Survey of the training needs of genetic assistants supports the creation of genetic assistant training programs.

Despite the increasing numbers of genetic assistants (GAs) in the genomics workforce, their training needs and how to best prepare GAs for their role have not been well defined. We sought to identify the current educational status of GAs, opinions on their training needs, and attitudes about GA training programs (GATPs). Survey links were emailed to NSGC members, 17 state genetic counseling (GC) professional organizations, and genomic medicine researchers. Respondents (n = 411) included GCs (n = 231) and GAs (n = 136). Like other studies, we found that the GA position is filled by a range of education levels and career aspirations. Most respondents supported the creation of GATPs, with 63% endorsing that GATPs would be helpful and half endorsing a short-term (3 months or less) program. Most believed GATPs should focus on general knowledge, with almost all practical skills learned on-the-job. If more GATPs are created, our survey provides evidence that graduates would be hired. Indeed, of those whose work setting required a bachelor's degree, the number of respondents who favored keeping that requirement was similar to the number who favored hiring a GA without a degree if they attended a GATP. However, there were concerns about GATPs. Many (44%) believed creating GATPs could discourage candidates from becoming GAs. We observed that there are two types of GAs: entry-level and bachelor's-level, with the entry-level being those who do not have and are not working to obtain a bachelor's degree and the bachelor's-level being those who do/are. GATPs could focus on the education of entry-level GAs, while gaps in the knowledge base of bachelor's-level GAs could be addressed by augmenting bachelor's curriculum or providing additional training after hire. Further research on the training needs of GAs and hiring practices of institutions will be vital to understanding their training needs and designing and implementing effective GATPs.

Development of Competency-based Online Genomic Medicine Training (COGENT).

The fields of genetics and genomics have greatly expanded across medicine through the development of new technologies that have revealed genetic contributions to a wide array of traits and diseases. Thus, the development of widely available educational resources for all healthcare providers is essential to ensure the timely and appropriate utilization of genetics and genomics patient care. In 2020, the National Human Genome Research Institute released a call for new proposals to develop accessible, sustainable online education for health providers. This paper describes the efforts of the six teams awarded to reach the goal of providing genetic and genomic training modules that are broadly available for busy clinicians.

Soy and Frequent Dairy Consumption with Subsequent Equol Production Reveals Decreased Gut Health in a Cohort of Healthy Puerto Rican Women.

The U.S. Hispanic female population has one of the highest breast cancer (BC) incidence and mortality rates, while BC is the leading cause of cancer death in Puerto Rican women. Certain foods may predispose to carcinogenesis. Our previous studies indicate that consuming combined soy isoflavones (genistein, daidzein, and glycitein) promotes tumor metastasis possibly through increased protein synthesis activated by equol, a secondary dietary metabolite. Equol is a bacterial metabolite produced in about 20-60% of the population that harbor and exhibit specific gut microbiota capable of producing it from daidzein. The aim of the current study was to investigate the prevalence of equol production in Puerto Rican women and identify the equol producing microbiota in this understudied population. Herein, we conducted a cross-sectional characterization of equol production in a clinically based sample of eighty healthy 25-50 year old Puerto Rican women. Urine samples were collected and evaluated by GCMS for the presence of soy isoflavones and metabolites to determine the ratio of equol producers to equol non-producers. Furthermore, fecal samples were collected for gut microbiota characterization on a subset of women using next generation sequencing (NGS). We report that 25% of the participants were classified as equol producers. Importantly, the gut microbiota from equol non-producers demonstrated a higher diversity. Our results suggest that healthy women with soy and high dairy consumption with subsequent equol production may result in gut dysbiosis by having reduced quantities (diversity) of healthy bacterial biomarkers, which might be associated to increased diseased outcomes (e.g., cancer, and other diseases).

Evaluation of the Pink Luminous Breast LED-Based Technology Device as a Screening Tool for the Early Detection of Breast Abnormalities.

