Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

Influences of −482C&gt;T and 3238G&gt;C polymorphisms of the <Emphasis Type="Italic">Apolipoprotein C3</Emphasis> gene on prevalence of metabolic syndrome

Apolipoprotein C3 (ApoC3) plays a regulatory role in triglyceride (TG) metabolism. The higher level of TG can be a cause in pathogenesis of the vascular diseases or metabolic syndrome (MetS). In this study, we examined the associations of ApoC3 polymorphisms (?482C>T rs2854117 and 3238G>C rs5128) with Korean MetS patients. A total of 835 subjects were investigated, including 320 patients with MetS and 515 healthy subjects. The genotype analysis of the ApoC3 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism methods. Of the two polymorphisms studied, we observed a significant difference in the ?482C>T polymorphism between the MetS and control groups. The TT genotype of the ?482C>T polymorphism was associated with increased risk for MetS, compared with the controls (OR 1.627, 95 % CI 1.075–2.463, P = 0.021). The association was female-specific. No associations were found for the risk of MetS in the 3238G>C polymorphism. Haplotypes composed of two polymorphisms, however, were associated with MetS susceptibility in only male group. The 3238G>C polymorphism was significantly associated with TG levels (P = 0.013). Our data suggest that the ApoC3 ?482C>T polymorphism is associated with increased MetS susceptibility in the Korean population.     

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice

New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (T_CM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R⁺ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R⁺ T_CM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R⁺ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.     

National Use of Safety-Net Clinics for Primary Care among Adults with Non-Medicaid Insurance in the United States

Objective

To describe the prevalence, characteristics, and predictors of safety-net use for primary care among non-Medicaid insured adults (i.e., those with private insurance or Medicare).

Methods

Cross-sectional analysis using the 2006–2010 National Ambulatory Medical Care Surveys, annual probability samples of outpatient visits in the U.S. We estimated national prevalence of safety-net visits using weighted percentages to account for the complex survey design. We conducted bivariate and multivariate logistic regression analyses to examine characteristics associated with safety-net clinic use.

Results

More than one-third (35.0%) of all primary care safety-net clinic visits were among adults with non-Medicaid primary insurance, representing 6,642,000 annual visits nationally. The strongest predictors of safety-net use among non-Medicaid insured adults were: being from a high-poverty neighborhood (AOR 9.53, 95% CI 4.65–19.53), being dually eligible for Medicare and Medicaid (AOR 2.13, 95% CI 1.38–3.30), and being black (AOR 1.97, 95% CI 1.06–3.66) or Hispanic (AOR 2.28, 95% CI 1.32–3.93). Compared to non-safety-net users, non-Medicaid insured adults who used safety-net clinics had a higher prevalence of diabetes (23.5% vs. 15.0%, p<0.001), hypertension (49.4% vs. 36.0%, p<0.001), multimorbidity (≥2 chronic conditions; 53.5% vs. 40.9%, p<0.001) and polypharmacy (≥4 medications; 48.8% vs. 34.0%, p<0.001). Nearly one-third (28.9%) of Medicare beneficiaries in the safety-net were dual eligibles, compared to only 6.8% of Medicare beneficiaries in non-safety-net clinics (p<0.001).

Conclusions

Safety net clinics are important primary care delivery sites for non-Medicaid insured minority and low-income populations with a high burden of chronic illness. The critical role of safety-net clinics in care delivery is likely to persist despite expanded insurance coverage under the Affordable Care Act.     

