Point-of-Care Testing for Sensitive Detection of the African Swine Fever Virus Genome.

African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.

Subclinical Impairment of Left Ventricular Function assessed by Speckle Tracking in Type 2 Diabetic Obese and Non-Obese Patients: Case Control Study.

Objectives: Type 2 diabetes mellitus (DM) and obesity are an independent risk factor for cardiovascular diseases, so early prediction of LV dysfunction carries better prognosis. So our aim was to assess the subclinical LV dysfunction in type 2 diabetic obese and non-obese patients using two-dimensional speckle tracking echocardiography (2DSTE). Materials and Methods: We studied 93 patients, including two groups of 31 each with type 2 diabetes mellitus (T2DM), divided by body mass index (BMI), and 31 non-diabetic non-obese controls. All these subjects underwent two-dimensional Echo (2DE) imaging with analysis of conventional parameters of systolic and diastolic function, as well as speckle tracking echocardiography s (STE) analysis of LV global and regional longitudinal strain. Results: We reported significant inter-group differences in parameters of diastolic function, but no significant differences in ejection fraction or fractional shortening. Nevertheless, we found significant differences in strain, which we interpreted as evidence of subclinical systolic dysfunction. Conclusion: 2DSTE is better than basic echocardiographic measurements in assessment of subclinical LV dysfunction in type 2 diabetic obese and non-obese patients which can be used to predict cardiomyopathic changes in the earlier course of type 2 DM and start earlier treatment with better prognosis.

Easy Express Extraction (TripleE)-A Universal, Electricity-Free Nucleic Acid Extraction System for the Lab and the Pen.

The complexity of the current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method (easy express extraction, called TripleE) as a centrifugation-free and electricity-free nucleic acid isolation method. The procedure is based on the well-established magnetic-bead extraction technology using an in-house self-made magnetic 8-channel and a rod cover. With this extraction system, nucleic acids can be isolated with two simple and universal protocols. One method was designed for the extraction of the nucleic acid in resource-limited "easy labs", and the other method can be used for RNA/DNA extraction in the field for so-called molecular "pen-side tests". In both scenarios, users can extract up to 8 samples in 6 to 10 min, without the need for any electricity, centrifuges or robotic systems. In order to evaluate and compare both methods, clinical samples from various viruses (African swine fever virus; lumpy skin disease virus; peste des petits ruminants virus; bluetongue virus), matrices and animals were tested and compared with standard magnetic-bead nucleic acid extraction technology based on the KingFisher platform. Hence, validation data were generated by evaluating two DNA viruses as well as one single-stranded and one double-stranded RNA virus. The results showed that the fast, easy, portable and electricity-free extraction protocols allowed rapid and reliable nucleic acid extraction for a variety of viruses and most likely also for other pathogens, without a substantial loss of sensitivity compared to standard procedures. The speed and simplicity of the methods make them ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings.

Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards.

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Swift and Reliable "Easy Lab" Methods for the Sensitive Molecular Detection of African Swine Fever Virus.

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

Comparative Pathology of Domestic Pigs and Wild Boar Infected with the Moderately Virulent African Swine Fever Virus Strain "Estonia 2014".

Endemically infected European wild boar are considered a major reservoir of African swine fever virus in Europe. While high lethality was observed in the majority of field cases, strains of moderate virulence occurred in the Baltic States. One of these, "Estonia 2014", led to a higher number of clinically healthy, antibody-positive animals in the hunting bag of North-Eastern Estonia. Experimental characterization showed high virulence in wild boar but moderate virulence in domestic pigs. Putative pathogenic differences between wild boar and domestic pigs are unresolved and comparative pathological studies are limited. We here report on a kinetic experiment in both subspecies. Three animals each were euthanized at 4, 7, and 10 days post infection (dpi). Clinical data confirmed higher virulence in wild boar although macroscopy and viral genome load in blood and tissues were comparable in both subspecies. The percentage of viral antigen positive myeloid cells tested by flow cytometry did not differ significantly in most tissues. Only immunohistochemistry revealed consistently higher viral antigen loads in wild boar tissues in particular 7 dpi, whereas domestic pigs already eliminated the virus. The moderate virulence in domestic pigs could be explained by a more effective viral clearance.

