We used a freely available algorithm to perform spectral rotation on the musical stimuli (http://www.fil.ion.ucl.ac.uk/~jcrinion/rotation/blesser3.m). This method has been described in previous works (Blesser, 1972; Scott et al., 2000; Warren et al., 2006; Abrams et al., 2012). The center frequency for spectral rotation was 5512 Hz. This center frequency was chosen so that the rotated frequencies would be within the frequency response range of the fMRI-compatible headphones (20–10 000 Hz). Phase-scrambling was performed by applying

a Fourier transform to each of the four symphonies that constitute the Natural Music stimulus and then randomizing its MK-2206 order phase response by adding a random phase shift at every frequency

(Prichard & Theiler, 1994). The phase shifts were obtained by randomly sampling in the interval (0, 2π). This process preserves the power spectrum of each of the four symphonies. Note that, by design, the Phase-Scrambled control stimulus preserves spectral density but not time-dependent fluctuations. We preferred this design as it facilitates a simple and interpretable result: brain structures that show greater ISS for Natural Music compared with the Phase-Scrambled condition are sensitive to the temporal structure of music. Our design therefore forms a necessary starting point for future investigations of more complex time-dependent attributes of musical structure that lead to synchronized responses among subjects, perhaps using a wavelet transform that preserves

both the Selleck PD0332991 spectral density and the time-dependent fluctuations in that density. Brain images were acquired on a 3T GE Signa scanner using a standard GE whole head coil (software Lx 8.3). For the Natural Music, Spectrally-Rotated and Phase-Scrambled conditions, images were acquired every 2 s in two runs that lasted 9 min 42 s. The sequence of these stimulus conditions was consistent across listeners: the Natural Music condition was presented first, the Phase-Scrambled condition Edoxaban was presented second and the Spectrally-Rotated condition was presented third. While it would have been preferable to have randomized the stimulus presentation order across subjects to control for attention and fatigue, we do not believe that this had a significant effect on the results given that there was vastly greater ISS for the final stimulus condition (Spectral-Rotation) relative to the penultimate stimulus condition (Phase-Scrambled), which would not have occurred had fatigue and attention negatively affected ISS results. Subjects were instructed to attend to all the music and music-like stimuli. To allow for a natural listening experience, we did not provide any additional instructions to the subjects. A custom-built head holder was used to prevent head movement. Twenty-eight axial slices (4.0 mm thick, 0.

We used a freely available algorithm to perform spectral rotation on the musical stimuli (http://www.fil.ion.ucl.ac.uk/~jcrinion/rotation/blesser3.m). This method has been described in previous works (Blesser, 1972; Scott et al., 2000; Warren et al., 2006; Abrams et al., 2012). The center frequency for spectral rotation was 5512 Hz. This center frequency was chosen so that the rotated frequencies would be within the frequency response range of the fMRI-compatible headphones (20–10 000 Hz). Phase-scrambling was performed by applying

a Fourier transform to each of the four symphonies that constitute the Natural Music stimulus and then randomizing its PLX-4720 solubility dmso phase response by adding a random phase shift at every frequency

(Prichard & Theiler, 1994). The phase shifts were obtained by randomly sampling in the interval (0, 2π). This process preserves the power spectrum of each of the four symphonies. Note that, by design, the Phase-Scrambled control stimulus preserves spectral density but not time-dependent fluctuations. We preferred this design as it facilitates a simple and interpretable result: brain structures that show greater ISS for Natural Music compared with the Phase-Scrambled condition are sensitive to the temporal structure of music. Our design therefore forms a necessary starting point for future investigations of more complex time-dependent attributes of musical structure that lead to synchronized responses among subjects, perhaps using a wavelet transform that preserves

both the click here spectral density and the time-dependent fluctuations in that density. Brain images were acquired on a 3T GE Signa scanner using a standard GE whole head coil (software Lx 8.3). For the Natural Music, Spectrally-Rotated and Phase-Scrambled conditions, images were acquired every 2 s in two runs that lasted 9 min 42 s. The sequence of these stimulus conditions was consistent across listeners: the Natural Music condition was presented first, the Phase-Scrambled condition GBA3 was presented second and the Spectrally-Rotated condition was presented third. While it would have been preferable to have randomized the stimulus presentation order across subjects to control for attention and fatigue, we do not believe that this had a significant effect on the results given that there was vastly greater ISS for the final stimulus condition (Spectral-Rotation) relative to the penultimate stimulus condition (Phase-Scrambled), which would not have occurred had fatigue and attention negatively affected ISS results. Subjects were instructed to attend to all the music and music-like stimuli. To allow for a natural listening experience, we did not provide any additional instructions to the subjects. A custom-built head holder was used to prevent head movement. Twenty-eight axial slices (4.0 mm thick, 0.

