Inhibition of GP130/STAT3 and EMT by combined bazedoxifene and paclitaxel treatment in ovarian cancer.

The interleukin 6 (IL­6)/glycoprotein 130 (GP130)/signal transducer and activator of transcription 3 (STAT3) signalling pathway, with GP130 as an intermediate membrane receptor, is involved in the survival, metastasis, and resistance of ovarian cancer. Bazedoxifene, an FDA­approved drug, is an inhibitor of GP130 and a selective estrogen modulator (SERM). We studied the mechanism of the combination therapy of bazedoxifene and paclitaxel in inhibiting the IL­6­mediated GP130/STAT3 signaling pathway in ovarian cancer. Surface plasmon resonance (SPR) was used to assess the binding of bazedoxifene to GP130. Migration, invasion, and apoptosis of ovarian cancer cells were assessed using bazedoxifene and paclitaxel. In addition, we determined the effects of bazedoxifene and paclitaxel alone or in combination on the GP130/STAT3 pathway and epithelial­mesenchymal transition (EMT). The results revealed that the combination of bazedoxifene and paclitaxel suppressed cell viability, migration, and invasion in the ovarian cancer cells. In addition, the combination treatment increased apoptosis. Furthermore, bazedoxifene combined with paclitaxel inhibited the growth of ovarian cancer cells in a xenograft tumour model. This combination reduced STAT3 phosphorylation and suppressed gene expression and EMT. In conclusion, inhibition of GP130/STAT3 signalling and EMT via a combination of bazedoxifene and paclitaxel could be used as a therapeutic strategy by which to overcome ovarian cancer.

Unraveling the mechanism of arbidol binding and inhibition of SARS-CoV-2: Insights from atomistic simulations.

The COVID-19 pandemic has spread rapidly and poses an unprecedented threat to the global economy and human health. Broad-spectrum antivirals are currently being administered to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). China's prevention and treatment guidelines suggest the use of an anti-influenza drug, arbidol, for the clinical treatment of COVID-19. Reports indicate that arbidol could neutralize SARS-CoV-2. Monotherapy with arbidol is superior to lopinavir-ritonavir or favipiravir for treating COVID-19. In SARS-CoV-2 infection, arbidol acts by interfering with viral binding to host cells. However, the detailed mechanism by which arbidol induces the inhibition of SARS-CoV-2 is not known. Here, we present atomistic insights into the mechanism underlying membrane fusion inhibition of SARS-CoV-2 by arbidol. Molecular dynamics (MD) simulation-based analyses demonstrate that arbidol binds and stabilizes at the receptor-binding domain (RBD)/ACE2 interface with a high affinity. It forms stronger intermolecular interactions with the RBD than ACE2. Analyses of the detailed decomposition of energy components and binding affinities revealed a substantial increase in the affinity between the RBD and ACE2 in the arbidol-bound RBD/ACE2 complex, suggesting that arbidol generates favorable interactions between them. Based on our MD simulation results, we propose that the binding of arbidol induces structural rigidity in the viral glycoprotein, thus restricting the conformational rearrangements associated with membrane fusion and virus entry. Furthermore, key residues of the RBD and ACE2 that interact with arbidol were identified, opening the door for developing therapeutic strategies and higher-efficacy arbidol derivatives or lead drug candidates.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 1. Production, milk fatty acid profile, and nutrient digestibility.

