Laser melting manufacturing of large elements of lunar regolith simulant for paving on the Moon.

The next steps for the expansion of the human presence in the solar system will be taken on the Moon. However, due to the low lunar gravity, the suspended dust generated when lunar rovers move across the lunar soil is a significant risk for lunar missions as it can affect the systems of the exploration vehicles. One solution to mitigate this problem is the construction of roads and landing pads on the Moon. In addition, to increase the sustainability of future lunar missions, in-situ resource utilization (ISRU) techniques must be developed. In this paper, the use of concentrated light for paving on the Moon by melting the lunar regolith is investigated. As a substitute of the concentrated sunlight, a high-power CO2 laser is used in the experiments. With this set-up, a maximum laser spot diameter of 100 mm can be achieved, which translates in high thicknesses of the consolidated layers. Furthermore, the lunar regolith simulant EAC-1A is used as a substitute of the actual lunar soil. At the end of the study, large samples (approximately 250 × 250 mm) with interlocking capabilities were fabricated by melting the lunar simulant with the laser directly on the powder bed. Large areas of lunar soil can be covered with these samples and serve as roads and landing pads, decreasing the propagation of lunar dust. These manufactured samples were analysed regarding their mineralogical composition, internal structure and mechanical properties.

MR-Guided Transurethral Ultrasound Ablation of Prostate Cancer: Initial Experience of Monitoring Tumor Response by Dynamic Apparent Diffusion Coefficient Measurements at 3.0 T.

INTRODUCTION: Diffusion-weighted imaging (DWI) as part of multiparametric magnetic resonance imaging (mpMRI) is an important sequence for the detection of prostate cancer (PCa). The objective of this retrospective analysis was to evaluate changes in apparent diffusion coefficient (ADC) measurements in biopsy-proven PCa undergoing TULSA-PRO (MR-guided transurethral ultrasound ablation of the prostate) at 3.0 T after 1, 3, and 6-12 months posttreatment. METHODS: Nineteen patients underwent follow-up examinations after 1, 3, and 6-12 months including mpMRI at 3.0 T and urological-clinical examinations with quantitative analysis of ADCs. RESULTS: In PCa, a significant increase of ADC values after 6-12 months was measured after TULSA-PRO treatment by 29.1% (pre-TULSA: 0.79 ± 0.16 × 10-3 mm2/s, 6-12 months: 1.02 ± 0.35 × 10-3 mm2/s), while the corresponding value in the reference tissue decreased by 48.5% (pre-TULSA: 1.20 ± 0.15 × 10-3 mm2/s, 6-12 months: 0.91 ± 0.29 × 10-3 mm2/s). The mean ADC values in the early follow-up groups at 1 and 3 months did not change significantly. CONCLUSION: DWI with ADC as part of mpMRI can serve as a biomarker to dynamically monitor the follow-up after TULSA after 6-12 months. For early posttreatment progression, it is not suitable due to too many confounding variables.

Pain management after robot-assisted minimally invasive esophagectomy.

Background: Adequate pain control after open esophagectomy is associated with reduced complications, earlier recovery and higher patient satisfaction. While further developing surgical procedures like robot-assisted minimally invasive esophagectomy (RAMIE) it is relevant to adapt postoperative pain management. The primary question of this observational survey was whether one of the two standard treatments, thoracic epidural analgesia (TEA) or intravenous patient-controlled analgesia (PCA), is superior for pain control after RAMIE as the optimal pain management for these patients still remains unclear. Use of additional analgesics, changes in forced expiratory volume in 1 s (FEV1), postoperative complications and duration of intensive care and hospital stay were also analyzed. Methods: This prospective observational pilot study analyzed 50 patients undergoing RAMIE (postoperative PCA with piritramide or TEA using bupivacaine; each n = 25). Patient reported pain using the numeric rating scale score and differences in FEV1 using a micro spirometer were measured at postoperative day 1, 3 and 7. Additional data of secondary endpoints were collected from patient charts. Results: Key demographics, comorbidity, clinical and operative variables were equivalently distributed. Patients receiving TEA had lower pain scores and a longer-lasting pain relief. Moreover, TEA was an independent predictive variable for reduced length of hospital stay (HR -3.560 (95% CI: -6.838 to -0.282), p = 0.034). Conclusions: Although RAMIE leads to reduced surgical trauma, a less invasive pain therapy with PCA appears to be inferior compared to TEA in case of sufficient postoperative analgesia and length of hospital stay. According to the results of this observational pilot study analgesia with TEA provided better and longer-lasting pain relief compared to PCA. Further randomized controlled trials should be conducted to evaluate the optimal postoperative analgesic treatment for RAMIE.

