Modeling SARS-CoV-2: Comparative Pathology in Rhesus Macaque and Golden Syrian Hamster Models.

Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.

Evaluation of the Association of Polymorphisms With Palbociclib-Induced Neutropenia: Pharmacogenetic Analysis of PALOMA-2/-3.

BACKGROUND: The most frequently reported treatment-related adverse event in clinical trials with the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib is neutropenia. Allelic variants in ABCB1 and ERCC1 might be associated with early occurrence (i.e., end of week 2 treatment) of grade 3/4 neutropenia. Pharmacogenetic analyses were performed to uncover associations between single nucleotide polymorphisms (SNPs) in these genes, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. MATERIALS AND METHODS: ABCB1 (rs1045642, rs1128503) and ERCC1 (rs3212986, rs11615) were analyzed in germline DNA from palbociclib-treated patients from PALOMA-2 (n = 584) and PALOMA-3 (n = 442). SNP, race, and cycle 1 day 15 (C1D15) absolute neutrophil count (ANC) data were available for 652 patients. Univariate and multivariable analyses evaluated associations between SNPs, patient baseline characteristics, and early occurrence of grade 3/4 neutropenia. Analyses were stratified by Asian (n = 122) and non-Asian (n = 530) ethnicity. Median progression-free survival (mPFS) was estimated using the Kaplan-Meier method. The effect of genetic variants on palbociclib pharmacokinetics was analyzed. RESULTS: ABCB1 and ERCC1_rs11615 SNP frequencies differed between Asian and non-Asian patients. Multivariable analysis showed that low baseline ANC was a strong independent risk factor for C1D15 grade 3/4 neutropenia regardless of race (Asians: odds ratio [OR], 6.033, 95% confidence interval [CI], 2.615-13.922, p < .0001; Non-Asians: OR, 6.884, 95% CI, 4.138-11.451, p < .0001). ABCB1_rs1128503 (C/C vs. T/T: OR, 0.57, 95% CI, 0.311-1.047, p = .070) and ERCC1_rs11615 (A/A vs. G/G: OR, 1.75, 95% CI, 0.901-3.397, p = .098) were potential independent risk factors for C1D15 grade 3/4 neutropenia in non-Asian patients. Palbociclib mPFS was consistent across genetic variants; exposure was not associated with ABCB1 genotype. CONCLUSION: This is the first comprehensive assessment of pharmacogenetic data in relationship to exposure to a CDK4/6 inhibitor. Pharmacogenetic testing may inform about potentially increased likelihood of patients developing severe neutropenia (NCT01740427, NCT01942135). IMPLICATIONS FOR PRACTICE: Palbociclib plus endocrine therapy improves hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer outcomes, but is commonly associated with neutropenia. Genetic variants in ABCB1 may influence palbociclib exposure, and in ERCC1 are associated with chemotherapy-induced severe neutropenia. Here, the associations of single nucleotide polymorphisms in these genes and baseline characteristics with neutropenia were assessed. Low baseline absolute neutrophil count was a strong risk factor (p < .0001) for grade 3/4 neutropenia. There was a trend indicating that ABCB1_rs1128503 and ERCC1_rs11615 were potential risk factors (p < .10) for grade 3/4 neutropenia in non-Asian patients. Pharmacogenetic testing could inform clinicians about the likelihood of severe neutropenia with palbociclib.

Exploring the Use of Serum-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Hepatic Cytochromes P450 and Organic Anion Transporting Polypeptides.

Liver-derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two-step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N = 3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold-increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5-4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1-6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22-0.34, P < 0.0001; and 0.17, 95% CI = 0.13-0.22, P < 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤ 1.5-fold). It was also possible to measure liver sEV-catalyzed dextromethorphan (DEX) O-demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r = 0.917, P = 0.0001; N = 10) and 3-hour plasma DXO-to-DEX concentration ratio (r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2-fold, P = 0.003) and CYP2D6 (3.7-fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

Dose-Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs.

To address the most appropriate endogenous biomarker for drug-drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate-3-glucuronide, glycochenodeoxycholate-3-sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose-dependent change in area under the plasma concentration-time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0-24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR-1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B-mediated drug-drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

A Solution Phase Platform to Characterize Chemical Reaction Compatibility with DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis. This approach includes the assessment of chemical fidelity (reaction yield and purity), encoding fidelity (ligation efficiency), and readability (DNA compatibility), revealing the fate of the DNA tag during DECL chemistry from a single platform.

Uncovering Hemocyanin Subunit Heterogeneity in Penaeid Shrimp using RNA-Seq.

Aquatic crustaceans can experience low levels of O2 alone but more often in combination with high levels of CO2 both in natural estuaries and in aquaculture ponds. Hemocyanin, the respiratory pigment in many crustacean species, facilitates O2 transport and is documented to change in abundance, structure, and function in response to low O2 The impacts of high CO2 on the respiratory pigment are less clear. In this synthesis we bring together data from recently published and new RNA-Seq studies toward the aims of defining the full repertoire of hemocyanin subunits, as well as their differential expression and regulation in the Penaeoidea family in response to low O2 with or without high CO2 RNA-Seq data were collected from the hepatopancreas tissues of aquacultured Pacific whiteleg shrimp Litopenaeus vannamei, wild-caught L. vannamei, and wild-caught Atlantic brown shrimp Farfantepenaeus aztecus. De novo assembly yielded high-quality stranded transcriptomes. Manual curation of the hemocyanin subunits from all three groups of penaeid shrimp confirmed the existence of a small γ-type hemocyanin subunit (HcS), greater sequence diversity in the large γ-type hemocyanin subunit than previously identified (HcL1-3 isoforms) and expression of a ß-type hemocyanin subunit (HcB) previously unidentified in Penaeid shrimp. Relative abundance of transcripts encoding these hemocyanin isoforms differed within and among the three species/strains. Exposure to low O2 induced expression of all of the subunits in aquacultured L. vannamei With concurrent exposure to high CO2, the number of γ-type hemocyanin transcripts decreased while the expression of ß-type transcripts remained unchanged. Together with functional data for hemocyanins in the same shrimp species/strains, the RNA-Seq approach shows great promise to provide new insights into the connection between sequence, protein structure, and physiological function of respiratory pigments in this decapod crustacean family.

