Novel non-linear models for clinical trial analysis with longitudinal data: A tutorial using SAS for both frequentist and Bayesian methods.

Longitudinal data from clinical trials are commonly analyzed using mixed models for repeated measures (MMRM) when the time variable is categorical or linear mixed-effects models (ie, random effects model) when the time variable is continuous. In these models, statistical inference is typically based on the absolute difference in the adjusted mean change (for categorical time) or the rate of change (for continuous time). Previously, we proposed a novel approach: modeling the percentage reduction in disease progression associated with the treatment relative to the placebo decline using proportional models. This concept of proportionality provides an innovative and flexible method for simultaneously modeling different cohorts, multivariate endpoints, and jointly modeling continuous and survival endpoints. Through simulated data, we demonstrate the implementation of these models using SAS procedures in both frequentist and Bayesian approaches. Additionally, we introduce a novel method for implementing MMRM models (ie, analysis of response profile) using the nlmixed procedure.

Assessment of health risks associated with prediction of vegetable inorganic arsenic concentrations given different soil properties.

Inorganic arsenic (iAs) is a carcinogen. Vegetables such as water spinach (Ipomoea aquatica Forssk.) and amaranth (Amaranthus mangostanus L.) are recognized as high-risk sources of iAs exposure because they can accumulate significant amounts of iAs and are widely consumed. To ensure safe cultivation conditions, this study aimed to establish prediction models for iAs concentration in the edible parts of water spinach and amaranth based on soil properties. Subsequently, health risk assessments associated with iAs exposure through the consumption of these vegetables were conducted using prediction models. Soil samples were collected from agricultural fields in Taiwan and used in the pot experiments. Pearson correlation and partial correlation analyses were used to explore the relationship between soil properties, including total As, clay, organic matter, iron oxides and available phosphates, and iAs concentration in edible parts of water spinach and amaranth. Prediction models based on soil properties were developed by stepwise multiple linear regression. Health risk assessments were conducted using the Monte Carlo algorithm. The results indicate that total As and organic matter contents in soil were major predictors of iAs concentration in water spinach, whereas those in amaranth were total As and clay contents. Therefore, higher health risks for consuming water spinach and amaranth are associated with higher levels of organic matter and clay contents in soil, respectively, and these are crucial factors to consider to ensure food safety. This study suggested that As-elevated soils enriched with organic matter and clay contents should be avoided when growing water spinach and amaranth, respectively.

Identification of potential SLA-I-specific T-cell epitopes within the structural proteins of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that mainly threatens newborn piglets and poses a potential broad cross-species transmission risk. The antigenic epitopes of PDCoV are currently unidentified, and no information about T cell epitopes is available. Here, T-cell epitopes of PDCoV structural proteins were predicted using computational methods. 17 epitope peptides were synthesized and then screened using ELIspot, intracellular cytokine staining (ICS), and RT-qPCR detection of IFN-γ mRNA to evaluate their ability to elicit interferon-gamma (IFN-γ) responses in peripheral blood mononuclear cells (PBMCs) from PDCoV-challenged pigs. Five peptides (M1, M2, M3, N6, and S4) elicited high levels of IFN-γ and were investigated further as potential T-cell epitope candidates. All five peptides were cytotoxic T lymphocyte (CTL) epitopes, and two peptides (M3, N6) were recognized simultaneously by CD8 + and CD4 + T cells. A multi-epitope peptide combining the five epitopes (designated "5T") was synthesized and its immune response and protection efficacy was evaluated in a piglet model. ELISpot assay results indicated that 5T induces robust epitope-specific cellular immune responses. Four epitopes (M1, M2, N6, S4) elicited IFN-γ responses in 5T-vaccinated piglets. No obvious protection efficacy was detected in piglets vaccinated with 5T alone. Our results provide valuable information concerning PDCoV-related antigenic epitopes and will be useful in the design of epitope-based vaccines.

Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus.

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Field experiments for evaluating the effects of water management and phosphate application on inorganic arsenic accumulation in water spinach (Ipomoea aquatica Forssk.).

Water spinach (Ipomoea aquatica Forssk.) is a commonly planted vegetable in the Southeast Asia; it is a semi-aquatic leafy vegetable with high inorganic arsenic (As) accumulation capability and can be planted under both upland and flooding cultivation conditions. To date, a limited number of field studies have investigated the effect of soil management on As phytotoxicity and accumulation of water spinach. Therefore, in this study, a field experiment was conducted to investigate the effects of water management and phosphate (P) application on the As phytotoxicity and accumulation of water spinach grown in As-contaminated fields (121 mg As kg-1). Water spinach was planted in the study field with two water management (flooding and upland cultivation) and two P application rates (90 and 180 kg P2O5 ha-1), and continuously harvested three times. Results reveal that the concentration and estimated daily intake (EDI) of inorganic As in the edible parts of water spinach under flooding cultivation were approximately twofold higher than those under upland cultivation. It was also found that the accumulation of As in the shoot of water spinach was strongly related to the As concentrations, rather than P/As molar ratio in pore water due to that P application rates were lower than the maximum capacity for P retention of the tested soil. Moreover, the As phytotoxicity and accumulation of water spinach were reduced at the third harvest relative to the first two harvests because of the increase in iron plaque formation on the root surface and the decrease in the growing temperature during the experimental period. Our results suggest that upland cultivation is the better practice than flooding cultivation for reducing inorganic As accumulation in the edible parts of water spinach grown in As-contaminated soils. Further, ratooning may be a feasible cultivation approach to reducing inorganic As accumulation in water spinach.

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis.

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-ß-cyclodextrin (MßCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Study of the inhibitory effect of STAT1 on PDCoV infection.

Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT, STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.