The objectives of this experiment were to determine the effects of increased diet fermentability and polyunsaturated fatty acids (FA) with or without supplemental 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), isoacids (IA; isobutyrate, 2-methylbutyrate, isovalerate, and valerate) or the combination of these on milk fat depression (MFD). Ten Holstein cows (194 ± 58 DIM, 691 ± 69 kg BW, 28 ± 5 kg milk yield) were used in a replicated 5 × 5 Latin square design. Treatments included a high-forage control diet (HF-C), a low-forage control diet (LF-C) causing MFD by increasing starch and decreasing neutral detergent fiber (NDF), the LF-C diet supplemented with HMTBa at 0.11% (28 g/d), the LF-C diet supplemented with IA at 0.24% of dietary dry matter (60 g/d), and the LF-C diet supplemented with HMTBa and IA. Preplanned contrasts were used to compare HF-C versus LF-C and to examine the main effects of HMTBa or IA and their interactions within the LF diets. Dry matter intake was greater for LF-C versus HF-C, but milk yield remained unchanged. The LF-C diet decreased milk fat yield (0.87 vs. 0.98 kg/d) but increased protein yield compared with HF-C. As a result, energy-corrected milk was lower (28.5 vs. 29.6 kg/d) for LF-C versus HF-C. Although the concentration of total de novo synthesized FA in milk fat was not affected, some short- and medium-chain FA were lower for LF-C versus HF-C, but the concentrations of C18 trans-10 isomers were not different. Total-tract NDF apparent digestibility was numerically lower (42.4 vs. 45.6%) for LF-C versus HF-C. As the main effects, the decrease in milk fat yield observed in LF-C was alleviated by supplementation of HMTBa through increasing milk yield without altering milk fat content and by IA through increasing milk fat content without altering milk yield so that HMTBa or IA, as the main effects, increased milk fat yield within the LF diets. However, interactions for milk fat yield and ECM were observed between HMTBa and IA, suggesting no additive effect when used in combination. Minimal changes were found on milk FA profile when HMTBa was provided. However, de novo synthesized FA increased for IA supplementation. We detected no main effect of HMTBa, IA, and interaction between those on total-tract NDF digestibility. In conclusion, the addition of HMTBa and IA to a low-forage and high-starch diet alleviated moderate MFD. Although the mechanism by which MFD was alleviated was different between HMTBa and IA, no additive effects of the combination were observed on milk fat yield and ECM.

A Protocol for Studying HIV-1 Envelope Glycoprotein Function.

HIV-1 envelope glycoproteins (Envs) bind to CD4 receptor and CCR5/CXCR4 coreceptor and mediate viral entry (Feng et al., 1996; Herschhorn et al., 2016, 2017; Kwong et al., 1998). HIV-1 Envs are the sole target of neutralizing antibodies and a main focus of vaccine development (Flemming et al., 2018). Here, we provide a step-by-step protocol to measure Env sensitivity to ligands, cold, and small molecules, as well as to study viral infectivity and to dissect parameters affecting HIV-1 Env function. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).

In Silico Screening of Potential Spike Glycoprotein Inhibitors of SARS-CoV-2 with Drug Repurposing Strategy.

OBJECTIVE: To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2 (ACE2) receptor and viral spike protein by virtual screening. METHODS: The three-dimensional (3D)-coordinate file of the receptor-binding domain (RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration (FDA)-approved drugs from DrugBank and natural compounds from Traditional Chinese Medicine Systems Pharmacology (TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine (CM) herb sources were listed in this paper. RESULTS: It was found that digitoxin, a cardiac glycoside in DrugBank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen (), a CM formula reportedly exerting activity against severe acute respiratory syndrome (SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. CONCLUSIONS: A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-CoV-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.

Short communication: A decrease in diameter of milk fat globules accompanies milk fat depression induced by conjugated linoleic acid supplementation in lactating dairy cows.

Milk fat is secreted from the mammary gland in the form of milk fat globules (MFG). Although milk fat depression has been studied since the beginning of the last century, the extent to which this phenomenon alters MFG synthesis is not fully understood. The aim of this study was to evaluate the effect of conjugated linoleic acid (CLA) on the size and distribution of MFG during milk fat depression in dairy cows. Twelve Holstein cows in mid lactation (145 ± 31 d in milk, 583 ± 34.6 kg of body weight, and 27.2 ± 2.4 kg of milk/d) were randomly assigned to a control diet or control plus Ca-protected CLA at 15 g/kg of dry matter for a 6-d period. The average diameter and particle size distribution of MFG were measured using a Mastersizer 3000 laser particle size analyzer (Malvern Instruments Ltd., Malvern, UK). Feeding CLA did not affect dry matter intake (16.2 ± 0.4 kg/d), milk production (28.4 ± 0.4 kg/d), milk protein, or lactose, but it decreased milk fat content (3.46 vs. 2.52%). In addition, surface area-related mean diameter of fat globules in cows fed CLA was lower compared with controls (3.02 vs. 3.45 µm). The percentage of large fat globules decreased and that of small fat globules increased in response to CLA. Overall, the data suggest that the milk fat depression induced by CLA is accompanied by a decrease in average diameter of MFG.

