Alpha to Omicron: Disease Severity and Clinical Outcomes of Major SARS-CoV-2 Variants.

BACKGROUND: Four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. METHOD: Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. RESULTS: In total 2779 patients identified with either Alpha (n 1153), Gamma (n 122), Delta (n 808), or Omicron variants (n 696) were selected for analysis. No difference in frequency of hospitalization, intensive care unit (ICU) admission, and death were found among Alpha, Gamma, and Delta variants. However, patients with Omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (2 12.8, P .001; 2 21.6, P .002; 2 9.6, P .01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with Delta or Omicron variants (218/808 [26.9] and 513/696 [73.7], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between Omicron subvariants BA.1 and BA.2. CONCLUSIONS: Disease severity associated with Alpha, Gamma, and Delta variants is comparable while Omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with Omicron infection.

Intra-host mutation rate of acute SARS-CoV-2 infection during the initial pandemic wave.

SARS-CoV-2 mutation is minimized through a proofreading function encoded by NSP-14. Most estimates of the SARS-CoV-2 mutation rate are derived from population based sequence data. Our understanding of SARS-CoV-2 evolution might be enhanced through analysis of intra-host viral mutation rates in specific populations. Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) ≥ 0.25, ≥ 0.5 and ≥ 0.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with (ΔNSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI 90.8-96.4], 40.7 (95%CI 38.9-42.6) and 34.7 (95%CI 33.0-36.4) substitutions/genome/year at AF ≥ 0.25, ≥ 0.5, ≥ 0.75 respectively. Mutation rate in ΔNSP-14 were significantly elevated at AF ≥ 0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.

Novel spirooxindole alkaloid derivatives from the medicinal insect Blaps japanensis and their biological evaluation.

Blapspirooxindoles A-C (1-3), three novel spirooxindole alkaloids with a unique spiro[chromane-4,3'-indoline]-2,2'-dione motif, blapcumaranons A and B (4 and 5), two new 2-cumaranon derivatives, blapoxindoles A-J (6-15), ten new oxindole alkaloid derivatives, along with one known compound (16), were isolated from the whole bodies of Blaps japanensis. Their structures including absolute configurations were determined by using spectroscopic, X-ray crystallographic, and computational methods. Compounds 1-11 and 13 exist as racemic mixtures in nature, and their (-)- and (+)-antipodes were separated by chiral HPLC. Biological evaluations of these compounds were determined with multiple assays including anti-tumor, anti-inflammatory, and renal protection activities in vitro. Several compounds displayed effective activity in one or more assays.

Analysis of metabolites in green tea during the roasting process using non-targeted metabolomics.

BACKGROUND: Roasting plays an important role in the formation of flavor of roasted green tea; however, the changes in chemicals during this process have not been systematically studied until now. To reveal the dynamic changes in chemicals in green tea during roasting, non-targeted metabolomics, coupled with chemometrics, was employed. RESULTS: A total of 101 non-volatile metabolites were identified in tea samples, and 29 metabolites were identified as characteristic metabolites of roasting. A significant increase in catechins and their derivatives, organic acids, and flavonoid glycosides was observed, while the content of some amino acids and their derivatives decreased over 50% during roasting. The content of theanine glucoside increased dramatically (by 21.23-fold at the roasting stage), and Maillard-derived compounds also increased to varying degrees. CONCLUSION: Glycosylation, oxidative polymerization, and pyrolysis were important reactions responsible for the formation and transformation of flavor compounds in roasted green tea during roasting. © 2022 Society of Chemical Industry.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Effect of red light on the composition of metabolites in tea leaves during the withering process using untargeted metabolomics.

BACKGROUND: Red light withering significantly improves the sensory flavor qualities of tea, although changes in metabolites during this process have not been systematically studied until now. The present study comprehensively analyzes metabolites in withered tea leaves at 2-h intervals up to 12 h under red light (630 nm) and dark conditions using ultra performance liquid chromatography-high resolution mass spectrometry (untargeted metabolomics). RESULTS: Ninety-four non-volatile compounds are identified and relatively quantified, including amino acids, catechins, dimeric catechins, flavonol glycosides, glycosidically-bound volatiles, phenolic acids and nucleosides. The results show that amino acids, catechins and dimeric catechins are most affected by red light treatment. Ten free amino acids, theaflavins and theasinensin A increase after red light irradiation, whereas epigallocatechin gallate and catechin fall. CONCLUSION: The present study provides a comprehensive and systematic profile of the dynamic effects of red light on withering tea and a rationale for its use in tea processing quality control. © 2021 Society of Chemical Industry.

Correction to: Hybridization capture-based next-generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis.

FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

Ansamycin derivatives from the marine-derived Streptomyces sp. SCSGAA 0027 and their cytotoxic and antiviral activities.