Assessing Seasonal and Inter-Annual Variations of Lake Surface Areas in Mongolia during 2000-2011 Using Minimum Composite MODIS NDVI

A minimum composite method was applied to produce a 15-day interval normalized difference vegetation index (NDVI) dataset from Moderate Resolution Imaging Spectroradiometer (MODIS) daily 250 m reflectance in the red and near-infrared bands. This dataset was applied to determine lake surface areas in Mongolia. A total of 73 lakes greater than 6.25 km²in area were selected, and 28 of these lakes were used to evaluate detection errors. The minimum composite NDVI showed a better detection performance on lake water pixels than did the official MODIS 16-day 250 m NDVI based on a maximum composite method. The overall lake area detection performance based on the 15-day minimum composite NDVI showed -2.5% error relative to the Landsat-derived lake area for the 28 evaluated lakes. The errors increased with increases in the perimeter-to-area ratio but decreased with lake size over 10 km². The lake area decreased by -9.3% at an annual rate of -53.7 km² yr^-1 during 2000 to 2011 for the 73 lakes. However, considerable spatial variations, such as slight-to-moderate lake area reductions in semi-arid regions and rapid lake area reductions in arid regions, were also detected. This study demonstrated applicability of MODIS 250 m reflectance data for biweekly monitoring of lake area change and diagnosed considerable lake area reduction and its spatial variability in arid and semi-arid regions of Mongolia. Future studies are required for explaining reasons of lake area changes and their spatial variability.     

Altered Gut Microbiota Composition Associated with Eczema in Infants

Eczema is frequently the first manifestation of an atopic diathesis and alteration in the diversity of gut microbiota has been reported in infants with eczema. To identify specific bacterial communities associated with eczema, we conducted a case-control study of 50 infants with eczema (cases) and 51 healthy infants (controls). We performed high-throughput sequencing for V3–V4 hypervariable regions of the 16S rRNA genes from the gut fecal material. A total of 12,386 OTUs (operational taxonomic units) at a 97% similarity level were obtained from the two groups, and we observed a difference in taxa abundance, but not the taxonomic composition, of gut microbiota between the two groups. We identified four genera enriched in healthy infants: Bifidobacterium, Megasphaera, Haemophilus and Streptococcus; and five genera enriched in infants with eczema: Escherichia/Shigella, Veillonella, Faecalibacterium, Lachnospiraceae incertae sedis and Clostridium XlVa. Several species, such as Faecalibacterium prausnitzii and Ruminococcus gnavus, that are known to be associated with atopy or inflammation, were found to be significantly enriched in infants with eczema. Higher abundance of Akkermansia muciniphila in eczematous infants might reduce the integrity of intestinal barrier function and therefore increase the risk of developing eczema. On the other hand, Bacteroides fragilis and Streptococcus salivarius, which are known for their anti-inflammatory properties, were less abundant in infants with eczema. The observed differences in genera and species between cases and controls in this study may provide insight into the link between the microbiome and eczema risk.     

Senecio changii (Asteraceae: Senecioneae), a New Species from Sichuan,China

Senecio changii (Asteraceae: Senecioneae), a new species from Muli, Sichuan, southwestern China, is described. It is distinguished in Chinese Senecio s.s. by having lyrate-pinnatisect to pinnatisect leaves and a single terminal large discoid capitulum which is somewhat nodding. Evidence from floral micromorphology, karyology and molecular phylogenetic analyses based on the nuclear ITS/ETS sequence data all support its membership within Senecio s.s.     

Dynamic Modelling of Tooth Deformation Using Occlusal Kinematics and Finite Element Analysis

Background

Dental biomechanics based on finite element (FE) analysis is attracting enormous interest in dentistry, biology, anthropology and palaeontology. Nonetheless, several shortcomings in FE modeling exist, mainly due to unrealistic loading conditions. In this contribution we used kinematics information recorded in a virtual environment derived from occlusal contact detection between high resolution models of an upper and lower human first molar pair (M¹ and M₁, respectively) to run a non-linear dynamic FE crash colliding test.

Methodology

MicroCT image data of a modern human skull were segmented to reconstruct digital models of the antagonistic right M¹ and M₁ and the dental supporting structures. We used the Occlusal Fingerprint Analyser software to reconstruct the individual occlusal pathway trajectory during the power stroke of the chewing cycle, which was applied in a FE simulation to guide the M₁ 3D-path for the crash colliding test.

