Comparative genomic hybridization analysis of Yersinia enterocolitica and Yersinia pseudotuberculosis identifies genetic traits to elucidate their different ecologies

Enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis are both etiological agents for intestinal infection known as yersiniosis, but their epidemiology and ecology bearmany differences. Swine are the only known reservoir for Y. enterocolitica 4/O:3 strains, which are the most common cause of human disease, while Y. pseudotuberculosis has been isolated from a variety of sources, including vegetables and wild animals. Infections caused by Y. enterocolitica mainly originate froms wine, but fresh produce has been the source for widespread Y. pseudotuberculosis outbreaks within recent decades. A comparative genomic hybridization analysis with a DNA microarray based on three Yersinia enterocolitica and four Yersinia pseudotuberculosis genomes was conducted to shed light on the genomic differences between enteropathogenic Yersinia. The hybridization results identified Y. pseudotuberculosis strains to carry operons linked with the uptake and utilization of substances not found in living animal tissues but present in soil, plants, and rotting flesh. Y. pseudotuberculosis also harbors a selection of type VI secretion systems targeting other bacteria and eukaryotic cells. These genetic traits are not found in Y. enterocolitica, and it appears that while Y. pseudotuberculosis has many tools beneficial for survival in varied environments, the Y. enterocolitica genome is more streamlined and adapted to their preferred animal reservoir.Peer reviewe

Stability condition for the drive bunch in a collinear wakefield accelerator

The beam breakup instability of the drive bunch in the structure-based collinear wakefield accel- erator is considered and a stabilizing method is proposed. The method includes using the specially designed beam focusing channel, applying the energy chirp along the electron bunch, and keeping energy chirp constant during the drive bunch deceleration. A stability condition is derived that defines the limit on the accelerating field for the witness bunch.Comment: 10 pages, 6 figure

Prolyl Hydroxylase PHD3 Enhances the Hypoxic Survival and G1 to S Transition of Carcinoma Cells

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells

Transportable system enabling multiple irradiation studies under simultaneous hypoxia in vitro

BackgroundCells in solid tumours are variably hypoxic and hence resistant to radiotherapy - the essential role of oxygen in the efficiency of irradiation has been acknowledged for decades. However, the currently available methods for performing hypoxic experiments in vitro have several limitations, such as a limited amount of parallel experiments, incapability of keeping stable growth conditions and dependence on CO2 incubator or a hypoxia workstation. The purpose of this study was to evaluate the usability of a novel portable system (Minihypoxy) in performing in vitro irradiation studies under hypoxia, and present supporting biological data.Materials and methodsThis study was conducted on cancer cell cultures in vitro. The cells were cultured in normoxic (similar to 21% O-2) or in hypoxic (1% O-2) conditions either in conventional hypoxia workstation or in the Minihypoxy system and irradiated at dose rate 1.28Gy/min2.9%. The control samples were sham irradiated. To study the effects of hypoxia and irradiation on cell viability and DNA damage, western blotting, immunostainings and clonogenic assay were used. The oxygen level, pH, evaporation rate and osmolarity of the culturing media on cell cultures in different conditions were followed.ResultsThe oxygen concentration in interest (5, 1 or 0% O-2) was maintained inside the individual culturing chambers of the Minihypoxy system also during the irradiation. The radiosensitivity of the cells cultured in Minihypoxy chambers was declined measured as lower phosphorylation rate of H2A.X and increased clonogenic capacity compared to controls (OER similar to 3).Conclusions The Minihypoxy system allows continuous control of hypoxic environment in multiple wells and is transportable. Furthermore, the system maintains the low oxygen environment inside the individual culturing chambers during the transportation and irradiation in experiments which are typically conducted in separate facilities.Peer reviewe

Transportable system enabling multiple irradiation studies under simultaneous hypoxia in vitro