Breast cancer is the leading cause of sex-specific female cancer deaths in the United States. Detection at earlier stages contributes to decreasing the mortality rate. The mammogram is the "Gold Standard" for breast cancer screening with an estimated sensitivity of 86.9% and a specificity of 88.9%. However, these values are negatively affected by the breast density considered a risk factor for developing breast cancer. Herein, we validate the novel LED-based medical device Pink Luminous Breast (PLB) by comparison with the mammogram using a double blinded approach. The PLB works by emitting a LED red light with a harmless spectrum of 640-800 nanometers. This allows the observation of abnormalities represented by dark or shadow areas. In this study, we evaluated the sensitivity and specificity of the PLB device as a screening tool for the early detection of breast abnormalities. Our results show that the PLB device has a high sensitivity (89.6%) and specificity (96.4%) for detecting breast abnormalities comparable to the adjusted mammogram values: 86.3 and 68.9%, respectively. The percentage of presence of breast density was 78.2% using PLB vs. 72.9% with the mammogram. Even with higher findings of breast density, the PLB is still capable of detecting 9.4% of calcifications compared to 6.2% in mammogram results and the reported findings for cysts, masses, or tumor-like abnormalities was higher using the PLB (6.5%) than the mammogram (5.6%). A 100% of the participants felt comfortable using the device without feeling pain or discomfort during the examination with 100% acceptability. The PLB positive validation shows its potential for routine breast screening at non-clinical settings. The PLB provides a rapid, non-invasive, portable, and easy-to-use tool for breast screening that can complement the home-based breast self-examination technique or the clinical breast examination. In addition, the PLB can be conveniently used for screening breasts with surgical implants. PLB provides an accessible and painless breast cancer screening tool. The PLB use is not intended to replace the mammogram for breast screening but rather to use it as an adjunct or complemental tool as part of more efficient earlier detection strategies contributing to decrease mortality rates.

Ganoderma lucidum extract (GLE) impairs breast cancer stem cells by targeting the STAT3 pathway.

The aggressive nature of triple negative breast cancer (TNBC) may be explained in part by the presence of breast cancer stem cells (BCSCs), a subpopulation of cells, which are involved in tumor initiation, progression, metastasis, recurrence, and therapy resistance. The signal transducer and activator of transcription 3 (STAT3) pathway participates in the development and progression of BCSCs, but its role in TNBC remains unclear. Here, we report that Ganoderma lucidum extract (GLE), a medicinal mushroom with anticancer activity, acts on BCSCs in vitro and in TNBC pre-clinical animal tumor models by downregulating the STAT3 pathway. We show that GLE significantly reduces TNBC cell viability, and down-regulates total and phosphorylated STAT3 expression. This is consistent with the reduction of OCT4, NANOG and SOX2 expression, reduction in the BCSC population by loss of the ALDH1 and CD44+/CD24- population, the deformation of mammospheres, and the strong reduction in animal tumor volume and tumor weight. Analysis of the BCSC compartment in tumors revealed that GLE decreases the STAT3 pathway and the expression of OCT4, NANOG, and SOX2 in BCSCs. These findings demonstrate that the anti-cancer activity of GLE targets BCSCs of TNBC through the downregulation of the STAT3 pathway.

Naturally Derived Anti-HIV Polysaccharide Peptide (PSP) Triggers a Toll-Like Receptor 4-Dependent Antiviral Immune Response.

AIM: Intense interest remains in the identification of compounds to reduce human immunodeficiency virus type 1 (HIV-1) replication. Coriolus versicolor's polysaccharide peptide (PSP) has been demonstrated to possess immunomodulatory properties with the ability to activate an innate immune response through Toll-like receptor 4 (TLR4) showing insignificant toxicity. This study sought to determine the potential use of PSP as an anti-HIV agent and whether its antiviral immune response was TLR4 dependent. MATERIALS AND METHODS: HIV-1 p24 and anti-HIV chemokine release was assessed in HIV-positive (HIV+) THP1 cells and validated in HIV+ peripheral blood mononuclear cells (PBMCs), to determine PSP antiviral activity. The involvement of TLR4 activation in PSP anti-HIV activity was evaluated by inhibition. RESULTS: PSP showed a promising potential as an anti-HIV agent, by downregulating viral replication and promoting the upregulation of specific antiviral chemokines (RANTES, MIP-1α/ß, and SDF-1α) known to block HIV-1 coreceptors in THP1 cells and human PBMCs. PSP produced a 61% viral inhibition after PSP treatment in HIV-1-infected THP1 cells. Additionally, PSP upregulated the expression of TLR4 and TLR4 inhibition led to countereffects in chemokine expression and HIV-1 replication. CONCLUSION: Taken together, these findings put forward the first evidence that PSP exerts an anti-HIV activity mediated by TLR4 and key antiviral chemokines. Elucidating these new molecular mediators may reveal additional drug targets and open novel therapeutic avenues for HIV-1 infection.

