Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps.

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright's staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied.

Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy.

Rabies is an acute and lethal encephalomyelitis caused by lyssaviruses, among which rabies virus (RABV) is the most prevalent and important for public health. Although preventable through the post-exposure administration of rabies vaccine and immunoglobulins (RIGs), the disease is almost invariably fatal since the onset of clinical signs. Two human neutralizing monoclonal antibodies (mAbs), RVC20 and RVC58, have been shown to be effective in treating symptomatic rabies. To better understand how these mAbs work, we conducted structural modeling and in vitro assays to analyze their mechanisms of action, including their ability to mediate Fc-dependent effector functions. Our results indicate that both RVC20 and RVC58 recognize and lock the RABV-G protein in its pre-fusion conformation. RVC58 was shown to neutralize more potently the extra-cellular virus, while RVC20 mainly acts by reducing viral spreading from infected cells. Importantly, RVC20 was more effective in promoting effector functions compared to RVC58 and 17C7-RAB1 mAbs, the latter of which is approved for human rabies post-exposure treatment. These results provide valuable insights into the multiple mechanisms of action of RVC20 and RVC58 mAbs, offering relevant information for the development of these mAbs as treatment for human rabies.

Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotypes, and evaluate the effects of viral evolution. This study provided a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection using different approaches (description of clinical signs, viral load, receptor profiling, and host immune response) and targeting four different organs (lungs, intestine, brain, and PBMCs). Our data showed that both male and female hamsters were susceptible to the infection and developed a disease similar to the one observed in patients with COVID-19 that included moderate to severe pulmonary lesions, inflammation, and recruitment of the immune system in the lungs and at the systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, which highlighted another crucial difference with the multiorgan impairment of severe COVID-19. When comparing male and female hamsters, we observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions, and triggered higher pulmonary inflammation. Overall, these data confirmed the Syrian hamster as a suitable model for mild to moderate COVID-19 and reflected sex-related differences in the response against the virus observed in humans.

Alternative Methods to Current In Vivo Procedures to Address the 3Rs Tenet in Rabies Proficiency Testing.

Canine rabies is responsible for an estimated 59,000 human deaths every year. In an attempt to reach the ZeroBy30 goal, robust disease surveillance coupled with improved diagnostics play a paramount role in ensuring reliable data and gradually attesting rabies control advancements. In this context, proficiency testing is organized to harmonize rabies diagnostic capacities. In most exercises, rabies-positive samples consist of brains collected from intracerebrally inoculated mice. This procedure causes distress and severe suffering to animals, raising important ethical concerns that can no longer be ignored. In the last decades, the 3Rs tenet (Replace, Reduce, Refine) has been successfully implemented in several scientific areas, and we strongly support its application in the framework of rabies proficiency testing. Here, we discuss cell-based technologies as innovative sustainable in vitro candidate systems to replace in vivo experiments for the production of proficiency testing samples. The application of these alternative methods can allow completely in vitro or ex vivo production of rabies proficiency testing panels, which would represent an important replacement or reduction/refinement for current in vivo procedures.

