Forced expression of FLO11 confers pellicle-forming ability and furfural tolerance on Saccharomyces cerevisiae in ethanol production

We constructed a plasmid that expresses FLO11 encoding a cell surface glycoprotein of Saccharomyces cerevisiae under the control of a constitutive promoter. This plasmid conferred pellicle-forming ability on the non-pellicle-forming industrial strain of S. cerevisiae at the air–liquid interface of the glucose-containing liquid medium. The induced pellicle-forming cells exhibited tolerance to furfural, which is a key toxin in lignocellulosic hydrolysates, in ethanol production.     

Zebrafish Polycomb group gene ph2alpha is required for epiboly and tailbud formation acting downstream of FGF signaling

We analyzed Polycomb group gene ph2alpha functionally in zebrafish embryos by a gene knock-down procedure using morpholino antisense oligos. Inhibition of ph2alpha message translation resulted in abnormal epibolic movements as well as a thick tailbud or incomplete covering of the yolk plug. At the 24hpf stage, morphants had short trunks and tails, phenotypes similar to those with disturbances in FGF signaling. Accordingly, we looked at the effects of ph2alpha expression upstream and downstream of the FGF pathway. Treatment with SU5402, an inhibitor of Fgfrs, or injection of dominant-negative Fgfr1 DNA markedly reduced ph2alpha expression in the tailbud. In addition, cells expressing mRNAs for no tail, spadetail, myoD, and papc, which are involved in FGF-related development of posterior mesoderm, were distributed abnormally. Collectively, the data argue that ph2alpha is required for epiboly and tailbud formation, acting downstream of the FGF signaling pathway.     

Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

Summary Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam^– strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam^– strand with the Dam⁺ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.     

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily,Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266^∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.     

Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).     

Static interaction between muscle mechanoreflex and arterial baroreflex in determining efferent sympathetic nerve activity

Elucidation of the interaction between the muscle mechanoreflex and the arterial baroreflex is essential for better understanding of sympathetic regulation during exercise. We characterized the effects of these two reflexes on sympathetic nerve activity (SNA) in anesthetized rabbits (n = 7). Under open-loop baroreflex conditions, we recorded renal SNA at carotid sinus pressure (CSP) of 40, 80, 120, or 160 mmHg while passively stretching the hindlimb muscle at muscle tension (MT) of 0, 2, 4, or 6 kg. The MT-SNA relationship at CSP of 40 mmHg approximated a straight line. Increase in CSP from 40 to 120 and 160 mmHg shifted the MT-SNA relationship downward and reduced the response range (the difference between maximum and minimum SNA) to 43 +/- 10% and 19 +/- 6%, respectively (P < 0.01). The CSP-SNA relationship at MT of 0 kg approximated a sigmoid curve. Increase in MT from 0 to 2, 4, and 6 kg shifted the CSP-SNA relationship upward and extended the response range to 133 +/- 8%, 156 +/- 14%, and 178 +/- 15%, respectively (P < 0.01). A model of algebraic summation, i.e., parallel shift, with a threshold of SNA functionally reproduced the interaction of the two reflexes (y = 1.00x - 0.01; r(2) = 0.991, root mean square = 2.6% between estimated and measured SNA). In conclusion, the response ranges of SNA to baroreceptor and muscle mechanoreceptor input changed in a manner that could be explained by a parallel shift with threshold.     

Structural and Functional Characterization of IS679 and IS66-Family Elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.