Epitranscriptomic regulation in fasting hearts: implications for cardiac health.

Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Safety and efficacy of nivolumab plus ipilimumab in patients with advanced renal cell carcinoma with brain metastases: CheckMate 920.

BACKGROUND: Nivolumab plus ipilimumab (NIVO + IPI) has demonstrated long-term efficacy and safety in patients with previously untreated, advanced renal cell carcinoma (aRCC). Although most phase 3 clinical trials exclude patients with brain metastases, the ongoing, multicohort phase 3b/4 CheckMate 920 trial (ClincalTrials.gov identifier NCT02982954) evaluated the safety and efficacy of NIVO + IPI in a cohort that included patients with aRCC and brain metastases, as reported here. METHODS: Patients with previously untreated aRCC and asymptomatic brain metastases received NIVO 3 mg/kg plus IPI 1 mg/kg every 3 weeks × 4 followed by NIVO 480 mg every 4 weeks. The primary end point was the incidence of grade ≥3 immune-mediated adverse events (imAEs) within 100 days of the last dose of study drug. Key secondary end points were progression-free survival and the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1 (both determined by the investigator). Exploratory end points included overall survival, among others. RESULTS: After a minimum follow-up of 24.5 months (N = 28), no grade 5 imAEs occurred. The most common grade 3 and 4 imAEs were diarrhea/colitis (n = 2; 7%) and hypophysitis, rash, hepatitis, and diabetes mellitus (n = 1 each; 4%). The objective response rate was 32% (95% CI, 14.9%-53.5%) with a median duration of response of 24.0 months; 4 of 8 responders remained without reported progression. Seven patients (25%) had intracranial progression. The median progression-free survival was 9.0 months (95% CI, 2.9-12.0 months), and the median overall survival was not reached (95% CI, 14.1 months to not estimable). CONCLUSIONS: In patients who had previously untreated aRCC and brain metastases-a population with a high unmet medical need that often is underrepresented in clinical trials-the approved regimen of NIVO + IPI followed by NIVO showed encouraging antitumor activity and no new safety signals.

T3P-Promoted Synthesis of a Series of 2-Aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones and Their Activity against the Kinetoplastid Parasite Trypanosoma brucei.

A series of fourteen 2-aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones was prepared at room temperature by T3P-mediated cyclization of N-phenyl-C-aryl imines with thionicotinic acid, two difficult substrates. The reactions were operationally simple, did not require specialized equipment or anhydrous solvents, could be performed as either two or three component reactions, and gave moderate-good yields as high as 63%. This provides ready access to N-phenyl compounds in this family, which have been generally difficult to prepare. As part of the study, the first crystal structure of neutral thionicotinic acid is also reported, and showed the molecule to be in the form of the thione tautomer. Additionally, the synthesized compounds were tested against T. brucei, the causative agent of Human African Sleeping Sickness. Screening at 50 µM concentration showed that five of the compounds strongly inhibited growth and killed parasites.

Functional assessments of foot strength: a comparative and repeatability study.

BACKGROUND: Evaluating the strength of the small muscles of the foot may be useful in a variety of clinical applications but is challenging from a methodology standpoint. Previous efforts have focused primarily on the functional movement of toe flexion, but clear methodology guidelines are lacking. A novel foot doming test has also been proposed, but not fully evaluated. The purposes of the present study were to assess the repeatability and comparability of several functional foot strength assessment techniques. METHODS: Forty healthy volunteers were evaluated across two testing days, with a two-week doming motion practice period between them. Seven different measurements were taken using a custom toe flexion dynamometer (seated), custom doming dynamometer (standing), and a pressure mat (standing). Measurements from the doming dynamometer were evaluated for reliability (ICCs) and a learning effect (paired t-tests), while measurements from the toe flexion dynamometer and pressure mat were evaluated for reliability and comparability (correlations). Electromyography was also used to descriptively assess the extent of muscle isolation in all measurements. RESULTS: Doming showed excellent within-session reliability (ICCs > 0.944), but a clear learning effect was present, with strength (p < 0.001) and muscle activity increasing between sessions. Both intrinsic and extrinsic muscles were engaged during this test. All toe flexion tests also showed excellent reliability (ICCs > 0.945). Seated toe flexion tests using the dynamometer were moderately correlated to standing toe flexion tests on a pressure mat (r > 0.54); however, there were some differences in muscle activity. The former may better isolate the toe flexors, while the latter appeared to be more functional for many pathologies. On the pressure mat, reciprocal motion appeared to display slightly greater forces and reliability than isolated toe flexion. CONCLUSIONS: This study further refines potential methodology for foot strength testing. These devices and protocols can be duplicated in the clinic to evaluate and monitor rehabilitation progress in clinical populations associated with foot muscle weakness.