Digestive and gastroprotective effects of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae): From traditional uses to preclinical studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae) is an alpine endemic plant whose aerial parts are harvested by the locals mainly for the digestive properties. Despite its widespread use, few studies have been conducted to date to verify its bioactivity. AIM OF THE STUDY: The purpose of the work was to meet the tradition confirming with experimental data the popular belief that the consumption of this species offers beneficial effects to the gastrointestinal system. MATERIALS AND METHODS: Using Soxhlet apparatus, the dried aerial parts of A. erba-rotta subsp. moschata were successively extracted with petroleum ether (PET), dichloromethane (DCM) and methanol (MeOH). The essential oil (EO) was obtained by hydrodistillation using a Clevenger apparatus while infusion (AE) was prepared following the traditional local recipe. Their chemical characterization was performed by various techniques including SPME-GC/MS, GC/MS and HPLC/MS-MS. An in vitro biological screening was carried out. The influence of AE on lipid digestion was monitored by titration of free fatty acids (FFA) during pancreatic lipase activity with the pH-stat method. For all extracts and EO, the anti-Helicobacter pylori activity was assessed by the broth microdilution method, the influence on cell viability was evaluated against NCI-N87, OE21 and Caco-2 cell lines and a preliminary toxicity evaluation was done using Brine Shrimp lethality (BSL) assay. The anti-inflammatory potential was evidenced by interleukin IL-1- induced IL8 expression on Caco-2 cells. RESULTS: AE increased by 15% the FFA releasing compared to the pancreatic lipase alone. PET, DCM and MeOH extracts as well as AE and EO were considered active against the growth of both antimicrobial susceptible and resistant strains of H. pylori with MIC values starting from 16 µg/mL. PET and DCM (IC50 = 89 µg/mL and 96 µg/mL, respectively, against Caco-2 cell line) extracts showed the high effect on cell viability while the EO reduced in 50% of cell viability at 1.48 µL/mL (NCI-N87 cells), 1.42 µL/mL (OE21 cells), and 3.44 µL/mL (Caco-2 cells) corroborating the BSL results. In different degrees, all extracts and EO inhibited the IL-1ß-stimulated IL-8 production in Caco-2 cells. CONCLUSIONS: The obtained data are encouraging and provide a scientific basis for the traditional use of A. erba-rotta subsp. moschata as a digestive agent although they need to be further corroborated by studies involving the investigation of both the in vivo activities and the role of the compounds detected in the extracts.

Identification of Dobrava-Belgrade Virus in Apodemus flavicollis from North-Eastern Italy during Enhanced Mortality.

Hantaviruses include several zoonotic pathogens that cause different syndromes in humans, with mortality rates ranging from 12 to 40%. Most commonly, humans get infected through the inhalation of aerosols or dust particles contaminated with virus-containing rodent excreta. Hantaviruses are specifically associated with the host species, and human cases depend on the presence and the dynamics of reservoir hosts. In this letter, we report the identification of Dobrava-Belgrade virus (DOBV) in the yellow-necked mouse (Apodemus flavicollis) from Italy. The virus was detected in the mountainous area of the province of Udine, bordering Austria and Slovenia, during an event of enhanced mortality in wild mice and voles. Despite serological evidence in rodents and humans that suggested the circulation of hantaviruses in Italy since 2000, this is the first virological confirmation of the infection. Phylogenetic analyses across the whole genome of the two detected viruses confirmed the host-specificity of DOBV sub-species and showed the highest identity with viruses identified in Slovenia and Croatia from both A. flavicollis and humans, with no signs of reassortment. These findings highlight the need for ecologists, veterinarians and medical doctors to come together in a coordinated approach in full compliance with the One Health concept.

Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors.

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

Sisymbrium officinale, the Plant of Singers: A Review of Its Properties and Uses.

Sisymbrium officinale (hedge mustard and formerly called Erysimum officinale) is a common plant in wild lands of Europe and Africa. It is also cultivated for its seeds and leaves to be used in salad or mustard. Sisymbrium officinale is useful not only in culinary preparations, but it also seems to possess interesting therapeutic properties, especially for throat diseases such as aphonia and hoarseness. For this reason, it is commonly called "herb of singers" (in Italian, "Erba dei cantanti"). Indeed a cup of Sisymbrium officinale infusion is frequently consumed by singers before artistic performance, even if its beneficial ability still needs to be scientifically demonstrated. Some preliminary data can be analyzed, but new efforts and resources should be devoted to study and investigate a plant with valuable therapeutic potential. This review summarizes the data available for Sisymbrium officinale.

Preclinical Pharmacological Activities of Epigallocatechin-3-gallate in Signaling Pathways: An Update on Cancer.

