Nonspecific Activity Research Articles

Gastrodin, a traditional Chinese medicine monomer compound, can be used as adjuvant to enhance the immunogenicity of melanoma vaccines

Gastrodin (GAS) is a Chinese medicine with wide application for the treatment of nervous system disease. Previous studies reported that GAS exhibited non-specific immunomodulatory activities. To explore the effects of GAS as a vaccine adjuvant, the expression levels of CD80, CD86, MHCI and MHCII activated markers were detected after GAS treatment in vitro and in vivo, and the expression levels of IL-2 and TNF-α in splenocytes were detected after GAS treatment in vivo. Besides, the expression levels of IL-2 and IFN-γ in CD4+T cells and perforin, TNF-α and IFN-γ in CD8+T cells were detected. The effects of GAS on the survival rate and tumor size of tumor-challenged mice and the effect of cytotoxicity on CD8+T cells were also investigated. Our data showed that GAS ameliorated CD8+T cell mediated immune response and significantly improved protection of tumor-challenged animals. The results demonstrated that GAS is a potential adjuvant contributing to anticancer immunomodulation.

Effects of dietary multi-strain probiotics supplementation in a low fishmeal diet on growth performance, nutrient utilization, proximate composition, immune parameters, and gut microbiota of juvenile olive flounder (Paralichthys olivaceus)

A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (Paralichthys olivaceus) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 108–109 CFU kg−1 of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the Lactobacillus abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder.

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1–Cas2 integrase

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

Abstract 554: Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules

Abstract Introduction: Trophoblast glycoprotein 5T4 is expressed on the cell surface of multiple cancers yet sparingly on normal adult tissues. CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that support cell activation, proliferation and survival. Previously we described bispecific tumor associated antigen x CD137 DART® molecules limiting CD137 mediated immunostimulation to the tumor microenvironment (AACR2017) as an approach to reduce the unwanted systemic CD137 effects associated with agonistic anti-CD137 mAbs. We subsequently identified a bispecific TRIDENT format bearing bivalent CD137 and monovalent tumor antigen engagement that provides maximal CD137 activation in a tumor antigen anchored manner. Here we apply that format to generate a 5T4 x CD137 x CD137 TRIDENT molecule that promotes CD137 activation in proximity to 5T4-expressing tumor cells leading to enhanced T-cell co-stimulation, proliferation and tumor cell killing activity when combined with CD3-engaging tumor-targeting bispecific DART molecules. Methods: TRIDENT molecules were constructed comprising bivalent CD137 binding and monovalent 5T4 binding. Binding properties were evaluated by surface plasmon resonance (SPR) and flow cytometry. Signaling was assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human or cynomolgus monkey T cells. T cells were incubated with or without antigen-expressing cells and submaximal concentrations of either anti-CD3 bead or CD3-engaging tumor-targeting bispecific DART molecules. Results: SPR and flow cytometry analyses demonstrate that 5T4 x CD137 x CD137 TRIDENT molecule binds human and monkey target antigens. The 5T4 x CD137 x CD137 TRIDENT molecule induces CD137 signaling in a reporter cell line and promotes cytokine release in primary human & cynomolgus monkey T-cell assays in the presence of 5T4+ tumor cells. In the absence of 5T4+ tumor cells, the TRIDENT molecule lacks agonistic activity. Furthermore, 5T4 x CD137 x CD137 TRIDENT molecule enhances T-cell proliferation and tumor cell cytolysis induced by CD3-targeted DART molecules. Consistent with preferential induction of CD137 by CD8 T-cells, 5T4 x CD137 x CD137 TRIDENT increases the fraction of CD8+ central memory and effector memory T cells in the presence 5T4+ tumor cells. Conclusions: 5T4 x CD137 x CD137 TRIDENT molecule promotes T-cell co-stimulation in a tumor antigen-dependent manner offering an opportunity to target CD137 immunostimulation, while potentially limiting non-specific systemic T-cell activation and related side effects. Citation Format: Liqin Liu, Ling Huang, Vatana Long, Yinhua Yang, Robert Burns, Jonathan Li, Jennifer DiChiara, Qihong Xu, Arin Whiddon, Chia-Ying K Lam, Jim Tamura, Valentina Ciccarone, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 554.

