Freezing and desiccation tolerance in the moss Physcomitrella patens: an in situ Fourier transform infrared spectroscopic study

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze-thaw cycle down to -80 degrees C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H(2)O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of alpha-helical structures compared to rapidly dried tissues.     

X-ray structures of the N- and C-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation

Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. We describe the structure of the two proteolytically resistant domains of the N protein from infectious bronchitis virus (IBV), a prototype coronavirus. These domains are located at its N- and C-terminal ends (NTD and CTD, respectively). The NTD of the IBV Gray strain at 1.3-A resolution exhibits a U-shaped structure, with two arms rich in basic residues, providing a module for specific interaction with RNA. The CTD forms a tightly intertwined dimer with an intermolecular four-stranded central beta-sheet platform flanked by alpha helices, indicating that the basic building block for coronavirus nucleocapsid formation is a dimeric assembly of N protein. The variety of quaternary arrangements of the NTD and CTD revealed by the analysis of the different crystal forms delineates possible interfaces that could be used for the formation of a flexible filamentous ribonucleocapsid. The striking similarity between the dimeric structure of CTD and the nucleocapsid-forming domain of a distantly related arterivirus indicates a conserved mechanism of nucleocapsid formation for these two viral families.     

Formation and maintenance of the shoot apical meristem

Development in higher plants is characterized by the reiterative formation of lateral organs from the flanks of shoot apical meristems. Because organs are produced continuously throughout the life cycle, the shoot apical meristem must maintain a pluripotent stem cell population. These two tasks are accomplished within separate functional domains of the apical meristem. These functional domains develop gradually during embryogenesis. Subsequently, communication among cells within the shoot apical meristem and between the shoot apical meristem and the incipient lateral organs is needed to maintain the functional domains within the shoot apical meristem.     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.     

Molecular discrimination of phytoseiids associated with the red palm mite Raoiella indica (Acari: Tenuipalpidae) from Mauritius and South Florida

Phytoseiid populations imported from Mauritius for evaluation for a classical biological control program in Florida, USA, were morphologically identified as Amblyseius largoensis Muma, a species associated with the red palm mite in south Florida and the Caribbean. Bayesian analysis and sequence divergences of the mitochondrial 12S rRNA and nuclear Elongation factor--I alpha (EF-Iα) genes and Neighbor-Joining analysis of High-fidelity-RAPD-PCR markers were used to discriminate between the south Florida and Mauritius populations. High-fidelity-RAPD-PCR markers in addition to Bayesian and sequence divergence analyses of the 12S rRNA sequences suggest that the Mauritius and south Florida populations are genetically different but whether these are species or population differences is unknown. The degenerate EF-Iα primers used to survey the phytoseiids amplified two different elongation factor sequences with distinct amino acid translations, the putative EF-Iα and an unknown elongation factor. Variability within the 12S gene was used to develop population-specific primers for identifying the Mauritius phytoseiids in the event they are released in south Florida.     

Sensitive Windows of Skeletal Development in Rabbits Determined by Hydroxyurea Exposure at Different Times throughout Gestation

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo–fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day,GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio‐facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.     

Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene

Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon''s indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student''s t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.