Fourteen ansamycin derivatives including seven new herbimycins G-L (1-6) and divergolide O (7), and seven known analogues were isolated from a culture broth of the marine-derived Streptomyces sp. SCSGAA 0027. Their complete structures were determined by detailed analysis of spectroscopic data and quantum chemical calculations. Compounds 1-5 and 7 featured an additional eight-membered O-heterocycle that has rarely been reported for ansamycins, and the Z,Z- and E,E-configurations for Δ2,Δ4 were reported for the first time in geldanamycin analogues. Compound 1 exhibited weak inhibition activity towards Hsp90α with an IC50 value of 96 µM, 2-5 showed mild cytotoxicity against four human cancer cell lines with IC50 values ranging from 13 µM to 86 µM, and 7 had moderate anti-HSV-1 activity with an IC50 value of 19 µM and very weak cytotoxicity towards Vero cell. The possible biosynthetic pathways for 1-5 were proposed. And their structure-bioactivity relationship was also discussed.

Targeted next generation sequencing (NGS) to classify melanocytic neoplasms.

This study piloted a pan-solid-tumor next generation sequence (NGS)-based laboratory developed test as a diagnostic aid in melanocytic tumors. 31 cases (4 "epithelioid" nevi, 5 blue nevi variants, 7 Spitz tumors [3 benign and 4 malignant] and 15 melanomas) were evaluated. All tumors [median diameter 7 mm (range 4-15 mm); median thickness 2.25 mm (range 0.25-12 mm)] yielded satisfactory results. The number of small nucleotide variants/tumor was significantly different between melanoma (median 18/tumor, range 4-71) and all other lesions (median 8/tumor, range 3-17) (P < 0.004) and malignant (median 16/tumor, range 4-71) vs benign lesions (median 7/tumor, range 3-14) (P = 0.01). BRAF, MET, NTRK1, and ROS fusions only occurred in benign Spitz tumors; EML4 fusion, BRAF, MAP2K1 and TERT mutations occurred in malignant Spitz tumors and/or melanoma. Amplifications and NRAS and NF1 mutations only occurred in melanoma. Most melanomas contained >1 pathogenic alteration. Developed NGS-based criteria correctly classified all malignant lesions in this series. 10/12 cases showed concordance with FISH; consensus diagnosis agreed with NGS classification in FISH-non-concordant cases. This pilot study suggests that NGS may be an effective diagnostic adjunct comparable to FISH, but further studies with larger numbers of cases are needed.

Sulfur and nitrogen-containing compounds from the whole bodies of Blaps japanensis.

Pipajiains H-J (1-3), three new phenolic derivatives with an unusual sulfone group, pipajiamides A-C (4-6), three new amide derivatives, pipajiaine A (7), one new imidazole analogue, and pipajiaine B (8), a pair of new pyrrolidine derivatives, along with three known compounds were isolated from the insect Blaps japanensis. Their structures were identified by spectroscopic and computational methods. Chiral HPLC was used to separate the (-)- and (+)-antipodes of 4 and 8. Biological activities of all the new compounds against extracellular matrix in rat renal proximal tubular cells, human cancer cells (A549, Huh-7, and K562), COX-2, ROCK1, and JAK3 were evaluated. The results show that compounds 2, (+)-4, and (-)-4 are active against kidney fibrosis, whereas, compound 9 is active toward human cancer cells, inflammation, and JAK3 kinase.

(±) Cochlearoids N-P: three pairs of phenolic meroterpenoids from the fungus Ganoderma cochlear and their bioactivities.

Three pairs of meroterpenoids (±) cochlearoids N-P (1-3) were isolated from the fruiting bodies of Ganoderma cochlear. Their structures including absolute configurations were assigned by spectroscopic techniques. All the isolated compounds were tested for their inhibitory activities toward BRD4, human cancer cells, and micro-organisms. The results show that the enantiomers of (±)-1 are BRD4 inhibitors, (-)-1 and (+)-3 are cytotoxic against human cancer cells (K562) with IC50 values of 7.68 and 6.68 µM, respectively. Besides compounds (±)-2 and (±)-3 exhibit potent inhibitory activity against Staphylococcus aureus with IC50 values in the range of 5.43-17.99 µM.

Spiro Meroterpenoids from Ganoderma applanatum.

Spiroapplanatumines A-Q (1-12, 14-16, 18, and 20), new spiro meroterpenoids respectively bearing a 6/5/7 or 6/5/5 ring system, along with three known compounds, spirolingzhines A, B, and D, were isolated from the fruiting bodies of the fungus Ganoderma applanatum. Their structures including absolute configurations were assigned by using spectroscopic methods, ECD and 13C NMR calculations, and single-crystal X-ray diffraction analysis. Biological evaluation of all the compounds disclosed that compounds 7 and 8 inhibited JAK3 kinase with IC50 values of 7.0 ± 3.2 and 34.8 ± 21.1 µM, respectively.

Molecular cytology genotyping of primary and metastatic GI stromal tumors by using a custom two-gene targeted next-generation sequencing panel with therapeutic intent.

BACKGROUND AND AIMS: In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. METHODS: DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. RESULTS: Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. CONCLUSIONS: Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

Kinase genotype analysis of gastric gastrointestinal stromal tumor cytology samples using targeted next-generation sequencing.

