Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities

The thermostable lipase (TliA) from Pseuodmonas fluorescens was functionally displayed on the surface of Escherichia coli using the ice-nucleation protein (INP) as an anchor. The INP–TliA fusion proteins were correctly synthesized and localized on the surface, confirmed by flow cytometer and halo forming activity on tributyrin emulsion agar plate. Lipase-displaying cells were used as an alternative immobilized biocatalyst to hydrolyze olive oil in aqueous–organic solvent two phases reaction. Furthermore, the randomly generated library of TliA was also displayed on E. coli. In order to be able to screen mutants showing increased specific activities, we optimized culture conditions, induction condition and host cell types. From more than 10⁵ members of library, top four mutants were selected. Selected clones of T48, T54, T61, and T68 showed 29-, 24-, 2-, and 19-fold increases, respectively, in whole-cell activities compared to wild-type enzyme. The DNA sequencing showed that one or three amino acids were exchanged and positions critical for increased activities were random. These results demonstrate that surface display provide a useful technology for directed evolution of industrially important lipases.     

Gap junction- and hemichannel-independent actions of connexins

Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.     

Virgin and recently mated males are equally able to achieve new matings in Neoleucinodes elegantalis

Lepidopteran male mating success is recognized to be directly related to physical and behavioural traits such as ability, vigour, activity and persistence in courtship. In the tomato fruit borer Neoleucinodes elegantalis Guenée, the mating system is known to be monogamic and therefore males' sexual investment is apparently low. The hypothesis that recently mated males have a remating probability equal to that of virgin males is tested. The impact of body size in remating success and the cost of remating are also analyzed. Mated males show as much propensity to remate as naïve ones. Copula duration and the time taken to copulate are similar in mated and virgin males. However, spermatophore size is not related to male size. The results suggest the ability of N. elegantalis males to remate within 24 h between mating events and their propensity to remate are not affected by copula investment. © 2013 The Royal Entomological Society     

Mitochondrial Targeting of the Arabidopsis F1-ATPase γ-Subunit via Multiple Compensatory and Synergistic Presequence Motifs

The majority of mitochondrial proteins are encoded in the nuclear genome and imported into mitochondria posttranslationally from the cytosol. An N-terminal presequence functions as the signal for the import of mitochondrial proteins. However, the functional information in the presequence remains elusive. This study reports the identification of critical sequence motifs from the presequence of Arabidopsis thaliana F1-ATPase γ-subunit (pFAγ). pFAγ was divided into six 10–amino acid segments, designated P1 to P6 from the N to the C terminus, each of which was further divided into two 5–amino acid subdivisions. These P segments and their subdivisions were substituted with Ala residues and fused to green fluorescent protein (GFP). Protoplast targeting experiments using these GFP constructs revealed that pFAγ contains several functional sequence motifs that are dispersed throughout the presequence. The sequence motifs DQEEG (P4a) and VVRNR (P5b) were involved in translocation across the mitochondrial membranes. The sequence motifs IAARP (P2b) and IAAIR (P3a) participated in binding to mitochondria. The sequence motifs RLLPS (P2a) and SISTQ (P5a) assisted in pulling proteins into the matrix, and the sequence motif IAARP (P2b) functioned in Tom20-dependent import. In addition, these sequence motifs exhibit complex relationships, including synergistic functions. Thus, multiple sequence motifs dispersed throughout the presequence are proposed to function cooperatively during protein import into mitochondria.     

Hybrid speciation in sparrows I: phenotypic intermediacy,genetic admixture and barriers to gene flow

Homoploid hybrid speciation is thought to require unusual circumstances to yield reproductive isolation from the parental species, and few examples are known from nature. Here, we present genetic evidence for this mode of speciation in birds. Using Bayesian assignment analyses of 751 individuals genotyped for 14 unlinked, nuclear microsatellite loci, we show that the phenotypically intermediate Italian sparrow (Passer italiae) does not form a cluster of its own, but instead exhibits clear admixture (over its entire breeding range) between its putative parental species, the house sparrow (P. domesticus) and the Spanish sparrow (P. hispaniolensis). Further, the Italian sparrow possesses mitochondrial (mt) DNA haplotypes identical to both putative parental species (although mostly of house sparrow type), indicating a recent hybrid origin. Today, the Italian sparrow has a largely allopatric distribution on the Italian peninsula and some Mediterranean islands separated from its suggested parental species by the Alps and the Mediterranean Sea, but co‐occurs with the Spanish sparrow on the Gargano peninsula in southeast Italy. No evidence of interbreeding was found in this sympatric population. However, the Italian sparrow hybridizes with the house sparrow in a sparsely populated contact zone in the Alps. Yet, the contact zone is characterized by steep clines in species‐specific male plumage traits, suggesting that partial reproductive isolation may also have developed between these two taxa. Thus, geographic and reproductive barriers restrict gene flow into the nascent hybrid species. We propose that an origin of hybrid species where the hybrid lineage gets geographically isolated from its parental species, as seems to have happened in this system, might be more common in nature than previously assumed.     

The WPA-WHO Global Survey of Psychiatrists�� Attitudes Towards Mental Disorders Classification

This article describes the results of the WPA-WHO Global Survey of 4,887 psychiatrists in 44 countries regarding their use of diagnostic classification systems in clinical practice, and the desirable characteristics of a classification of mental disorders. The WHO will use these results to improve the clinical utility of the ICD classification of mental disorders through the current ICD-10 revision process. Participants indicated that the most important purposes of a classification are to facilitate communication among clinicians and to inform treatment and management. They overwhelmingly preferred a simpler system with 100 or fewer categories, and over two-thirds preferred flexible guidance to a strict criteria-based approach. Opinions were divided about how to incorporate severity and functional status, while most respondents were receptive to a system that incorporates a dimensional component. Significant minorities of psychiatrists in Latin America and Asia reported problems with the cross-cultural applicability of existing classifications. Overall, ratings of ease of use and goodness of fit for specific ICD-10 categories were fairly high, but several categories were described as having poor utility in clinical practice. This represents an important focus for the ICD revision, as does ensuring that the ICD-11 classification of mental disorders is acceptable to psychiatrists throughout the world.