and 81% were needed, comprising 24 unique assays. Despite the 33-fold difference in dose levels, we found relative consistency in the key mechanistic groups in rule clusters, namely (i) activities against Cytochrome P, (ii) immunological responses, and (iii) nuclear receptor activities. Less specific effects, such as oxidative stress and cell cycle arrest, were used more by rules to describe toxicity at the level of 500 mg/kg/day. Although the endocrine disruption through nuclear receptor activity formulated an essential cluster of rules, this bioactivity was not covered in four commercial assay setups for hepatotoxicity. Using an external set of 29 drugs with drug-induced liver injury (DILI) labels, we found that promiscuity over important assays discriminates between compounds with different levels of liver injury. In vitro-in vivo associations were also improved by incorporating physicochemical properties especially for the potent, 15 mg/kg/day toxicity level as well for assays describing nuclear receptor activity and phenotypic changes. The most frequently used physicochemical properties, predictive for hepatotoxicity in combination with assay activities, are linked to bioavailability, which were the number of rotatable bonds (less than 7) at a of level of 15 mg/kg/day and the number of rings (of less than 3) at level of 500 mg/kg/day. In summary, hepatotoxicity cannot very well be captured by single assay end points, but better by a combination of bioactivities in relevant assays, with the likelihood of hepatotoxicity increasing with assay promiscuity. Together, these findings can be used to prioritize assay combinations that are appropriate to assess potential hepatotoxicity.