Deterioration of ovary plays a key role in heat stress-induced spikelet sterility in sorghum.

In sorghum (Sorghum bicolor [L.] Moench), the impact of heat stress during flowering on seed set is known, but mechanisms that lead to tolerance are not known. A diverse set of sorghum genotypes was tested under controlled environment and field conditions to ascertain the impact of heat stress on time-of-day of flowering, pollen viability, and ovarian tissue. A highly conserved early morning flowering was observed, wherein >90% of spikelets completed flowering within 30 min after dawn, both in inbreds and hybrids. A strong quantitative impact of heat stress was recorded before pollination (reduced pollen viability) and post pollination (reduced pollen tube growth and linear decline in fertility). Although viable pollen tube did reach the micropylar region, 100% spikelet sterility was recorded under 40/22°C (day/night temperatures), even in the tolerant genotype Macia. Heat stress induced significant damage to the ovarian tissue near the micropylar region, leading to highly condensed cytoplasmic contents and disintegrated nucleolus and nucleus in the susceptible genotype RTx430. Whereas, relatively less damages to ovarian cell organelles were observed in the tolerant genotype Macia under heat stress. Integrating higher tolerance in female reproductive organ will help in effective utilization of the early morning flowering mechanism to enhance sorghum productivity under current and future hotter climate.

High night temperature induced changes in grain starch metabolism alters starch, protein, and lipid accumulation in winter wheat.

Unlike sporadic daytime heat spikes, a consistent increase in night-time temperatures can potentially derail the genetic gains being achieved. Ten winter wheat genotypes were exposed to six different night-time temperatures (15-27°C) during flowering and grain-filling stages in controlled environment chambers. We identified the night-time temperature of 23o C as the critical threshold beyond which a consistent decline in yields and quality was observed. Confocal laser scanning micrographs of central endosperm, bran, and germ tissue displayed differential accumulation of protein, lipid, and starch with increasing night-time temperatures. KS07077M-1 recorded a decrease in starch and an increase in protein and lipid in central endosperm with increasing night-time temperatures, whereas the same was significantly lower in the tolerant SY Monument. Expression analysis of genes encoding 21 enzymes (including isoforms) involved in grain-starch metabolism in developing grains revealed a high night-time temperature (HNT)-induced reduction in transcript levels of adenosine diphosphate glucose pyrophosphorylase small subunit involved in starch synthesis and a ≥2-fold increase in starch degrading enzymes isoamylase III, alpha-, and beta-amylase. The identified critical threshold, grain compositional changes, and the key enzymes in grain starch metabolism that lead to poor starch accumulation in grains establish the foundational knowledge for enhancing HNT tolerance in wheat.

Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development.

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.

Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium.

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.

Mimecan/osteoglycin-deficient mice have collagen fibril abnormalities.

PURPOSE: To study the role of mimecan, a member of the small leucine-rich proteoglycans (SLRPs) gene family and one of the major components of the cornea and other connective tissues, mice that lack a functional mimecan gene were generated and characterized. METHODS: Mimecan-deficient mice were generated by gene-targeting using standard techniques. Mice were genotyped by Southern blot analysis. The absence of mimecan transcripts was confirmed by Northern blot analysis. Corneal clarity was examined by slit lamp biomicroscopy. The strength of the skin was evaluated using a biomechanical skin fragility test. Collagen morphology in cornea and skin preparations from mimecan-null and control wild-type mice was analyzed by transmission electron microscopy. The diameter of collagen fibrils in these tissues was determined by morphometric analysis. RESULTS: Mice lacking mimecan appear to develop normally, are viable and fertile. In a controlled laboratory environment they do not display an evident pathological phenotype compared to wild type mice. Examination of corneal clarity and measurements of corneal thickness show no significant changes in the cornea. However, a skin fragility test revealed a moderate reduction in the tensile strength of skin from mutant mice. Ultrastructural analyses show, on average, thicker collagen fibrils in both corneal and skin preparations from mimecan-null mice. Collagen fibrils from the cornea of mutant mice show an average diameter of 31.84+/-0.322 nm, versus 22.40+/-0.296 nm in their wild type litter-mates. The most pronounced increase in collagen fibril diameter was found in the skin of mimecan-null mice, who demonstrated an average diameter of 130.33+/-1.769 nm, versus 78.82+/-1.157 nm in the wild type mice. In addition, size variability and altered collagen morphology was detected in dorsal and tail skin preparations from the mutant mice. CONCLUSIONS: The results of the present study demonstrate that mimecan, similar to other members of the SLRP gene family, has a role in regulating collagen fibrillogenesis in vivo. Further studies, such as functional challenges, an evaluation of potential compensation by other proteins (including members of the SLRP family), and generation of double-knockouts will be necessary to fully uncover physiological functions of mimecan in mice.

Naturally occurring thallium: a hidden geoenvironmental health hazard?

