Bacterial chemotaxis in static gradients quantified in a biopolymer membrane-integrated microfluidic platform.

Chemotaxis is a fundamental bacterial response mechanism to changes in chemical gradients of specific molecules known as chemoattractant or chemorepellent. The advancement of biological platforms for bacterial chemotaxis research is of significant interest for a wide range of biological and environmental studies. Many microfluidic devices have been developed for its study, but challenges still remain that can obscure analysis. For example, cell migration can be compromised by flow-induced shear stress, and bacterial motility can be impaired by nonspecific cell adhesion to microchannels. Also, devices can be complicated, expensive, and hard to assemble. We address these issues with a three-channel microfluidic platform integrated with natural biopolymer membranes that are assembled in situ. This provides several unique attributes. First, a static, steady and robust chemoattractant gradient was generated and maintained. Second, because the assembly incorporates assembly pillars, the assembled membrane arrays connecting nearby pillars can be created longer than the viewing window, enabling a wide 2D area for study. Third, the in situ assembled biopolymer membranes minimize pressure and/or chemiosmotic gradients that could induce flow and obscure chemotaxis study. Finally, nonspecific cell adhesion is avoided by priming the polydimethylsiloxane (PDMS) microchannel surfaces with Pluronic F-127. We demonstrated chemotactic migration of Escherichia coli as well as Pseudomonas aeruginosa under well-controlled easy-to-assemble glucose gradients. We characterized motility using the chemotaxis partition coefficient (CPC) and chemotaxis migration coefficient (CMC) and found our results consistent with other reports. Further, random walk trajectories of individual cells in simple bright field images were conveniently tracked and presented in rose plots. Velocities were calculated, again in agreement with previous literature. We believe the biopolymer membrane-integrated platform represents a facile and convenient system for robust quantitative assessment of cellular motility in response to various chemical cues.

Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Synthesis of dehydro-oogoniol, a female-activating hormone of Achlya: the progesterone route.

The structure of dehydro-oogoniol (3 beta,11 alpha,15 beta,29-tetrahydroxystigmasta-5,24(28)(E)-dien-7-one), a female-activating hormone of the water mold Achlya, has been confirmed by synthesis. The starting material was progesterone, which was converted to the 11 alpha, 15 beta-dihydroxy derivative by microbiological hydroxylation with Aspergillus giganteus (ATCC 10059). The side chain was constructed in a stepwise manner by means of Wittig and Horner-Emmons reactions, and the C-7 ketone was then introduced by allylic oxidation. The biological activity of the synthetic compound was the same as that of the natural hormone.

Genetic and environmental control of diabetes induction by multi-dose streptozotocin in two BALB/c substrains.

BALB/cJ male mice were resistant and BALB/cByJ males were susceptible to induction of diabetes by multi-dose streptozotocin (MSz). Although both closely-related BALB/c substrains expressed H-2d haplotype, they could be differentiated by allelic differences at three genetic loci [Qa-2 (Chr 17), Bcd-1 (Chr 5), and Afr-1]. (BALB/cJ X BALB/cByJ)F1 males inherited the BALB/cJ resistance phenotype in a dominant fashion, thereby eliminating the BALB/cJ-expressed Afr-1b (recessive) allele as the susceptibility locus. Backcross of F1 mice to the susceptible BALB/cByJ strain produced a 1:1 segregation of susceptible and resistant (F1-like) phenotypes, suggesting that susceptibility was controlled by a single recessive gene. No linkage was found between the putative susceptibility gene and the mutant BALB/cByJ Qa-2,3 gene linked to the H-2 complex or with the mutant Bcd-1c allele. Since the resistant F1 males expressed low levels of androgen-dependent mouse urinary protein characteristic of the resistant BALB/cJ parental strain, the possibility was discussed that the alleles controlling sensitivity to MSz also controlled tissue sensitivity to endogenous androgens. An environmental effect on phenotype expression was indicated when BALB/cByJ males obtained from a colony free of pneumonia virus of mice (PVM) showed an attenuated rate of response to hyperglycemia induction in comparison to males obtained previously from an enzootically infected colony.

Conformational analysis of 9beta,19-cyclopropyl sterols: Detection of the pseudoplanar conformer by nuclear Overhauser effects and its functional implications.

Nuclear Overhauser difference spectroscopy and variable temperature studies of the 9beta,19-cyclopropyl sterols 24,25-dehydropollinastanol (4,4-desmethyl-5alpha-cycloart-24-en-3beta-ol) and cyclolaudenol [(24S)-24-methyl-5alpha-cycloart-25(27)-en-3beta-ol] have shown the solution conformation of the B/C rings to be twist-chair/twist-boat rather than boat/chair as suggested in the literature. This is very similar to the known crystal structure conformation of 9beta,19-cyclopropyl sterols. The effect of these conformations on the molecular shape is highly significant: the first conformation orients into a pseudoplanar or flat shape analogous to lanosterol, whereas the latter conformation exhibits a bent shape. The results are interpreted to imply that, for conformational reasons, cyclopropyl sterols can be expected to maintain the pseudoplanar shape in membrane bilayers.

Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated, MHC-unlinked traits.

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs. B6-H-2b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 X BKs)F1 and F2 db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F1 generation, and were transmitted to F2 db/db males without apparent segregation. No association between H-2b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2b congenic stock. However, an apparent interaction between H-2b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F2 db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F1 hybrids exhibit the higher activity associated with the BKs strain.

Sterol composition and biosynthesis in sorghum: Importance to developmental regulation.

Sterol composition and biosynthesis have been examined in seeds, germinating seeds and blades from fally matured leaves ofSorghum bicolor in various stages of development'from seedlings (seven-day plants) to flowering (66-day) plants. The profile of the dominant free sterols of seeds was similar to that of leaf blades; both contained cholesterol, 24α-methylcholesterol (campesterol), 24ß-methylcholesterol (dihydrobrassicasterol), 24α-ethylcholesterol (sitosterol) and 24α-ethylcholesta-5,22-dienol (stigmasterol). Sufficient sterol intermediates were identified in the plant to indicate separate post-cycloartenol pathways to sterolic end products. The total free sterol content of the seed (µg/seed) increased somewhat during the 20 hr germination period. However, as the plant developed (seven to 48 days), there was a logarithmic increase in the leaf blade sterol content (µg/leaf blade) which plateaued at the onset of floral differentiation (ca. day 41). Over the next 18 days (48 to 66 days-period of inflorescense development), the sterol content rapidly decreased. In the early stages of plant development, the leaf blade pentacyclic triterpenoid (PT) content was negligible. With the onset of floral differentiation, PT content increased logarithmically, reaching a plateau level that surpassed the sterol content as flowering progressed. These results imply that a critical mass of sterol is associated with sorghum for floral induction. Sterol loss from the leaves of the flowering plants presumably was compensated for by the diversion of 2,3-oxidosqualene (SO) from sterol synthesis to PT production. Additional feeding and trapping experiments with [2-(14)C]mevalonic acid, [2-(3)H]cycloartenol, [24-(3)H]lanosterol [4-(14)C]sitosterol and [4-(14)C]cholesterol fed to germinating seeds and leaves from flowering plants demonstrated that sorghum possessed a cycloartenolbased pathway; germinating seeds synthesized 24-alkylsterols but not cholesterol, although cholesterol was identified in both dry and germinating seeds by gas chromatography-mass spectroscopy (GC-MS); and mature leaves synthesized cholesterol and 24α-alkylsterols but not 24ß-methylcholesterol.

Genetic control of susceptibility to streptozotocin diabetes in inbred mice: effect of testosterone and H-2 haplotype.

Males of certain mouse strains are more susceptible than females to the diabetogenic effect of multiple low doses of streptozotocin (MSZ, 40 mg/kg BW.day X 5). Several investigators linked sensitivity to the potentiating action of androgens and genes of the major histocompatibility (H-2) complex. Our studies were designed to investigate the role of testosterone in MSZ-diabetes induction in males of three C3H stocks: C3H X SW/SnJ (H-2b), C3HeB/FeJ (H-2k), and C3H/OuJ (H-2k). Serum testosterone levels in gonad-intact animals correlated inversely with SZ sensitivity, the more resistant C3HeB/FeJ males having a higher mean level than the other two stocks. Males from each group were castrated at 4 weeks of age and implanted with either testosterone or cholesterol; 4 weeks later they were given MSZ. C3H.SW/SnJ and C3H/OuJ castrates implanted with either testosterone or cholesterol were as sensitive to the hyperglycemic effect of MSZ as the intact controls, whereas C3HeB/FeJ castrates implanted with cholesterol lost sensitivity; this sensitivity could be fully restored by testosterone implants. Surprisingly, there was no difference in the residual pancreatic insulin content (90% reduced) between the SZ-resistant cholesterol-implanted vs. the SZ-sensitive testosterone-implanted C3HeB/FeJ castrates. This demonstrated that the androgen was not potentiating SZ destruction of the beta-cells, but rather was antagonizing the ability of the residual insulin to maintain glycemic control. The present study also indicated that the H-2 complex was not a significant factor predisposing to SZ sensitivity as reflected by marked sensitivity of the C3H/OuJ and C3H.SW/SnJ males vs. the relative resistance of C3HeB/FeJ males sharing the same H-2 haplotype as C3H/OuJ.