Epigallocatechin gallate (EGCG) is the main bioactive component of catechins predominantly present in svarious types of teas. EGCG is well known for a wide spectrum of biological activity as an anti-oxidative, anti-inflammatory, and anti-tumor agent. The effect of EGCG on cell death mechanisms via the induction of apoptosis, necrosis, and autophagy has been documented. Moreover, its anti-proliferative and chemopreventive action has been demonstrated in many cancer cell lines. It was also involved in the modulation of cyclooxygenase-2, in oxidative stress and inflammation of different cell processes. EGCG has been reported as a promising target for plasma membrane proteins, such as epidermal growth factor receptor (EGFR). In addition, it has been demonstrated a mechanism of action relying on the inhibition of ERK1/2, p38 MAPK, NF-κB, and vascular endothelial growth factor (VEGF). EGCG and its derivatives were used in proteasome inhibition and they were involved in epigenetic mechanisms. In summary, EGCG is the most predominant and bioactive constituent of teas and it has a pivotal role in cancer prevention. Its preclinical pharmacological activities are associated with complex molecular mechanisms that involve numerous signaling pathways.

Biological Effects of EF24, a Curcumin Derivative, Alone or Combined with Mitotane in Adrenocortical Tumor Cell Lines.

BACKGROUND: Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R. METHOD AND RESULTS: EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 µM and 4.9 ± 2.8 µM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/ß-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines. CONCLUSION: This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

Investigation of the molecular reactivity of bioactive oxiranylmethyloxy anthraquinones.

The design of a multitarget and multifunctional small molecule containing two functional groups reacting through different mechanisms represents an attractive goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy anthraquinones, previously investigated as anticancer agents, is described here. These compounds combine a planar, DNA-intercalating and pro-oxidant anthraquinone scaffold and the alkylating epoxide functions which can covalently react with the nucleic acid. Their multilevel molecular reactivity was studied through a combination of analytical techniques: The DNA-binding properties were investigated using a mass spectrometry-based binding assay and by nuclear magnetic resonance, highlighting the formation of a covalent adduct with a nucleobase. Moreover, the contribution of the pro-oxidant redox cycling was evaluated.

Crude extract of Origanum vulgare L. induced cell death and suppressed MAPK and PI3/Akt signaling pathways in SW13 and H295R cell lines.

Oregano (Origanum vulgare L.) is a common aromatic plant used in Mediterranean and Asian Regions for treating respiratory diseases, painful menstruation, rheumatoid arthritis, etc. Recently its role as an anticancer plant has been suggested, although oregano has been never evaluated into adrenocortical tumour cell models. This study analysed for the first time the anticancer effects of a crude extract of wild mountain oregano (Origanum vulgare L.) in SW13 and H295R cell lines. The crude extract was characterised by GC/MS and the toxic effects of oregano were first analysed by brine shrimp lethality assay. Our findings demonstrated that oregano decreased cell viability, survival, modified cell cycle and induced cell death (through necrotic process) and that the effects can be attributed to a blockade of MAPK and PI3 K/Akt pathways. These results suggest that oregano extract exerts anticancer activities in adrenocortical tumour cell lines, providing evidence for further research in higher models.

Anticancer Effects of Wild Mountain Mentha longifolia Extract in Adrenocortical Tumor Cell Models.