Gastric gastrointestinal stromal tumors (GISTs) usually contain the mast/stem cell growth factor receptor Kit gene (KIT) or platelet-derived growth factor receptor A (PDGFRA) mutations that can be targeted by, or mediate resistance to, imatinib. Diagnostic material often is obtained by endoscopic ultrasound-guided fine-needle aspiration, which often is unsuitable for molecular analysis. We investigated whether targeted next-generation sequencing (NGS) can be used in multiplex genotype analysis of cytology samples collected by endoscopic ultrasound-guided fine-needle aspiration. We used the Ion AmpliSeq V2 Cancer Hotspot NGS Panel (Life Technologies, Carlsbad, CA) to identify mutations in more than 2800 exons from 50 cancer-associated genes in GIST samples from 20 patients. We identified KIT mutations in 58% of samples (91% in exon 11 and 9% in exon 17) and PDGFRA mutations in 26% (60% in exon 18 and 40% in exon 12); 16% of samples had no mutations in KIT or PDGFRA. No pathogenic alterations were found in PIK3CA, BRAF, KRAS, NRAS, or FGFR3. We predicted that 32% of patients would have primary resistance to imatinib, based on mutations in exon 17 of KIT, exon 18 of PDGFRA (D842V), or no mutation in either gene. Targeted NGS of cytology samples from GISTs is feasible and provides clinically relevant data about kinase genotypes that can help guide individualized therapy.

Frequency of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathway pathogenic alterations in EUS-FNA sampled malignant lymph nodes in rectal cancer with theranostic potential.

BACKGROUND: Targeted next-generation sequencing has the potential to stratify a tumor by molecular subtype and aid the development of a biomarker profile for prognostic risk stratification and theranostic potential. OBJECTIVE: To assess the frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens. DESIGN: Multigene molecular profiling of archived malignant EUS-FNA lymph node cytology specimens using the Ion Ampliseq Cancer Hotspot Panel v2, which targets at least 2855 possible mutations within 50 cancer-associated genes. SETTING: Single tertiary referral center. PATIENTS: Sporadic, treatment naive, locally advanced primary rectal cancer by EUS-FNA (n = 76) who subsequently completed neoadjuvant therapy with on-site oncologic surgery. MAIN OUTCOME MEASUREMENTS: The frequency and distribution of pathogenic alterations in malignant lymph node cytology specimens by the mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling pathways, by KRAS or NRAS wild-type lymph node status, by extramesenteric lymph node status, and by a complete pathologic response status. RESULTS: Eleven patients (14.5%) were 50-gene panel wild-type. Sixty-five patients had 139 pathogenic alterations (2 [1-3] per patient) in 13 of 50 evaluated genes. The following represent a spectrum of identified alterations: TP53 (n = 52; 68.4%), APC (n = 36; 47.4%), KRAS (n = 22; 28.9%), FBXW7 (n = 8; 10.5%), NRAS (n = 6; 7.9%), PIK3CA (n = 4; 5.3%), SMAD4 (n = 3; 3.9%), and BRAF (n = 3; 3.9%). Pathogenic alterations were identified in the MAPK and PI3K signaling pathways in 41% and 5% of patients, respectively. LIMITATIONS: Findings were limited to a 50 cancer-associated gene analysis. CONCLUSIONS: Molecular EUS lymph node assessments using cancer "hotspot" panels can identify pathogenic alteration frequency and distribution and have theranostic potential for individualized patient care.

Compounds from the insect Blaps japanensis with COX-1 and COX-2 inhibitory activities.

Blapsols A-D (1-4), four new compounds possessing a 2,3-dihydrobenzo[b][1,4]dioxin group, together with five known N-acetyldopamine dimers (5-9), were isolated from Blaps japanensis. Their structures including the absolute configuration of (+)-1 were determined by means of spectroscopic and X-ray crystallographic methods. Chiral HPLC was used to separate (-)- and (+)-enantiomers of compounds 1-4, which were isolated from this insect as racemic mixtures. All the compounds were found to have inhibitory effects towards COX-2 with IC50 values in the range of 1.3-17.8µM.

Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter, tylosin.

Antimicrobials have been used extensively as growth promoters (AGPs) in agricultural animal production. However, the specific mechanism of action for AGPs has not yet been determined. The work presented here was to determine and characterize the microbiome of pigs receiving one AGP, tylosin, compared with untreated pigs. We hypothesized that AGPs exerted their growth promoting effect by altering gut microbial population composition. We determined the fecal microbiome of pigs receiving tylosin compared with untreated pigs using pyrosequencing of 16S rRNA gene libraries. The data showed microbial population shifts representing both microbial succession and changes in response to the use of tylosin. Quantitative and qualitative analyses of sequences showed that tylosin caused microbial population shifts in both abundant and less abundant species. Our results established a baseline upon which mechanisms of AGPs in regulation of health and growth of animals can be investigated. Furthermore, the data will aid in the identification of alternative strategies to improve animal health and consequently production.