HIV-1 Gp120 clade B/C induces a GRP78 driven cytoprotective mechanism in astrocytoma.

HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

A decade of viral mutations and associated drug resistance in a population of HIV-1+ Puerto Ricans: 2002-2011.

Puerto Rico has one of the highest rates of HIV/AIDS seen for any US state or territory, and antiretroviral therapy has been a mainstay of efforts to mitigate the HIV/AIDS public health burden on the island. We studied the evolutionary dynamics of HIV-1 mutation and antiretroviral drug resistance in Puerto Rico by monitoring the population frequency of resistance-associated mutations from 2002 to 2011. Whole blood samples from 4,475 patients were analyzed using the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System in the Immunoretrovirus Research Laboratory at Universidad Central del Caribe. Results show that 64.0% of female and 62.9% of male patients had HIV-1 mutations that confer resistance to at least one antiretroviral medication. L63P and M184V were the dominant mutations observed for the protease (PRO) and reverse transcriptase (RT) encoding genes, respectively. Specific resistance mutations, along with their associated drug resistance profiles, can be seen to form temporal clusters that reveal a steadily changing landscape of resistance trends over time. Both women and men showed resistance mutations for an average of 4.8 drugs over the 10-year period, further underscoring the strong selective pressure exerted by antiretrovirals along with the rapid adaptive response of HIV. Nevertheless, both female and male patients showed a precipitous decrease for overall drug resistance, and for PRO mutations in particular, over the entire course of the study, with the most rapid decrease in frequency seen after 2006. The reduced HIV-1 mutation and drug resistance trends that we observed are consistent with previous reports from multi-year studies conducted around the world. Reduced resistance can be attributed to the use of more efficacious antiretroviral drug therapy, including the introduction of multi-drug combination therapies, which limited the ability of the virus to mount rapid adaptive responses to antiretroviral selection pressure.

Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis.

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

The proteome signature of the inflammatory breast cancer plasma membrane identifies novel molecular markers of disease.

Inflammatory Breast Cancer (IBC) is the most lethal form of breast cancer with a 35% 5-year survival rate. The accurate and early diagnosis of IBC and the development of targeted therapy against this deadly disease remain a great medical challenge. Plasma membrane proteins (PMPs) such as E-cadherin and EGFR, play an important role in the progression of IBC. Because the critical role of PMPs in the oncogenic processes they are the perfect candidates as molecular markers and targets for cancer therapies. In the present study, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) followed by mass spectrometry analysis was used to compare the relative expression levels of membrane proteins (MP) between non-cancerous mammary epithelial and IBC cells, MCF-10A and SUM-149, respectively. Six of the identified PMPs were validated by immunoblotting using the membrane fractions of non-IBC and IBC cell lines, compared with MCF-10A cells. Immunohistochemical analysis using IBC, invasive ductal carcinoma or normal mammary tissue samples was carried out to complete the validation method in nine of the PMPs. We identified and quantified 278 MPs, 76% of which classified as PMPs with 1.3-fold or higher change. We identified for the first time the overexpression of the novel plasminogen receptor, PLGRKT in IBC and of the carrier protein, SCAMP3. Furthermore, we describe the positive relationship between L1CAM expression and metastasis in IBC patients and the role of SCAMP3 as a tumor-related protein. Overall, the membrane proteomic signature of IBC reflects a global change in cellular organization and suggests additional strategies for cancer progression. Together, this study provides insight into the specialized IBC plasma membrane proteome with the potential to identify a number of novel therapeutic targets for IBC.

Ganoderma lucidum Combined with the EGFR Tyrosine Kinase Inhibitor, Erlotinib Synergize to Reduce Inflammatory Breast Cancer Progression.