Mint [Mentha longifolia (L.) Hudson] is an aromatic plant that belongs to Lamiaceae family. It is traditionally used as herbal tea in Europe, Australia and North Africa and shows numerous pharmacological effects, such as spasmolytic, antioxidant, antimicrobial and anti-hemolytic. Recently, its antiproliferative role has been suggested in a small number of tumor cell models, but no data are available on adrenocortical carcinoma, a malignancy with a survival rate at 5 years of 20%-30% which frequently metastasize. This work aimed to study the effects of Mentha longifolia L. crude extract (ME) on two adrenocortical tumor cell models (H295R and SW13 cells). Chemical composition of ME was assessed by gas-chromatography/mass spectrometry and NMR spectroscopy analysis. Brine shrimp lethality assay showed ME effects at >0.5 µg/µl (p < 0.05). Cell viability and vitality were determined by MTT, SRB, and trypan blue assays in H295R and SW13 cells. The anti-proliferative effects of ME were more evident in SW13 cells at 72 h (ME > 0.5 µg/µl, p < 0.05). Combination of ME with mitotane (approved drug for adrenocortical carcinoma) seemed not to reinforce the efficacy of the herb. As control, human fibroblasts were treated with ME with no effect on cell viability. Clonogenic assay was concordant with previous cell viability tests (ME > 0.5 µg/µl, p < 0.05), while Wright staining demonstrated the presence of both necrotic and apoptotic cells. Cell cycle analysis showed a strong increase in subG0/G1 phase, related to cell death. Furthermore, MAPK and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with ME alone or combined with mitotane. The crude methanolic extract of wild mountain mint can decrease cell viability, vitality and survival of adrenocortical tumor cell models, in particular of SW13 cells. These data show the potential anticancer effects of ME, still more work is needed to corroborate these findings.

Antiproliferative activity of Juglone derivatives on rat glioma.

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term survival to patients. Natural bioactive compounds represent a precious source of molecules with antiproliferative activity, potentially effective also against glioma cells. Among these, Juglone is a known allelopathic compound extracted from the eastern black walnut (Juglans nigra) whose antimitotic effect has been extensively described in mammalian cells. We investigated the antiproliferative effect of a synthetic derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We compared this molecule and its effect with the natural reference compound and with newly synthesised derivatives to build a preliminar structure-activity relationship. Biological assays and NMR-based redox experiments confirmed that DiNAF is a promising lead and supported the hypothesis of a redox mechanism underlying its cytotoxic activity.

Semi-synthetic derivatives of natural isoflavones from Maclura pomifera as a novel class of PDE-5A inhibitors.

Natural (iso)flavonoids have been recently reported to inhibit cyclic nucleotide phosphodiesterases (PDEs) and induce vasorelaxation, albeit the results described in the literature are discordant. The cGMP-selective isoform PDE-5A, in particular, represents the target of sildenafil and its analogues in the treatment of erectile dysfunction (ED) and pulmonary hypertension by promoting relaxation in vascular smooth muscle through the activation of the NO/cGMP pathway. We undertook this study to verify if osajin and pomiferin, two natural prenylated isoflavones and major constituents of Maclura pomifera extracts previously investigated for their anticancer, antibacterial and antidiabetic properties, show inhibitory activity on PDE-5A. These two isoflavones were isolated from the plant extracts and then synthetically modified to obtain a set of semi-synthetic derivatives with slight and focused modifications on the natural scaffold. The compounds were at first screened against PDE-5A in vitro and, based on the encouraging results, further tested for their relaxant effect on isolated rat artery rings. Computational docking studies were also carried out to explore the mode of interaction with the target protein. The obtained data were compared to the behaviour of the well-known PDE-5A inhibitor sildenafil. Our results demonstrate that semi-synthetic derivatives of osajin and pomiferin show an inhibitory effect on the isolated enzyme that, for some of the compounds, is accompanied by a vasorelaxant activity. Based on our findings, we propose the here described isoflavones as potential lead compounds for the development, starting from natural scaffolds, of a new class of PDE-5A inhibitors with vasorelaxant properties.

Molecular targets in glioblastoma.

Glioblastoma is the most lethal brain tumor. The poor prognosis results from lack of defined tumor margins, critical location of the tumor mass and presence of chemo- and radio-resistant tumor stem cells. The current treatment for glioblastoma consists of neurosurgery, followed by radiotherapy and temozolomide chemotherapy. A better understanding of the role of molecular and genetic heterogeneity in glioblastoma pathogenesis allowed the design of novel targeted therapies. New targets include different key-role signaling molecules and specifically altered pathways. The new approaches include interference through small molecules or monoclonal antibodies and RNA-based strategies mediated by siRNA, antisense oligonucleotides and ribozymes. Most of these treatments are still being tested yet they stay as solid promises for a clinically relevant success.