Selective growth inhibition of human malignant melanoma cells by syringic acid-derived proteasome inhibitors.

BACKGROUND: It has been shown that proteasome inhibition leads to growth arrest in the G1 phase of the cell cycle and/or induction of apoptosis. However, it was found that some of these inhibitors do not induce apoptosis in several human normal cell lines. This selective activity makes proteasome inhibition a promising target for new generation of anticancer drugs. Clinical validation of the proteasome, as a therapeutic target in oncology, has been provided by the dipeptide boronic acid derivative; bortezomib. Bortezomib has proven to be effective as a single agent in multiple myeloma and some forms of non-Hodgkin's lymphoma. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid, 1), a known phenolic acid, was isolated from the methanol extract of Tamarix aucheriana and was shown to possess proteasome inhibitory activity. METHODS: Using Surflex-Dock program interfaced with SYBYL, the docking affinities of syringic acid and its proposed derivatives to 20S proteasome were studied. Several derivatives were virtually proposed, however, five derivatives: benzyl 4-hydroxy-3,5-dimethoxybenzoate (2), benzyl 4-(benzyloxy)-3,5-dimethoxybenzoate (3), 3'-methoxybenzyl 3,5-dimethoxy-4-(3'-methoxybenzyloxy)benzoate (4), 3'-methoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (5) and 3',5'-dimethoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (6), were selected based on high docking scores, synthesized, and tested for their anti-mitogenic activity against human colorectal, breast and malignant melanoma cells as well as normal human fibroblast cells. RESULTS: Derivatives 2, 5, and 6 showed selective dose-dependent anti-mitogenic effect against human malignant melanoma cell lines HTB66 and HTB68 with minimal cytotoxicity on colorectal and breast cancer cells as well as normal human fibroblast cells. Derivatives 2, 5 and 6 significantly (p ≤ 0.0001) inhibited the various proteasomal chymotrypsin, PGPH, and trypsin like activities. They growth arrested the growth of HTB66 cells at G1 and G2-phases. They also arrested the growth of HTB68 cells at S- and G2-phase, respectively. Moreover, derivatives 2, 5, and 6 markedly induced apoptosis (≥ 90%) in both HTB66 and HTB68. CONCLUSIONS: Computer-derived syringic acid derivatives possess selective anti-mitogenic activity on human malignant melanoma cells that may be attributed to perturbation of cell cycle, induction of apoptosis and inhibition of various 26S proteasomal activities.

In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

In-cell fluorescence activation and labeling of proteins mediated by FRET-quenched split inteins.

Methods to visualize, track, and modify proteins in living cells are central for understanding the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We report in this work the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins to control their cellular localization and potentially alter their biological activity.

Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.

Design of semisynthetic analogues and 3D-QSAR study of eunicellin-based diterpenoids as prostate cancer migration and invasion inhibitors.

Prostrate cancer constitutes the second leading cause of cancer deaths in men in United States. Eunicellin-based diterpenoids are important bioactive marine natural products isolated from corals of alcyonaria species. The bioactivities of eunicellin diterpenes were correlated with their chemical structures. Recently eunicellin diterpenes from the Red Sea soft coral Cladiella pachyclados showed significant anti-migratory and anti-invasive activities against prostate cancer in wound-healing and Cultrex(®) invasion models. These results encouraged the semisynthetic and 3D-QSAR studies of this unique marine natural product class as possible hits for the control of metastatic prostate cancer. Ten new semisynthetic analogues of cladiellisin (1) were prepared. These include C-6 carbamoylation and ∆(11-17) epoxidation. Carbamate analogues of 1 showed potent anti-migratory and anti-invasive activities against PC-3 cells. Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were performed using SYBYL 8.1 program package to create a valid 3D-QSAR model to guide future design of potent eunicellin diterpenes cancer migration inhibitors. Eunicellin-based diterpenes are potential marine natural hits appropriate for optimization as inhibitors of metastatic prostate cancer.