High CO2 alters the hypoxia response of the Pacific whiteleg shrimp (Litopenaeus vannamei) transcriptome including known and novel hemocyanin isoforms.

Acclimation to low O2 in many organisms involves changes at the level of the transcriptome. Here we used high-throughput RNA sequencing (RNA-Seq) to explore the global transcriptomic response and specific involvement of a suite of hemocyanin (Hc) subunits to low O2 alone and in combination with high CO2, which naturally co-occurs with low O2. Hepatopancreas mRNA of juvenile L. vannamei exposed to air-saturated water, low O2, or low O2/high CO2 for 4 or 24 h was pooled, sequenced (HiSeq 2500) and assembled (Trinity: 52,190 contigs) to create a deep strand-specific reference transcriptome. Annotation of the assembly revealed sequences encoding the previously described small Hc subunit (HcS), and three full-length isoforms of the large subunit (HcL1-3). In addition to this, a previously unidentified full-length Hc subunit was discovered. Phylogenetic analysis demonstrated the subunit to be a ß-type Hc subunit (denoted HcB), making this the first report of a ß-type hemocyanin subunit in the Penaeoidea. RNAs of individual shrimp were sequenced; regulated genes identified from pairwise comparisons demonstrated a distinct pattern of regulation between prolonged low O2 and low O2/high CO2 treatments by GO term enrichment analysis (Roff-Bentzen, P < 0.0001), showcasing the stabilization of energetically costly translational machinery, mobilization of energy stores, and downregulation of the ubiquitin/proteasomal degradation machinery. Exposure to hypoxia for 24 h resulted in an increase in all of the full-length hemocyanin subunits (HcS, HcL1, HcL2, HcL3, and HcB). The addition of CO2 to hypoxia muted the transcriptomic response of all the Hc subunits to low O2, except for the ß-type subunit.

Caspase-like activity during aging and cell death in the toxic dinoflagellate Karenia brevis.

The observation of caspase-like activity during cell death has provided a new framework for understanding the evolutionary and ecological contexts of programmed cell death in phytoplankton. However, additional roles for this caspase-like activity, the enzymes responsible, and the targets of this enzyme activity in phytoplankton remain largely undefined. In the present study, the role of caspase-like activity in aging and ROS-mediated cell death were investigated and death programs both dependent on and independent of caspase-like activity were observed in the toxic dinoflagellate, Karenia brevis. The dual use of in situ caspase 3/7 and TUNEL staining identified previously undescribed death-associated morphotypes in K. brevis. In silico motif analysis identified several enzymes with predicted caspase-like activity in the K. brevis transcriptome, although bona fide caspases are absent. Lastly, computational prediction of downstream caspase substrates, using sequence context and predicted secondary structure, identified proteins involved in a wide range of biological processes including regulation of protein turnover, cell cycle progression, lipid metabolism, coenzyme metabolism, apoptotic and autophagic death. To confirm the computational predictions, a short peptide was designed around the predicated caspase cleavage site in a predicted novel K. brevis caspase 3/7-like target, S-adenosylmethionine synthetase (KbAdoMetS). Cleavage of the peptide substrate with recombinant caspase 3 enzyme was determined by MALDI-TOF MS, confirming that KbAdoMetS is indeed a bona fide caspase substrate. These data identify the involvement of caspase-like activity in both aging and cell death in K. brevis and identify novel executioner enzymes and downstream targets that may be important for bloom termination.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.

Transcriptome remodeling associated with chronological aging in the dinoflagellate, Karenia brevis.

The toxic dinoflagellate, Karenia brevis, forms dense blooms in the Gulf of Mexico that persist for many months in coastal waters, where they can cause extensive marine animal mortalities and human health impacts. The mechanisms that enable cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. To gain an understanding of processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out to identify changes in the global transcriptome that accompany the entry and maintenance of stationary phase up to the onset of cell death. The transcriptome of K. brevis was assayed using a custom 10,263 feature oligonucleotide microarray from mid-logarithmic growth to the onset of culture demise. A total of 2958 (29%) features were differentially expressed, with the mid-stationary phase timepoint demonstrating peak changes in expression. Gene ontology enrichment analyses identified a significant shift in transcripts involved in energy acquisition, ribosome biogenesis, gene expression, stress adaptation, calcium signaling, and putative brevetoxin biosynthesis. The extensive remodeling of the transcriptome observed in the transition into a quiescent non-dividing phase appears to be indicative of a global shift in the metabolic and signaling requirements and provides the basis from which to understand the process of chronological aging in a dinoflagellate.

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition.

BACKGROUND: The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' trans-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of K. brevis is responsive to nitrogen and phosphorus and is informative of nutrient status. RESULTS: Microarray analysis of N-depleted K. brevis cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. CONCLUSIONS: Microarray analysis provided transcriptomic evidence for N- but not P-limitation in K. brevis. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.