001). The estimates for calendar year were unaffected by the choice of lagging window (6–12, 12–24 or 24–36

months) for the introduction of new drugs and classes. Similarly, the introduction of an additional variable coding for long delays of >6 months between viral load determinations did not alter the findings. INK-128 This study of a large national observational cohort demonstrated a continuous improvement of virological and immunological effectiveness of ART over recent years. Between 2000 and 2008, the proportion of participants with three consecutive viral load values <50 copies/mL increased from 37 to 64% and the proportion with CD4 counts >500 cells/μL rose from 40 to >50%. In our study we were able to adjust for adherence, treatment interruptions, stable partnership and active hepatitis virus coinfections without

appreciable effects on the time trends, but the improvements Bleomycin chemical structure could only partially be attributed to the numerous predictors tested, including the use of new drugs. Of note, we did not find a relevant dilution effect through new participants entering our open clinical cohort over time. Assigning the most unfavourable outcome to individuals who were lost to follow-up or died did attenuate but not offset the time trends. Because, by definition, the number of individuals lost to follow-up increases, a favourable time trend for virological effectiveness is artificially reduced. Further, in a resource-rich country with universal health care, most individuals will continue to receive adequate care and ART outside the cohort. Our findings are consistent with the results from a collaboration of five HIV clinics analysing time trends of virological success during the early years of combination ART from 1996 to 2002 [11]. The authors attributed some of the observed improvements to better starting regimens, and concluded that additional factors, such as increasing clinical experience, may have played an important role.

Clearly, the experience of care providers continues to improve, and greater physician experience is related to better survival [12], earlier adoption of new treatments [13] and increased adherence 4-Aminobutyrate aminotransferase to treatment [14]. In addition, societal factors such as further reductions of HIV-related stigma and improvement in knowledge of patients may also have played a role [15]. In addition to the superior virological outcome, we found that there was an improvement in immunological status over time, especially after 2004. Contrary to our expectations, time trends for the proportion of individuals with CD4 lymphocyte counts >500 cells/μL did not differ between the open and closed cohorts despite the constant influx of new patients with median CD4 counts of 360 cells/μL in 2001 and 420 cells/μL in 2007 (data not shown). This supports observations from the analyses of the virological endpoint suggesting a negligible bias of time trend analyses by cohort design.

Wheat blast pathogen M. oryzae strain Br48 was grown on oatmeal

agar media at 24 °C for 7 days, and the mycelia were then rubbed and incubated for a further 3 days at 24 °C under near-UV light (360 nm, 40 W) to promote sporulation. Conidia were harvested with distilled water and the Selleckchem Alectinib concentration of spore suspension was adjusted to 2 × 104 conidia mL−1 for experimental assay. The wheat plant (Triticum aestivum cv. Norin 4), which is susceptible to M. oryzae strain Br48, was sown in a seedling case (5.5 × 15 × 10 cm) with vermiculite and grown in a 12-h photoperiod room at 22 °C. Primary leaves were used 10 days after planting for inoculation. To degrade polysaccharides, β-1,3-glucanase (1 U mL−1; Wako), α-glucosidase from Saccharomyces cerevisiae (10 U mL−1; Sigma), α-mannosidase