Chemical Transfers Occurring Through Oenococcus oeni Biofilm in Different Enological Conditions.

Chardonnay wine malolactic fermentations were carried out to evaluate the chemical transfers occurring at the wood/wine interface in the presence of two different bacterial lifestyles. To do this, Oenococcus oeni was inoculated into must and wine in its planktonic and biofilm lifestyles, whether adhering or not to oak chips, leading to three distinct enological conditions: (i) post-alcoholic fermentation inoculation in wine in the absence of oak chips, (ii) post-alcoholic fermentation inoculation in wine in the presence of oak chips, and (iii) co-inoculation of both Saccharomyces cerevisiae and O. oeni directly in Chardonnay musts in the presence of oak chips. Classical microbiological and physico-chemical parameters analyzed during the fermentation processes confirmed that alcoholic fermentation was completed identically regardless of the enological conditions, and that once O. oeni had acquired a biofilm lifestyle in the presence or absence of oak, malolactic fermentation occurred faster and with better reproducibility compared to planktonic lifestyles. Analyses of volatile components (higher alcohols and wood aromas) and non-volatile components (Chardonnay grape polyphenols) carried out in the resulting wines revealed chemical differences, particularly when bacterial biofilms were present at the wood interface. This study revealed the non-specific trapping activity of biofilm networks in the presence of wood and grape compounds regardless of the enological conditions. Changes of concentrations in higher alcohols reflected the fermentation bioactivity of bacterial biofilms on wood surfaces. These chemical transfers were statistically validated by an untargeted approach using Excitation Emission Matrices of Fluorescence combined with multivariate analysis to discriminate innovative enological practices during winemaking and to provide winemakers with an optical tool for validating the biological and chemical differentiations occurring in wine that result from their decisions.

Proteolytic Activity of Edible Spruce Morchella esculenta

Abstract This study focused on the determination of non-specific proteolytic activity of edible spruce Morchella esculenta in water extract, phosphate-buffered saline (PBS) solution (pH = 7.5) extract and a suspension prepared from 200 mg DW (dry weight) of edible spruce in PBS solution (pH 7.5). A clear casein solution was used as a substrate. The absorbances were measured in quartz cuvettes at the wavelength of 280 nm against a blank with zero concentration of trypsin. Non-specific proteolytic activity was expressed as trypsin equivalents per kilogram of mushroom dry weight (mg.kg−1 DW). All of the extracts demonstrated non-specific enzymatic activity. The highest activity was observed in the PBS suspension and the lowest enzymatic activity was measured in the water extract of the Morchella esculenta fungi. The non-specific proteolytic activity decreased in the following order: PBS suspension extract (pH 7.5; 22.9 mg.kg−1 DW), followed by PBS extract (pH 7.5; 13.6 mg.kg−1 DW) and finally the water extract (10.94 mg.kg−1 DW).

S877 SAFETY AND CLINICAL ACTIVITY OF AMV564, A CD33/CD3 T‐CELL ENGAGER, IN PATIENTS WITH RELAPSED/REFRACTORY ACUTE MYELOID LEUKEMIA (AML): UPDATED RESULTS FROM THE PHASE 1 FIRST‐IN‐HUMAN TRIAL

