Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.     

Mating,births, and transitions: a flexible two-sex matrix model for evolutionary demography

Models of sexually-reproducing populations that consider only a single sex cannot capture the effects of sex-specific demographic differences and mate availability. We present a new framework for two-sex demographic models that implements and extends the birth-matrix mating-rule approach of Pollak. The model is a continuous-time matrix model that explicitly includes the processes of mating (which is nonlinear but homogeneous), offspring production, and demographic transitions and survival. The resulting nonlinear model converges to exponential growth with an equilibrium population composition. The model can incorporate age- or stage-structured life histories and flexible mating functions. As an example, we apply the model to analyze the effects of mating strategies (polygamy or monogamy, and mated unions composed of males and females, of variable duration) on the response to sex-biased harvesting. The combination of demographic complexity with the interaction of the sexes can have major population dynamic effects and can change the outcome of evolution on sex-related characters.     

Effects of grazing on plant species diversity and carbon partitioning in semiarid rangelands of northeastern China

Grasslands are one of the most widespread landscapes worldwide, covering approximately one-fifth of the world’s land surface, where grazing is a common practice. How carbon storage responds to grazing in steppes remains poorly understood. We quantified the effects of grazing on community composition and species diversity, and carbon storage in two typical grasslands of northeastern China, one in Horqin and the other one in Hulunbeier. In both grasslands, grazing did not influence plant species diversity. However, it substantially decreased aboveground carbon by 31% and 54% in Horqin and Hulunbeier, respectively. Fenced and grazing treatments showed a similar belowground carbon at both locations. The predominant carbon pool in the study grassland ecosystem was found in the upper 100 cm soil depth, from 98.2 to 99.1% of the total carbon storage. There were no significant effects of grazing on soil carbon neither in the whole profile nor in the uppermost 20 cm soil depth in the two study grasslands. Studies on the effects of varying rangeland management, such as region disparity and grazing systems, may have important consequences on species diversity and carbon partitioning, and thus on rangeland stability and ecosystem functioning.     

Computational Study of Evolutionary Selection Pressure on Rainbow Trout Estrogen Receptors

Molecular dynamics simulations were used to determine the binding affinities between the hormone 17-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout, Oncorhynchus mykiss. Previous phylogenetic analysis indicates that a whole genome duplication prior to the divergence of ray-finned fish led to two distinct ER isoforms, ER and ER, and the recent whole genome duplication in the ancestral salmonid created two ER isoforms, ER and ER. The objective of our computational studies is to provide insight into the underlying evolutionary pressures on these isoforms. For the ER subtype our results show that E2 binds preferentially to ER over ER. Tests of lineage specific N/S ratios indicate that the ligand binding domain of the ER gene is evolving under relaxed selection relative to all other ER genes. Comparison with the highly conserved DNA binding domain suggests that ER may be undergoing neofunctionalization possibly by binding to another ligand. By contrast, both ER and ER bind similarly to E2 and the best fitting model of selection indicates that the ligand binding domain of all ER genes are evolving under the same level of purifying selection, comparable to ER.     

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.     

Outcomes of Anterolateral Thigh Flap Reconstruction for Salvage Laryngopharyngectomy for Hypopharyngeal Cancer after Concurrent Chemoradiotherapy

Objective

To evaluate the functional and oncological outcomes of anterolateral thigh flap reconstruction for salvage laryngopharyngectomy after concurrent chemoradiotherapy for patients with hypopharyngeal carcinoma.

Materials/Methods

A retrospective review was conducted on patients who underwent pharyngoesophageal reconstruction using anterolateral thigh flap after salvage laryngopharyngectomy for recurrent hypopharyngeal carcinoma between June 2003 and May 2010 at Chang Gung Memorial Hospital. The perioperative morbidity, mortality, functional outcomes, and oncological outcomes were evaluated.

Results

33 patients were entered into the study. The mean follow-up time was 19.5±12.3 months. Recurrent pathological TNM stages included 3 (9.1%), 2 (6.1%), and 28 (84.8%) patients with stage II, III, and IV disease, respectively. Mean ICU stay was 10.3 days and the mean hospital stay was 39.9 days. Peri-operative mortality occurred in one patient (3%). 16 patients (48.5%) developed recipient site complications. Among them, 14 patients (42.4%) developed fistulas and 9 patients (27.3%) developed strictures. Except for 4 patients (12.1%), all achieved varying degree of oral intake with 29 patients (60.6%) being completely independent from tube feeding. The mean interval to start oral intake was 15.1 days. The 5-year overall survival and disease–free survival rates were 51.8% and 53.7%, respectively. The pN status is an independent predictor of overall survival and disease–free survival (P = 0.027 and 0.038, respectively).

Conclusion

Pharyngoesophageal reconstruction after salvage laryngopharyngectomy remains challenging even in the experienced hands. Reconstructive microsurgeons who are prepared to take on these cases should be equally well prepared to manage the potential postoperative complications.