Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Identification of the binding pocket for the TRH peptide in the melanocortin 1 receptor

We found recently that thyrotropin-releasing hormone (TRH) acts as a selective agonist on the melanocortin MC₁ receptor. While the TRH was capable of fully activating the MC₁ receptor, it did not interact with any of the other MSH peptide binding G-protein coupled melanocortin receptor subtypes MC_3-5. The MC₁ receptor is a promising target for the development of anti-inflammatory and immuno-modulatory drugs, and it was of wide interest to explore the binding site of the TRH in this receptor. Here we have investigated the binding of TRH to MC₁/MC₃ chimeric receptors and used a partial least squares (PLS) modelling approach for the data evaluation. Statistically valid PLS models (R² = 0.80; Q² = 0.66) were obtained explaining the contribution of the amino acid sequence parts of the receptor chimeras for the binding of TRH. By using the variable importances in the projection (VIPs) deduced from the PLS-model, it was revealed that the transmembrane (TM) regions TM1 and TM2/TM3 contribute the most to the TRH binding. The TM6/TM7 also had a significant influence on the binding. Moreover, it was revealed that an interaction between TM1 and TM6/TM7 of the receptor contributed to the binding of TRH. The data are in full agreement with a 3D model of a TRH peptide and MC₁ receptor complex and validates the location of the TRH ligand binding pocket between the TM1, TM2 and TM7 of the receptor.     

Erotylidae (Insecta, Coleoptera) of Poland - problematic taxa, updated keys and new records

New data concerning the occurrence of pleasing fungus beetles (Coleoptera: Erotylidae) in Poland are given, with a focus on rare and difficult to identify Central European taxa. Cryptophilus cf. integer (Heer) (Cryptophilinae) is reported from the Polish territory for the first time based on adult and larval specimens collected in the Wielkopolska-Kujawy Lowland. Identification problems concerning species of Cryptophilus introduced to Europe are discussed. Triplax carpathica Reitter (Erotylinae) is recorded from the Białowieża Primeval Forest, which is the first known non-Carpathian finding of this species, located in the close proximity of the Polish-Belarussian UNESCO World Heritage Site “Białowieża Forest”. Discussion of Triplax carpathica being conspecific with Siberian Triplax rufiventris Gebler is provided. New Polish localities of several other Erotylidae are reported, and an updated key to Central European species of Triplax is given. The Triplax key is supplemented with dorsal and ventral habitus images of all treated Triplax species. One of the rarest Central European erotyline species Combocerus glaber (Schaller) is recorded from xerothermic grasslands in North-West Poland.     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

A supertree of temnospondyli: cladogenetic patterns in the most species-rich group of early tetrapods

As the most diverse group of early tetrapods, temnospondyls provide a unique opportunity to investigate cladogenetic patterns among basal limbed vertebrates. We present five species-level supertrees for temnospondyls, built using a variety of methods. The standard MRP majority rule consensus including minority components shows slightly greater resolution than other supertrees, and its shape matches well several currently accepted hypotheses of higher-level phylogeny for temnospondyls as a whole. Also, its node support is higher than those of other supertrees (except the combined standard plus Purvis MRP supertree). We explore the distribution of significant as well as informative changes (shifts) in branch splitting employing the standard MRP supertree as a reference, and discuss the temporal distribution of changes in time-sliced, pruned trees derived from this supertree. Also, we analyse those shifts that are most relevant to the end-Permian mass extinction. For the Palaeozoic, shifts occur almost invariably along branches that connect major Palaeozoic groups. By contrast, shifts in the Mesozoic occur predominantly within major groups. Numerous shifts bracket narrowly the end-Permian extinction, indicating not only rapid recovery and extensive diversification of temnospondyls over a short time period after the extinction event (possibly less than half a million years), but also the role of intense cladogenesis in the late part of the Permian (although this was counteracted by numerous 'background' extinctions).     

Critical role of fine needle aspiration cytology and immunocytochemistry in preoperative diagnosis of pediatric renal tumors

OBJECTIVE: To evaluate accuracy and role of immunocytochemistry (ICC) in cytologic diagnosis of pediatric renal tumors. STUDY DESIGN: Fine needle aspirates from 75 cases of pediatric renal tumors were studied. Radiologic-guided aspirations were performed, with 6-7 smears stained with Papanicolaou and Giemsa stains. Smears were screened without the knowledge of final histologic diagnosis. Subsequently, clinical details, final histology and diagnosis rendered by the original cytologist were noted to judge accuracy of diagnosis by a sensitized cytologist. Five neuroblastomas that entered close differentials for Wilms tumor were also evaluated. ICC studies were also performed after staining. RESULTS: Of 58 Wilms tumors, 5 were misdiagnosed; 3 renal rhabdoid tumors and 1 clear cell sarcoma were missed on cytology. Non-Hodgkin's lymphomas presenting as renal masses were accurately diagnosed on cytology, but primitive neuroectodermal tumors (n = 3) and renal cell carcinomas (n = 2) were not accurately diagnosed. Accuracy rate improved from 65% to 92% on review by a cytologist aware of cytologic features of pediatric renal tumors. CONCLUSION: A good accuracy rate of diagnosis of pediatric renal tumors can be achieved by priming pathologists to typical features of tumors. Immunocytochemistry plays a supportive role in cases with atypical morphology or unusual presentations.