Recent decline in extratropical lower stratospheric ozone attributed to circulation changes.

1998-2016 ozone trends in the lower stratosphere (LS) are examined using the Modern-Era Retrospective Analysis for Research and Applications Version 2 (MERRA-2) and related NASA products. After removing biases resulting from step-changes in the MERRA-2 ozone observations, a discernible negative trend of -1.67±0.54 Dobson units per decade (DU/decade) is found in the 10-km layer above the tropopause between 20°N and 60°N. A weaker but statistically significant trend of -1.17±0.33 DU/decade exists between 50°S and 20°S. In the Tropics, a positive trend is seen in a 5-km layer above the tropopause. Analysis of an idealized tracer in a model simulation constrained by MERRA-2 meteorological fields provides strong evidence that these trends are driven by enhanced isentropic transport between the tropical (20°S-20°N) and extratropical LS in the past two decades. This is the first time that a reanalysis dataset has been used to detect and attribute trends in lower stratospheric ozone.

Aspartate ß-hydroxylase modulates cellular senescence through glycogen synthase kinase 3ß in hepatocellular carcinoma.

UNLABELLED: Aspartate ß-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second-generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3ß and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3ß and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3ß, enhanced p16 expression in tumor cells, and promoted cellular senescence. CONCLUSIONS: We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC.

The Forty-Sixth Euro Congress on Drug Synthesis and Analysis: Snapshot †.

The 46th EuroCongress on Drug Synthesis and Analysis (ECDSA-2017) was arranged within the celebration of the 65th Anniversary of the Faculty of Pharmacy at Comenius University in Bratislava, Slovakia from 5-8 September 2017 to get together specialists in medicinal chemistry, organic synthesis, pharmaceutical analysis, screening of bioactive compounds, pharmacology and drug formulations; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topic of the conference, "Drug Synthesis and Analysis," meant that the symposium welcomed all pharmacists and/or researchers (chemists, analysts, biologists) and students interested in scientific work dealing with investigations of biologically active compounds as potential drugs. The authors of this manuscript were plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

The Length of Child Anxiety Treatment in a Regional Health System.

Anxiety disorders are often undertreated due to unsuccessful dissemination of evidence-based treatments (EBTs). Lack of empirical data regarding the typical length of treatment in clinical settings may hamper the development of clinically relevant protocols. The current study examined billing records for 335 children ages 7-17 years to quantify the treatment received for newly diagnosed anxiety disorders within a regional health system. The vast majority of patients did not receive a sufficient number of appointments to complete the typical cognitive behavioral therapy protocol or reach the sessions introducing exposure. Although half of the sample received pharmacotherapy, the vast majority received fewer follow-up appointments than participants in pharmacotherapy research studies. Further, the type of treatment (i.e., number of sessions and medication) differed depending on utilization of specialty care. These results underscore the need to develop brief and flexible EBT protocols that can be standardized and implemented in community practice.

The Eighth Central European Conference "Chemistry towards Biology": Snapshot.

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on August 28-September 1, 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

A cell-surface ß-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-ß-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of ß-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. CONCLUSIONS: These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced ß-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

Confinement dependent chemotaxis in two-photon polymerized linear migration constructs with highly definable concentration gradients.