Results

FE analysis results showed that the stress pattern changes considerably during the power stroke, demonstrating that knowledge about chewing kinematics in conjunction with a morphologically detailed FE model is crucial for understanding tooth form and function under physiological conditions.

Conclusions/Significance

Results from such advanced dynamic approaches will be applicable to evaluate and avoid mechanical failure in prosthodontics/endodontic treatments, and to test material behavior for modern tooth restoration in dentistry. This approach will also allow us to improve our knowledge in chewing-related biomechanics for functional diagnosis and therapy, and it will help paleoanthropologists to illuminate dental adaptive processes and morphological modifications in human evolution.     

Examination of Duct Physiology in the Human Mammary Gland

BackgroundThe human breast comprise several ductal systems, or lobes, which contain a small amount of fluid containing cells, hormones, proteins and metabolites. The complex physiology of these ducts is likely a contributing factor to the development of breast cancer, especially given that the vast majority of breast cancers begin in a single lobular unit.MethodsWe examined the levels of total protein, progesterone, estradiol, estrone sulfate, dehydroepiandrosterone sulfate, and macrophages in ductal fluid samples obtained from 3 ducts each in 78 women, sampled twice over a 6 month period. Samples were processed for both cytological and molecular analysis. Intraclass correlation coefficients and mixed models were utilized to identify significant data.ResultsWe found that the levels of these ductal fluid components were generally uncorrelated among ducts within a single breast and over time, suggesting that each lobe within the breast has a distinct physiology. However, we also found that estradiol was more correlated in women who were nulliparous or produced nipple aspirate fluid.ConclusionsOur results provide evidence that the microenvironment of any given lobular unit is unique to that individual unit, findings that may provide clues about the initiation and development of ductal carcinomas.     

Cross-Modal and Intra-Modal Characteristics of Visual Function and Speech Perception Performance in Postlingually Deafened,Cochlear Implant Users

Evidence of visual-auditory cross-modal plasticity in deaf individuals has been widely reported. Superior visual abilities of deaf individuals have been shown to result in enhanced reactivity to visual events and/or enhanced peripheral spatial attention. The goal of this study was to investigate the association between visual-auditory cross-modal plasticity and speech perception in post-lingually deafened, adult cochlear implant (CI) users. Post-lingually deafened adults with CIs (N = 14) and a group of normal hearing, adult controls (N = 12) participated in this study. The CI participants were divided into a good performer group (good CI, N = 7) and a poor performer group (poor CI, N = 7) based on word recognition scores. Visual evoked potentials (VEP) were recorded from the temporal and occipital cortex to assess reactivity. Visual field (VF) testing was used to assess spatial attention and Goldmann perimetry measures were analyzed to identify differences across groups in the VF. The association of the amplitude of the P1 VEP response over the right temporal or occipital cortex among three groups (control, good CI, poor CI) was analyzed. In addition, the association between VF by different stimuli and word perception score was evaluated. The P1 VEP amplitude recorded from the right temporal cortex was larger in the group of poorly performing CI users than the group of good performers. The P1 amplitude recorded from electrodes near the occipital cortex was smaller for the poor performing group. P1 VEP amplitude in right temporal lobe was negatively correlated with speech perception outcomes for the CI participants (r = -0.736, P = 0.003). However, P1 VEP amplitude measures recorded from near the occipital cortex had a positive correlation with speech perception outcome in the CI participants (r = 0.775, P = 0.001). In VF analysis, CI users showed narrowed central VF (VF to low intensity stimuli). However, their far peripheral VF (VF to high intensity stimuli) was not different from the controls. In addition, the extent of their central VF was positively correlated with speech perception outcome (r = 0.669, P = 0.009). Persistent visual activation in right temporal cortex even after CI causes negative effect on outcome in post-lingual deaf adults. We interpret these results to suggest that insufficient intra-modal (visual) compensation by the occipital cortex may cause negative effects on outcome. Based on our results, it appears that a narrowed central VF could help identify CI users with poor outcomes with their device.