CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.     

Structure of Pinus sylvestris L. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: Provenance tests

In the present study we investigated the genetic structure and genetic diversity of Pinus sylvestris populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. We were interested in addressing the following questions: (1) can population structure in Scots pine be detected via chloroplast microsatellites markers and terpenes; (2) are there differences in population differentiation between the two analyses; and (3) how are the patterns related to geographic distances. Twelve provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using six primer pairs. Between 4 to 8 size variants were identified at each locus. A total of 35 size variants at the six loci were identified, 11 occurring at low frequencies (<1%). They were combined in 134 different haplotypes, of which seven represent 1/3 of the genetic structure. AMOVA analysis revealed that 10.99% of the variation was found among populations, while 89.01% was expressed within populations. The cpSSR analysis divided Scots pine populations into two groups, the first represented by populations located in the south-western part of the Rhodopes and Pirin mountains, while the second group is located in the northeast of Rhodopes and Rila mountains. Terpene analysis revealed that on average, 53% of the monoterpene pool in P. sylvestris was accounted for by -pinene (range 47–59%) followed by β-pinene (range 6–12%). The presence of two distinct groups is weekly consistent with physical distances between populations, similar significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance (determined by terpenes) was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in Scots pine populations is the consequence of historical biogeographic processes.     

The influence of habitat and adults on the spatial distribution of juvenile corals

Population distributions are affected by a variety of spatial processes, including dispersal, intraspecific dynamics and habitat selection. Within reef‐building coral communities, these processes are especially important during the earliest life stages when reproduction provides mobility among sessile organisms and populations experience the greatest mortality bottlenecks both before and immediately after settlement. Here, we used large‐area imaging to create photomosaics that allowed us to identify and map the location of 4681 juvenile (1–5 cm diameter) and 25 902 adult (>5 cm diameter) coral colonies from eight 100‐m² plots across the forereef of Palmyra Atoll. Using metrics of density, percent cover and the relative location of each colony within each plot, we examined abundance and spatial relationships between juvenile and adult coral taxa. Within coral taxa, juvenile density was generally positively related to the numerical density and percent cover of adults. Nearest neighbor analyses showed aggregation of juveniles near adults of the same taxon for two of the focal taxa (Pocillopora and Fungiids), while all other taxa showed random spatial patterning relative to adults. Three taxa had clustered distributions of juveniles overall. Additionally, we found that on a colony level, juveniles for five of nine focal taxa (accounting for >98% of all identified juveniles) associated with a specific habitat type, with four of those five taxa favoring unconsolidated (e.g. rubble) over consolidated substrata. The general lack of clustering in juvenile corals contrasts with consistent clustering patterns seen in adult corals, suggesting that adult spatial patterns are largely driven by processes occurring after maturity such as partial colony mortality, including fission and fragmentation. The association of many taxa with unconsolidated habitat also suggests that corals may play an important role in colonizing natural rubble patches that could contribute to reef stabilization over time.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.     

Composition of Archaea in Seawater,Sediment, and Sponges in the Kepulauan Seribu Reef System,Indonesia

Coral reefs are among the most diverse and productive ecosystems in the world. Most research has, however, focused on eukaryotes such as corals and fishes. Recently, there has been increasing interest in the composition of prokaryotes, particularly those inhabiting corals and sponges, but these have mainly focused on bacteria. There have been very few studies of coral reef Archaea, despite the fact that Archaea have been shown to play crucial roles in nutrient dynamics, including nitrification and methanogenesis, of oligotrophic environments such as coral reefs. Here, we present the first study to assess Archaea in four different coral reef biotopes (seawater, sediment, and two sponge species, Stylissa massa and Xestospongia testudinaria). The archaeal community of both sponge species and sediment was dominated by Crenarchaeota, while the seawater community was dominated by Euryarchaeota. The biotope explained more than 72 % of the variation in archaeal composition. The number of operational taxonomic units (OTUs) was highest in sediment and seawater biotopes and substantially lower in both sponge hosts. No “sponge-specific” archaeal OTUs were found, i.e., OTUs found in both sponge species but absent from nonhost biotopes. Despite both sponge species hosting phylogenetically distinct microbial assemblages, there were only minor differences in Kyoto Encyclopedia of Genes and Genomes (KEGG) functional pathways. In contrast, most functional pathways differed significantly between microbiomes from sponges and nonhost biotopes including all energy metabolic pathways. With the exception of the methane and nitrogen metabolic pathway, all energy metabolic pathways were enriched in sponges when compared to nonhost biotopes.     