from jack beans (0.5 mg mL−1; Sigma), and β-mannosidase from Helix pomatia (0.5 U mL−1; Sigma) were used. For degrading proteins, α-chymotrypsin from bovine pancreas (1 U mL−1; Sigma), pepsin from porcine gastric mucosa (1 U mL−1; Selleck Roxadustat Sigma), protease (10 μg mL−1; MP Biomedicals), pronase E (50 μg mL−1; Merck), and trypsin from bovine pancreas (1 U mL−1; Sigma) were used. Lipase (1 mg mL−1; Wako) was used to degrade lipids. To degrade glycoproteins [with matrix metalloproteinases (MMPs)], collagenases from Clostridium histolyticum, consisting of crude type (50 μg mL−1; Wako), size-fractionated Gefitinib chemical structure type I (10 μg mL−1; Wako), size-fractionated type 4 (50 μg mL−1; MP Biomedicals), size-fractionated type V (50 μg mL−1; Wako), and size-fractionated type X (1 mg mL−1; Wako), collagenases from Streptomyces purvulus, consisting of size-fractionated type N-2 (50 μg mL−1; Nitta Gelatin) and size-fractionated type S-1 (670 μg mL−1; Nitta Gelatin); and recombinant gelatinase B from medaka (10 μg mL−1; Hokudo) were used. Each enzyme was dissolved in 50 mM Tris-HCl (pH 7.4) and the highest concentrations used that were not affected on spore germination were determined. Each 8-μL

drop of spore suspension (2 × 104 spores mL−1) was placed on the hydrophobic surface of a flexible vinyl plastic coverglass (Fisher Scientific) at 24 °C in a closed Petri dish containing water-absorbed filter paper. To trace the area of spore inoculation, circles were drawn with an oil pen on the plastic cover glass at opposite sides to the spores. The respective droplets were added with 2 μL of the individual enzymes, adjusted to the above-described concentration, at 0 hpi (ungerminated spore), 1 hpi (starting-to-germinate spore), and 6 hpi (about to form appressorium). The time course of morphological development has been described previously (Hamer et al., 1988; Inoue et al., 2007).

, 2006, 2007; Petkun et al., 2010). Surprisingly, several CBM3s appeared not to be associated with the cellulolytic system of this bacterium. Among these proteins, we discovered that Cthe_0059, Cthe_0267 and Cthe_0404 shared similar N-terminal segments (∼165 residues) Z-VAD-FMK datasheet that resembled those of the B. subtilisσI-modulating factor RsgI (Fig. S1) and RsgI-like proteins in certain Firmicutes species

(data not shown). These ∼165-residue domains of the C. thermocellum hypothetical proteins were termed ‘RsgI-like domains’ here, and their sequences were used further in this study as queries to sequence similarity searches against the C. thermocellum genome databases (see next section). In lieu of a signal peptide motif, all nine RsgI-like proteins were predicted to contain three subdomains

– an ∼50- to 60-residue N-terminal region located inside the cell, followed by a single transmembrane helix (TMH) and a C-terminal region predicted to be localized on the cell exterior (Fig. 1). Putative TMHs were found to be located approximately at residues 55–85 in eight RsgI-like proteins. In one exception (Cthe_0260), a TMH carrying an ∼95 amino learn more acid (aa) insert was located at residues 150–172, and the gene encoding this protein is likely to be monocistronic without an upstream sigI-like gene (Fig. 2). Comparative sequence analysis of the RsgI-like domains from C. thermocellum with those of RsgI-like proteins from Bacillus and several other Clostridium species revealed a relatively high sequence divergence. Nevertheless, the three abovementioned subdomains were consistently predicted in all N-terminal sequences of the identified RsgI-like proteins (Fig. S1). Within the context of the present work, the N-terminal sequences that constitute the intracellular domain of approximately 40 different RsgI-like proteins were aligned, in order to establish a novel Pfam family, designated PF12791 or RsgI_N. Using this motif, approximately 150 RsgI-like proteins can be found in public protein databases (data not shown). Two other N-terminal subdomains of the RsgI-like proteins, a

TMH and a part of the predicted extracellular-sensing domain, also share a very weak, SB-3CT but recognizable conservation (Fig. S1). Analysis of the C. thermocellum ATCC 27405 genome (GenBank accession numbers CP000568 and NC_009012), using the ∼165 aa N-terminal sequences of the B. subtilis RsgI and its three C. thermocellum homologues as blast queries, revealed the presence of six additional ORFs (Fig. S1). Eight of the nine rsgI-like genes appeared to form bicistronic operons downstream of genes encoding proteins, which bear strong similarity to the B. subtilisσI factor (Fig. 2). Similar findings for the sigI- and corresponding rsgI-like genes were evident from analysis of the genomes of two other C. thermocellum strains: DSM 4150 (JW20) and DSM 2360 (LQR1). Extensive analysis of the B. subtilisσI and its putative C. thermocellum homologues revealed an atypical domain organization.

