Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Multi‐factor climate change effects on insect herbivore performance

The impact of climate change on herbivorous insects can have far‐reaching consequences for ecosystem processes. However, experiments investigating the combined effects of multiple climate change drivers on herbivorous insects are scarce. We independently manipulated three climate change drivers (CO₂, warming, drought) in a Danish heathland ecosystem. The experiment was established in 2005 as a full factorial split‐plot with 6 blocks × 2 levels of CO₂ × 2 levels of warming × 2 levels of drought = 48 plots. In 2008, we exposed 432 larvae (n = 9 per plot) of the heather beetle (Lochmaea suturalis Thomson ), an important herbivore on heather, to ambient versus elevated drought, temperature, and CO₂ (plus all combinations) for 5 weeks. Larval weight and survival were highest under ambient conditions and decreased significantly with the number of climate change drivers. Weight was lowest under the drought treatment, and there was a three‐way interaction between time, CO₂, and drought. Survival was lowest when drought, warming, and elevated CO₂ were combined. Effects of climate change drivers depended on other co‐acting factors and were mediated by changes in plant secondary compounds, nitrogen, and water content. Overall, drought was the most important factor for this insect herbivore. Our study shows that weight and survival of insect herbivores may decline under future climate. The complexity of insect herbivore responses increases with the number of combined climate change drivers.     

Observations on Bostrychia radicosa comb. nov. (Rhodomelaceae, Rhodophyta)

Only two genera in the Rhodomelaceae share the morphological character of transverse division of periaxial cells into two or more tier cells in which the pit connection is retained between the lower cell and the axial cell: Bostrychia and Rhodolachne. One species, Rhodolachne radicosa Itono, has been reported from mangroves, a common habitat for Bostrychia. Many collections of an entity similar to Rhodolachne radicosa have been made from localities around the Indo‐Pacific. Culture observations show a Polysiphonia‐type sexual life history in Malaysia and New Caledonia isolates that produce self‐compatible bisexual gametophytes. The New Caledonia isolate also has unisexual gametophytes. An isolate from New South Wales (Australia) reproduces asexually through successive generations of tetrasporophytes. The Thailand isolate has successive generations of mixed‐phase tetrasporophytes. The tetrasporangial stichidia also bear male spermatangial sectors, but female structures are lacking. Western Australia and Madagascar isolates do not reproduce in culture. Molecular evidence, based on sequencing of the rbcL and the large subunit ribosomal RNA genes, shows that these isolates belong to the genus Bostrychia. Low molecular weight carbohydrate analysis reveals high levels of digeneaside in all isolates. The sugar hexitol sorbitol, an osmolyte characteristic of Bostrychia, occurs in all isolates, whereas the Madagascar and New Caledonia isolates have very low levels of dulcitol. Molecular, low molecular weight carbohydrate and morphological evidence show that Rhodolachne radicosa belongs within the genus Bostrychia. We transfer Rhodolachne radicosa to Bostrychia radicosa (Itono) West, Zuccarello and Hommersand.     

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.     

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Zur Spezifität des histochemischen Carboanhydratase-Nachweises im inselorgan der Bauchspeicheldrüse

Zusammenfassung Die enzymatische Aufspaltung von Kobalthydrogenkarbonaten durch das Ferment CAH führt beim Häuslerschen Fermentnachweis über eine sekundäre Visualisation der Kobaltkarbonatniederschläge durch Umwandlung in Kobaltsulfid zur Markierung der Fermentaktivität am Schnitt. Untersuchungen am Pankreas zeigen, daß die Aussagekraft fermentmarkierender Niederschläge durch eine unspezifische Fällung zweiwertiger, nichtfermentgebundener Zinkionen beeinträchtigt wird. Das inkretorische Parenchym des Pankreas enthält keine CAH, wird aber intensiv imprägniert durch seinen Reichtum an nichtfermentgebundenen Zinkionen. Nach Abfangen dieser Metallionen durch Metallchelatbildner (Dithizon, NDDC) fehlt eine Kobaltsulfidschwärzung der Inselzellen.Im exkretorischen Parenchym führt dagegen die Bebrütung von Kryostatschnitten zu spezifischer Fermentmarkierung. Aus sterischen Gründen (tertiäre Bindung des zweiwertigen Metalls im Enzymmolekül) ist das CAH-Zink einer Chelatbindung mit Dithizon oder NDDC nicht zugänglich, die Aktivität der CAH in den Gangepithelien der Bauchspeicheldrüse wird durch Chelatbildner nicht beeinflußt.Die Spezifität des Häuslerschen Fermentnachweises ist unbestritten. Zu fordern ist aufgrund der vorliegenden Ergebnisse aber eine Kontrolle der Fermentreaktion durch Chelatbildner, um eine gleichzeitige unspezifische Imprägnation zweiwertiger Metallionen (insbesondere des Zinks) im Kryostatschnitt auszuschließen.

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Summary In the Häusler incubation method the demonstration of the catalytic activity of carbonic anhydrase in tissue sections involves an enzymatic splitting of cobalt hydrogencarbonates. The resulting carbonate precipitates are secondarily visualized by being transformed into cobalt sulfide. Examination of the pancreas by the modified Häusler reaction indicates that the specificity of the precipitates marking the enzyme is masked by an unspecific precipitation of bivalent zinc ions that are not bound to the enzyme. The endocrine parenchyma of the pancreas does not contain any carbonic anhydrase, but is intensely impregnated because of its abundance of zinc ions bound to insulin or glucagon. If these metallic ions are sequestered by the use of metal chelating agents (dithiozone, sodium diethyldithiocarbamate), the unspecific precipitates in the islets of Langerhans are eliminated.In the exocrine parenchyma, however, the incubation of cryostat sections results in a specific demonstration of the enzyme. The steric arrangement of zinc ions within the enzymic molecules (trivalent complexes of bivalent metal ions) prevents a complex linkage of dithiozone or sodium diethyldithiocarbamate to carbonic anhydrase. The activity of carbonic anhydrase in the epithelium of the ducts is not at all inhibited by chelating agents.The specificity of the Häusler incubation method for demonstrating carbonic anhydrase is uncontested. The results show, however, that in histochemical studies of the enzyme the use of chelating agents is necessary as a control to exclude a simultaneous unspecific precipitation of bivalent metal ions (especially zinc) in cryostat sections.

     

Untersuchungen über das Vorkommen und die Zahl von Muskelspindeln im M. Vocalis des Menschen

Zusammenfassung Die für das Problem der Stimmbandschwingungen wichtige Frage nach dem Vorkommen und der Zahl von Muskelspindeln im M. vocalis des Menschen wurde an Hand von Schnittserien an den Mm. vocales eines Neugeborenen und eines 10jährigen Knaben nachgeprüft. Nur in einem der vier untersuchten Stimmuskeln wurde eine einzige Muskelspindel nachgewiesen. Dieser Befund steht in Übereinstimmung mit der unimodalen Verteilung der Faserkaliber im motorischen Ast des N. recurrens.Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.