A Single Divergent Exon Inhibits Ankyrin-B Association with the Plasma Membrane

Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU5₂-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase inhibitors. Structure–activity relationships led to the discovery of sulfonamide, carbamate and urea series of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13 cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13 xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses improved pharmacologic and selectivity profiles and could be more effective than previously disclosed FLT3 inhibitors in the treatment of acute myeloid leukemia.     

Risk Factors for Progressive Visual Field Loss in Primary Angle-Closure Glaucoma: A Retrospective Cohort Study

Purpose

To investigate risk factors associated with progressive visual field (VF) loss in primary angle closure glaucoma (PACG).

Methods

We retrospectively reviewed medical record of PACG patients who had ≥5 reliable VF examinations (central 24-2 threshold test, Humphrey Field Analyzer) and ≥2 years of follow-up. Each VF was scored using Collaborative Initial Glaucoma Treatment Study system. Progression was defined if 3 consecutive follow-up VF tests had an increased score of ≥3 above the mean of the first 2 VF scores. Factors associated with VF progression were evaluated by Cox proportional hazards models.

Results

A total of 89 eyes from 89 patients (mean age, 69.8 ± 7.9 years), who received a mean of 6.9 ± 2.3 VF tests (mean deviation at initial, -8.1 ± 4.4 dB) with a mean follow-up of 63.9 ± 23.9 months were included. VF progression was detected in 9 eyes (10%). The axial length (AL), anterior chamber depth, and intraocular pressure (IOP) in patients with and without progression were 22.5 ± 0.6 and 23.1 ± 0.9 mm, 2.5 ± 0.3 and 2.5 ± 0.3 mm, 14.8 ± 2.4 and 14.3 ± 2.3 mm Hg, respectively. AL was the only factor associated with progression in both Cox proportional hazards univariate (p = 0.031) and multivariate models (p = 0.023).

Conclusion

When taking into account age, IOP, follow-up period, and number of VF tests, a shorter AL is the only factor associated with VF progression in this cohort of Chinese patients with PACG. Further studies are warranted to verify the role of AL in progressive VF loss in PACG.     

Research on a Community-based Platform for Promoting Health and Physical Fitness in the Elderly Community

This study aims to assess the acceptability of a fitness testing platform (iFit) for installation in an assisted living community with the aim of promoting fitness and slowing the onset of frailty. The iFit platform develops a means of testing Bureau of Health Promotion mandated health assessment items for the elderly (including flexibility tests, grip strength tests, balance tests, and reaction time tests) and integrates wireless remote sensors in a game-like environment to capture and store subject response data, thus providing individuals in elderly care contexts with a greater awareness of their own physical condition. In this study, we specifically evaluated the users’ intention of using the iFit using a technology acceptance model (TAM). A total of 101 elderly subjects (27 males and 74 females) were recruited. A survey was conducted to measure technology acceptance, to verify that the platform could be used as intended to promote fitness among the elderly. Results indicate that perceived usefulness, perceived ease of use and usage attitude positively impact behavioral intention to use the platform. The iFit platform can offer user-friendly solutions for a community-based fitness care and monitoring of elderly subjects. In summary, iFit was determined by three key drivers and discussed as follows: risk factors among the frail elderly, mechanism for slowing the advance frailty, and technology acceptance and support for promoting physical fitness.     

Bacterial Communities of Three Saline Meromictic Lakes in Central Asia

Meromictic lakes located in landlocked steppes of central Asia (~2500 km inland) have unique geophysiochemical characteristics compared to other meromictic lakes. To characterize their bacteria and elucidate relationships between those bacteria and surrounding environments, water samples were collected from three saline meromictic lakes (Lakes Shira, Shunet and Oigon) in the border between Siberia and the West Mongolia, near the center of Asia. Based on in-depth tag pyrosequencing, bacterial communities were highly variable and dissimilar among lakes and between oxic and anoxic layers within individual lakes. Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Firmicutes were the most abundant phyla, whereas three genera of purple sulfur bacteria (a novel genus, Thiocapsa and Halochromatium) were predominant bacterial components in the anoxic layer of Lake Shira (~20.6% of relative abundance), Lake Shunet (~27.1%) and Lake Oigon (~9.25%), respectively. However, few known green sulfur bacteria were detected. Notably, 3.94% of all sequencing reads were classified into 19 candidate divisions, which was especially high (23.12%) in the anoxic layer of Lake Shunet. Furthermore, several hydro-parameters (temperature, pH, dissolved oxygen, H₂S and salinity) were associated (P< 0.05) with variations in dominant bacterial groups. In conclusion, based on highly variable bacterial composition in water layers or lakes, we inferred that the meromictic ecosystem was characterized by high diversity and heterogenous niches.     

Dynamic observation and quantification of type I/II collagen in chondrogenesis of mesenchymal stem cells by second‐order susceptibility microscopy

Second‐order susceptibility (SOS) microscopy is used to image and characterize chondrogenesis in cultured human mesenchymal stem cells. SOS analysis shows that the SOS tensor ratios can be used to characterize type I and II collagens in living tissues and that both collagen types are produced at the onset of chondrogenesis. Time‐lapse analysis shows a modulation of extracellular matrix results in a higher rate in increase of type II collagen, as compared to type I collagen. With time, type II collagen content stabilizes at the composition of 70% of total collagen content. SOS microscopy can be used to continuously and noninvasively monitor the production of collagens I and II. With additional development, this technique can be developed into an effective quality control tool for monitoring extracellular matrix production in engineered tissues.