SCLM was used to quantify biofilm development on the glass bottom of microscope dishes (WillCo Wells

BV, the Netherlands, diameter 40 mm, thickness of a glass bottom 0.16–0.19 mm). Bacterial strains were grown overnight in MMA, then 1 : 100 dilutions were prepared in the same medium and 3 mL aliquots of this cell suspension were placed in dishes and incubated at 25 °C. After a given incubation period the medium was removed and the biofilm, which had developed on the bottom of the dish, was washed three times with 10 mM MgSO4. A solution of acridine orange (10 μg mL−1 in 10 mM MgSO4) was then added to the dish. After 30 min incubation, the biofilm was rinsed twice with 10 mM MgSO4. SCLM was conducted using a Nikon Eclipse Ti (A1) microscope equipped with a × 60, 1.4 NA oil immersion http://www.selleckchem.com/products/r428.html phase-contrast lens. An argon laser with a maximum-emission line at 488 nm was used as the excitation source.

Horizontal optical thin sections were collected at 4.0-μm intervals from the outer surface of the biofilm to the bottom of the glass plate. These images were captured by nis-elements interactive software and three-dimensional reconstructions (3D) were created. The reciprocal effect of ompR mutation on invasin expression and motility in Y. enterocolitica identified in previous studies (Brzostek et al., 2007; Raczkowska et al., 2011) raised important questions about selleck products the physiological meaning Montelukast Sodium of these observations. To evaluate the influence of

the OmpR regulatory function, the ability to adhere to and invade human epithelial HEp-2 cells was examined using Y. enterocolitica ompR, flhDC and inv mutant strains. The three mutants varied in their motility as judged by swimming assays: the ompR mutant (strain AR4) and the flhDC mutant (strain DN1) were nonmotile, whereas the inv mutant (strain DC2) exhibited wild-type motility (Fig. 2). In order to separate the effects of the nonmotile phenotype and increased invasin expression in the ompR mutant strain on cellular adhesion–invasion, tissue culture assays were performed with and without centrifugation (see Materials and methods). The centrifugation step artificially brings bacteria into contact with host cells, which bypasses the need for flagellar motility. Cell culture assays performed with the centrifugation step showed that the adhesion abilities of the ompR strain AR4 were decreased compared with the wild-type strain Ye9 and the nonmotile flhDC mutant DN1 (Fig. 3a). The inv mutant DC2 exhibited the weakest adherence phenotype, confirming the role of invasin as a major adhesive-invasion factor in Y. enterocolitica (Pepe & Miller, 1993).

Interestingly, this pattern of amplitude differences reversed during the extinction phase, leading to a CS– specific enhancement [F1,25 = 12.73, P = 0.001,  = 0.34]. The suitability of the temporal window used for the overall anovas above (the final 3200 ms of each segment) was tested in an additional method check, with discrete Fourier analyses conducted for 1-s segments across the time-domain averages for each condition. The normalized amplitude (divided by the Everolimus in vitro number of time points) at the reversal rates was extracted from the spectrum in each time window and averaged

across participants to result in time-course data for each condition, across the viewing epoch. These data are shown for the acquisition phase in Fig. 6. They suggest that, in line with earlier reports, the differential ssVEP amplification for the CS+ increased over the viewing epoch and tended to reach a maximum around the termination of the CSs. In the present study, this pattern was specific to the luminance stimulus. These findings confirm that the segment chosen for the main analyses appropriately

reflects the desired variability among threat and safety cues. To control for potential confounds of stimulation type and the kind of contrast underlying the ssVEP, and to more closely parallel the Z-VAD-FMK purchase luminance stimulus condition in which the Gabor patches were reversed in anti-phase, we conducted an experiment with the chromatic TCL condition in a separate group of individuals (n = 12), where the same chromatic Gabor patches were reversed at 14 Hz, but red and green Gabor patches were presented in anti-phase, not in-phase as in the main study. Although strong

driving was observed with anti-phase chromatic reversal on an isoluminant background, no differences emerged between safe (CS–) and threat (CS+) cues; all F < 2.12, all P > 0.22. The present study examined the extent to which low-spatial-frequency luminance vs. high-spatial-frequency chromatic visual information is critical for the acquisition of low-level visual sensory biases towards threat cues. Using a differential classical conditioning design with Gabor patch stimuli designed to preferentially activate either the luminance or the chromatic-driven human visual pathways, we found that an isoluminant stimulus that relied purely on chromatic contrast did not lead to an enhancement of threat-evoked visuocortical responses. By contrast, stimulating the luminance pathway by means of grayscale low-contrast, low-spatial-frequency pattern reversal resulted in pronounced conditioning effects. Specifically, we observed selectively enhanced neural response amplitudes for the CS+ relative to CS– during the acquisition phase of the experiment. This difference between the conditioned threat and safety signals was no longer present, and was in fact reversed, during extinction.