Chilling outweighs photoperiod in preventing precocious spring development

It is well known that increased spring temperatures cause earlier onset dates of leaf unfolding and flowering. However, a temperature increase in winter may be associated with delayed development when species' chilling requirements are not fulfilled. Furthermore, photosensitivity is supposed to interfere with temperature triggers. To date, neither the relative importance nor possible interactions of these three factors have been elucidated. In this study, we present a multispecies climate chamber experiment to test the effects of chilling and photoperiod on the spring phenology of 36 woody species. Several hypotheses regarding their variation with species traits (successional strategy, floristic status, climate of their native range) were tested. Long photoperiods advanced budburst for one‐third of the studied species, but magnitudes of these effects were generally minor. In contrast to prior hypotheses, photosensitive responses were not restricted to climax or oceanic species. Increased chilling length advanced budburst for almost all species; its effect greatly exceeding that of photoperiod. Moreover, we suggest that photosensitivity and chilling effects have to be rigorously disentangled, as the response to photoperiod was restricted to individuals that had not been fully chilled. The results indicate that temperature requirements and successional strategy are linked, with climax species having higher chilling and forcing requirements than pioneer species. Temperature requirements of invasive species closely matched those of native species, suggesting that high phenological concordance is a prerequisite for successful establishment. Lack of chilling not only led to a considerable delay in budburst but also caused substantial changes in the chronological order of species' budburst. The results reveal that increased winter temperatures might impact forest ecosystems more than formerly assumed. Species with lower chilling requirements, such as pioneer or invasive species, might profit from warming winters, if late spring frost events would in parallel occur earlier.     

Vertical distribution of bacterial community is associated with the degree of soil organic matter decomposition in the active layer of moist acidic tundra

The increasing temperature in Arctic tundra deepens the active layer, which is the upper layer of permafrost soil that experiences repeated thawing and freezing. The increasing of soil temperature and the deepening of active layer seem to affect soil microbial communities. Therefore, information on soil microbial communities at various soil depths is essential to understand their potential responses to climate change in the active layer soil. We investigated the community structure of soil bacteria in the active layer from moist acidic tundra in Council, Alaska. We also interpreted their relationship with some relevant soil physicochemical characteristics along soil depth with a fine scale (5 cm depth interval). The bacterial community structure was found to change along soil depth. The relative abundances of Acidobacteria, Gammaproteobacteria, Planctomycetes, and candidate phylum WPS-2 rapidly decreased with soil depth, while those of Bacteroidetes, Chloroflexi, Gemmatimonadetes, and candidate AD3 rapidly increased. A structural shift was also found in the soil bacterial communities around 20 cm depth, where two organic (upper Oi and lower Oa) horizons are subdivided. The quality and the decomposition degree of organic matter might have influenced the bacterial community structure. Besides the organic matter quality, the vertical distribution of bacterial communities was also found to be related to soil pH and total phosphorus content. This study showed the vertical change of bacterial community in the active layer with a fine scale resolution and the possible influence of the quality of soil organic matter on shaping bacterial community structure.     

6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Macrophage colony-stimulating factor and its receptor signaling augment glycated albumin-induced retinal microglial inflammation in vitro

Background  

Microglial activation and the proinflammatory response are controlled by a complex regulatory network. Among the various candidates, macrophage colony-stimulating factor (M-CSF) is considered an important cytokine. The up-regulation of M-CSF and its receptor CSF-1R has been reported in brain disease, as well as in diabetic complications; however, the mechanism is unclear. An elevated level of glycated albumin (GA) is a characteristic of diabetes; thus, it may be involved in monocyte/macrophage-associated diabetic complications.     

Modulation of Rac localization and function by dynamin

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin(-2) inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin(-2) depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.