(-)-Oleocanthal as a c-Met inhibitor for the control of metastatic breast and prostate cancers.

The proto-oncogene receptor tyrosine kinase c-Met encodes the high-affinity receptor for hepatocyte growth factor (HGF). Dysregulation of the HGF-c-Met pathway plays a significant oncogenic role in many tumors. Overexpression of c-Met is a prognostic indicator for some transitional cell carcinomas. Extra-virgin olive oil (EVOO) provides a variety of minor phenolic compounds with beneficial properties. (-)-Oleocanthal (1) is a naturally occurring minor secoiridoid isolated from EVOO, which showed potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2. It altered the structure of neurotoxic proteins believed to contribute to the debilitating effects of Alzheimer's disease. Computer-Assisted Molecular Design (CAMD) identified 1 as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with an IC (50) range of 10-20 µM, and demonstrated anti-angiogenic activity via downregulating the expression of the microvessel density marker CD31 in endothelial colony forming cells with an IC (50) of 4.4 µM. It inhibited the phosphorylation of c-Met kinase IN VITRO in the Z'-LYTE™ assay, with an IC (50) value of 4.8 µM. (-)-Oleocanthal and EVOO can have potential therapeutic use for the control of c-Met-dependent malignancies.

Redox-silent tocotrienol esters as breast cancer proliferation and migration inhibitors.

Tocotrienols are vitamin E members with potent antiproliferative activity against preneoplastic and neoplastic mammary epithelial cells with little or no effect on normal cell growth or functions. However, physicochemical and pharmacokinetic properties greatly limit their use as therapeutic agents. Tocotrienols' chemical instability, poor water solubility, NPC1L1-mediated transport, and rapid metabolism are examples of such obstacles which hinder the therapeutic use of these valuable natural products. Vitamin E esters like α-tocopheryl succinate were prepared to significantly improve chemical and metabolic stability, water solubility, and potency. Thus, 12 semisynthetic tocotrienol ester analogues 4-15 were prepared by direct esterification of natural tocotrienol isomers with various acid anhydrides or chlorides. Esters 4-15 were evaluated for their ability to inhibit the proliferation and migration of the mammary tumor cells +SA and MDA-MB-231, respectively. Esters 5, 9, and 11 effectively inhibited the proliferation of the highly metastatic +SA rodent mammary epithelial cells with IC(50) values of 0.62, 0.51, and 0.86µM, respectively, at doses that had no effect on immortalized normal mouse CL-S1 mammary epithelial cells. Esters 4, 6, 8-10, and 13 inhibited 50% of the migration of the human metastatic MDA-MB-231 breast cancer cells at a single 5µM dose in wound-healing assay. The most active ester 9 was 1000-fold more water-soluble and chemically stable versus its parent α-tocotrienol (1). These findings strongly suggest that redox-silent tocotrienol esters may provide superior therapeutic forms of tocotrienols for the control of metastatic breast cancer.

Pachycladins A-E, prostate cancer invasion and migration inhibitory Eunicellin-based diterpenoids from the red sea soft coral Cladiella pachyclados.

Alcyonaria species are among the important marine invertebrate classes that produce a wealth of chemically diverse bioactive diterpenes. Examples of these are the potent microtubule disruptor sarcodictyins and eleutherobin. The genus Cladiella has proven to be a rich source of cytotoxic eunicellin-based diterpenoids. Five new eunicellin diterpenes, pachycladins A-E (1-5), were isolated from the Red Sea soft coral Cladiella pachyclados. The known sclerophytin A Cladiellisin, 3-acetylcladiellisin, 3,6-diacetylcladiellisin, (+)-polyanthelin A, klysimplexin G, klysimplexin E, sclerophytin F methyl ether, (6Z)-cladiellin (cladiella-6Z,11(17)-dien-3-ol), sclerophytin B, and patagonicol were also identified. The structures of the isolated compounds were elucidated by extensive interpretation of their spectroscopic data. These compounds were evaluated for their ability to inhibit growth, proliferation, invasion, and migration of the prostate cancer cells PC-3. Some of the new metabolites exhibited significant anti-invasive activity.

