In vitro radiosensitization of breast cancer with hypoxia‐activated prodrugs

KP167 is a novel hypoxia‐activated prodrug (HAP), targeting cancer cells via DNA intercalating and alkylating properties. The single agent and radiosensitizing efficacy of KP167 and its parental comparator, AQ4N, were evaluated in 2D and 3D cultures of luminal and triple negative breast cancer (TNBC) cell lines and compared against DNA damage repair inhibitors. 2D normoxic treatment with the DNA repair inhibitors, Olaparib or KU‐55933 caused, as expected, substantial radiosensitization (sensitiser enhancement ratio, SER_0.01 of 1.60–3.42). KP167 induced greater radiosensitization in TNBC (SER_0.01 2.53 in MDAMB‐231, 2.28 in MDAMB‐468, 4.55 in MDAMB‐436) and luminal spheroids (SER_0.01 1.46 in MCF‐7 and 1.76 in T47D cells) compared with AQ4N. Significant radiosensitization was also obtained using KP167 and AQ4N in 2D normoxia. Although hypoxia induced radioresistance, radiosensitization by KP167 was still greater under 2D hypoxia, yielding SER_0.01 of 1.56–2.37 compared with AQ4N SER_0.01 of 1.13–1.94. Such data show KP167 as a promising single agent and potent radiosensitiser of both normoxic and hypoxic breast cancer cells, with greater efficacy in TNBCs.     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Larval and early juvenile development of Lithodes santolla (Molina, 1782) (Decapoda: Anomura: Lithodidae) reared at different temperatures in the laboratory

The southern king crab, Lithodes santolla Molina, is distributed in cold-temperate and subantarctic waters ranging from the southeastern Pacific island of Chiloé (Chile) and the deep Atlantic waters off Uruguay, south to the Beagle Channel (Tierra del Fuego, Argentina/Chile). Recent investigations have shown that its complete larval development from hatching to metamorphosis, comprising three zoeal stages and a megalopa, is fully lecithotrophic, i.e. independent of food. In the present study, larvae were individually reared in the laboratory at seven constant temperatures ranging from 1 to 18 °C, and rates of survival and development through successive larval and early juvenile stages were monitored throughout a period of 1 year. The highest temperature (18 °C) caused complete mortality within 1 week; only a single individual moulted under this condition, 2 days after hatching, to the second zoeal stage, while all other larvae died later in the zoea I stage. At the coldest condition (1 °C), 71% of the larvae reached the zoea III stage, but none of these moulted successfully to a megalopa. A temperature of 3 °C allowed for some survival to the megalopa stage (17-33% in larvae obtained from two different females), but only a single individual passed successfully, 129 days after hatching, through metamorphosis to the first juvenile crab instar. At all other experimental conditions (6, 9, 12 and 15 °C), survival through metamorphosis varied among temperatures and two hatches from 29% to 90% without showing a consistent trend. The time of nonfeeding development from hatching to metamorphosis lasted, on average, from 19 days at 15 °C to 65 days at 6 °C. The relationship between the time of development through individual larval or juvenile stages (D) and temperature (T) was described as a power function (D=aT^b, or log[D]=log[a]blog[T]). The same model was also used to describe the temperature dependence of cumulative periods of development from hatching to later larval or juvenile stages. One year after hatching, the 7th (6 °C) to 9th (15 °C) crab instar was reached. Under natural temperature conditions in the region of origin of our material (Beagle Channel, Argentina), L. santolla should reach metamorphosis in October-December, i.e. ca. 2 months after hatching (taking place in winter and early spring). Within 1 year from hatching, the crabs should grow approximately to juvenile instars VII-VIII. Our results indicate that the early life-history stages of L. santolla tolerate moderate cold stress as well as planktonic food-limitation in winter, implying that this species is well adapted to subantarctic environments with low temperatures and a short seasonal plankton production.     

Predators and priority effects suggested as potential drivers of microfauna communities in a community transplantation experiment along an elevational gradient

Transplantation experiments are a useful method to identify responses of organisms to environmental change. However, they are typically restricted to single or few species. Our experiment was carried out using entire bromeliad‐inhabiting microfauna communities which were transplanted along an elevational gradient, simulating environmental change acting on the communities. Additionally, we manipulated trophic interactions, i.e. resource availability and predator presence, thus combining abiotic and biotic effects in a full‐factorial experimental design. Using this experiment, we found a strong signal of original elevation in microfauna community structure (abundance, evenness, functional composition) with a shift from amoeba‐dominated to flagellate‐dominated communities with increasing original elevation. Surprisingly, the transplantation of communities along the elevational gradient did not affect community structure, indicating strong priority effects. Predation decreased microfauna abundance and increased microfauna evenness, specifically in higher original elevation and high resource levels. In summary, our results suggest that microfauna communities in bromeliads might be primarily shaped by priority effects and predator presence. However, interacting effects (between predator presence and resource availability, as well as between predator presence and original elevation) highlight the usefulness of studies with full‐factorial experimental designs to understand community‐structuring processes. Bromeliads and other micro‐ecosystems provide convenient study systems for community level approaches that could be used in future studies concerning the effects of environmental change, for example climate change on community structure.     

Description of Leptopharynx bromelicola n. sp. and characterization of the genus Leptopharynx Mermod, 1914 (Protista, Ciliophora)

Using morphological, morphometrical, and molecular methods, we describe Leptopharynx bromelicola n. sp. from tank bromeliads of Jamaica. We add significant data to Leptopharynx costatus and briefly characterize and review the genus Leptopharynx Mermod, 1914, including four new combinations. Nine species can be distinguished when applying the following main features and assuming that most or all have the ability to produce macrostomes (MAs): distinct ridges along the right side ciliary rows; special features like spines or wings on the body and of the oral basket; dikinetids present vs. absent from somatic kinety 3; number of kinetids in kinety 6 as two for the costatus pattern and ≥ five for the bromelicola pattern; beginning and structure of kinety 9 as either underneath or far underneath the adoral membranelles and with or without dikinetids; postoral complex present vs. absent; and preoral kinety 4 continuous vs. discontinuous. The 18S rDNA sequences of L. bromelicola and L. costatus differ by 1.7% and show that Leptopharynx forms a distinct clade within the Nassophorea Small & Lynn, 1981. Leptopharynx bromelicola is possibly closely related to Leptopharynx euglenivora Kahl, 1926, which, however, lacks the basket nose so typical of the former. Leptopharynx forms thin-walled, non-kinetosome-resorbing resting cysts maintaining most of the trophic organelles.     

Conjugation morphology of Zygogonium ericetorum (Zygnematophyceae,Charophyta) from a high alpine habitat

Reproductive characteristics are important for defining taxonomic groups of filamentous Zygnematophyceae, but they have not been fully observed in the genus Zygogonium. Specimens of Z. ericetorum previously studied and used to clarify the generic concept lacked fertile material, which was obtained recently. This study illustrates for the first time, using color light microscopic and fluorescence images, a consequent conjugation stage in Z. ericetorum, including completely developed zygospores and purple cytoplasmic residue content left outside the zygospores, similar to aplanospore formation. Structures confirmed earlier reports and provided new observation informative regarding phylogenetically relevant reproductive characters of Z. ericetorum.