HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-β(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ～5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.     

PavB is a surface‐exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections

The genomic analysis of Streptococcus pneumoniae strains identified the Pneumococcal adherence and virulence factor B (PavB), whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with human fibronectin. Here, we showed the gene in all tested pneumococci and identified that the observed differences in the molecular mass of PavB rely on the number of repeats, ranging from five to nine SSURE. PavB interacted with fibronectin and plasminogen in a dose‐dependent manner as shown by using various SSURE peptides. In addition, we identified PavB as colonization factor. Mice infected intranasally with ΔpavB pneumococci showed significantly increased survival times compared with wild‐type bacteria. Importantly, the pavB‐mutant showed a delay in transmigration to the lungs as observed in real‐time using bioluminescent pneumococci and decreased colonization rates in a nasopharyngeal carriage model. In co‐infection experiments the wild‐type out‐competed the pavB‐mutant and infections of epithelial cells demonstrated that PavB contributes to adherence to host cell. Blocking experiments suggested a function of PavB as adhesin, which was confirmed by direct binding of SSURE peptides to host cells. Finally, PavB may represent a new vaccine candidate as SSURE peptides reacted with human sera. Taken together, PavB is a surface‐exposed adhesin, which contributes to pneumococcal colonization and infections of the respiratory airways.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.     

A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.     

Stamens are not essential as an attractant for pollinators in females of cryptically dioecious Thalictrum pubescens Pursch. (Ranunculaceae)

 It has been hypothesized that females of some dioecious species maintain stamens that produce sterile pollen as a means of attracting pollinators to promote greater seed set. However, this hypothesis has rarely been tested. This paper examines the role of pollinators in the maintenance of sterile stamens in the cryptically dioecious species, Thalictrum pubescens. Wind was found to contribute to pollination; branches of female T. pubescens enclosed in cheesecloth to exclude insects but not wind were still able to set seed. Therefore, females may not need the stamens for pollination. In 1994, insects were found to discriminate against emasculated female flowers, but this discrimination did not lead to a significant difference in either the amount of pollen received or seed set. In 1995, emasculation was combined with the addition of pollen to determine if emasculated females were pollen limited. No significant effect of pollen addition or emasculation was found. Emasculated branches in the pollen-addition treatment appeared to have slightly lower seed set than non-emasculated branches that received pollen, indicating that the slight reduction in seed set was caused by emasculation rather than pollen limitation. Since stamens do not appear to be maintained as pollinator attractants, other explanations, such as genetic constraints, must be investigated. Received: 4 February 1997 / Revision accepted: 20 May 1997     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.     

Response to hyperoxia is associated with similar ho-1 gene expression level in lungs of aging CBA mice of both sexes

Beside classical antioxidative enzymes, the response to hyperoxia might be mediated via regulation of other systems, such as heme oxygenase (HO). Ho-1 gene expression is found to be upregulated by hyperoxia in all groups of mice, while HO-1 protein isoform was increased only in 4 months old male mice. In steady-state conditions ho-1 and ho-2 gene expression remained unchanged irrespective of sex or age, which was not the case with protein level of both isoforms. This study suggests that in lungs of CBA mice the response to oxidative stress may be mediated through the interaction of other systems such as heme oxygenase, primarily via upregulation of ho-1 gene expression in both sexes. Contrary to our previous study in liver of hyperoxia treated mice, current results might imply that at conventional oxygen conditions lungs of female mice with the emphasis on aging females, are better prepared for oxidative stress conditions through the increase of HO-activity.