Long-Acting BMS-378806 Analogues Stabilize the State-1 Conformation of the Human Immunodeficiency Virus Type 1 Envelope Glycoproteins.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)3] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

The antifungal peptide CGA-N12 inhibits cell wall synthesis of Candida tropicalis by interacting with KRE9.

CGA-N12, an antifungal peptide derived from chromogranin A, has specific antagonistic activity against Candida spp., especially against Candida tropicalis, by inducing cell apoptosis. However, the effect of CGA-N12 on the Candida cell wall is unknown. The Candida protein KRE9, which possesses ß-1,6-glucanase activity, was screened by affinity chromatography after binding to CGA-N12. In this study, the effect of CGA-N12 on KRE9 and the interaction between CGA-N12 and KRE9 was studied to clarify the effect of CGA-N12 on C. tropicalis cell wall synthesis. The effect of CGA-N12 on recombinant KRE9 ß-1,6-glucanase activity was investigated by analyzing the consumption of glucose. The results showed that CGA-N12 inhibited the activity of KRE9. After C. tropicalis was treated with CGA-N12, the structure of the C. tropicalis cell wall was damaged. The interaction between CGA-N12 and KRE9 was analyzed by isothermal titration calorimetry (ITC). The results showed that their interaction process was involved an endothermic reaction, and the interaction force was mainly hydrophobic with a few electrostatic forces. The results of the fluorescence resonance energy transfer (FRET) assay showed that the distance between CGA-N12 and KRE9 was 7 ∼ 10 nm during their interaction. Therefore, we concluded that the target of CGA-N12 in the C. tropicalis cell membrane is KRE9, and that CGA-N12 weakly binds to KRE9 within a 7 ∼ 10 nm distance and inhibits KRE9 activity.

Impact of sublethal exposure to synthetic and natural acaricides on honey bee (Apis mellifera) memory and expression of genes related to memory.

Acaricides are used by beekeepers in honey bee (Apis mellifera L.) colonies to control parasitic mites, but may also have adverse effects to honey bees. In this study, five commonly used acaricides were tested for their sublethal effects on memory and expression of neural-related genes in honey bees. Memory measured with the proboscis extension reflex (PER) assay was significantly reduced by topical treatment of bees with a single LD05 dose of formic acid at 2 and 24 h post treatment (hpt). However, tau-fluvalinate, amitraz, coumaphos, and formic acid, but not thymol, resulted in memory loss at 48 hpt. The LD05 doses of the acraricides did not affect expression of neuroligin-1, related to memory, or expression of major royal jelly protein-1, related to both memory and development, although expression of both genes was affected at LD50 doses. The LD05 doses of thymol, formic acid, amitraz and coumaphos increased defensin-1 expression, which is related to both memory and immunity. The effect of thymol, however, may have been due to its impact on the immune response rather than memory. This study demonstrates that acaricides vary in their effects on bee's memory, and that the widely used acaricide, formic acid, is particularly damaging.

Identifying small molecule probes of ENTPD5 through high throughput screening.

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation.

Maduramicin induces apoptosis through ROS-PP5-JNK pathway in skeletal myoblast cells and muscle tissue.

Our previous work has shown that maduramicin, an effective coccidiostat used in the poultry production, executed its toxicity by inducing apoptosis of skeletal myoblasts. However, the underlying mechanism is not well understood. Here we show that maduramicin induced apoptosis of skeletal muscle cells by activating c-Jun N-terminal kinase (JNK) pathway in murine C2C12 and L6 myoblasts as well as skeletal muscle tissue. This is supported by the findings that inhibition of JNK with SP600125 or ectopic expression of dominant negative c-Jun attenuated maduramicin-induced apoptosis in C2C12 cells. Furthermore, we found that treatment with maduramicin reduced the cellular protein level of protein phosphatase 5 (PP5). Overexpression of PP5 substantially mitigated maduramicin-activated JNK and apoptosis. Moreover, we noticed that treatment with maduramicin elevated intracellular reactive oxygen species (ROS) level. Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed maduramicin-induced inhibition of PP5 and activation of JNK as well as apoptosis. The results indicate that maduramicin induction of ROS inhibits PP5, which results in activation of JNK cascade, leading to apoptosis of skeletal muscle cells. Our finding suggests that manipulation of ROS-PP5-JNK pathway may be a potential approach to prevent maduramicin-induced apoptotic cell death in skeletal muscle.