HSP90 inhibitors 17-AAG and VER-82576 inhibit porcine deltacoronavirus replication in vitro.

Porcine deltacoronavirus (PDCoV) is highly pathogenic to piglets, and no specific drugs or vaccines are available for the prevention and treatment of PDCoV infection, the need for antiviral therapies is pressing. HSP90 inhibitors have potent inhibitory effects against the replication of numerous viruses, hence we evaluated three HSP90 inhibitors, 17-AAG, VER-82576, and KW-2478, for their effects on PDCoV infection in vitro. We evaluated their effectivenesses at suppressing PDCoV by qRT-PCR, western blot, and TCID50 assay, and found that 17-AAG and VER-82576 inhibited PDCoV at the early stage of replication, while KW-2478 showed no significant antiviral activity at any stage of infection. These results indicated that the PDCoV-inhibitory effects of 17-AAG and VER-82576 might be exerted by targeting host cell factor HSP90AB1 but not HSP90AA1. Further study showed that HSP90AB1 mRNA and protein levels were not significantly different in 17-AAG and VER-82576-treated cells versus control cells. 17-AAG and VER-82576 were also evaluated for their effects on the expressions of TNF-α, IL-6, and IL-12, which are PDCoV-induced proinflammatory cytokines. We found that both 17-AAG and VER-82576 inhibited the expressions of TNF-α, IL-6, and IL-12 to varying degrees, but in a dose dependent manner. From our data we can conclude that the HSP90 inhibitors 17-AAG and VER-82576 are promising candidates for the treatment of PDCoV infection.

Intensity-dependent angular distribution of low-energy electrons generated by intense high-frequency laser pulse.

By solving the three-dimensional time-dependent Schrödinger equation, we investigate the angular distributions of the low-energy electrons when an intense high-frequency laser pulse is applied to the hydrogen atom. Our numerical results show that the angular distributions of the low-energy electrons which generated by the nonadiabatic transitions sensitively depend on the laser intensity. The angular distributions evolve from a two-lobe to a four-lobe structure as the laser intensity increases. By analyzing nonadiabatic process in the Kramers-Henneberger frame, we illustrate that this phenomenon is attributed to the intensity-dependent adiabatic evolution of the ground state wavefunction. When the laser intensity further increases, the pathway of nonadiabatic transition from the ground state to the excited state and then to the continuum states is non-negligible, which results in the ring-like structure in the photoelectron momentum distribution. The angular distributions of the low-energy electrons provide a way to monitor the evolution of the electron wavefunction in the intense high frequency laser fields.

Comparison of As accumulation and speciation in water spinach (Ipomoea aquatica Forssk.) grown in As-elevated soils under flooding versus upland conditions.

Water spinach (Ipomoea aquatica Forssk.) is a popular semi-aquatic vegetable with high arsenic (As) accumulation capability in tropical and subtropical areas, and it can grow under both flooding and upland conditions. Because only few studies addressed the dietary As risk of vegetable consumption, this study investigated the accumulation and speciation of As in the water spinach, and the human health risk of consuming this crop grown in different water managements. Pot experiments were conducted in two soils with different As levels under both flooded and upland conditions. The results showed that As concentrations, mainly inorganic As, in soil pore water and shoots under flooded conditions increased 2.05- to 5.11-fold and 1.30- to 2.55-fold respectively as compared with upland conditions. Even though Fe plaque formation on the root surface was higher under flooded conditions than under upland conditions, it still cannot act as an effective barrier to sequester As released from flooded soils into plant tissue. The dietary risk of consuming inorganic As through water spinach planted in flooded soils was higher than in upland soils, especially in high-As-level soils. This suggests that in As-elevated soils, water spinach should be planted in upland conditions rather than in flooding conditions.

Bempegaldesleukin (NKTR-214) plus Nivolumab in Patients with Advanced Solid Tumors: Phase I Dose-Escalation Study of Safety, Efficacy, and Immune Activation (PIVOT-02).

This single-arm, phase I dose-escalation trial (NCT02983045) evaluated bempegaldesleukin (NKTR-214/BEMPEG), a CD122-preferential IL2 pathway agonist, plus nivolumab in 38 patients with selected immunotherapy-naïve advanced solid tumors (melanoma, renal cell carcinoma, and non-small cell lung cancer). Three dose-limiting toxicities were reported in 2 of 17 patients during dose escalation [hypotension (n = 1), hyperglycemia (n = 1), metabolic acidosis (n = 1)]. The most common treatment-related adverse events (TRAE) were flu-like symptoms (86.8%), rash (78.9%), fatigue (73.7%), and pruritus (52.6%). Eight patients (21.1%) experienced grade 3/4 TRAEs; there were no treatment-related deaths. Total objective response rate across tumor types and dose cohorts was 59.5% (22/37), with 7 complete responses (18.9%). Cellular and gene expression analysis of longitudinal tumor biopsies revealed increased infiltration, activation, and cytotoxicity of CD8+ T cells, without regulatory T-cell enhancement. At the recommended phase II dose, BEMPEG 0.006 mg/kg plus nivolumab 360 mg every 3 weeks, the combination was well tolerated and demonstrated encouraging clinical activity irrespective of baseline PD-L1 status. SIGNIFICANCE: These data show that BEMPEG can be successfully combined with a checkpoint inhibitor as dual immunotherapy for a range of advanced solid tumors. Efficacy was observed regardless of baseline PD-L1 status and baseline levels of tumor-infiltrating lymphocytes, suggesting therapeutic potential for patients with poor prognostic risk factors for response to PD-1/PD-L1 blockade.See related commentary by Rouanne et al., p. 1097.This article is highlighted in the In This Issue feature, p. 1079.