Increased sympathetic modulation in breast cancer survivors determined by measurement of heart rate variability.

Experimental and clinical studies have shown that the sympathetic nervous system (SNS) stimulates cancer progression and reduces the efficacy of oncological treatment. These effects may be reduced by pharmacological and psychotherapeutical approaches attenuating SNS tone. Therefore, it is necessary to identify those cancer survivors whose sympathetic modulation is excessively increased. For determination of SNS modulation, non-invasive method of heart rate variability (HRV) is widely used. In our study, HRV was determined from 5-min heartbeat recordings in healthy volunteers and in women with benign or malignant breast neoplasias, both in newly diagnosed patients and in women after initial treatment. We showed impaired cardio-vagal regulation in breast cancer patients (linear methods) and also found the increased sympathetic modulation indicated by the non-linear (the symbolic dynamics 0V%) parameter. This non-linear HRV analysis seems to be more sensitive than the linear one, indicating significant differences also in survivors after initial therapy in comparison to healthy controls. The lower sample entropy revealed reduced complexity in heart rate control in both breast cancer survivors groups. These findings suggest that HRV detection represents an inexpensive, easy, and reliable method for identification of those patients with breast cancer whose sympathetic modulation is significantly increased and in which the interventions, aimed at normalizing the balance in the autonomic nervous system (e.g. psychotherapy, biofeedback, treatment by ß-blockers) may be the most effective.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species.

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by 'Candidatus B. rudakovii' and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

The Role of Peridomestic Animals in the Eco-Epidemiology of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.

Increased Stromal Infiltrating Lymphocytes Are Associated with the Risk of Disease Progression in Mesenchymal Circulating Tumor Cell-Positive Primary Breast Cancer Patients.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, p < 0.001 for CD3high; HR = 6.17, 95% CI 2.75-13.80, p < 0.001 for CD8high; HR = 6.93, 95% CI 2.86-16.81, p < 0.001 for CD45ROhigh). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.

Hedgehogs, Squirrels, and Blackbirds as Sentinel Hosts for Active Surveillance of Borrelia miyamotoi and Borrelia burgdorferi Complex in Urban and Rural Environments.

Lyme borreliosis (LB), caused by spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex, is one of the most common vector-borne zoonotic diseases in Europe. Knowledge about the enzootic circulation of Borrelia pathogens between ticks and their vertebrate hosts is epidemiologically important and enables assessment of the health risk for the human population. In our project, we focused on the following vertebrate species: European hedgehog (Erinaceus europaeus), Northern white-breasted hedgehog (E. roumanicus), Eurasian red squirrel (Sciurus vulgaris), and Common blackbird (Turdus merula). The cadavers of accidentally killed animals used in this study constitute an available source of biological material, and we have confirmed its potential for wide monitoring of B. burgdorferi s.l. presence and genospecies diversity in the urban environment. High infection rates (90% for E. erinaceus, 73% for E. roumanicus, 91% for S. vulgaris, and 68% for T. merula) were observed in all four target host species; mixed infections by several genospecies were detected on the level of individuals, as well as in particular tissue samples. These findings show the usefulness of multiple tissue sampling as tool for revealing the occurrence of several genospecies within one animal and the risk of missing particular B. burgdorferi s.l. genospecies when looking in one organ alone.

LmxM.22.0250-Encoded Dual Specificity Protein/Lipid Phosphatase Impairs Leishmania mexicana Virulence In Vitro.

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host's phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have been implicated in human infections before. In this work, we have identified and characterized the dual specificity protein/lipid phosphatase LmDUSP1 as a novel virulence factor governing Leishmania mexicana infection. The LmDUSP1-encoding gene (LmxM.22.0250 in L. mexicana) has been acquired from bacteria via horizontal gene transfer. Importantly, its orthologues have been associated with virulence in several bacterial species, such as Mycobacterium tuberculosis and Listeria monocytogenes. Leishmania mexicana with ablated LmxM.22.0250 demonstrated severely attenuated virulence in the experimental infection of primary mouse macrophages, suggesting that this gene facilitates Leishmania pathogenicity in vertebrates. Despite significant upregulation of LmxM.22.0250 expression in metacyclic promastigotes, its ablation did not affect the ability of mutant cells to differentiate into virulent stages in insects. It remains to be further investigated which specific biochemical pathways involve LmDUSP1 and how this facilitates the parasite's survival in the host. One of the interesting possibilities is that LmDUSP1 may target host's substrate(s), thereby affecting its signal transduction pathways.