Design and preliminary structure-activity relationship of redox-silent semisynthetic tocotrienol analogues as inhibitors for breast cancer proliferation and invasion.

Vitamin E (VE) is a generic term that represents a family of compounds composed of various tocopherol and tocotrienol isoforms. Tocotrienols display potent anti-angiogenic and antiproliferative activities. Redox-silent tocotrienol analogues also display potent anticancer activity. The ultimate objective of this study was to develop semisynthetically C-6-modified redox-silent tocotrienol analogues with enhanced antiproliferative and anti-invasive activities as compared to their parent compound. Examples of these are carbamate and ether analogues of alpha-, gamma-, and delta-tocotrienols (1-3). Various aliphatic, olefinic, and aromatic substituents were used. Steric limitation, electrostatic, hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) properties were varied at this position and the biological activities of these derivatives were tested. Three-dimensional quantitative structure-activity relationship (3D QSAR) studies were performed using Comparative Molecular Field (CoMFA) and Comparative Molecular Similarity Indices Analyses (CoMSIA) to better understand the structural basis for biological activity and guide the future design of more potent VE analogues.

Design and pharmacophore modeling of biaryl methyl eugenol analogs as breast cancer invasion inhibitors.

Cell invasion and migration are required for the parent solid tumor cells to metastasize to distant organs. Microtubules form a polarized network, enabling organelle and protein movement throughout the cell. Cytoskeletal elements coordinately regulate cell's motility, adhesion, migration, exocytosis, endocytosis, and division. Thus, microtubule disruption can be a useful target to control cancer cell invasion and metastasis. The phenolic ether methyl eugenol (1), the major component of the essential oil of the leaves of Melaleuca ericifolia Sm. (Myrtaceae), was used as a starting scaffold to design eleven new and three known anti-tubulin agents 2-15 using carbon-carbon coupling reactions. A computer-assisted approach was used to design these new biaryl derivatives using colchicine-binding site of tubulin as the molecular target and colchicine as an active ligand. Several derivatives showed potent inhibitory activity against MDA-MB-231 cell migration at the 1-4microM dose range. The Z isomers, 4 and 15 were more active as invasion inhibitors compared to their structurally related E isomers, 2 and 14. The cytotoxic activities of compounds 2-15 against two breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated. Anti-invasive activity of the semisynthetic derivatives is not due to a direct cytotoxic effect on MDA-MB-231. Analogs 2-15 may promote their anti-invasive activity through the induction of changes in cell morphology. A pharmacophore model was generated involving seven essential features for activity, which was consistent with a previously generated colchicine site inhibitors model.

Rational design and semisynthesis of betulinic acid analogues as potent topoisomerase inhibitors.

Chemical transformation studies were conducted on betulinic acid (1), a common plant-derived lupane-type triterpene. Eleven new rationally designed derivatives of 1 (2-5 and 7-13) were synthesized based on docking studies and tested for their topoisomerase I and IIalpha inhibitory activity. Semisynthetic reactions targeted C-3, C-20, and C-28 in 1. Structures of the new compounds were confirmed by spectroscopic methods (1D and 2D NMR and MS). Compound 9, 3-O-[N-(phenylsulfonyl)carbamoyl-17beta-N-(phenylsulfonyl)amide]betulinic acid, showed 1.5-fold the activity of CPT in a topoisomerase I DNA relaxation assay. Four out of 14 betulinic acid analogues (5, 9, 11, and 12) showed 1.5-fold the activity of etoposide in a topoisomerase II assay. The new analogues exhibited better cytotoxic activities against the human colon cancer cells SW948 and HCT-116 and the breast cancer cell line MDA-MB-231 compared to the parent (1). Betulinic acid (1) is a potential scaffold for the design of new topoisomerase I and IIalpha inhibitors.