The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression

Sorghum Genome Sequencing by Methylation Filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis

Access to spine care for the poor and near poor.

BACKGROUND CONTEXT: Access to care for poor/near poor patients is a concerning and growing problem within the American system of medical care. PURPOSE: The objective of this study was to examine the relationship between health insurance status and access to spine care among patients below 65 years of age eventually receiving treatment at our tertiary academic medical center. STUDY DESIGN: Descriptive study based on chart review and telephone interviews. PATIENT SAMPLE: Two groups of 64 patients each with surgical pathology of limited complexity and limited comorbidities, one with Medicaid insurance and one with private, commercial insurance. OUTCOME MEASURES: Reasons for referral, travel distance, travel time, frequency of visits, and proximity of fellowship-trained spinal surgeons. METHODS: Two groups, each with 64 consecutive spine surgical patients, were studied and compared. Group One had Medicaid coverage and Group Two was privately insured. All patients (both groups) were treated surgically for similar pathology of limited complexity and had limited comorbidities. They were assessed to determine the difficulties they encountered in receiving care before referral to our medical center including factors such as referral by a local provider based on insurance status alone and travel time/distance/frequency to eventually receive care at our center. The availability of local care for these patients (fellowship-trained spine surgeons in their local area) was also assessed. RESULTS: The great majority (78%) of poor/near poor patients with Medicaid coverage from counties at some distance from (and local to) our center were referred/deferred on the basis of insurance status alone given surgical problems which could have comfortably been addressed by orthopedic surgeons, neurosurgeons, or fellowship-trained spine surgeons local to the patient. This difficulty in access to care results in a significant burden (measured in time/travel/costs) for these patients. CONCLUSIONS: The poor/near poor with Medicaid insurance have less access to local spine care than those with private, commercial health insurance. The implications (from both surgeon and patient perspectives) of this dilemma are discussed

Missing Excitons: How Energy Transfer Competes with Free Charge Generation in Dilute-Donor/Acceptor Systems

Energy transfer across the donor–acceptor interface in organic photovoltaics is usually beneficial to device performance, as it assists energy transport to the site of free charge generation. Here, we present a case where the opposite is true: dilute donor molecules in an acceptor host matrix exhibit ultrafast excitation energy transfer (EET) to the host, which suppresses the free charge yield. We observe an optimal photochemical driving force for free charge generation, as detected via time-resolved microwave conductivity (TRMC), but with a low yield when the sensitizer is excited. Meanwhile, transient absorption shows that transferred excitons efficiently produce charge-transfer states. This behavior is well described by a competition for the excited state between long-range electron transfer that produces free charge and EET that ultimately produces only localized charge-transfer states. It cannot be explained if the most localized CT states are the intermediate between excitons and the free charge in this system

Groundwater vulnerability to pesticides in Northwest Bangladesh

The transport and leaching potential hazards of various pesticides were studied in a shallow unconfined aquifer located in Northwest Bangladesh. Pesticide leaching potential was quantified using a one-dimensional advective–dispersive transport equation for a non-conservative chemical that follows first-order decay and linear adsorption in soils. Leaching potential index (LPI) was calculated for 69 sites in the study area to evaluate the relative vulnerability to pesticide leaching and to prioritize sites for model study and soil sampling. The numerical ranks of computed LPI were grouped by quantiles into very high, high, moderate, low and very low categories; and based on these rankings, the most vulnerable site was selected. The fate and transport of pesticides in this most vulnerable site was modeled using MT3D. The model results indicate that pesticides with high sorptivity and moderate to high persistence have low potential impact on groundwater. Top soils are found to be particularly vulnerable to the accumulation of organochlorine pesticides. Results also revealed that decreasing the soil organic matter and increasing the half-life of the pesticides at deeper depths did not make any significant change. Finally, six soil samples were collected from the same site at depths of 0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 m for the analysis of pesticide residues. The soil–water was extracted from the samples following standard extraction technique and tested using gas chromatography (GC) and high-performance liquid chromatography (HPLC) for pesticide residues. Results showed no trace of pesticide residues in the soil–water; however, a few unknown peaks were detected indicating the use of some unknown brand of chemicals in the study area

MARCKS mutant expression and localization in U87 glioma cells.

<p><b>A)</b> Diagram depicting the domains of the engineered MARCKS wild-type (WT) and effector domain deleted (ΔED) lentiviral constructs. Starting at the N-terminus there is a myristoylation domain (N-MYR), MH2 domain (N-MH2), an effector domain (WT-ED) with amino acid sequence indicated, and lastly a C-terminus V-5 tag. <b>B)</b> Western blot showing doxycycline inducible MARCKS mutant over-expression in U87 cells. Nuclear and cytoplasmic fractions were prepared and separated by SDS-PAGE and probed for V-5 (for MARCKS expression), lamin and tubulin for nuclear and cytoplasmic fraction, respectively. <b>C)</b> Confocal microscopy is shown for U87 WT-MARCKS (WT) and ΔED-MARCKS (ΔED) cells were fixed and stained for MARCKS (V5), nucleus (DAPI), and PIP₂ with the merged image shown on the right. <b>D)</b> High magnification of the PIP2 staining of the nucleus with punctate staining marked with red arrows.</p

Changes in RNA expression and protein levels with ED-deleted MARCKS.

<p><b>A)</b> After overnight doxycycline-induction of ΔED-MARCKS or WT-MARCKS U87 cells, RNA was collected and ran on NanoString nCounter GX Human Cancer Reference Kit. Data was analyzed on nSolver software. Violin plot displays > 1.5 log fold increase in GBM cells overexpressing an ΔED-MARCKS protein as compared to WT MARCKS. <b>B)</b> Changes in total protein levels as well as phosphorylated protein levels between the ΔED and WT- MARCKS expressing U87 GBM cell lines were determined by Kinex KAM-850 microarray. Data is displayed as z-ratio of ΔED results compared to WT-MARCKS results. Increase in z-ratio is indicated in green dots while decrease is indicated in red dots. Total protein targets are listed in standard font while phosphorylated proteins are listed in italicized font with phosphorylated amino acids designated.</p

MARCKS localizes to the nucleus in D54 and U251 glioma cells.

<p><b>A)</b> D54 and U251 cells were fixed and stained for MARCKS and DAPI with the merged image shown on the right side as indicated. <b>B)</b> D54 and U251 cell lysates were separated into nuclear and cytoplasmic fractions. The proteins were then were separated by 8% SDS-PAGE and probed for MARCKS, the cytoplasmic marker tubulin, and the nuclear marker, lamin.</p