1C), we trained TMZ/saline-treated rats in VLD eyeblink conditioning, a task that is also dependent on an intact hippocampus. We then trained the same rats in trace eyeblink conditioning, to examine whether learning VLD conditioning would facilitate learning this more complex hippocampus-dependent task. In the last experiment (Fig. 1D), rats were trained in

trace eyeblink conditioning until they acquired a robust conditioned response, and then tested for the memory of the conditioned response 3 weeks later. This experiment was conducted to control RG7204 ic50 for the effects of acute, non-specific side effects of TMZ and to further assess the effects of chemotherapy on retention of trace memories. Each rat undergoing eyeblink conditioning was acclimated to

the conditioning chamber by being placed inside for 1 h with the headstage secured. On the next day, training was begun by giving 10 presentations of the white noise (83 dB, 250 ms) to determine whether the rats showed any sensitised responses to the noise. Eyeblink conditioning was then started. White noise was used as a CS, and a 100-ms periorbital shock (0.65 mA) as a US. A trace conditioning trial consisted of a 250-ms CS followed by a 500-ms stimulus-free time interval that separated the CS from the presentation of the US. A delay conditioning trial consisted of an 850-ms CS that overlapped and coterminated with the US. Finally, a VLD conditioning trial consisted of a 1500-ms CS that overlapped and coterminated with the US. Trials were presented with an intertrial interval of 25 ± 5 s. The number of trials per day and the number Regorafenib of days of training for each variation of eyeblink Phospholipase D1 conditioning were determined on the basis of the difficulty of the task evaluated in light of previous experience in our laboratory. Trace conditioning is harder to learn than VLD conditioning (Nokia et al.,

2012), whereas VLD conditioning is harder to learn than delay conditioning. Thus, for trace conditioning, 200 trials/day for up to 6 days were given, for VLD conditioning, 200 trials/day for 4 days were given, and for delay conditioning, 100 trials/day for 4 days were given (Fig. 1). During training, electromyographic (EMG) signals from the upper eyelid and local-field potentials from the hippocampus were recorded. The EMG signal was bandpass filtered between 300 and 500 Hz (1700 Differential AC amplifier; A-M Systems). The local-field potentials were filtered between 1 and 500 Hz (PGA16; MultiChannel Systems, Reutlingen, Germany). All signals were sampled at a rate of 2000 Hz and recorded continuously (Digidata1440 and AxoScope; Molecular Devices, Sunnyvale, CA, USA). Matlab (MathWorks, Natick, MA, USA) was used for data analyses. To determine learned responding from the EMG signals, the signal amplitude was derived with Hilbert transformation. Next the mean and the standard deviation (SD) of the signal during a 250-ms period immediately preceding the onset of the CS were obtained.

0-GHz irradiated cells and with antibiotics used were observed (P < 0.015 and P < 0.01 for ceftriaxone and kanamycin, respectively). However, compared with irradiated cells, the antibiotics used had no marked effects on DCCD-sensitive ATPase activity (Fig. 3b). This is associated with the F0F1 activity reported by Trchounian & Kobayashi (1998). These data regarding the combined effects of EMI and antibiotics on F0F1-ATPase activity with membrane vesicles seem to be different

from those for H+ transport activity obtained with whole cells (compare Figs 2 and 3). It is possible that antibiotics affected cells differently from FOF1. They might therefore have mediated effects on this ATPase. Alternatively, the decreased ATPase activity values were so low and almost residual that the effects of different antibiotics could not be seen. The results obtained indicate that the action Crizotinib nmr of low-intensity extremely high-frequency EMI in combination with different antibiotics – ceftriaxone and kanamycin – enhances the EMI antibacterial effects. This is clearly observed in the longer duration of En. hirae lag phase growth and increased inhibition of specific growth rate. These effects might be due to changes in membrane properties such as of H+ and K+ transport caused by EMI and enhanced by antibiotics. These