Background:AMV564 is a novel bivalent, bispecific (2:2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3ε on T‐cell receptors. AMV564 creates an immune synapse between CD33‐expressing cells and T cells, initiating T‐cell directed lysis of CD33 expressing cells, and inducing activation, differentiation and proliferation of T cells. In preclinical studies, AMV564 eliminated myeloid blasts with picomolar potency and demonstrated broad activity independent of cytogenetic or molecular abnormalities, CD33 expression level, and disease stage, with no nonspecific activation of T cells (Reusch U et al. Clin Cancer Res 2016; 22:5829–38).Aims:The primary objectives of this study are to characterize the safety, tolerability, and preliminary anti‐leukemic activity of AMV564. Evaluation of pharmacokinetics (PK), assessment of cytokine changes, and immunophenotyping of T cells and myeloid cells are secondary objectives.Methods:This is an ongoing Phase 1 study with a 3+3 dose‐escalation followed by a dose expansion (NCT03144245). The key inclusion/exclusion criteria are: adults with relapsed and/or refractory AML after 1–2 prior induction regimens (with a standard anthracycline‐based regimen or hypomethylating agent) and no more than 2 prior salvage regimens. AMV564 is administered by continuous intravenous infusion (CIV) for 14 consecutive days in a 28 day cycle. Responding patients are eligible to receive additional cycles until disease progression or unacceptable toxicity. AMV564 and cytokine concentrations are measured by validated immunoassays and immunophenotyping is performed by flow cytometry.Results:To date, 30 patients (16 male/14 female) with a median age of 72 years (range 24–84 years) have been enrolled in 8 dose cohorts from 0.5 to 200 mcg/day. Twenty‐two patients (73%) had secondary AML and/or adverse cytogenetics, including 9 patients (30%) with a p53 mutation. Nineteen patients (63%) had received at least 1 prior salvage regimen and 18 (60%) had received prior intensive chemotherapy, including 9 patients (30%) who had received a high‐dose (≥ 1 g/m2) cytarabine‐based regimen. At the time of this analysis, 28 patients were evaluable for safety and 27 patients were evaluable for activity. No dose‐limiting toxicity or treatment‐related Grade ≥ 3 adverse events (AE) were reported. Using a lead‐in dose regimen, Grade 2 CRS was observed in 1 patient (treated at 200 mcg/day) and no Grade 3 or higher CRS has been observed. The most common Grade ≥3 treatment‐emergent AE has been febrile neutropenia, reported in 9 (30%) of 30 patients. No patient has died within 30 days of treatment initiation. Bone marrow blast reductions were observed in 17 (63%) of 27 efficacy evaluable patients. At doses of 100 mcg or higher, objective responses (1 complete response [CR], 1 CR with incomplete hematologic recovery, and 1 partial response) were observed in 3 of 9 evaluable patients. AMV564 PK was dose proportional with a terminal half‐life of 2–3 days. Serum concentrations increased gradually, with times to steady‐state concentration of 3–7 days. Marked increases in serum IL6, IL8, and IL10 were observed with evidence of T‐cell activation in both blood and bone marrow.Summary/Conclusion:AMV564 is well‐tolerated and demonstrates anti‐leukemic activity through T‐cell engagement using a 14‐day administration schedule. AMV564 has a unique PK profile with a gradual increase in drug concentrations and thus the potential for better controlled T‐cell activation.

Enhanced bath immersion vaccination through microbubble treatment in the cyprinid loach

Immunization by bath immersion is likely the simplest method of fish vaccination. Although the route of immunogenicity has not been fully identified, immersion vaccination is clearly a useful labor-saving technique. In this study, microbubble (MB) treatment was assessed for its ability to improve the efficacy of bath immersion vaccination in the cyprinid loach. MBs are commonly defined as minute particles of gas with a diameter of less than 100 μm, which generated free radicals. Here, the efficacy of MB treatment for vaccination enhancement in the cyprinid loach was assessed in direct challenge experiments using the virulent Aeromonas hydrophila JUNAH strain; assessments comprised agglutination titer assay and non-specific parameter analysis. Agglutination titers were high in loaches that were immunized via injection with inactivated cells (FKC group); however, non-specific immune activation parameters (e.g., lysozyme, superoxide dismutase, and phagocytic activity) were more increased in loaches that were immunized via bath immersion with MB treatment. Moreover, MB-treated loaches showed comparable survival rates, relative to loaches immunized via injection with formalin inactivated cells. Thus, higher levels of non-specific immune parameters suggest increased efficacy of this vaccine approach. Improving the effectiveness of bath immersion vaccine will increase its affordability and ease of application in aquaculture.

Cometabolic biotransformation and microbial-mediated abiotic transformation of sulfonamides by three ammonia oxidizers

