Integrative taxonomy reveals a new Gammarus species (Crustacea,Amphipoda) surviving in a previously unknown southeast European glacial refugium

The multidisciplinarity of integrative taxonomy is particularly useful for clarifying the systematics of speciose groups that are poorly differentiated morphologically, and this approach can also illuminate their evolutionary history and biogeography. Here, we utilize it to examine the systematics and taxonomy of a newly recognized amphipod species, Gammarus hamaticornis n. sp., which belongs to a highly diverse genus of endemic freshwater crustaceans that show very limited morphological differentiation. Since this species is endemic to northern Dobrogea, a region at the northwestern Black Sea coast devoid of permafrost during the Last Glacial Maximum, we hypothesized that it survived in situ during the Quaternary climatic oscillations. We first examined the phylogenetic position of Gammarus hamaticornis n. sp. within the genus and then compared its morphology, phylogeography, distribution, and climatic niche with that of its sister species. Results indicate that G. hamaticornis n. sp. is most closely related to its widely distributed northern neighbor, G. kischineffensis, and a remarkable agreement was observed among morphological, multilocus coalescent and climatic analyses which supported the distinctiveness of both taxa. These apparently diverged during the Pliocene from a common ancestor that likely colonized freshwaters from the adjacent brackish basins of the shrinking Paratethys. The results indicate that G. hamaticornis n. sp. has persisted throughout the Pleistocene in northern Dobrogea, a hitherto hypothesized refugium confirmed for the first time with molecular genetic data. Due to its narrow geographical range, rarity in the local communities and highly fluctuating nature of the streams it inhabits, this species should be in the focus of future conservation priorities.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

Ticks and mosquitoes as vectors of Borrelia burgdorferi s. l. in the forested areas of Szczecin

The aim of the study was to determine the infection level of adult forms and larvae of ticks and mosquitoes with Borrelia burgdorferi in the forested areas of Szczecin. A total of 1699 ticks Ixodes ricinus, including 1422 nymphs, 277 adult forms and 2862 mosquito females representing the genera Aedes (89.6%) and Culex (10.4%) were collected between the years 2004 and 2005. A further 3746 larvae and 1596 pupae of Culex pipiens pipiens were colleted from water bodies. Borrelia burgdorferi s. l. was detected in the arthropods by the method of indirect immunofluorescence assay (IFA). A positive immunological reaction was detected in 16.6% of the adult forms and in 16.5% of the nymphs of Ixodes ricinus. Spirochetes were also detected in 1.7% of mosquito females, 3.2% of larvae and in 1.6% of pupae of Culex pipiens pipiens. The results of the present study confirm that contact with ticks constitutes the main risk of contracting Lyme disease, although mosquitoes play a role as vectors as well.     

Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.     

Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.     

Determination of treosulfan in plasma and urine by HPLC with refractometric detection; pharmacokinetic studies in children undergoing myeloablative treatment prior to haematopoietic stem cell transplantation

A direct and selective HPLC method with refractometric detection was worked out for determination of treosulfan in plasma and urine of children. Before injection onto reverse phase column plasma samples with treosulfan and barbital (I.S.) were clarified using filtration. The mobile phase was composed of phosphate buffer, pH 5 and acetonitrile. The linear range of the standard curve of treosulfan spanned concentrations of 10.0-2000.0 microg/ml and 50.0-10000.0 microg/ml in plasma and urine, respectively, and covered the levels found in biological fluids after infusion of the drug. The limit of detection amounted to 5 microg/ml for plasma and 25 microg/ml for urine. Intra- and inter-day precision and accuracy of the measurement fulfilled analytical criteria accepted in pharmacokinetic studies. Recovery of treosulfan as well as stability in biological fluids was also calculated. The validated method was successfully applied in pharmacokinetic studies of treosulfan administered to children prior to haematopoietic stem cell transplantation. Differences between pharmacokinetics of treosulfan in children and adults were also studied.     

Reduction of IL-2 fragmentation during manufacturing of a novel immunocytokine by DoE process optimization

Interleukin-2 (IL-2) is a potent molecule in cancer therapy. Clinical application, however, is limited due to its strong side effects during the treatment. We developed an IL-2 variant (IL-2v) immunocytokine to circumvent the drawbacks of the current IL-2 therapy. During the production of the IL-2v immunocytokine in Chinese hamster ovary (CHO) cells, molecules with fragmented IL-2v and therefore reduced cytokine activity can be observed. To control product fragmentation different production process conditions were investigated. By shifting temperature or pH after the cell growth phase to lower values, fragmented species can be reduced from 10% to 12% to about 4%. However, with the adopted process conditions, the effective titer is decreased concomitantly. Moreover, fermentation length and inoculation cell density are parameters to adjust fragmentation and effective titer. A suitable method for efficient process optimization is the design of experiment approach. With this procedure, novel optimal values for temperature, pH value, harvest day, and inoculation cell densities were proposed and tested subsequently. In comparison to the former process, the improved process reduces fragmentation by 66% while keeping the effective titer comparable. In summary, these findings will help to control fragmentation in CHO production processes of different IL-2v or IL-2 containing therapeutic proteins.     

Human Sperm Characteristics with Regard to Cobalt,Chromium, and Lead in Semen and Activity of Catalase in Seminal Plasma

Biological Trace Element Research - We analyzed cobalt (Co), chromium (Cr), and lead (Pb) concentrations in human semen and catalase CAT activity in seminal plasma and the effects of their...     

The Emergence of the Spike Furin Cleavage Site in SARS-CoV-2

Compared with other SARS-related coronaviruses (SARSr-CoVs), SARS-CoV-2 possesses a unique furin cleavage site (FCS) in its spike. This has stimulated discussion pertaining to the origin of SARS-CoV-2 because the FCS has been observed to be under strong selective pressure in humans and confers the enhanced ability to infect some cell types and induce cell–cell fusion. Furthermore, scientists have demonstrated interest in studying novel cleavage sites by introducing them into SARSr-CoVs. We review what is known about the SARS-CoV-2 FCS in the context of its pathogenesis, origin, and how future wildlife coronavirus sampling may alter the interpretation of existing data.     

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.