A miniaturized device for the measurement of sheet-metal formability using digital image correlation.

A laboratory-scale device to obtain forming-limit diagram data was designed to utilize the Marciniak and Kuczynski (MK) sample geometry. The design uses a high-resolution photographic camera, automatic trigger, and light-emitting diode (LED) lighting to record the time history of deformation calculated with the digital-image correlation technique. Because the testing device was miniaturized, it was possible to halt the forming experiments at intermediate strains and recrystallize the MK carrier blank. This permits large formability strains to be obtained without cracks developing at the carrier blank's central hole, an advantage over full-size specimens and conventional testing rates. A number of initial experiments were performed on a zinc alloy sheet (Zn-Cu-Ti) over the entire forming-limit range (-0.5 ≤ ε2/ε1 ≤ 1), and the strain fields reduced employing the Bragard criterion to obtain limit strains. These results are compared favorably to previous data of this material obtained with a hemispherical, Nakazima, punch and a circle-grid pattern.

Design, construction, and performance of a device for directional recrystallization of metallic alloys.

A device was designed to apply the directional recrystallization method to Fe-based alloys in order to obtain bamboo-like microstructures. This microstructure is suitable for improving creep properties and resistance to fatigue in some alloys and for enhancing pseudoelastic properties in shape memory alloys. The design and construction of a flat coil are described in detail. In addition, we developed an electromechanical system to control the movement of a wire within the flat coil. The construction details and system performance are presented. Furthermore, metallographic studies taken from the directionally recrystallized low-carbon steel samples are shown. Nearly monocrystalline and bamboo-like microstructures were achieved in the steel wires.

Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841.

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.

The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.

New ratiometric fluorescent calcium indicators with moderately attenuated binding affinities.

Mono-halogenated derivatives of the calcium indicators fura-2 and indo-1 were synthesized and their spectroscopic properties evaluated. Halogenation ortho or para to the bridging oxygen in the BAPTA nucleus had a more pronounced weakening effect on binding affinity than in the meta position in the fura derivatives. Two new excitation ratioable fluorescent calcium indicators, benzothiaza-1 and 2, were also synthesized. Kd values of 400 nM to 5.3 microM [Ca2+] were observed in these families of new probes.

Identification of a putative LPS-associated cation exporter from Rhizobium leguminosarum bv. viciae.

A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transferase while LpcB is a 2-keto-3-deoxyoctonate transferase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpaA::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.

Effects of muscle contraction on the load-strain properties of frog aponeurosis and tendon.

The mechanical properties of the frog semitendinosus (ST) tendon and aponeurosis were measured during passive tensile loading to a force equal to ST maximum tetanic tension and during active isometric muscle contraction. During active contraction, both the tendon and aponeurosis regions initially strained at rates exceeding 400%/s while near the end of the muscle contraction, strain rates were nearly zero. At this point, the strain in the tendon region was equal to that observed during slow passive loading to the same tension level. However, for the aponeurosis, even near the zero strain rate, strain at the end of the active contraction was significantly below that observed during slow passive loading (p < 0. 001). Specifically, when aponeurosis strain rate was almost zero, aponeurosis strain was 13.8 +/- 3% (means +/- SEM, n = 10), which was significantly below that measured during passive loading (23.7 +/- 5%) suggesting that active contraction actually altered aponeurosis material properties. These data demonstrate that, while the tendon and aponeurosis regions have different passive biomechanical properties and both demonstrate viscosity typical of other connective tissues, the aponeurosis region of the frog ST actually changed its intrinsic properties during muscle contraction. Thus, extrapolation of biomechanical data obtained at nonphysiological strain rates or under conditions where the muscle-tendon junction has been interrupted should be made with caution.

Frog semitendinosis tendon load-strain and stress-strain properties during passive loading.

The mechanical properties of the frog semitendinosis (ST) tendon, bone-tendon junction, and aponeurosis were measured during passive loading to a tension equal to maximum isometric tension (Po). Stiffness and strain in these regions continuously increased as load increased. Tendon stiffness was approximately four times the aponeurosis stiffness. Tendon Young's modulus at Po was only 188 MPa, which is approximately 10 times less than the modulus reported for most mammalian tendons. Similarly, tendon stress at Po was only approximately 3 MPa, which is also less than that predicted for many tendons. Tendon strain at Po was approximately 2% after passive loading. We conclude that different regions of the frog ST tendon have different mechanical properties and that the frog ST tendon operates physiologically in the "toe" region of the stress-strain curve with a variable stiffness that increases with load. Taken together, these results have significant implications in understanding muscle-tendon design and neuromotor control strategies.

Role of the vagus nerve and its recurrent laryngeal branch in the development of the human ductus arteriosus.

The reason that the normal ductus arteriosus has a muscular media, contrasting with the elastic lamellar structure of the adjacent great arteries, is unknown. We examined the hypothesis that the anatomic relationship of the ductus arteriosus to the vagus and recurrent laryngeal nerves during early development might be of importance in influencing ductal morphology. Normal human embryos from the Carnegie Embryological Collection and embryos and fetuses from the Hopkins Pathology Collection were studied microscopically, by reconstructions made from serial histologic sections, or by gross dissection. At Carnegie stage 16 the recurrent laryngeal nerves pass medially from the vagus nerve to the laryngeal area and are caudal to the bilaterally symmetric sixth aortic arches. By stage 18 the right sixth aortic arch has disappeared and the left sixth aortic arch is in a more caudal position relative to the larynx. The left vagus nerve and its recurrent laryngeal branch form a sling supporting the distal (or ductus arteriosus component) of the left sixth aortic arch. In subsequent development there is greater relative separation of the larynx and ductus arteriosus. The media of the ductus arteriosus beneath the supporting nerves is thinner and has less elastic fiber formation than the elastic lamellar media of the adjacent aortic arches. The study shows that the vagus and recurrent laryngeal nerves are in a position to provide mechanical support to the ductus arteriosus during its development and that the morphology of the media of the supported ductus arteriosus differs from that of the adjacent unsupported aortic arches. It is suggested that this local mechanical support may be the reason that the normal ductus arteriosus differentiates as a muscular artery and is therefore able to obliterate its lumen in postnatal life. Without such support the ductal media could develop the abundant elastic fibers characteristic of the normal unsupported aorta and pulmonary trunk and become an abnormal, persistently patent ductus arteriosus.