Prolactin Elevations and Permeability Glycoprotein.

OBJECTIVE: To examine the role of permeability glycoprotein (P-gp) and its substrates in the mechanism of hyperprolactinemia. DATA SOURCES: PubMed and Google Scholar were queried with the search term permeability glycoprotein crossed with antipsychotic, neuroleptic, prolactin, risperidone, paliperidone, and amisulpride. The searches were performed in early 2018 with no date restrictions. STUDY SELECTION: All references cited in PubMed were examined (108), but only the first 40 references of each search in Google Scholar (total of approximately 30,000 hits) for a total of 240 were examined. Approximately 100 references were felt to be relevant. DATA EXTRACTION: Information regarding mechanism of action and clinical relevance was extracted as appropriate for the discussion. RESULTS: Risperidone, paliperidone, and amisulpride are associated with higher prolactin levels than would be anticipated from striatal dopamine receptor occupancy studies. This elevation occurs because the levels of these antipsychotics are higher in the anterior pituitary than in parts of the brain protected by the blood-brain barrier and P-gp. P-gp has high affinity to all these antipsychotics and selectively removes them from the brain and concentrates them in the blood draining the hypothalamus, allowing greater dopamine receptor blockade in the cells in the anterior pituitary that produce prolactin. CONCLUSIONS: The anatomy of the portal circulation, the presence of P-gp, and the high affinity of this protein to risperidone, paliperidone, and amisulpride all conspire to concentrate the antipsychotic concentration in the anterior pituitary to levels higher than in other parts of the brain, with consequent increase of prolactin above expectations.

Myostatin regulates pituitary development and hepatic IGF1.

Circulating myostatin-attenuating agents are being developed to treat muscle-wasting disease despite their potential to produce serious off-target effects, as myostatin/activin receptors are widely distributed among many nonmuscle tissues. Our studies suggest that the myokine not only inhibits striated muscle growth but also regulates pituitary development and growth hormone (GH) action in the liver. Using a novel myostatin-null label-retaining model (Jekyll mice), we determined that the heterogeneous pool of pituitary stem, transit-amplifying, and progenitor cells in Jekyll mice depletes more rapidly after birth than the pool in wild-type mice. This correlated with increased levels of GH, prolactin, and the cells that secrete these hormones, somatotropes and lactotropes, respectively, in Jekyll pituitaries. Recombinant myostatin also stimulated GH release and gene expression in pituitary cell cultures although inhibiting prolactin release. In primary hepatocytes, recombinant myostatin blocked GH-stimulated expression of two key mediators of growth, insulin-like growth factor (IGF)1 and the acid labile subunit and increased expression of an inhibitor, IGF-binding protein-1. The significance of these findings was demonstrated by smaller muscle fiber size in a model lacking myostatin and liver IGF1 expression (LID-o-Mighty mice) compared with that in myostatin-null (Mighty) mice. These data together suggest that myostatin may regulate pituitary development and function and that its inhibitory actions in muscle may be partly mediated by attenuating GH action in the liver. They also suggest that circulating pharmacological inhibitors of myostatin could produce unintended consequences in these and possibly other tissues.

BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation.

Bpifa1 (BPI fold-containing group A member 1) is an airway host-protective protein with immunomodulatory properties that binds to LPS and is regulated by infectious and inflammatory signals. Differential expression of Bpifa1 has been widely reported in lung disease, yet the biological significance of this observation is unclear. We sought to understand the role of Bpifa1 fluctuations in modulating lung inflammation. We treated wild-type (WT) and Bpifa1-/- mice with intranasal LPS and performed immunological and transcriptomic analyses of lung tissue to determine the immune effects of Bpifa1 deficiency. We show that neutrophil (polymorphonuclear cells, PMNs) lung recruitment and transmigration to the airways in response to LPS is impaired in Bpifa1-/- mice. Transcriptomic analysis revealed a signature of 379 genes that differentiated Bpifa1-/- from WT mice. During acute lung inflammation, the most downregulated genes in Bpifa1-/- mice were Cxcl9 and Cxcl10. Bpifa1-/- mice had lower bronchoalveolar lavage concentrations of C-X-C motif chemokine ligand 10 (Cxcl10) and Cxcl9, interferon-inducible PMN chemokines. This was consistent with lower expression of IFNγ, IFNλ, downstream IFN-stimulated genes, and IFN-regulatory factors, which are important for the innate immune response. Administration of Cxcl10 before LPS treatment restored the inflammatory response in Bpifa1-/- mice. Our results identify a novel role for Bpifa1 in the regulation of Cxcl10-mediated PMN recruitment to the lungs via IFNγ and -λ signaling during acute inflammation.

