Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Early senescence and production of senescence-associated cytokines are major determinants of radioresistance in head-and-neck squamous cell carcinoma

Resistance against radio(chemo)therapy-induced cell death is a major determinant of oncological treatment failure and remains a perpetual clinical challenge. The underlying mechanisms are manifold and demand for comprehensive, cancer entity- and subtype-specific examination. In the present study, resistance against radiotherapy was systematically assessed in a panel of human head-and-neck squamous cell carcinoma (HNSCC) cell lines and xenotransplants derived thereof with the overarching aim to extract master regulators and potential candidates for mechanism-based pharmacological targeting. Clonogenic survival data were integrated with molecular and functional data on DNA damage repair and different cell fate decisions. A positive correlation between radioresistance and early induction of HNSCC cell senescence accompanied by NF-κB-dependent production of distinct senescence-associated cytokines, particularly ligands of the CXCR2 chemokine receptor, was identified. Time-lapse microscopy and medium transfer experiments disclosed the non-cell autonomous, paracrine nature of these mechanisms, and pharmacological interference with senescence-associated cytokine production by the NF-κB inhibitor metformin significantly improved radiotherapeutic performance in vitro and in vivo. With regard to clinical relevance, retrospective analyses of TCGA HNSCC data and an in-house HNSCC cohort revealed that elevated expression of CXCR2 and/or its ligands are associated with impaired treatment outcome. Collectively, our study identifies radiation-induced tumor cell senescence and the NF-κB-dependent production of distinct senescence-associated cytokines as critical drivers of radioresistance in HNSCC whose therapeutic targeting in the context of multi-modality treatment approaches should be further examined and may be of particular interest for the subgroup of patients with elevated expression of the CXCR2/ligand axis.Subject terms: Radiotherapy, Head and neck cancer, Senescence, Tumour heterogeneity

     

Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors

Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium-related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2-(3′-dimethylaminopyrazinio)ethylidene-1,1-bisphosphonic acid betaine (VS-6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal-cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix-maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal-cell proliferation, and on cell-mediated mineralization. These BPs differentially interact with cell-associated phosphohydrolysis, particularly at a concentration of 10⁻² of ALP K_m, in which alendronate inhibits whereas VS-6 did not inhibit phosphatase activity. VS-6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186–194, 1998. © 1998 Wiley-Liss, Inc.     

Effects of climate change on four New England groundfish species

Multiple groundfish stocks in New England remain depleted despite management measures that have been effective elsewhere. A growing body of research suggests that environmental change driven by increasing concentrations of carbon dioxide in the atmosphere and ocean is unfolding more rapidly in New England than elsewhere, and is an important factor in the failure of these stocks to respond to management. We reviewed research on effects of changes in temperature, salinity, dissolved oxygen, pH, and ocean currents on pelagic life stages, post-settlement life stages, and reproduction of four species in the New England groundfish fishery: Atlantic cod (Gadus morhua), haddock (Melanogrammus aeglefinus), winter flounder (Pseudopleuronectes americanus), and yellowtail flounder (Limanda ferruginea). The volume of research on cod was nearly equal to that on the other three species combined. Similarly, many more studies examined effects of temperature than other factors. The majority of studies suggest adverse outcomes, with less evidence for mixed or positive effects. However, for all of the factors other than temperature, there are more knowledge gaps than known effects. Importantly, most work to date examines impacts in isolation, but effects might combine in nonlinear ways and cause stronger reductions in stock productivity than expected. Management strategies will need to account for known effects, nonlinear interactions, and uncertainties if fisheries in New England are to adapt to environmental change.     

High spatial resolution mass spectrometry imaging reveals the genetically programmed,developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix‐assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single‐cell resolution. Here we applied 5‐ and 10 μm high spatial resolution MALDI‐MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient from four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell‐specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1‐containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0‐containing PGs. Furthermore, PG 32:0 shows genotype‐specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. This study demonstrates the power of MALDI‐MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single‐cell resolution.     

Effect of fertilization pulses on the production of <Emphasis Type="Italic">Gracilaria birdiae</Emphasis> seedlings under laboratory and field conditions

The genus Gracilaria is one of the most important sources of agar in the world. In Brazil, Gracilaria birdiae is the main commercially exploited species; however, overexploitation has contributed to the depletion of natural beds. In order to obtain more information so as to consolidate G. birdiae cultivation, studies under laboratory (indoor and outdoor) and field (sea and shrimp pond) conditions were conducted to evaluate the effects of fertilizer pulses on biomass and relative growth rate (RGR) of this species. The following nutrient sources used were (T₁) shrimp-pond effluent, (T₂) fertilizer for aquarium plants (Mbreda), and (T₃) fertilizer extract of Ascophyllum nodosum (Acadian). Significant differences for growth were recorded over time for all treatments in both outdoor and field conditions (p < 0.001). The highest RGRs were recorded for treatments that used pulses of commercial fertilizers (T₂ and T₃) and the lowest for treatment using shrimp-pond effluent pulses (T₁). The analysis of the nutrient content in tissue also showed a relationship between growth and nitrogen and phosphorus accumulation in the algal tissues. The N/P ratio indicated a significant effect on the growth of G. birdiae and the highest RGRs were registered for seedlings with a N/P ratio ≥16 (T₂ and T₃). In conclusion, the best results were recorded for the Mbreda and Acadian commercial fertilizers. However, although no significant differences were detected between growth and the two fertilizers (T₂ and T₃), the seedlings cultivated under Acadian pulses showed a better performance against environmental stress caused by reduced salinity.     

Functional relatedness in the Inv/Mxi‐Spa type III secretion system family

Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi‐Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane‐integral pore, and the hydrophilic ‘tip complex’ translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food‐borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi‐Spa family. We used invasion‐deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi‐Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in‐depth survey of the functional interchangeability of Inv/Mxi‐Spa T3SS proteins acting directly at the host‐pathogen interface.