Dendritic cell chemotaxis is known to follow chemoattractant concentration gradients through tissue of heterogeneous pore sizes, but the dependence of migration velocity on pore size and gradient steepness is not fully understood. We enabled chemotaxis studies for at least 42 hours at confinements relevant to tissue models by two-photon polymerization of linear channel constructs with cross-sections from 10 × 10 µm(2) to 20 × 20 µm(2) inside commercially available chemotaxis analysis chips. Faster directed migration was observed with decreasing channel dimensions despite substantial cell deformation in the narrower channels. Finite element modeling of a cell either partly or fully obstructing chemokine diffusion in the narrow channels revealed strong local accentuation of the chemokine concentration gradients. The modeled concentration differences across a cell correlated well with the observed velocity dependence on channel cross-section. However, added effects due to spatial confinement could not be excluded. The design freedom offered by two-photon polymerization was exploited to minimize the accentuated concentration gradients in cell-blocked channels by introducing "venting slits" to the surrounding medium at a length scale too small (≤500 nm) for the cells to explore, thereby decoupling effects of concentration gradients and spatial confinement. Studies in slitted 10 × 10 µm(2) channels showed significantly reduced migration speeds indistinguishable from speeds observed in unslitted 20 × 20 µm(2) channel. This result agrees with model predictions of very small concentration gradient variations in slitted channels, thus indicating a strong influence of the concentration gradient steepness, not the channel size, on the directed migration velocity.

Piperidine-3,4-diol and piperidine-3-ol derivatives of pyrrolo[2,1-f][1,2,4]triazine as inhibitors of anaplastic lymphoma kinase.

The diastereoselective synthesis and biological activity of piperidine-3,4-diol and piperidine-3-ol-derived pyrrolotriazine inhibitors of anaplastic lymphoma kinase (ALK) are described. Although piperidine-3,4-diol and piperidine-3-ol derivatives showed comparable in vitro ALK activity, the latter subset of inhibitors demonstrated improved physiochemical and pharmacokinetic properties. Furthermore, the stereochemistry of the C3 and C4 centers had a marked impact on the in vivo inhibition of ALK autophosphorylation. Thus, trans-4-aryl-piperidine-3-ols (22) were more potent than the cis diastereomers (20).

Aspartate ß-hydroxylase Regulates Expression of Ly6 Genes.

Background: Overexpression of aspartate ß-hydroxylase (ASPH) in human tumors contributes to their progression by stimulating cell proliferation, migration, and invasion. Several signaling pathways affected by ASPH have been identified, but the high number of potential targets of ASPH hydroxylation suggests that additional mechanisms may be involved. This study was performed to reveal new targets of ASPH signaling. Methods: The effect of ASPH on the oncogenicity of three mouse tumor cell lines was tested using proliferation assays, transwell assays, and spheroid invasion assays after inhibition of ASPH with the small molecule inhibitor MO-I-1151. ASPH was also deactivated with the CRISPR/Cas9 system. A transcriptomic analysis was then performed with bulk RNA sequencing and differential gene expression was evaluated. Expression data were verified by quantitative PCR and immunoblotting. Results: Inhibition or abrogation of ASPH reduced proliferation of the cell lines and their migration and invasiveness. Among the genes with differential expression in more than one cell line, two members of the lymphocyte antigen 6 (Ly6) family, Ly6a and Ly6c1, were found. Their downregulation was confirmed at the protein level by immunoblotting, which also showed their reduction after ASPH inhibition in other mouse cell lines. Reduced production of the Ly6D and Ly6K proteins was shown after ASPH inhibition in human tumor cell lines. Conclusions: Since increased expression of Ly6 genes is associated with the development and progression of both mouse and human tumors, these results suggest a novel mechanism of ASPH oncogenicity and support the utility of ASPH as a target for cancer therapy.

Heterogeneous Response of Tumor Cell Lines to Inhibition of Aspartate ß-hydroxylase.