Multiple Lineages of Usutu Virus (Flaviviridae, Flavivirus) in Blackbirds (Turdus merula) and Mosquitoes (Culex pipiens, Cx. modestus) in the Czech Republic (2016-2019).

Usutu virus (USUV) is a flavivirus (Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages (Europe 1, 2 and Africa 3) of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.

Anaplasma phagocytophilum evolves in geographical and biotic niches of vertebrates and ticks.

BACKGROUND: Anaplasma phagocytophilum is currently regarded as a single species. However, molecular studies indicate that it can be subdivided into ecotypes, each with distinct but overlapping transmission cycle. Here, we evaluate the interactions between and within clusters of haplotypes of the bacterium isolated from vertebrates and ticks, using phylogenetic and network-based methods. METHODS: The presence of A. phagocytophilum DNA was determined in ticks and vertebrate tissue samples. A fragment of the groEl gene was amplified and sequenced from qPCR-positive lysates. Additional groEl sequences from ticks and vertebrate reservoirs were obtained from GenBank and through literature searches, resulting in a dataset consisting of 1623 A. phagocytophilum field isolates. Phylogenetic analyses were used to infer clusters of haplotypes and to assess phylogenetic clustering of A. phagocytophilum in vertebrates or ticks. Network-based methods were used to resolve host-vector interactions and their relative importance in the segregating communities of haplotypes. RESULTS: Phylogenetic analyses resulted in 199 haplotypes within eight network-derived clusters, which were allocated to four ecotypes. The interactions of haplotypes between ticks, vertebrates and geographical origin, were visualized and quantified from networks. A high number of haplotypes were recorded in the tick Ixodes ricinus. Communities of A. phagocytophilum recorded from Korea, Japan, Far Eastern Russia, as well as those associated with rodents had no links with the larger set of isolates associated with I. ricinus, suggesting different evolutionary pressures. Rodents appeared to have a range of haplotypes associated with either Ixodes trianguliceps or Ixodes persulcatus and Ixodes pavlovskyi. Haplotypes found in rodents in Russia had low similarities with those recorded in rodents in other regions and shaped separate communities. CONCLUSIONS: The groEl gene fragment of A. phagocytophilum provides information about spatial segregation and associations of haplotypes to particular vector-host interactions. Further research is needed to understand the circulation of this bacterium in the gap between Europe and Asia before the overview of the speciation features of this bacterium is complete. Environmental traits may also play a role in the evolution of A. phagocytophilum in ecotypes through yet unknown relationships.

Genotoxic Effects of Tributyltin and Triphenyltin Isothiocyanates, Cognate RXR Ligands: Comparison in Human Breast Carcinoma MCF 7 and MDA-MB-231 Cells.

The cytotoxicity of two recently synthesized triorganotin isothiocyanate derivatives, nuclear retinoid X receptor ligands, was tested and compared in estrogen-receptor-positive MCF 7 and -negative MDA-MB-231 human breast carcinoma cell lines. A 48 h MTT assay indicated that tributyltin isothiocyanate (TBT-ITC) is more cytotoxic than triphenyltin isothiocyanate (TPT-ITC) in MCF 7 cells, and the same trend was observed in the MDA-MB-231 cell line. A comet assay revealed the presence of both crosslinks and increasing DNA damage levels after the 17 h treatment with both derivatives. Differences in cytotoxicity of TBT-ITC and TPT-ITC detected by FDA staining correspond to the MTT data, communicating more pronounced effects in MCF 7 than in the MDA-MB-231 cell line. Both derivatives were found to cause apoptosis, as shown by the mitochondrial membrane potential (MMP) depolarization and caspase-3/7 activation. The onset of caspase activation correlated with MMP dissipation and the total cytotoxicity more than with the amount of active caspases. In conclusion, our data suggest that the DNA damage induced by TBT-ITC and TPT-ITC treatment could underlie their cytotoxicity in the cell lines studied.

CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1.

Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.