The abilities of three phylogenetically distant ammonia oxidizers, Nitrososphaera gargensis, an ammonia-oxidizing archaeon (AOA); Nitrosomomas nitrosa Nm90, an ammonia-oxidizing bacterium (AOB); and Nitrospira inopinata, the only complete ammonia oxidizer (comammox) available as a pure culture, to biotransform seven sulfonamides (SAs) were investigated. The removals and protein-normalized biotransformation rate constants indicated that the AOA strain N. gargensis exhibited the highest SA biotransformation rates, followed by N. inopinata and N. nitrosa Nm90. The transformation products (TPs) of sulfadiazine (SDZ), sulfamethazine (SMZ) and sulfamethoxazole (SMX) and the biotransformation mechanisms were evaluated. Based on the analysis of the TP formulas and approximate structures, it was found that during biotransformation, i) the AOA strain carried out SA deamination, hydroxylation, and nitration; ii) the AOB strain mainly performed SA deamination; and iii) the comammox isolate participated only in deamination reactions. It is proposed that deamination was catalyzed by deaminases while hydroxylation and nitration were mediated by nonspecific activities of the ammonia monooxygenase (AMO). Additionally, it was demonstrated that among the three ammonia oxidizers, only AOB contributed to the formation of pterin-SA conjugates. The biotransformation of SDZ, SMZ and SMX occurred only when ammonia oxidation was active, suggesting a cometabolic transformation mechanism. Interestingly, SAs could also be transformed by hydroxylamine, an intermediate of ammonia oxidation, suggesting that in addition to enzymatic conversions, a microbially induced abiotic mechanism contributes to SA transformation during ammonia oxidation. Overall, using experiments with pure cultures, this study provides important insights into the roles played by ammonia oxidizers in SA biotransformation.

Influences of immersion bathing in Bacillus velezensis DY-6 on growth performance, non-specific immune enzyme activities and gut microbiota of Apostichopus japonicus

In this study, the influences of immersion bathing in different concentrations of Bacillus velezensis DY-6 on the body weight gain rate and non-specific immune enzyme activities of the coelom fluid of sea cucumber (Apostichopus japonicus) were determined in order to obtain the optimum bacterial concentration. The gut microbiota change in A. japonicus was then analyzed through high-throughput sequencing during the immersion bathing in B. velezensis DY-6 at the optimum concentration for 49 d. The results illustrate that the body weight growth rate of all bathing groups was higher than that of the control. The highest growth rate (25.3%) was achieved when the bacterial concentration was 1×103 CFU/mL. The activities of non-specific immune enzymes (ACP, AKP, SOD and LZM) of all bathing groups increased, and the activities of the enzymes of groups bathed with the bacterium at 1×103 and 1×104 CFU/mL reached the highest on day 21 and day 28. Taking the growth rate and economic cost into consideration, the optimum concentration of B. velezensis DY-6 was 1×103 CFU/mL. The influences of immersion bathing in B. velezensis DY-6 at 1×103 CFU/mL on the gut microbiota of A. japonicus were then evaluated through 16S rDNA sequencing analysis. Results showed that the gut microbiota changed with the addition of B. velezensis DY-6, and the richness and diversity of the gut microbiota peaked twice on day 14 and day 21, respectively. In association with the non-specific immune enzyme activities and if day 28 was selected as the dividing point, the community structure of the gut microbiota could be obviously divided into two types. The correlation analysis revealed that the non-specific immune enzyme activities were correlated significantly to some gut bacteria (in the phyla Firmicutes, Proteobacteria, and Bacteroidetes) after immersion bathing in B. velezensis DY-6. Our findings will provide the theoretical foundation for probiotic application in sea cucumber farming.

Studies on vascular response to full superantigens and superantigen derived peptides: Possible production of novel superantigen variants with less vasodilation effect for tolerable cancer immunotherapy

Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K+ channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K+ channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61–100), (101–140) and (181–220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181–220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes.

