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During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC − defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called \"amidases\" to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.

In this study, the effect of plasma treatment on glass-cloth-containing polytetrafluoroethylene (GC–PTFE) was investigated. Previous plasma studies investigated pure PTFE (which does not contain glass cloth) but not GC–PTFE. The effect of Ar + H2O plasma treatment on GC–PTFE was investigated. The Ar + H2O plasma-treated GC–PTFE sheets were thermally compressed to stainless steel (SUS304) foils without using adhesive, and the GC–PTFE/SUS304 adhesion strengths were measured using a 90° peel test. The adhesion strength increased with the increase in the plasma treatment time (0.8 and 1.0 N/mm at 20 s and 300 s, respectively). Thus, strong adhesion between GC–PTFE/SUS304 was achieved without adhesive. This improvement in the adhesion properties of GC–PTFE can be attributed to the generation of oxygen-containing functional groups and the decrease in the surface roughness of the samples. Thereafter, the adhesion properties of GC–PTFE and pure PTFE were compared. Because, unlike pure PTFE, GC–PTFE has no weak boundary layer, GC–PTFE exhibited better adhesion properties than pure PTFE under short plasma treatment times.

Lipopolysacharride (LPS) forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. In this study, we established a method for monitoring the LPS biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Escherichia coli. Metabolites from compound-treated cells and genetically-perturbed cells were extracted from whole cells and concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior to analysis by normal phase (NP)LC-MS/MS. Data was normalized to cell density and an internal standard prior to comparison against untreated cells in order to determine fold accumulation and depletion for affected metabolites. Using this LC-MS/MS method, we were able to reliably monitor changes in levels of the LPS intermediates in response to compound-treatment and genetic modification. In addition, we found that deletion of periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased levels of the UDP-containing LPS intermediates, suggesting the enzymatic breakdown during sample preparation. This assay allows for probing a key essential pathway in Gram-negative bacteria in an effort to discover antibacterial agents that inhibit enzymes in the LPS biosynthetic pathway.

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We report here, the effects of extended competency on larval survival, metamorphosis, and postlarval juvenile growth of four closely related species of tropical sea urchins, Echinometra sp. A (Ea), E. mathaei (Em), Echinometra sp. C (Ec), and E. oblonga (Eo). Planktotrophic larvae of all four species fed on cultured phytoplankton (Chaetoceros gracilis) attained metamorphic competence within 22–24 days after fertilization. Competent larvae were forced to delay metamorphosis for up to 5 months by preventing them from settling in culture bottles with continuous stirring on a set of 10 rpm rotating rollers and larval survival per monthly intervals was recorded. Larval survival was highest at 24 days, when competence was attained (0 delayed period), and there were no significant differences among the four species. Larvae that had experienced a prolonged delay had reduced survival rate, metamorphosis success, and juvenile survival, but among older larvae, Em had the highest success followed by Ea, Eo, and Ec. Juveniles from larvae of all four species that metamorphosed soon after becoming competent tended to have higher growth rates (test diameter and length of spines) than juveniles from larvae that metamorphosed after a prolonged period of competence with progressively slower growth the longer the prolonged period. Despite the adverse effects of delaying metamorphosis on growth parameters, competent larvae of all four species were able to survive up to 5 months and after metamorphosis grew into 1-month-old juveniles in lab condition. Overall, delayed larvae of Em showed significantly higher larval survival, metamorphosis, and juvenile survival than Ea and Eo, while Ec showed the lowest values in these performances. Em has the most widespread distribution of these species ranging from Africa to Hawaii, while Ec probably has the most restricted distribution. Consequently, differences in distribution may be related to differences in the ability to delay metamorphosis.

The temperature tolerances of embryonic and early larval development stages of Tripneustes gratilla were investigated from 13-34°C under laboratory conditions. Zygotes showed unequal cleavage at 13°C, whereas cleavage did not occurred at 34°C. Hatching was observed between 16–31°C with maximum hatching rates observed at 22–29°C. The lower and higher temperature limits for embryonic development were approximately 22°C and 29°C, respectively. Outside of this temperature range, embryos showed abnormality at different incubation times. Early larvae of this species have the ability to survive the higher temperature limit for short periods of time. Prism and 2 arm pluteus larvae survived at temperatures between 30 and 33°C, whereas 4 arm pluteus larvae survived at temperatures between 30 and 36°C for 2 h. These results suggest that the larval temperature tolerance capability of T. gratilla is stage dependent. These findings are important for understanding the life history strategy of this sea urchin in the shallow open water environment.