BackgroundCells in solid tumours are variably hypoxic and hence resistant to radiotherapy - the essential role of oxygen in the efficiency of irradiation has been acknowledged for decades. However, the currently available methods for performing hypoxic experiments in vitro have several limitations, such as a limited amount of parallel experiments, incapability of keeping stable growth conditions and dependence on CO2 incubator or a hypoxia workstation. The purpose of this study was to evaluate the usability of a novel portable system (Minihypoxy) in performing in vitro irradiation studies under hypoxia, and present supporting biological data.Materials and methodsThis study was conducted on cancer cell cultures in vitro. The cells were cultured in normoxic (similar to 21% O-2) or in hypoxic (1% O-2) conditions either in conventional hypoxia workstation or in the Minihypoxy system and irradiated at dose rate 1.28Gy/min2.9%. The control samples were sham irradiated. To study the effects of hypoxia and irradiation on cell viability and DNA damage, western blotting, immunostainings and clonogenic assay were used. The oxygen level, pH, evaporation rate and osmolarity of the culturing media on cell cultures in different conditions were followed.ResultsThe oxygen concentration in interest (5, 1 or 0% O-2) was maintained inside the individual culturing chambers of the Minihypoxy system also during the irradiation. The radiosensitivity of the cells cultured in Minihypoxy chambers was declined measured as lower phosphorylation rate of H2A.X and increased clonogenic capacity compared to controls (OER similar to 3).Conclusions The Minihypoxy system allows continuous control of hypoxic environment in multiple wells and is transportable. Furthermore, the system maintains the low oxygen environment inside the individual culturing chambers during the transportation and irradiation in experiments which are typically conducted in separate facilities

Hypoxia-inducible factor (HIF)-prolyl hydroxylase 3 (PHD3) maintains high HIF2A mRNA levels in clear cell renal cell carcinoma

Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor -subunits (HIF-) and their downstream targets. HIF-2 expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2 protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2 protein and mRNA. Depletion of other PHD family members had no effect on HIF-2 expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2 protein expression. Accordingly, PHD3 knockdown decreased HIF-2 target gene expression in ccRCC cells and expression was restored upon forced HIF-2 expression. The effect of PHD3 depletion was pinpointed to HIF2A mRNA stability. In line with these in vitro results, a strong positive correlation of PHD3 and HIF2A mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2 in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2 expression and that of its target genes, which may enhance kidney cancer aggressiveness

A three-feature prediction model for metastasis-free survival after surgery of localized clear cell renal cell carcinoma

After surgery of localized renal cell carcinoma, over 20% of the patients will develop distant metastases. Our aim was to develop an easy-to-use prognostic model for predicting metastasis-free survival after radical or partial nephrectomy of localized clear cell RCC. Model training was performed on 196 patients. Right-censored metastasis-free survival was analysed using LASSO-regularized Cox regression, which identified three key prediction features. The model was validated in an external cohort of 714 patients. 55 (28%) and 134 (19%) patients developed distant metastases during the median postoperative follow-up of 6.3 years (interquartile range 3.4-8.6) and 5.4 years (4.0-7.6) in the training and validation cohort, respectively. Patients were stratified into clinically meaningful risk categories using only three features: tumor size, tumor grade and microvascular invasion, and a representative nomogram and a visual prediction surface were constructed using these features in Cox proportional hazards model. Concordance indices in the training and validation cohorts were 0.755 +/- 0.029 and 0.836 +/- 0.015 for our novel model, which were comparable to the C-indices of the original Leibovich prediction model (0.734 +/- 0.035 and 0.848 +/- 0.017, respectively). Thus, the presented model retains high accuracy while requiring only three features that are routinely collected and widely available.Peer reviewe

Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors : Results from a Phase I/II Clinical Trial

Purpose: Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8 T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment. Patients and Methods: In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer. Results: Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8(+)T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients. Conclusions: Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer.Peer reviewe

A three-feature prediction model for metastasis-free survival after surgery of localized clear cell renal cell carcinoma

After surgery of localized renal cell carcinoma, over 20% of the patients will develop distant metastases. Our aim was to develop an easy-to-use prognostic model for predicting metastasis-free survival after radical or partial nephrectomy of localized clear cell RCC. Model training was performed on 196 patients. Right-censored metastasis-free survival was analysed using LASSO-regularized Cox regression, which identified three key prediction features. The model was validated in an external cohort of 714 patients. 55 (28%) and 134 (19%) patients developed distant metastases during the median postoperative follow-up of 6.3 years (interquartile range 3.4-8.6) and 5.4 years (4.0-7.6) in the training and validation cohort, respectively. Patients were stratified into clinically meaningful risk categories using only three features: tumor size, tumor grade and microvascular invasion, and a representative nomogram and a visual prediction surface were constructed using these features in Cox proportional hazards model. Concordance indices in the training and validation cohorts were 0.755 +/- 0.029 and 0.836 +/- 0.015 for our novel model, which were comparable to the C-indices of the original Leibovich prediction model (0.734 +/- 0.035 and 0.848 +/- 0.017, respectively). Thus, the presented model retains high accuracy while requiring only three features that are routinely collected and widely available