Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.     

Temperature-induced changes in treatment efficiency and microbial structure of aerobic granules treating landfill leachate

This paper investigates the effect of temperature on nitrogen and carbon removal by aerobic granules from landfill leachate with a high ammonium concentration and low concentration of biodegradable organics. The study was conducted in three stages; firstly the operating temperature of the batch reactor with aerobic granules was maintained at 29 °C, then at 25 °C, and finally at 20 °C. It was found that a gradual decrease in operational temperature allowed the nitrogen-converting community in the granules to acclimate, ensuring efficient nitrification even at ambient temperature (20 °C). Ammonium was fully removed from leachate regardless of the temperature, but higher operational temperatures resulted in higher ammonium removal rates [up to 44.2 mg/(L h) at 29 °C]. Lowering the operational temperature from 29 to 20 °C decreased nitrite accumulation in the GSBR cycle. The highest efficiency of total nitrogen removal was achieved at 25 °C (36.8 ± 10.9 %). The COD removal efficiency did not exceed 50 %. Granules constituted 77, 80 and 83 % of the biomass at 29, 25 and 20 °C, respectively. Ammonium was oxidized by both aerobic and anaerobic ammonium-oxidizing bacteria. Accumulibacter sp., Thauera sp., cultured Tetrasphaera PAO and Azoarcus–Thauera cluster occurred in granules independent of the temperature. Lower temperatures favored the occurrence of denitrifiers of Zooglea lineage (not Z. resiniphila), bacteria related to Comamonadaceae, Curvibacter sp., Azoarcus cluster, Rhodobacter sp., Roseobacter sp. and Acidovorax spp. At lower temperatures, the increased abundance of denitrifiers compensated for the lowered enzymatic activity of the biomass and ensured that nitrogen removal at 20 °C was similar to that at 25 °C and significantly higher than removal at 29 °C.     

The LORE1 insertion mutant resource

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) ( http://lotus.au.dk ). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG‐hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost‐efficient strategy for generation of non‐transgenic mutant collections for unrestricted use in plant research.     

Rapid Screening of Antimicrobial Synthetic Peptides

Increasing resistance to conventional antibiotics among microorganisms is one of the leading problems of medicine nowadays. Antimicrobial peptides are compounds exhibiting both antibacterial and antifungal activities. However, it is difficult to predict whether a designed new compound would exhibit any biological activity. Moreover, purification of the peptides is one of the most time-consuming and expensive steps of the synthesis that sometimes leads to unnecessary loss of solvents and reagents. In our study we have developed a thin-layer chromatography (TLC) direct bioautography technique for rapid determination of antimicrobial activity of peptides without the necessity of high-performance liquid chromatography purification. In this assay, crude peptides were applied and separated on a TLC plate. Then, pre-prepared plates were dipped into microbial suspension and incubated under optimum conditions for bacteria and fungi as well. The activity of the tested compounds was visualized by spraying the TLC plates with a cell viability reagent, resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide). Effectiveness of this assay was compared with minimal inhibitory concentration results obtained by broth microdilution assay. Interestingly, so far such a screening method has not been applied for this group of compounds.     

Transferrins,haptoglobins, and ceruloplasmins among tribal groups of Madagascar

Transferrin phenotypes of 403 Malagasy were determined by autoradiography after electrophoresis of plasma or serum. Three phenotypes, Tf C, Tf CD, and Tf D, were found. The frequency of Tf^D was 0.051 in the total sample, 0.041 in tribes of the Madagascar plateau, and 0.075 in tribes of the coastal and lowland regions. Haptoglobin phenotypes of 402 Malagasy were determined. The frequency of Hp¹ was 0.41 in the total sample, 0.38 in tribes of the plateau, and 0.49 in coastal-lowland tribes. There is a significant difference in the frequency of Hp¹ between the tribes of the plateau and those of the coastal and lowland regions. This confirms earlier observations of significant differences in frequencies of haptoglobins and hemoglobin S. Plasma or serum samples from 405 Malagasy were examined by electrophoresis to determine ceruloplasmin phenotypes. Seven distinct ceruloplasmin bands were observed. These were A, B, Galveston, Bridgeport, New Haven, Tananarive, and X, the last two described for the first time. Ten different phenotypes composed of one or two of the bands were observed. The frequency of the allele for ceruloplasmin B (CpB) was 0.78 in the total sample, 0.73 in tribes of the plateau, and 0.92 in coastal and lowland tribes. There is a significant difference in the frequency of this allele in the two major tribal groups, and this provides additional evidence of genetic differences between the peoples of the plateau and those of the coast and lowland regions of Madagascar.     

X-linked,polymorphic genetic variation of thyroxin-binding globulin (TBG) in baboons and screening of additional primates

X-linked polymorphic variation of thyroxin-binding globulin (TBG) is observed in several human groups. Isoelectric focusing of plasma samples labeled in vitro with [¹²⁵I]thyroxin, followed by autoradiography, also reveals genetically determined polymorphic electrophoretic variation in baboon TBG. The protein detected by this method in baboon plasma is immunologically similar to human TBG and is distinct from the other thyroxin-binding proteins, albumin and prealbumin. The isoelectric patterns of human and baboon TBG are very similar and both have an isoelectric range of pH 4.1 to 4.5. The baboon TBG polymorphism is inherited in a two-allele X-linked fashion, with a frequency of 72% for the common allele and 28% for the slow allele. A survey of seven other primate species including African green monkey, bonnet macaque, chimpanzee, crab-eating macaque, gorilla, rhesus monkey, and spider monkey revealed no polymorphic variation in TBG, although isoelectric patterns were similar to the human and baboon patterns. In addition, samples from pregnant chimpanzees demonstrate a pronounced quantitative anodal shift in relative band densities, a shift also observed in pregnant humans. This shift was not observed in samples from pregnant baboons. TBG should prove to be a useful X-linked genetic marker in baboons and provides a model of serum protein changes in pregnancy, at least in humans and chimpanzees.This research was supported by NIH Grant 2R01-EY-02388 and a Biomedical Research Support Grant from the Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston.     

High rates of energy expenditure and water flux in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea

We measured water flux and energy expenditure in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea by using the doubly labeled water method. Previous laboratory investigations have suggested weak urinary concentrating ability, high rates of water flux, and low basal metabolic rates in this species. However, free-ranging measurements from hygric mammals are rare, and it is not known how these features interact in the environment. Rates of water flux (210+/-32 mL d(-1)) and field metabolic rates (1,488+/-486 kJ d(-1)) were 159% and 265%, respectively, of values predicted by allometric equations for similar-sized herbivores. Mountain beavers can likely meet their water needs through metabolic water production and preformed water in food and thus remain in water balance without access to free water. Arginine-vasopressin levels were strongly correlated with rates of water flux and plasma urea : creatinine ratios, suggesting an important role for this hormone in regulating urinary water loss in mountain beavers. High field metabolic rates may result from cool burrow temperatures that are well below lower critical temperatures measured in previous laboratory studies and suggest that thermoregulation costs may strongly influence field energetics and water flux in semifossorial mammals.