Background To assess the degree of hepatic fat content, simple and noninvasive methods with high objectivity and reproducibility are required. Magnetic resonance imaging (MRI) is one such candidate, although its accuracy remains unclear. We aimed to validate an MRI method for quantifying hepatic fat content by calibrating MRI reading with a phantom and comparing MRI measurements in human subjects with estimates of liver fat content in liver biopsy specimens. Methods The MRI method was performed by a combination of MRI calibration using a phantom and double-echo chemical shift gradient-echo sequence (double-echo fast low-angle shot sequence) that has been widely used on a 1.5-T scanner. Liver fat content in patients with nonalcoholic fatty liver disease (NAFLD, n = 26) was derived from a calibration curve generated by scanning the phantom. Liver fat was also estimated by optical image analysis. The correlation between the MRI measurements and liver histology findings was examined prospectively. Results Magnetic resonance imaging measurements showed a strong correlation with liver fat content estimated from the results of light microscopic examination (correlation coefficient 0.91, P < 0.001) regardless of the degree of hepatic steatosis. Moreover, the severity of lobular inflammation or fibrosis did not influence the MRI measurements. Conclusions This MRI method is simple and noninvasive, has excellent ability to quantify hepatic fat content even in NAFLD patients with mild steatosis or advanced fibrosis, and can be performed easily without special devices.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis , this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development. SEDS proteins are core peptidoglycan polymerases involved in bacterial cell wall elongation and division. SEDS proteins key to bacterial cell wall integrity It has been generally accepted that the cell wall peptidoglycans of the bacterial exoskeleton are synthesized by penicillin binding proteins (PBPs) known as class A PBPs. Now, using genetic manipulation, phylogenetic analysis and functional experiments in Bacillus subtilis , David Rudner and colleagues have identified SEDS family proteins as the main peptidoglycan polymerases more broadly conserved than class A PBPs. Specifically in B. subtilis , they show that the SEDS protein RodA, a widely conserved component of the Rod complex involved in elongation of rod-shaped bacteria, acts with class B PBPs as the core cell wall synthase of the cell elongation and division machinery. The authors conclude that B. subtilis and probably most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. This work also suggests that SEDS family proteins should be attractive targets for antibiotic development.

Two reef margin species of tropical sea urchins,Echinometra sp.C（Ec） and Echinometra oblonga（Eo）,occur sympatrically on Okinawa intertidal reefs in southern Japan.Hybridization between these species was examined through a series of cross-fertilization experiments.At limited sperm concentrations,where conspecific crosses reached near 100% fertilization,both heterospecific crosses showed high fertilization rates（81%-85%）.The compatibility of the gametes demonstrated that if gamete recognition molecules are involved in fertilization of these species,they are not strongly species-specific.We found that conspecific crosses reached peak fertilization levels much faster than did heterospecific crosses,indicating the presence of a prezygotic barrier to hybridization in the gametes.Larval survival,metamorphosis,and juvenile and adult survival of hybrid groups were nearly identical to those of their parent species.Hybrids from crosses in both directions developed normally through larval stages to sexually mature adults,indicating that neither gametic incompatibility nor hybrid inviability appeared to maintain reproductive isolation between these species.In adults,Ec×Ec crosses gave the highest live weight,followed by Eo（ova）×Ec（sperm）,Ec（ova）×Eo（sperm）,and Eo×Eo.Other growth performance measures（viz.,test size,Aristotle＇s lantern length,and gonad index） of hybrid groups and their parental siblings showed the same trends.The phenotypic color patterns of the hybrids were closer to the maternal coloration,whereas spine length,tube-foot and gonad spicule characteristics,pedicellaria valve length,and gamete sizes showed intermediate features.Adult F 1 hybrids were completely fertile and displayed high fertilization success in F 1 backcrosses,eliminating the likelihood that hybrid sterility is a postzygotic mechanism of reproductive isolation.Conversely,intensive surveys failed to find hybrid individuals in the field,suggesting the lack or rarity of natural hybridization.This strongly suggests that reproductive isolation is achieved by prezygotic isolating mechanism（s）.Of these mechanisms,habitat segregation,gamete competition,differences in spawning times,gametic incompatibility or other genetic and non-genetic factors appear to be important in maintaining the integrity of these species.