Combining TruCulture and OptiMAP to Profile Human Immune Immunity

Abstract TruCulture® is a whole blood collection and culture system designed for use at clinical sites. TruCulture outperforms traditional peripheral blood mononuclear cell (PBMC) culturing methods, with improved reproducibility and higher levels of stimulation (Duffy et. al. Clinical Immunology 2017). The TruCulture proprietary culture medium ensures minimal non-specific immune activation, delivering a steady baseline and decreased variation during stimulating parameters. OptiMAP is a multiplex immunoassay panel that measures 13 cytokines of the major immune response pathways (TH1, TH2, TH17, and monocyte/macrophage). Using TruCulture and OptiMAP, we analyzed healthy donor whole blood responses to common stimulants: T cell activators, TLR agonists, and superantigens. The subjects ranged from 24 to 70 years old and consisted of 48% males and 52% females. The results indicated that healthy donors responded similarly to stimulation with anti-CD3/CD28 and Staphylococcal enterotoxin type B (SEB) in production of T cell cytokines (IFN-γ, IL-2, and IL-17). Response to SEB is positively correlated to the monocyte population in whole blood. T cell responses to SEB and CytoStim (an antibody based alternative to SEB) are similar in production of IFN-γ and IL-17, but CytoStim had a reduced stimulation of IL-2. All healthy donors responded to a TLR-4 agonist (LPS) by producing high levels of TNF-α, IL- 6, and IL-1β. LPS stimulated production of IFN-γ was similar to SEB, most likely due to bystander activation. The TLR-7 agonist (gardiquimod, GDQ) stimulated IL-10 levels at a level comparable to LPS. These data illustrate the utility of combining TruCulture and OptiMAP to investigate human immune stimulation pathways for clinical research.

Dietary Carotenoids Lutein and Zeaxanthin Change Brain Activation in Older Adult Participants: A Randomized, Double-Masked, Placebo-Controlled Trial.

Steady-state visual evoked potentials (SSVEP) can be used to test the topological response of cortical neurons. Studies have shown that a lutein (L) preferentially accumulates within cortical tissue. L, zeaxanthin (Z), and their isomers can be measured directly in retina (macular pigment optical density, MPOD), and retinal L+Z correlate highly with L+Z levels in cortical visual processing areas. The purpose of this study was to determine the relation between MPOD and SSVEP signal power, cross-sectionally and after supplementation with L+Z. SSVEP to three different driving frequencies of stimulation (5, 10, and 16.6 Hz) were obtained for community-dwelling older adults, at baseline and after 12 months of supplementation with either 12 mg L+Z or placebo. Power was quantified at the driving frequencies. Non-specific activation was quantified within the 10-15 Hz band. MPOD was measured psychophysically. Subjects with low MPOD had reduced power at 16.6 Hz and reduced non-specific activation, compared with subjects with high MPOD. Supplementation significantly improved signal power at 5 and 10 Hz. Past research suggests that L+Z can improve visual memory, visual processing speeds, etc. One possible mechanism for that improvement may be improving signal-to-noise ratio throughout the vision system.

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.

Compounds based on 5-(perylen-3-ylethynyl)uracil scaffold: High activity against tick-borne encephalitis virus and non-specific activity against enterovirus A

Rigid amphipathic fusion inhibitors (RAFIs) are potent antivirals based on a perylene core linked with a nucleoside moiety. Sugar-free analogues of RAFIs, 5-(perylen-3-ylethynyl)uracil-1-acetic acid 1 and its amides 2, were synthesized using combined protection group strategy. Compounds 1 and 2 appeared to have low toxicity on porcine embryo kidney (PEK) or rhabdomiosarcoma (RD) cells together with remarkable activity against enveloped tick-borne encephalitis virus (TBEV): EC50 values vary from 0.077 μM to subnanomolar range. Surprisingly, 3-pivaloyloxymethyl (Pom) protected precursors 7 and 8 showed even more pronounced activity. All the compounds showed no activity against several non-enveloped enteroviruses, except 4-hydroxybutylamides 2d,g, which inhibited the reproduction of enterovirus A71 with EC50 50–100 μM, with a non-specific mode of action. The results suggest that the carbohydrate moiety of RAFI nucleosides does not play a crucial role in their antiviral action, and biological activity of the 5-(perylen-3-ylethynyl)uracil scaffold can be effectively modulated by substituents in positions 1 and 3. The high antiviral activity of these new compounds, coupled with low toxicity advocate their potential role in antiviral therapy.

High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity.

MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. It has been proved that the aberrant expression of miRNAs is related to disease and miRNAs can serve as potential biomarkers for early tumor diagnosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a is a recently discovered CRISPR-RNA (crRNA) guided RNA manipulation tool. The recognition of target RNA can morphologically activate the robust nonspecific trans ribonuclease activity of Cas13a. This unique property makes Cas13a ideal for nucleic acid detection. Herein, we first exploited CRISPR/LbuCas13a to directly detect miRNAs with high specificity and simplicity. A limit of detection (LOD) as low as 4.5 amol was achieved by this one-step assay within 30 min, and the dynamic range spanned 4 orders of magnitude from 10 amol to 100 fmol. More importantly, single nucleotide variation, even at the end of target miRNA, can be discriminated by rationally programmed crRNA. In addition, the practical application ability of this Cas13a/crRNA-based signal amplification strategy was demonstrated by miRNA quantification in complex biological samples (total small RNA). With excellent reliability, sensitivity, and simple to implement features, this method promises a great potential for early diagnosis of miRNA-related disease. Moreover, the systematic analysis of the crRNA design could provide guidance to further develop Cas13a-based molecular diagnoses.