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows.

The aim of this study was to use meta-analytical methods to estimate effects of adding exogenous fibrolytic enzymes (EFE) to dairy cow diets on their performance and to determine which factors affect the response. Fifteen studies with 17 experiments and 36 observations met the study selection criteria for inclusion in the meta-analysis. The effects were compared by using random-effect models to examine the raw mean difference (RMD) and standardized mean difference between EFE and control treatments after both were weighted with the inverse of the study variances. Heterogeneity sources evaluated by meta-regression included experimental duration, EFE type and application rate, form (liquid or solid), and method (application to the forage, concentrate, or total mixed ration). Only the cellulase-xylanase (C-X) enzymes had a substantial number of observations (n = 13 studies). Application of EFE, overall, did not affect dry matter intake, feed efficiency but tended to increase total-tract dry matter digestibility and neutral detergent fiber digestibility (NDFD) by relatively small amounts (1.36 and 2.30%, respectively, or <0.31 standard deviation units). Application of EFE increased yields of milk (0.83 kg/d), 3.5% fat-corrected milk (0.55 kg/d), milk protein (0.03 kg/d), and milk lactose (0.05 kg/d) by moderate to small amounts (<0.30 standard deviation units). Low heterogeneity (I 2 statistic <25%) was present for yields and concentrations of milk fat and protein and lactose yield. Moderate heterogeneity (I 2 = 25 to 50%) was detected for dry matter intake, milk yield, 3.5% fat-corrected milk, and feed efficiency (kg of milk/kg of dry matter intake), whereas high heterogeneity (I 2 > 50%) was detected for total-tract dry matter digestibility and NDFD. Milk production responses were higher for the C-X enzymes (RMD = 1.04 kg/d; 95% confidence interval: 0.33 to 1.74), but were still only moderate, about 0.35 standardized mean difference. A 24% numerical increase in the RMD resulting from examining only C-X enzymes instead of all enzymes (RMD = 1.04 vs. 0.83 kg/d) suggests that had more studies met the inclusion criteria, the C-X enzymes would have statistically increased the milk response relative to that for all enzymes. Increasing the EFE application rate had no effect on performance measures. Application of EFE to the total mixed ration improved only milk protein concentration, and application to the forage or concentrate had no effect. Applying EFE tended to increase dry matter digestibility and NDFD and increased milk yield by relatively small amounts, reflecting the variable response among EFE types.

Gene expression and proliferation biomarkers for antidepressant treatment resistance.

The neurotrophic hypothesis of depression suggests an association between effects on neuroplasticity and clinical response to antidepressant drug therapy. We studied individual variability in antidepressant drug effects on cell proliferation in lymphoblastoid cell lines (LCLs) from n=25 therapy-resistant patients versus n=25 first-line therapy responders from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. Furthermore, the variability in gene expression of genes associated with cell proliferation was analyzed for tentative candidate genes for prediction of individual LCL donor's treatment response. Cell proliferation was quantified by EdU (5-ethynyl-2'-deoxyuridine) assays after 21-day incubation of LCLs with fluoxetine (0.5 ng µl-1) and citalopram (0.3 ng µl-1) as developed and described earlier. Gene expression of a panel of candidate genes derived from genome-wide expression analyses of antidepressant effects on cell proliferation of LCLs from the Munich Antidepressant Response Signature (MARS) study was analyzed by real-time PCR. Significant differences in in vitro cell proliferation effects were detected between the group of LCLs from first-line therapy responders and LCLs from treatment-resistant patients. Gene expression analysis of the candidate gene panel revealed and confirmed influence of the candidate genes ABCB1, FZD7 and WNT2B on antidepressant drug resistance. The potential of these genes as tentative biomarkers for antidepressant drug resistance was confirmed. In vitro cell proliferation testing may serve as functional biomarker for individual neuroplasticity effects of antidepressants.

Myocilin expression is regulated by retinoic acid in the trabecular meshwork-derived cellular environment.

