Genetic differentiation between Mediterranean and Atlantic populations of the common prawn Palaemon serratus (Crustacea: Palaemonidae) reveals uncommon phylogeographic break

The Atlantic-Mediterranean transition zone between the Alboran Sea and the Gulf of Cadiz constitutes the most prominent marine geographic barrier in European waters and includes known phylogeographic breaks such as the Strait of Gibraltar and the Almeria-Oran Front. A genetic shift in this area has been previously documented for the European littoral shrimp Palaemon elegans. Here we carried out a phylogeographic analysis with the congeneric and sympatric species Palaemon serratus to test for similar intraspecific genetic differentiation and geographic structure. This littoral prawn is distributed in the Northeastern Atlantic Ocean, the Mediterranean Sea and the Black Sea. We compared DNA sequences from the mitochondrial genes Cox1 and to a lesser extent from 16S rRNA of several Atlantic and Mediterranean populations. Furthermore, sequences from the nuclear gene Enolase were included for corroborating differences between Mediterranean and Atlantic individuals. A pronounced genetic differentiation was detected between the Mediterranean and Atlantic populations, amounting to 10.14% in Cox1 and 2.0% in 16S, indicating the occurrence of two independent evolutionary lineages. Interestingly, specimens from the Atlantic Gulf of Cadiz cluster together with the Mediterranean individuals, indicating that a biogeographic barrier appears to be located west of the Strait of Gibraltar

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Leibniz AssociationEU Horizon 2020Indiana University Graduate Training Program in Quantitative and Chemical Biolog

Therapeutic benefit of Salmonella attributed to LPS and TNF-α is exhaustible and dictated by tumor susceptibility.

The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella. We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8+ T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella. In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT

Epithelial-Mesenchymal Transition during Metastasis of HPV-Negative Pharyngeal Squamous Cell Carcinoma

In epithelial tumors, a shift towards a mesenchymal phenotype has been associated with increased invasiveness and metastasis. It is assumed that this phenomenon plays a major role in disease progression and ultimately prognosis. This study investigated epithelial-mesenchymal transition (EMT) in human papillomavirus- (HPV-) negative pharyngeal squamous cell carcinoma. Tissue was obtained from one hypopharyngeal primary tumor and a regional lymph node metastasis during surgery with curative intention. A cell culture was established from the primary tumor and mesenchymal growth conditions were emulated. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition), applying gene set expression analysis (GSEA). Immunohistochemical staining and flow cytometry of CD44 and E-cadherin were compared in primary tumor, metastasis, and cell cultures. Primary tumor and metastasis were highly positive for CD44. A loss of E-cadherin occurred in the metastasis. Flow cytometry showed the appearance of a population without E-cadherin in spheroid colonies. In GSEA, the EMT phenotype was enriched in the primary tumor compared to metastasis and cell cultures (FDR < 25%, p < 5%). EMT showed variable expression during metastasis. It may thereby be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active only during the process of metastasis itself. Thereby, the primary tumor as well as the metastasis may exhibit fewer EMT properties

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization

Modeling the Ecological Responses of Tree Species to the Flood Pulse of the Amazon Negro River Floodplains

The large flood pulse of the Amazon basin is a principal driver of environmental heterogeneity with important implications for ecosystem function and the assembly of natural communities. Understanding species ecological response to the flood pulse is thus a key question with implications for theories of species coexistence, resource management, and conservation. Yet these remain largely undescribed for most species, and in particular for trees. The large flood pulse and high tree diversity of the Negro River floodplain makes it an ideal system to begin filling this knowledge gap. We merged historical hydrologic data with 41 forest inventories under variable flooding conditions distributed across the Negro River basin, comprising a total area of 34 ha, to (i) assess the importance of flood duration as a driver of compositional variation, (ii) model the response curve shapes of 111 of the most frequent tree species in function of flood duration, and (iii) derive their niche properties (optima and tolerance). We found that flood duration is a strong driver of compositional turnover, although the majority site-to-site variation in forest composition still remains unexplained. About 73% of species responded to the flood duration gradient, exhibiting a diversity of shapes, but most frequently skewed. About 29% of species were clearly favored by flood durations >120 days year–1, and 44% of species favored by shorter floods. The median niche breadth was 85 flood days year–1, corresponding to approximately 30% of the flood duration gradient. A significant subset of species (27%) did not respond to flooding, but rather exhibited wide tolerance to the flood gradient. The response models provided here offer valuable information regarding tree species differential capacity to grow, survive, and regenerate along an ecologically important gradient and are spatially valid for the Amazon Negro basin. These attributes make them an appealing tool with wide applicability for field and experimental studies in the region, as well as for vegetation monitoring and simulation models of floodplain forest change in the face of hydrologic alteration