PSECMAC intelligent insulin schedule for diabetic blood glucose management under nonmeal announcement.

Therapeutically, the closed-loop blood glucose-insulin regulation paradigm via a controllable insulin pump offers a potential solution to the management of diabetes. However, the development of such a closed-loop regulatory system to date has been hampered by two main issues: 1) the limited knowledge on the complex human physiological process of glucose-insulin metabolism that prevents a precise modeling of the biological blood glucose control loop; and 2) the vast metabolic biodiversity of the diabetic population due to varying exogneous and endogenous disturbances such as food intake, exercise, stress, and hormonal factors, etc. In addition, current attempts of closed-loop glucose regulatory techniques generally require some form of prior meal announcement and this constitutes a severe limitation to the applicability of such systems. In this paper, we present a novel intelligent insulin schedule based on the pseudo self-evolving cerebellar model articulation controller (PSECMAC) associative learning memory model that emulates the healthy human insulin response to food ingestion. The proposed PSECMAC intelligent insulin schedule requires no prior meal announcement and delivers the necessary insulin dosage based only on the observed blood glucose fluctuations. Using a simulated healthy subject, the proposed PSECMAC insulin schedule is demonstrated to be able to accurately capture the complex human glucose-insulin dynamics and robustly addresses the intraperson metabolic variability. Subsequently, the PSECMAC intelligent insulin schedule is employed on a group of type-1 diabetic patients to regulate their impaired blood glucose levels. Preliminary simulation results are highly encouraging. The work reported in this paper represents a major paradigm shift in the management of diabetes where patient compliance is poor and the need for prior meal announcement under current treatment regimes poses a significant challenge to an active lifestyle.

A citrate-inducible gene, encoding a putative tricarboxylate transporter, is downregulated by the organic solvent DMSO in Agrobacterium tumefaciens.

AIMS: To investigate the effects of the organic solvent dimethyl sulfoxide (DMSO) on the expression of a citrate-inducible gene, encoding a putative tricarboxylate transporter, in Agrobacterium tumefaciens. METHODS AND RESULTS: By two-dimensional gel electrophoresis, we discovered a putative tricarboxylate transporter named ActC, whose expression was downregulated by DMSO. The expression of actC is also induced by tricarboxylates but not affected by other organic acids of the TCA cycle. Intriguingly, transcriptional activation of actC by citrate is compromised in the presence of DMSO. Furthermore, expression of actC is abolished by deletion of actDE, encoding a putative two-component regulatory system upstream of the actCBA gene cluster. CONCLUSIONS: actC is a citrate-inducible gene that is repressed by DMSO and whose expression is likely regulated by a two-component system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information as to a potential DMSO-regulatory system of A. tumefaciens or other soil bacteria when encountering DMSO in nature. In addition, DMSO-regulated genes should be taken into account for studies in which bacterial cultures were treated with compounds dissolved in DMSO.

Hierarchically clustered adaptive quantization CMAC and its learning convergence.

The cerebellar model articulation controller (CMAC) neural network (NN) is a well-established computational model of the human cerebellum. Nevertheless, there are two major drawbacks associated with the uniform quantization scheme of the CMAC network. They are the following: (1) a constant output resolution associated with the entire input space and (2) the generalization-accuracy dilemma. Moreover, the size of the CMAC network is an exponential function of the number of inputs. Depending on the characteristics of the training data, only a small percentage of the entire set of CMAC memory cells is utilized. Therefore, the efficient utilization of the CMAC memory is a crucial issue. One approach is to quantize the input space nonuniformly. For existing nonuniformly quantized CMAC systems, there is a tradeoff between memory efficiency and computational complexity. Inspired by the underlying organizational mechanism of the human brain, this paper presents a novel CMAC architecture named hierarchically clustered adaptive quantization CMAC (HCAQ-CMAC). HCAQ-CMAC employs hierarchical clustering for the nonuniform quantization of the input space to identify significant input segments and subsequently allocating more memory cells to these regions. The stability of the HCAQ-CMAC network is theoretically guaranteed by the proof of its learning convergence. The performance of the proposed network is subsequently benchmarked against the original CMAC network, as well as two other existing CMAC variants on two real-life applications, namely, automated control of car maneuver and modeling of the human blood glucose dynamics. The experimental results have demonstrated that the HCAQ-CMAC network offers an efficient memory allocation scheme and improves the generalization and accuracy of the network output to achieve better or comparable performances with smaller memory usages. Index Terms-Cerebellar model articulation controller (CMAC), hierarchical clustering, hierarchically clustered adaptive quantization CMAC (HCAQ-CMAC), learning convergence, nonuniform quantization.

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens.

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.

The T-pilus of Agrobacterium tumefaciens.

T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.

Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens.

The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A. tumefaciens strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A. tumefaciens rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes. A search of the vir genes involved in controlling this biphasic reaction in induced A. tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A. tumefaciens.

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens.

Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107-1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A. tumefaciens.

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.

Transcriptional activation of flanking sequences by Tn1000 insertion.

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of beta-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of beta-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the "-35"-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoter-less tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.

Analysis of the gene cluster encoding carotenoid biosynthesis in Erwinia herbicola Eho13.

Erwinia herbicola is known to synthesize carotenoids and gives an orange-coloured phenotype. These carotenoids play a role in the protection of the cells from the damage caused by near-UV irradiation in nature. The genes encoding these carotenoids in E. herbicola Eho13 are clustered in a 7 kb DNA fragment. The complete sequence of this fragment has been determined. DNA sequence analysis revealed that the entire sequence contains at least five genes, which are transcribed in the same direction. These five genes are organized in the order crtE-crtX-crtY-crtI-crtB. A gene fusion study showed that two different regions in this 7 kb gene cluster contain promoter activity. Primer-extension analysis identified two transcription start sites, located 147 bp upstream from the first gene of the cluster, crtE, and within the last gene of the cluster, crtB. An RNA-PCR study suggested that the five crt genes were organized in an operon and were transcribed from the promoter upstream from crtE.