Hyaluronan (HA) is a simple but diverse glycosaminoglycan. It plays a major role in aging, cellular senescence, cancer, and tissue homeostasis. In which way HA affects the surrounding tissues greatly depends on the molecular weight of HA. Whereas high molecular weight HA is associated with homeostasis and protective effects, HA fragments tend to be linked to the pathologic state. Furthermore, the interaction of HA with its binding partners, the hyaladherins, such as CD44, is essential for sustaining tissue integrity and is likewise related to cancer. The naked mole rat, a rodent species, possesses a special form of very high molecular weight (vHMW) HA, which is associated with the extraordinary cancer resistance and longevity of those animals. This review addresses HA and its diverse facets: from HA synthesis to degradation, from oligomeric HA to vHMW-HA and from its beneficial properties to the involvement in pathologies. We further discuss the functions of HA in the naked mole rat and compare them to human conditions. Though intensively researched, this simple polymer bears some secrets that may hold the key for a better understanding of cellular processes and the development of diseases, such as cancer.

It is well accepted that liver diseases and their outcomes are associated with intestinal microbiota but causality is difficult to establish. The intestinal microbiota is altered in patients with hepatitis C. As chronic HCV infection can now be cured in almost all patients, it is an ideal model to study the influence of liver disease on the microbiota. We aimed to analyze prospectively the changes in the gut microbiome in patients who received direct acting antivirals (DAA) and achieved sustained virological response (SVR). Amplicon sequencing of the V1-V2 region in the 16S rRNA gene was performed in stool samples of patients with chronic hepatitis C. Patients in the treatment group received direct acting antivirals (n=65) whereas in the control group no DAA were given (n=33). Only patients achieving SVR were included. The alpha diversity increased numerical but not significantly from baseline to SVR24/48 (2.784±0.248 vs. 2.846±0.224; p= 0.057). When stratifying for the presence of liver cirrhosis, a significant increase in diversity was only seen in patients without cirrhosis. Differences in the microbial community structure induced by the achievement of SVR were only observed in patients without liver cirrhosis. In patients with liver cirrhosis and in the control group, no significant differences were observed. In conclusion, the achievement of SVR24/48 in patients with chronic HCV was associated with changes in the intestinal microbiota. However, these changes were only seen in patients without liver cirrhosis. A major role of liver remodeling on the intestinal microbiota is indicated by the dynamics of the intestinal microbial community structure depending on the stage of fibrosis in patients resolving chronic hepatitis C.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSV Viruses 2019, 11, x FOR PEER REVIEW and Proteomics Core Facility of the German Cancer Research Center, Heidelberg (Table 1). B viruses were produced and purified, as described in Section 2.5. Soluble ectodomains of GN a were stably expressed in drosophila S2 cells and purified according to a standard procedure Using these protocols, the protein purity reached over 90%. Antisera were prepared in guinea by injecting 100 µg of Triton X-100-inactivated purified viruses or soluble ectodomains of GN a in Freund's complete adjuvant. The priming was followed by three booster injections of 100 µg week intervals, the first one in Freund's incomplete adjuvant, and the others in phosphate-bu saline (PBS). Animals were bled before the first immunization (control pre-immune serum) days after the last injection. The purified rabbit polyclonal antibodies against the TOSV nucleop N (T4) and non-structural protein NSs (T5) were developed by GenScript (Piscataway Netherlands). Rabbits were immunized with either a peptide corresponding to the C termin amino acid residues of NSs or the full-length N protein with a C-terminal His-tag. The m immune ascitic fluid against all TOSV structural proteins was a generous gift from R.B. (University of Texas, Galveston, Texas, USA). s were constructed by GenScript. Briefly, the cDNAs encoding the full-length S, M, and f TOSV, flanked by an upstream T7 polymerase promoter sequence and a downstream ta virus (HdV) ribozyme sequence ( Figure S1), were synthesized. The gene synthesis e subjected to blunt-end ligation into EcoRV-linearized pUC57 plasmid, or alternatively, into the pCC1 plasmid, using the CloneEZ PCR Cloning Kit (Genscript), resulting in C1-M, and pUC57-L. In addition, a pUC57-SɸNSs was created in which the S segment mRNA, with the 18 first AUG codons replaced by UAG stop codons ( Figure S2). Viruses from Plasmid DNAscells expressing T7 polymerase were seeded in 6-well plates (2.5 × 10 5 cells per well). g day, rTOSV was rescued from cells by transfection with 1 µg each of the plasmids C1-M, and pUC57-L. Alternatively, the recovery of rTOSVɸNSs was achieved following thod but using the plasmid pUC57-SɸNSs instead of the pUC57-S. Transfection was ith Lipofectamine 2000 (Thermo Fisher Scientific), using a ratio of 1 µL Lipofectamine of plasmids in 400 µL of complete GMEM without antibiotics. Supernatants were fresh culture medium containing antibiotics and 2% serum 4 h post-transfection. Five nsfection, supernatants were harvested, c...