Background: Cancer development involves alterations in key cellular pathways, with aspartate ß-hydroxylase (ASPH) emerging as an important player in tumorigenesis. ASPH is upregulated in various cancer types, where it promotes cancer progression mainly by regulating the Notch1 and SRC pathways. Methods: This study explored the responses of various human cervical, pharyngeal, and breast tumor cell lines to second- and third-generation ASPH inhibitors (MO-I-1151 and MO-I-1182) using proliferation, migration, and invasion assays; western blotting; and cell cycle analysis. Results: ASPH inhibition significantly reduced cell proliferation, migration, and invasion and disrupted both the canonical and noncanonical Notch1 pathways. The noncanonical pathway was particularly mediated by AKT signaling. Cell cycle analysis revealed a marked reduction in cyclin D1 expression, further confirming the inhibitory effect of ASPH inhibitors on cell proliferation. Additional analysis revealed G0/G1 arrest and restricted progression into S phase, highlighting the regulatory impact of ASPH inhibitors on the cell cycle. Furthermore, ASPH inhibition induced distinctive alterations in nuclear morphology. The high heterogeneity in the responses of individual tumor cell lines to ASPH inhibitors, both quantitatively and qualitatively, underscores the complex network of mechanisms that are regulated by ASPH and influence the efficacy of ASPH inhibition. The effects of ASPH inhibitors on Notch1 pathway activity, cyclin D1 expression, and nuclear morphology contribute to the understanding of the multifaceted effects of these inhibitors on cancer cell behavior. Conclusion: This study not only suggests that ASPH inhibitors are effective against tumor cell progression, in part through the induction of cell cycle arrest, but also highlights the diverse and heterogeneous effects of these inhibitors on the behavior of tumor cells of different origins.

Synthesis and crystal structures of two racemic 2-heteroaryl-3-phenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-ones.

3-Phenyl-2-(thio-phen-3-yl)-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one (C17H12N2OS2, 1) and 2-(1H-indol-3-yl)-3-phenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one 0.438-hydrate (C21H15N3OS·0.438H2O, 2) crystallize in space groups P21/n and C2/c, respectively. The asymmetric unit in each case is comprised of two parent mol-ecules, albeit of mixed chirality in the case of 1 and of similar chirality in 2 with the enanti-omers occupying the neighboring asymmetric units. Structure 2 also has water mol-ecules (partial occupancies) that form continuous channels along the b -axis direction. The thia-zine rings in both structures exhibit an envelope conformation. Inter-molecular inter-actions in 1 are defined only by C-H⋯O and C-H⋯N hydrogen bonds between crystallographically independent mol-ecules. In 2, hydrogen bonds of the type N-H⋯O between independent mol-ecules and C-H⋯N(π) type, and π-π stacking inter-actions between the pyridine rings of symmetry-related mol-ecules are observed.

Parent-coached exposure therapy versus cognitive behavior therapy for childhood anxiety disorders.

Cognitive-behavioral therapy (CBT) is the strongest evidenced-based therapy for childhood anxiety disorders (CADs). However, CBT's impact is limited by its lack of clear superiority over treatment as usual, excessive length, and greater than 50% of patients remaining symptomatic. Parent-coached exposure therapy (PCET) is designed to treat CADs more effectively and efficiently through a focus on exposure and working with parents and youth together. In a randomized controlled trial, 78 patients (78% female) aged 7 to 17 with CADs were assigned to PCET or the gold-standard CBT. The primary outcome was independent evaluator ratings of anxiety severity at mid- and post-treatment. Secondary outcomes were parent- and child-reported symptoms. Patients receiving PCET had significantly lower mean scores than those receiving CBT on the primary outcome measure at mid-treatment (3.03 ± 0.14, 95% CI, 2.75-3.32 vs. 3.77 ± 0.16 95% CI, 3.45-4.08, p = 0.0010) and post-treatment (2.79 ± 0.14, 95% CI, 2.50-3.07 vs. 3.33 ± 0.16, 95% CI, 2.02-3.64, p = 0.0153). Similar significant results were found with the secondary parent- and child-reported outcomes. These superior results were achieved in PCET with fewer sessions (6.62, SD = 2.8) than those in CBT (8.00, SD = 3.1), p = 0.041. The superior effectiveness and efficiency of PCET likely results from the greater focus on implementing exposure exercises compared to traditional CBT.