changes disturb membrane proteins forming H+ secretion pathways and of F0F1, leading to effects on the structure and activity of other membrane proteins. Namely, KtrI activity is affected via changes in F0F1 or in the system itself. However, in both cases an interaction between these transport and enzyme systems learn more of the bacterial membrane might be destroyed. As

the interaction might be implemented through dithiol–disulfide transitions (Trchounian, 2004; Poladyan & Glycogen branching enzyme Trchounian, 2006), it is possible to suggest destruction of intra- or intermolecular bridges between membrane proteins. The molecular structure of proteins forming KtrI is not yet clear (Trchounian & Kobayashi, 1998; Trchounian, 2004), and its interaction mechanisms with F0F1 are not established (Trchounian & Kobayashi, 1998; Trchounian, 2004; Poladyan & Trchounian, 2006; Vardanyan & Trchounian, 2010), but our data may be useful in revealing the mechanisms of operation of KtrI and F0F1. In all cases, the membrane property changes by EMI suggest membranotrophic mechanisms in the antibacterial effects of this factor. Moreover, changes in these membrane proteins, in turn, might enhance the antibacterial effects of antibiotics used even if they act differently in cells (Kohanski et al., 2007; Tadevosyan et al., 2008; Lee et al., 2009; Torgomyan et al., 2011a). However, different effects with ceftriaxone and kanamycin may be of particular interest. Despite differences, the combined effects of extremely high-frequency EMI and antibiotics on En. hirae are similar to those with E. coli (Tadevosyan et al.

, 1957; Girodeau et al., 1986; Lloyd et al., 2004). Attempts to express the Mt-dapF (Rv 2726c) in E. coli failed, in spite of the highly efficient T7 promoter in the pET28 vector. It was reasoned out that the lack of dapF expression was related to poor translation (Usha et al., 2006). Mt-dapF was subsequently cloned and over-expressed using a novel codon

alteration strategy and the purified recombinant enzyme functionally characterized (Usha et al., 2006). The Km for meso-DAP was determined to be 1217 μM. Mt-DapF exists as a monomer. Dithiothreitol is required for Mt-DapF activity, consistent with its requirement for two reduced active site thiols (Usha et al., 2006). Mt-DapF activity is inactivated in the presence of nanomolar selleck inhibitor concentrations of the three different thiol-specific alkylating agents (Usha et al., 2008). Site-directed mutagenesis confirmed that the two conserved Cys87 and Cys226 residues were involved in catalysis (Usha et al., 2008). The crystal structure of

the unliganded form of Mt-DapF has been refined to 2.6 Ǻ resolution. Mt-DapF is made up of two pseudosymmetrical α/β domains (Usha et al., 2009). The active site is located in the cleft between domains I and II. The ribbon model of Mt-DapF learn more is shown in Fig. 2. Tyr76 is unique to suborder Corynebacterineae DapF, suggesting a route to the design of a species-specific inhibitor (Usha et al., 2009). In mycobacteria, and most Gram-negative bacteria, the third residue in the peptidoglycan (PG) pentapeptide is d,l (meso)-diaminopimelic acid (Schleifer

& Kandler, 1972). During exponential phase, mycobacteria cross-link the third (meso-DAP) residue and the fourth (d-Ala) residue of adjacent stem peptides (Schleifer & Kandler, 1972; Wietzerbin et al., 1974). On entering stationary phase, mycobacteria incorporate increasing amounts of meso-DAPmeso-DAP linkages, which results in an unusually high DAP content (Wietzerbin et al., 1974; Cirillo et al., 1994a). meso-DAP next is essential for both types of mycobacterial PG cross-linking. The percentage of cross-linking is very high (70–80%) in Mycobacterium species compared to E. coli (20–30%) (Cirillo et al., 1994b; Matsuhashi, 1994). meso-DAP is introduced into the PG network as part of the cross-linking moiety between the polysaccharide fibres (Ghuysen, 1980) (Fig. 3). In addition, the synthesis of meso-DAP is required for protein synthesis, because after decarboxylation, it yields l-lysine. Orthologues in M. tuberculosis of most of the DAP biosynthesis enzymes have been stably expressed in soluble form and functionally characterized. The crystal structures of most of the DAP biosynthesis enzymes have been solved and the chemical mechanisms studied.