The high incidence of resistance to Tyrosine Kinase Inhibitors (TKIs) targeted against EGFR and downstream pathways has increased the necessity to identify agents that may be combined with these therapies to provide a sustained response for breast cancer patients. Here, we investigate the therapeutic potential of Ganoderma lucidum extract (GLE) in breast cancer, focusing on the regulation of the EGFR signaling cascade when treated with the EGFR TKI, Erlotinib. SUM-149, or intrinsic Erlotinib resistant MDA-MB-231 cells, and a successfully developed Erlotinib resistant cell line, rSUM-149 were treated with increasing concentrations of Erlotinib, GLE, or their combination (Erlotinib/GLE) for 72h. Treatment effects were tested on cell viability, cell proliferation, cell migration and invasion. To determine tumor progression, severe combined immunodeficient mice were injected with SUM-149 cells and then treated with Erlotinib/GLE or Erlotinib for 13 weeks. We assessed the protein expression of ERK1/2 and AKT in in vitro and in vivo models. Our results show that GLE synergizes with Erlotinib to sensitize SUM-149 cells to drug treatment, and overcomes intrinsic and developed Erlotinib resistance. Also, Erlotinib/GLE decreases SUM-149 cell viability, proliferation, migration and invasion. GLE increases Erlotinib sensitivity by inactivating AKT and ERK signaling pathways in our models. We conclude that a combinatorial therapeutic approach may be the best way to increase prognosis in breast cancer patients with EGFR overexpressing tumors.

Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen.

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.

Novel KCNJ10 Gene Variations Compromise Function of Inwardly Rectifying Potassium Channel 4.1.

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 µmSPM. At 100 µmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.

Soy Isoflavone Genistein-Mediated Downregulation of miR-155 Contributes to the Anticancer Effects of Genistein.

We previously reported that dietary genistein inhibits mammary tumor growth and metastasis of the highly metastatic MDA-MB-435 cancer cells in immunocompromised mice. The purpose herein was to characterize the role of the novel oncogenic microRNA (miRNA) miR-155 in the anticancer effects of genistein in metastatic breast cancer. The effect of genistein was determined on breast cancer cell viability, apoptosis, and expression of miR-155 and its targets. At low physiologically relevant concentrations, genistein inhibits cell viability and induces apoptosis in metastatic MDA-MB-435 and Hs578t breast cancer cells, without affecting the viability of nonmetastatic MCF-7 breast cancer cells. In parallel with reduced cell viability, miR-155 is downregulated, whereas proapoptotic and anticell proliferative miR-155 targets FOXO3, PTEN, casein kinase, and p27 are upregulated in MDA-MB-435 and Hs578t cells in response to genistein treatment. However, miR-155 levels remain unchanged in response to genistein in the MCF-7 cells. Ectopic expression of miR-155 in MDA-MB-435 and Hs578t cells decreases the effects of genistein on cell viability and abrogates the effects of genistein on apoptosis and expression of proapoptotic genes. Therefore, genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein in metastatic breast cancer.

Chemical profile and in vivo hypoglycemic effects of Syzygium jambos, Costus speciosus and Tapeinochilos ananassae plant extracts used as diabetes adjuvants in Puerto Rico.

BACKGROUND: The increasing numbers of people who use plant-based remedies as alternative or complementary medicine call for the validation of less known herbal formulations used to treat their ailments. Since Puerto Rico has the highest rate of Type 2 diabetes within all the states and territories of the United States, and Puerto Ricans commonly use plants as diabetes adjuvants, it is important to study the plants' physiological effects, and identify their bioactive compounds to understand their role in modulation of blood glucose levels. We present the phytochemical profiles and hypoglycemic effects of Tapeinochilus ananassae, Costus speciosus and Syzygium jambos. METHODS: Phytochemicals in methanolic and aqueous extracts were analyzed by thin layer chromatography (TLC). Alkaloids (Bromocresol green, λ=470 nm), flavonoids (AlCl3, λ=415 nm), saponins (DNS, λ=760 nm), tannins (FeCl3/K4Fe(CN)6, λ=395 nm) and phenolics (Folin-Ciocalteau, λ=765 nm) were quantified. Male C57BLKS/J (db/db) and C57BL/J (ob/ob) genetically obese mice were orally gavaged with aqueous extracts of lyophilized plant decoctions for 10 wks. RESULTS: Our results show that T. ananassae had significantly greater amounts of flavonoids and tannins, while S. jambos showed the greatest concentration of phenolics and C. speciosus exhibited higher amounts of alkaloids. C57BLKS/J db/db treated with plant extracts show better glucose modulation when the extracts are administered in complement with an insulin injection. Finally, C57BL/J ob/ob mice on T. ananassae and S. jambos treatments show better blood glucose modulation over time. CONCLUSION: These results document for the first time the chemical profile of T. ananassae and provide evidence for a potential anti-diabetic efficacy of T. ananassae and S. jambos.

Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

Up-regulation of TREK-2 potassium channels in cultured astrocytes requires de novo protein synthesis: relevance to localization of TREK-2 channels in astrocytes after transient cerebral ischemia.

Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.