Clemastine/tamoxifen hybrids as easily accessible antileishmanial drug leads

A library of hybrid molecules is developed based on the common chemical features shared by clemastine and tamoxifen both of which are well known for their antileishmanial activities. In the initial screening against Leishmania major and L. amazonensis promastigotes, as well as cytotoxicity assays using HepG2 cells, several hybrids showed submicromolar activity against the parasite and no toxicity against human cells. The compounds with an EC50 10 were further characterized against intracellular amastigotes as well as promastigotes of species that cause both visceral and cutaneous leishmaniasis, such as L. infantum and L. braziliensis, respectively. These sequential screenings revealed the high pan-activity of this class of molecules against these species, with several compounds displaying an EC50 ≤ 2 μM against both promastigotes and intracellular amastigotes. Two of them were identified as the potential templates for lead optimization of this series having shown the highest activities against all species in both stages of parasite. The present findings can serve as a good starting point in the search for novel antileishmanial compounds that are easy to access and highly active

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays

Prognostic significance of germline BRCA mutations in patients with HER2-POSITIVE breast cancer.

Background: HER2-positive breast cancers are rare amongst BRCA mutation carriers. No data exist regarding clinicopathological characteristics and prognosis of this subgroup of patients. Materials and methods: Using a retrospective matched cohort design, we collected data from 700 women who were diagnosed with operable invasive breast cancer from January 2006 to December 2016 and were screened for germline BRCA mutations. Clinicopathological features and survival rates were analyzed by BRCA and HER2 status. Results: One hundred and fifteen HER2-positive/BRCA mutated cases were evaluated in comparison to the three control groups: HER2-positive/BRCA wild type (n = 129), HER2-negative/BRCA mutated (n = 222), HER2-negative/BRCA wild type (n = 234). HER2-positive breast cancers were more likely to have high histologic grade and high proliferation rate than HER2-negative neoplasms, regardless of BRCA mutation status. An interaction between BRCA mutations and HER2-positive status was found to correlate with worse survival after adjusting for prognostic variables (HR = 3.4; 95% CI: 1.3-16.7). Conclusions: Co-occurrence of BRCA mutations and HER2-positive status is a poor prognostic factor in patients with early or locally advanced breast cancer. This finding may be a proof of concept that a combined pharmacological intervention directed to these targets could be synergistic

Effective genome editing in Leishmania (Viannia) braziliensis stably expressing Cas9 and T7 RNA polymerase

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite

Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Single-celled Leishmania parasites, transmitted by sand flies, infect humans and other mammals in many tropical and sub-tropical regions, giving rise to a spectrum of diseases called the leishmaniases. Species of parasite within the Leishmania genus can be divided into two groups (referred to as sub-genera) that are separated by up to 100 million years of evolution yet are highly related at the genome level. Our research is focused on identifying gene differences between these sub-genera that may identify proteins that impact on the transmission and pathogenicity of different Leishmania species. Here we report the presence of a highly-variant genomic locus (OHL) that was previously described as absent in parasites of the L. (Viannia) subgenus (on the basis of lack of key genes) but is present and well-characterised (as the LmcDNA16 locus) in all members of the alternative subgenus, L. (Leishmania). We demonstrate that the proteins encoded within the LmcDNA16 and OHL loci are similar in their structure and surface localisation in mammalian-infective amastigotes, despite significant differences in their DNA sequences. Most importantly, we demonstrate that the OHL locus proteins, like the HASP proteins from the LmcDNA16 locus, contain highly variable amino acid repeats that are antigenic in man and may therefore contribute to future vaccine development

Tamoxifen Is Effective in the Treatment of Leishmania amazonensis Infections in Mice

Leishmaniasis is an antropozoonotic disease with a wide range of clinical manifestations. In humans, signs of disease vary from skin and mucosal ulcers to enlargement of internal organs such as the liver and spleen. The unicellular parasite Leishmania amazonensis is able to infect humans and cause localized or diffuse skin lesions. The treatment for this disease is difficult, as it requires prolonged and painful applications of toxic drugs that are poorly tolerated. Therefore, a key area in leishmaniasis research is the study of new therapeutic schemes and less toxic drugs. The present report is based on the investigation of tamoxifen's activity (a compound that has been in clinical use since the 1970s for the treatment of breast cancer) in the treatment of mice experimentally infected with L. amazonensis. We observed that infected mice treated with 20 mg/kg/day of tamoxifen for 15 days showed a significant clinical and parasitological response, with reduction in the size of lesions and ulcers and decreased numbers of parasites. These promising results pave the way for further testing of this drug as a new alternative in the chemotherapy of leishmaniasis

Long-Lasting Efficacy of Radio Electric Asymmetric Conveyer Neuromodulation Treatment on Functional Dysmetria, an Adaptive Motor Behavior

BackgroundFluctuating asymmetry (FA) is widely defined as the deviation from perfect bilateral symmetry and is considered an epigenetic measure of environmental stress. Rinaldi and Fontani hypothesized that the FA morpho-functional changes originate from an adaptive motor behavior determined by functional alterations in the cerebellum and neural circuits, not caused by a lesion, but induced by environmental stress. They called this phenomenon functional dysmetria (FD). On this premise, they developed the radio electric asymmetric conveyer (REAC) technology, a neuromodulation technology aimed at optimizing the best neuro-psycho-motor strategies in relation to environmental interaction.AimsPrevious studies showed that specific REAC neuro postural optimization (NPO) treatment can induce stable FD recovery. This study aimed to verify the duration of the NPO effect in inducing the stable FD recovery over timeMaterials and methodsData were retrospectively collected from a population of 29,794 subjects who underwent a specific semiological FD assessment and received the NPO treatment, regardless of the pathology referred.ResultsThe analysis of the data collected by the various participants in the study led us to ascertain the disappearance of FD in 100% of the cases treated, with a stability of the result detected up to 18 years after the single administration of the REAC NPO treatment.ConclusionsThe REAC NPO neurobiological modulation treatment consisting of a single administration surprisingly maintains a very long efficacy in the correction of FD. This effect can be explained as the long-lasting capacity of the NPO treatment to induce greater functional efficiency of the brain dynamics as proven in previous studies