Human Astrovirus 1-8 Seroprevalence Evaluation in a United States Adult Population.

Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1-8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes.

Protein Disulfide Isomerase A4 Is Involved in Genome Uncoating during Human Astrovirus Cell Entry.

Although human astroviruses (HAstVs) are important agents of gastroenteritis in young children, the studies aimed at characterizing their biology have been limited, in particular regarding their cell entry process. It has been shown that HAstV serotype 8 enters human cells by a classical clathrin-mediated endocytosis pathway; however, the cell receptor or other cell entry factors that may be relevant for an efficient viral infection are unknown. In this work we used a far-Western blotting approach to identify cellular proteins that interact with the recombinant capsid spike proteins of HAstV serotypes 1, 2, and 8, synthesized in Escherichia coli. We identified the 72 kDa protein disulfide isomerase A4 (PDIA4) as a binding partner for HAstV-1 and -8 spikes, but not for the HAstV-2 spike. In agreement with this observation, the PDI inhibitor 16F16 strongly blocked infection by HAstV serotypes 1 and 8, but not serotype 2. RNA interference of PDIA4 expression selectively blocked HAstV-8 infectivity. We also showed that the PDI activity does not affect virus binding or internalization but is required for uncoating of the viral genome.

A simplified workflow for monoclonal antibody sequencing.

The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.

Inhibition of Inflammasome-Dependent Interleukin 1ß Production by Streptococcal NAD+-Glycohydrolase: Evidence for Extracellular Activity.

Group A Streptococcus (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD+-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease. NADase can be translocated into host cells by a SLO-dependent mechanism, and cytosolic NADase has been assigned multiple properties such as protection of intracellularly located GAS bacteria and induction of host cell death through energy depletion. Here, we used a set of isogenic GAS mutants and a macrophage infection model and report that streptococcal NADase inhibits the innate immune response by decreasing inflammasome-dependent interleukin 1ß (IL-1ß) release from infected macrophages. Regulation of IL-1ß was independent of phagocytosis and ensued also under conditions not allowing SLO-dependent translocation of NADase into the host cell cytosol. Thus, our data indicate that NADase not only acts intracellularly but also has an immune regulatory function in the extracellular niche.IMPORTANCE In the mid-1980s, the incidence and severity of invasive infections caused by serotype M1 GAS suddenly increased. The results of genomic analyses suggested that this increase was due to the spread of clonal bacterial strains and identified a recombination event leading to enhanced production of the SLO and NADase toxins in these strains. However, despite its apparent importance in GAS pathogenesis, the function of NADase remains poorly understood. In this study, we demonstrate that NADase inhibits inflammasome-dependent IL-1ß release from infected macrophages. While previously described functions of NADase pertain to its role upon SLO-mediated translocation into the host cell cytosol, our data suggest that the immune regulatory function of NADase is exerted by nontranslocated enzyme, identifying a previously unrecognized extracellular niche for NADase functionality. This immune regulatory property of extracellular NADase adds another possible explanation to how increased secretion of NADase correlates with bacterial virulence.

A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control Type VI system-mediated secretion.

The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an ΔiglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, ΔiglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-α, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first ∼ 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.

Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication.

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, ΔpdpE and ΔiglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ΔiglA and ΔiglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection.

BACKGROUND: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system. RESULTS: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1ß, albeit lower than LVS, and markedly induced LDH release. CONCLUSIONS: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection.

Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM ß-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native ß-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.

Link between intraphagosomal biotin and rapid phagosomal escape in Francisella.

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.

DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity.

The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both ΔvgrG and ΔdotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.

IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS.

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.