This paper illustrates a real environmental concern and draws attention to the fact that natural processes can mobilize thallium (Tl), a highly toxic metal, which may enter the food chain as a "hidden health killer" with severe health impacts on local human population. Natural processes may be exacerbated by human activities such as mining and farming, and may cause enrichment of Tl in the environment. In geochemically anomalous areas with concentrated levels of Tl in the surface environment (bedrocks, waters, soils, and crops), such as the Lanmuchang area in southwestern Guizhou Province, China, it is essential to establish base-level values and to pay heed to the geological context of "natural contamination," as high concentrations of Tl in bedrocks/ores (6-35,000 mg/kg) can lead to enrichment of Tl in the aquatic system (0.005-1100 microg/l in groundwaters and 0.07-31 microg/l in surface waters) and soil layers (1.5-124 mg/kg). In sensitive areas such as the Yanshang area of southwestern Guizhou, elevated natural levels of Tl from bedrocks may also cause higher concentrations of Tl in the surface environment, and thus more attention must be paid to geoenvironmental management of human activities if socio-economic catastrophes are to be avoided. Due to high uptake of Tl by crops, Tl can be transferred from soils to crops and remarkably concentrated in food crops. Concentrations of 1-500 mg/kg Tl based on dry weight (DW) were determined in many food crops growing on Tl-contaminated arable soils from the Lanmuchang area. The daily intake of 1.9 mg of Tl from consumed food crops was estimated for the local adult inhabitant of Lanmuchang. Thus, Tl is regarded as a latent health hazard with potential risk of toxicity in humans within areas of "natural" contamination by Tl.

Environmental concerns related to high thallium levels in soils and thallium uptake by plants in southwest Guizhou, China.

Thallium (Tl) contamination in soils poses a significant threat to human health due to the high toxicity of Tl and its ready assimilation by crops. This study is focused on high concentrations of Tl in soils in the Lanmuchang area of southwest Guizhou, China, which is related to natural processes of Tl-rich sulfide mineralization. Thallium contents range from 40 to 124 mg/kg in soils originating from the mining area, from 20 to 28 mg/kg in slope wash materials, from 14 to 62 mg/kg in alluvial deposits downstream, from 1.5 to 6.9 mg/kg in undisturbed natural soils and <0.2 to 0.5 mg/kg Tl in soils from the background area. These values indicate that both the erosion of natural soils from the Tl mineralized area and the mining activity are responsible for the distribution of high Tl concentrations in soils. Two other important toxic metals of interest, mercury and arsenic, also show high contents in soils, and are generally higher than Tl concentrations. Thallium concentration in plants exhibit species-dependent preferences. Thus, the enrichment of Tl in the edible parts of crop species decreases in the following order: green cabbage>carrot>chili>Chinese cabbage>rice>corn. The highest level of Tl in green cabbage is up to 500 mg/kg as dry wt., surpassing the values of Tl in the soils in which the green cabbages grow. In contrast, Hg and As are relatively less concentrated in local plants. The average daily uptake of Tl by the villagers of the Lanmuchang area through consumption of locally planted crops has been estimated to be 1.9 mg/person, which is 50 times the daily ingestion of individuals from the Tl-free background area. The daily ingestion of As and Hg from the study area are 0.03 and 0.01 mg, respectively. This indicates that Tl in the contaminated soils related to the natural Tl mineralization is being readily transferred to the human body through the food chain, and poses a significant threat to the health of the local villagers. Arsenic may pose a lesser health hazard, but mercury has an insignificant health risk. This study illustrates a real environmental concern related to land use and human health in areas containing high contents of Tl in soils associated with the natural occurrence of Tl-rich sulfides and coals, with or without mining activities. Thallium contamination in soils should be a critical parameter for proper land use and health related environmental planning and regulations.

Differentiation of C2D macrophage cells after adoptive transfer.

C2D macrophage cells protect immunocompromised mice from experimentally induced pneumonias after intraperitoneal (i.p.) adoptive transfer. These macrophage cells are immature and display minimal activity in vitro. Therefore, we wanted to understand how adoptive transfer affected these cells. We believe that the in vivo environment affects the phenotypic and functional characteristics of macrophages that help maintain the physiological integrity of the host. To test this hypothesis, we characterized the trafficking patterns and cellular changes of the established macrophage C2D cell line after adoptive transfer. We examined phenotypic changes of the C2D macrophage cells in vivo with and without stimulation with gamma interferon (IFN-gamma). After in vivo i.p. adoptive transfer, C2D macrophage cells trafficked to the lungs, spleen, lymph nodes, and bone marrow of recipient mice. The cells were detected for as long as 2 months, and the cells expressed increased levels of CD11b, c-fms, and F4/80 on their surface, becoming more differentiated macrophages compared to cells maintained in vitro. Upon in vivo stimulation with IFN-gamma, c-fms levels decreased while Gr-1 levels increased compared to in vivo, unstimulated, phosphate-buffered saline-injected controls. These responses were independent of the genetic backgrounds of the recipient mice. These data support the hypothesis and indicate that C2D macrophage cells respond to in vivo signals that are absent during in vitro culture.