Crystal structures of the sulfones of 2,3-diphenyl-2,3-di-hydro-4H-1,3-benzo-thia-zin-4-one and 2,3-diphenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one.

The title sulfones, 2,3-diphenyl-2,3-di-hydro-4H-1,3-benzo-thia-zine-1,1,4-trione, C20H15NO3S, and 2,3-diphenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zine-1,1,4-trione, C19H14N2O3S, crystallize in space group P21/n with two mol-ecules in each of the asymmetric units and have almost identical unit cells and extended structures. In both structures, the thia-zine rings exhibit a screw-boat pucker. The inter-molecular inter-actions observed are C-H⋯O-type hydrogen bonds and parallel partial π-π stacking between the fused aromatic rings (benzo- or pyrido-) of the core of the mol-ecules within each asymmetric unit, and also connecting to mol-ecules with translational periodicity in the a-axis direction in what can be described as columns (two per asymmetric unit) of stacked mol-ecules with alternating chirality. The pendant phenyl groups of both mol-ecules do not participate in aromatic ring inter-actions.

Sub-100 nm Nanoparticle Upconcentration in Flow by Dielectrophoretic Forces.

This paper presents a novel microfluidic chip for upconcentration of sub-100 nm nanoparticles in a flow using electrical forces generated by a DC or AC field. Two electrode designs were optimized using COMSOL Multiphysics and tested using particles with sizes as low as 47 nm. We show how inclined electrodes with a zig-zag three-tooth configuration in a channel of 20 µm width are the ones generating the highest gradient and therefore the largest force. The design, based on AC dielectrophoresis, was shown to upconcentrate sub-100 nm particles by a factor of 11 using a flow rate of 2-25 µL/h. We present theoretical and experimental results and discuss how the chip design can easily be massively parallelized in order to increase throughput by a factor of at least 1250.

Investigation of monotherapy and combined anticoronaviral therapies against feline coronavirus serotype II in vitro.

OBJECTIVES: Feline infectious peritonitis (FIP), caused by genetic mutants of feline enteric coronavirus known as FIPV, is a highly fatal disease of cats with no currently available vaccine or US Food and Drug Administration-approved cure. Dissemination of FIPV in affected cats results in a range of clinical signs, including cavitary effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and perivasculitis, with or without central nervous system or ocular involvement. The objectives of this study were to screen an array of antiviral compounds for anti-FIPV (serotype II) activity, determine cytotoxicity safety profiles of identified compounds with anti-FIPV activity and strategically combine identified monotherapies to assess compound synergy against FIPV in vitro. Based upon clinically successful combination treatment strategies for human patients with HIV and hepatitis C virus infections, we hypothesized that a combined anticoronaviral therapy approach featuring concurrent multiple mechanisms of drug action would result in an additive or synergistic antiviral effect. METHODS: This study screened 90 putative antiviral compounds for efficacy and cytotoxicity using a multimodal in vitro strategy, including plaque bioassays, real-time RT-PCR viral inhibition and cytotoxicity assays. RESULTS: Through this process, we identified 26 compounds with effective antiviral activity against FIPV, representing a variety of drug classes and mechanisms of antiviral action. The most effective compounds include GC376, GS-441524, EIDD2081 and EIDD2931. We documented antiviral efficacy for combinations of antiviral agents, with a few examined drug combinations demonstrating evidence of limited synergistic antiviral activity. CONCLUSIONS AND RELEVANCE: Although evidence of compound synergy was identified for several combinations of antiviral agents, monotherapies were ultimately determined to be the most effective in the inhibition of viral transcription.