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles.

Protein kinase C-delta inhibitor (PKCδi) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKCδi. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKCδi (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

Cognitive stimulation, training, and rehabilitation

Cognition-focused interventions for people with cognitive impairment including mild-to-moderate dementia may be classified into three categories: cognitive stimulation, training, and rehabilitation. Cognitive stimulation, consisting of nonspecific cognition-enhancing activities often in group format, produces small improvements on some aspects of cognitive ability which can be accompanied by gains in quality of life, everyday functioning, and social interaction. Cognitive training which involves repeated practice of tasks that target specific cognitive functions, often using computers, provides improvements on trained tasks that usually do not translate to nontrained tasks and have little impact on real life. Cognitive rehabilitation represents an individual approach that focuses on disability rather than cognition per se and is helpful in terms of reaching personally relevant goals and improving day-to-day performance. Data are largely lacking to support commercial claims regarding the efficacy of video games or virtual reality and augmented reality devices. There is insufficient evidence from randomized controlled trials whether cognition-focused interventions can delay or prevent cognitive decline. However, data from prospective cohort studies strongly suggest that late-life cognitive activity is associated with a reduced risk of dementia.

Evaluation of an individualized vs non-specific standardized patient activity in improving communication skills amongst pharmacy students

Background and purposeTo evaluate the impact of two standardized patient (SP) approaches on student communication skills and self-perceived confidence. Educational activity and settingSecond professional year pharmacy students were assessed on overall and area of improvement (AOI) communication skills and self-perceived confidence. Students were invited to participate in a SP intervention activity, following a fall and before a spring class assessment, where they were randomized to an individualized or non-specific SP group. Communication skills were assessed using a four component 64-point rubric; confidence was assessed using an 11-question Likert-type survey. FindingsNineteen students participated in the SP intervention activity; 11 students comprised the individualized group and eight students comprised the non-specific group. Students in the individualized group demonstrated a significantly higher median change in AOI communication scores compared to the non-specific group [five points vs. three points, respectively (p = 0.033)]. There was a significantly higher median change in overall communication skills in the individualized compared to the non-specific group of 12 points and 6.5 points, respectively (p = 0.017). Student self-perceived confidence in the individualized and non-specific group showed similar improvements overall from pre- to post assessment [seven points vs. eight points, respectively (p = 0.62)]. SummaryThe use of SPs in pharmacy curricula can improve student communication skills. However, tailoring the activity to students' needs significantly improves their communication AOI and overall communication skill scores more than a non-specific activity. Student self-perceived confidence improved similarly in both groups, although no statistically significant differences were found between groups.

Psychoactive Substance Abuse in Adolescence and Social Work in the School: A Point a View from Professionals and Students in Castelo Branco, Portugal

This paper explores the relevance of preventive social work-focused programs implemented in schools in order to reduce psychoactive substance abuse by adolescents. The research was implemented in the city-district school of Castelo Branco, a Portuguese central inland territory. By using a qualitative research methodology, it was possible to learn about adolescent students’ life experience and their peers related with the drug abuse and their perceptions about their experience in school preventive social programs. Although this case study does not allow generalization, it confirms: (1) the existence of psychoactive substance abuse behaviors by young students in the school; (2) the social work intervention carried out in the school did not have the desired effects, because drug abuse continues to assume significant importance in this and in other schools. Given these conclusions, the authors emphasize the importance of nonspecific preventive activities at an early life stages that alter or prevent drug abuse through practices that do not focus only on drugs and consumption behaviors. The results argue the need to rethink the social work intervention programs carried out in elementary schools and to create new intervention forms that prioritize drug abuse prevention in a more holistic way. So this article is divided into four parts: purpose, method, results, and discussion.