Fabrication of protein microarrays using the electrospray deposition (ESD) method: Application of microfluidic chips in immunoassay

In this study, antibody-based protein microarrays for high-throughput immunoassay were fabricated on an aldehydemodified indium-tin oxide glass plate using the electrospray deposition (ESD) method and their characteristics were evaluated immunochemically. The microarrays were also integrated into microfluidic chips with a polydimethylsiloxane (PDMS) micro-channel to detect human cytokines, which were quantitatively analyzed with a high resolution chargecoupled device. Simultaneous detection of various antigens was performed using the microarrays with considerable sensitivity (ca. 100 pg/mL). The results of this study indicate that microfluidic chip comprising a protein microarray formed by the ESD method and a PDMS micro-channel could be easy to handle, and offers high-throughput detection of molecular biomarkers.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (T_m) of the mutants over the native BcEST that has a T_m of 50°C was outperformed by TtEST, a naturally occurring homologue with a T_m of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.     

Fatty acid ethyl esters in the liverwort Conocephalum conicum

A series of fatty acid ethyl esters ranging from C₁₄ to C₂₄ was isolated from a hexane extract of the liverwort Conocephalum conicum, these esters accounted for 77% of the extract. The ethyl esters consisting of even-numbered fatty acids were predominant and ethyl palmitate was the major constituent.     

Effect of photoperiod on winter and summer diapause of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae)

To understand the geographical differences between diapause systems and synchronization of adult occurrence in the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae), we examined the timing of winter diapause termination and intensity of summer diapause using univoltine and potentially bivoltine individuals in Iwate, Japan. In laboratory rearing experiments of mature larvae maintained at constant temperature (20 °C), winter diapause intensity weakened by January without photoperiodic responses. Meanwhile, summer diapause was maintained by the long day length and presumably terminated with the photoperiodic transition from long to short day length. The intensity of summer diapause was stronger for cocoons that transitioned from a 16 h light to 8 h dark (LD 16:8) to a LD 15:9 photoperiod than for those that transitioned from LD 15:9 to LD 14:10. These results suggest that populations distributed in relatively low-latitude areas, with partly or potentially bivoltine individuals, would have a weaker summer diapause or none at all. Moreover, sexual differences in the number of days to emergence were not detected when individuals experienced a photoperiodic transition from long to short day length, suggesting that the summer diapause system may function to synchronize the emergence of males and females in the population examined.     

Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey

Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara''s owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.     

A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance

In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.     

Meningitis and bacteremia by nonhemolytic Group B Streptococcus strain: A whole genome analysis

Group B streptococcus (GBS) is a leading cause of neonatal infections. Most isolates are β-hemolytic, and their activity is considered to be pivotal for GBS pathogenicity. We report a case of a neonate with meningitis caused by nonhemolytic GBS. The patient developed meningitis 3 days after birth. Genotyping was performed and the characteristics of the strain (GCMC97051) identified by whole genome sequence using next generation sequencing. GCMC97051 possesses genetic alterations such as disruption of cylA by IS1381A insertion and a frameshift mutation in cylE, resulting in a lack of hemolysis. Thus, nonhemolytic GBS can retain the potential to cause invasive infections.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.