Glaucoma is the leading cause of irreversible blindness and is usually classified as angle closure and open angle glaucoma (OAG). Primary open angle glaucoma represents the most frequent clinical presentation leading to ganglion cell death and optic nerve degeneration as a main consequence of an intraocular pressure' (IOP) increase. The mechanisms of this IOP increase in such pathology remain unclear but one protein called Myocilin could be a part of the puzzle in the trabecular meshwork (TM). Previously described to be transcriptionally regulated by glucocorticoids, the comprehension of the trabecular regulation of Myocilin' expression has only weakly progressed since 15 years. Due to the essential molecular and cellular implications of retinoids' pathway in eye development and physiology, we investigate the potential role of the retinoic acid in such regulation and expression. This study demonstrates that the global retinoids signaling machinery is present in immortalized TM cells and that Myocilin (MYOC) expression is upregulated by retinoic acid alone or combined with a glucocorticoid co-treatment. This regulation by retinoic acid acts through the MYOC promoter which contains a critical cluster of four retinoic acid responsive elements (RAREs), with the RARE-DR2 presenting the strongest effect and binding the RARα/RXRα heterodimer. All together, these results open up new perspectives for the molecular understanding glaucoma pathophysiology and provide further actionable clues on Myocilin gene regulation.

Antidiabetic effects of Brucea javanica seeds in type 2 diabetic rats.

BACKGROUND: Brucea javanica (B. javanica) seeds, also known as "Melada pahit" in Indo-Malay region are traditionally used to treat diabetes. The objective of this study was to determine antidiabetic, antioxidant and anti-inflammatory effects of B. javanica seeds on nicotinamide (NA)-streptozotocin (STZ) induced type 2 diabetic (T2D) rats and to analyze its chemical composition that correlate with their pharmacological activities. METHODS: A hydroethanolic extract of B. javanica seeds was fractionated with n-hexane, chloroform and ethyl acetate. An active fraction was selected after screening for its ability to inhibit α-glucosidase and glycogen phosphorylase α (GP-α). Isolation and characterization were carried out by using column chromatography, NMR and LCMS/MS. All isolates were assayed for inhibition of GP-α and α-glucosidase. Antidiabetic effect of active fraction was further evaluated in T2D rat model. Blood glucose and body weight were measured weekly. Serum insulin, lipid profile, renal function, liver glycogen and biomarkers of oxidative stress and inflammation were analyzed after 4-week treatment and compared with standard drug glibenclamide. RESULTS: Ethyl acetate fraction (EAF) exerted good inhibitory potential for α-glucosidase and GP-α compared with other fractions. Chromatographic isolation of the EAF led to the identification of seven compounds: vanillic acid (1), bruceine D (2), bruceine E (3), parahydroxybenzoic acid (4), luteolin (5), protocatechuic acid (6), and gallic acid (7). Among them, Compound (5) was identified as the most potent inhibitor of GP-α and α-glucosidase and its GP-α inhibitory activity (IC50 = 45.08 µM) was 10-fold higher than that of caffeine (IC50 = 457.34 µM), and α-glucosidase inhibitory activity (IC50 = 26.41 µM) was 5.5-fold higher than that of acarbose (IC50 = 145.83 µM), respectively. Compounds (4), (6), and (7) inhibited GP-α activity in a concentration-dependent manner with IC50 values of 357.88, 297.37, and 214.38 µM, and their inhibitory effect was higher than that of caffeine. These compounds exhibited weak potency on α-glucosidase compared with acarbose. Compounds (1), (2), and (3) showed no inhibition on both GP-α and α-glucosidase. In vivo study showed that EAF treatment significantly reduced blood glucose level, increased insulin and glycogen contents, decreased markers of oxidative stress and inflammation, and lipid levels in T2D rats compared with untreated group. CONCLUSIONS: The EAF has potential therapeutic value for the treatment of T2D via acting as GP-α and α-glucosidase inhibitors by improving hepatic glucose and carbohydrate metabolism, suppressing oxidative stress, and preventing inflammation in T2D rats. According to the results, the efficacy of EAF could be due to the presence of luteolin along with synergistic effect of multiple compounds such as parahydroxybenzoic acid, protocatechuic acid, and gallic acid in B. javanica seeds.

Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus.

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection.