Comparison of mechanistic and hybrid modeling approaches for characterization of a CHO cultivation process: Requirements,pitfalls and solution paths

Despite the advantages of mathematical bioprocess modeling, successful model implementation already starts with experimental planning and accordingly can fail at this early stage. For this study, two different modeling approaches (mechanistic and hybrid) based on a four-dimensional antibody-producing CHO fed-batch process are compared. Overall, 33 experiments are performed in the fractional factorial four-dimensional design space and separated into four different complex data partitions subsequently used for model comparison and evaluation. The mechanistic model demonstrates the advantage of prior knowledge (i.e., known equations) to get informative value relatively independently of the utilized data partition. The hybrid approach displayes a higher data dependency but simultaneously yielded a higher accuracy on all data partitions. Furthermore, our results demonstrate that independent of the chosen modeling framework, a smart selection of only four initial experiments can already yield a very good representation of a full design space independent of the chosen modeling structure. Academic and industry researchers are recommended to pay more attention to experimental planning to maximize the process understanding obtained from mathematical modeling.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Prediction of B-cell epitopes forSalmonella typhi OmpC

The possible B-cell epitopes of the outer membrane porin OmpC ofSalmonella typhi have been identified, using the primary structure of the protein, by means of multiple sequence alignment and the known molecular structure of two other porins. From the analysis, 8 regions were identified as immunodominant and these were ranked based on antigenic index and the ratio of the number of nonconserved residues to the fragment length. Model building of the top two ranked regions show the tendency to form loop structures supporting the possibility of these being candidate epitopes.     

The application of molecular markers to the study and conservation of fish populations, with special reference to Salmo

The main molecular techniques which can be used to generate genetic markers, and the applications of these markers to studies of fish populations are outlined. Published and ongoing studies, in the authors' laboratories, on brown trout and Atlantic salmon are used to compare the resolution and applicability of allozyme, mitochondrial DNA and minisatellite (variable number of tandem repeats) markers for studies on population structuring, genetic variation within populations, and the impact of the accidental and deliberate introduction of non-native salmonids on the genetic make-up of natural populations.     

Image analysis, neural networks, and the taxonomic impediment to biodiversity studies

The taxonomic impediment to biodiversity studies may be influenced radically by the application of new technology, in particular, desktop image analysers and neural networks. The former offer an opportunity to automate objective feature measurement processes, and the latter provide powerful pattern recognition and data analysis tools which are able to 'learn' patterns in multivariate data. The coupling of these technologies may provide a realistic opportunity for the automation of routine species identifications. The potential benefits and limitations of these technologies, along with the development of automated identification systems are reviewed.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase of Haloferax volcanii: role of histidine 398 and attenuation of activity by introduction of negative charge at position 404.

Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine. To first verify the sequence-based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG-CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403-residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii-hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild-type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.     

光合细菌H3菌株色素分析

H3菌株系由盐田微生物层中分离获得的光合细菌株。具有丰富的天然色素。经活细胞色素光谱吸收峰值测定,色素经有机溶剂提取、硅胶薄板层析、SDS－PAGE电泳等,结果表明H3菌株的主要色素包括细菌叶绿素a、细菌脱镁叶绿素（Bacteriophaeophytin）和三种类胡萝卜素。总胡萝卜素含量占细胞于重的0．6％,胡萝卜素蛋白复合体的分子量约11,000.培养条件的差异对色素形成及相对含量有不同程度的影响。     

Sex estimation using the first cervical vertebra

The articular surfaces and vertebral foramen area of the first cervical vertebra are sexually dimorphic and can be used to sex complete or fragmentary specimens. Eight measurements were taken from the articular regions (superior and inferior) of 100 first cervical vertebrae from Terry collection specimens housed at the Smithsonian Institution. Seven regression and seven discriminant function equations were created that predict sex with 77–85% and 75–85% accuracy, respectively. In separate control tests, measurements from 100 first cervical vertebrae from Hamann-Todd collection individuals (Cleveland Museum of Natural History) and from 34 archaeological specimens were used with the Terry equations. The control samples were sexed with 60—85% accuracy. © 1995 Wiley-Liss, Inc.