FindDNAFusion: An Analytical Pipeline with Multiple Software Tools Improves Detection of Cancer-Associated Gene Fusions from Genomic DNA.

Detection of cancer-associated gene fusions is crucial for diagnosis, prognosis, and treatment selection. Many bioinformatics tools are available for the detection of fusion transcripts by RNA sequencing, but there are fewer well-validated software tools for DNA next-generation sequencing (NGS). A 542-gene solid tumor NGS panel was designed, with exonic probes supplemented with intronic bait probes against genes commonly involved in oncogenic fusions, with a focus on lung cancer. Three software tools for the detecting gene fusions in this DNA-NGS panel were selected and evaluated. The performance of these tools was compared after a pilot study, and each was configured for optimal batch analysis and detection rate. A blacklist for filtering common tool-specific artifacts, and criteria for selecting clinically reportable fusions, were established. Visualization tools for annotating and confirming somatic fusions were applied. Subsequently, a full clinical validation was used for comparing the results to those from in situ hybridization and/or RNA sequencing. With JuLI, Factera, and GeneFuse, 94.1%, 88.2%, and 66.7% of expected fusions were detected, respectively. With a combinatorial pipeline (termed FindDNAFusion), developed by integrating fusion-calling tools with methods for fusion filtering, annotating, and flagging reportable calls, the accuracy of detection of intron-tiled genes was improved to 98.0%. FindDNAFusion is an accurate and efficient tool in detecting somatic fusions in DNA-NGS panels with intron-tiled bait probes when RNA is unavailable.

Isolation and characterization of a SARS-CoV-2 variant with a Q677H mutation in the spike protein.

We isolated 20 SARS-CoV-2 strains from positive clinical samples collected in Columbus, Ohio, and investigated the replication of one pair of isolates: a clade 20G strain and a variant of this strain carrying a Q677H mutation in the spike protein and six other amino acid mutations. The OSU.20G variant replicated to a higher peak infectious titer than the 20G base strain in Vero-E6 cells, but the titers were similar when both strains were grown in Calu-3 cells. These results suggest that the OSU.20G variant has increased replication fitness compared to the 20G base strain. This may have contributed to its emergence in December 2020-January 2021.

The Cold-Adapted, Temperature-Sensitive SARS-CoV-2 Strain TS11 Is Attenuated in Syrian Hamsters and a Candidate Attenuated Vaccine.

Live attenuated vaccines (LAVs) replicate in the respiratory/oral mucosa, mimic natural infection, and can induce mucosal and systemic immune responses to the full repertoire of SARS-CoV-2 structural/nonstructural proteins. Generally, LAVs produce broader and more durable protection than current COVID-19 vaccines. We generated a temperature-sensitive (TS) SARS-CoV-2 mutant TS11 via cold-adaptation of the WA1 strain in Vero E6 cells. TS11 replicated at >4 Log10-higher titers at 32 °C than at 39 °C. TS11 has multiple mutations, including those in nsp3, a 12-amino acid-deletion spanning the furin cleavage site of the S protein and a 371-nucleotide-deletion spanning the ORF7b-ORF8 genes. We tested the pathogenicity and protective efficacy of TS11 against challenge with a heterologous virulent SARS-CoV-2 D614G strain 14B in Syrian hamsters. Hamsters were randomly assigned to mock immunization-challenge (Mock-C) and TS11 immunization-challenge (TS11-C) groups. Like the mock group, TS11-vaccinated hamsters did not show any clinical signs and continuously gained body weight. TS11 replicated well in the nasal cavity but poorly in the lungs and caused only mild lesions in the lungs. After challenge, hamsters in the Mock-C group lost weight. In contrast, the animals in the TS11-C group continued gaining weight. The virus titers in the nasal turbinates and lungs of the TS11-C group were significantly lower than those in the Mock-C group, confirming the protective effects of TS11 immunization of hamsters. Histopathological examination demonstrated that animals in the Mock-C group had severe pulmonary lesions and large amounts of viral antigens in the lungs post-challenge; however, the TS11-C group had minimal pathological changes and few viral antigen-positive cells. In summary, the TS11 mutant was attenuated and induced protection against disease after a heterologous SARS-CoV-2 challenge in Syrian hamsters.

Wastewater SARS-CoV-2 monitoring as a community-level COVID-19 trend tracker and variants in Ohio, United States.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 129 million confirm cases. Many health authorities around the world have implemented wastewater-based epidemiology as a rapid and complementary tool for the COVID-19 surveillance system and more recently for variants of concern emergence tracking. In this study, three SARS-CoV-2 target genes (N1 and N2 gene regions, and E gene) were quantified from wastewater influent samples (n = 250) obtained from the capital city and 7 other cities in various size in central Ohio from July 2020 to January 2021. To determine human-specific fecal strength in wastewater samples more accurately, two human fecal viruses (PMMoV and crAssphage) were quantified to normalize the SARS-CoV-2 gene concentrations in wastewater. To estimate the trend of new case numbers from SARS-CoV-2 gene levels, different statistical models were built and evaluated. From the longitudinal data, SARS-CoV-2 gene concentrations in wastewater strongly correlated with daily new confirmed COVID-19 cases (average Spearman's r = 0.70, p < 0.05), with the N2 gene region being the best predictor of the trend of confirmed cases. Moreover, average daily case numbers can help reduce the noise and variation from the clinical data. Among the models tested, the quadratic polynomial model performed best in correlating and predicting COVID-19 cases from the wastewater surveillance data, which can be used to track the effectiveness of vaccination in the later stage of the pandemic. Interestingly, neither of the normalization methods using PMMoV or crAssphage significantly enhanced the correlation with new case numbers, nor improved the estimation models. Viral sequencing showed that shifts in strain-defining variants of SARS-CoV-2 in wastewater samples matched those in clinical isolates from the same time periods. The findings from this study support that wastewater surveillance is effective in COVID-19 trend tracking and provide sentinel warning of variant emergence and transmission within various types of communities.

Gastrointestinal Pathology in Samples From Coronavirus Disease 2019 (COVID-19)-Positive Patients.

CONTEXT.­: Although primarily considered a respiratory illness, coronavirus disease 2019 (COVID-19) can cause gastrointestinal manifestations. OBJECTIVE.­: To evaluate histopathology and in situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in gastrointestinal samples from patients with recent and remote COVID-19. DESIGN.­: Patients with positive SARS-CoV-2 nasopharyngeal tests and a gastrointestinal tissue specimen were included. SARS-CoV-2 in situ hybridization (ISH) was performed on each sample. A subset had SARS-CoV-2 next-generation sequencing (NGS) performed. RESULTS.­: Twenty-five patients met inclusion criteria. Five had positive SARS-CoV-2 nasopharyngeal tests within 7 days of their gastrointestinal procedure. Two were ulcerative colitis patients on steroid therapy who lacked typical COVID-19 symptoms. Their colectomies showed severe ulcerative colitis; one demonstrated SARS-CoV-2 by NGS but a negative ISH. Another had an ischemic colon resected as a complication of the COVID-19 course; however, both ISH and NGS were negative. A fourth had a normal-appearing terminal ileum but positive ISH and NGS. The fifth patient had ileal ulcers with SARS-CoV-2 negativity by both modalities. The remaining 20 patients had positive nasopharyngeal tests an average of 53 days prior to procedure. None of their samples demonstrated SARS-CoV-2 ISH positivity, but one was positive on NGS despite a negative nasopharyngeal test. CONCLUSIONS.­: Gastrointestinal findings from SARS-CoV-2-infected patients ranged from normal with virus detected by ISH and NGS to bowel ischemia secondary to systemic viral effects without evidence of virus in the tissue. No distinct histologic finding was identified in those with gastrointestinal tissue specimens demonstrating SARS-CoV-2 positivity in this cohort.

Mutation-Specific SARS-CoV-2 PCR Screen: Rapid and Accurate Detection of Variants of Concern and the Identification of a Newly Emerging Variant with Spike L452R Mutation.

The emergence of more transmissible and/or more virulent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has triggered intensive genomic surveillance, which is costly and difficult to sustain operationally over the long term. To address this problem, we developed a set of four multiplex mutation-specific PCR-based assays with same-day reporting that can detect five VOC and three variants of interest (VOI), as defined in the March 2021 guidelines from the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/coronavirus/2019-ncov/). The screening results were compared to the whole-genome sequencing (WGS) and showed 100% concordance for strain typing for B.1.1.7 (n = 25) and P.1 (n = 5) variants using spike (S) mutation S-N501Y, S-E484K, and S-H69-V70del assays. The S-L450R assay, designed to detect the B.1.427/429 VOC, also identified multiple isolates of a newly emerging multiply mutated B.1.526.1 variant that is now rapidly increasing in the eastern United States. PCR approaches can be easily adopted in clinical laboratories, providing rapid screening methods to allow early detection of newly emergent variants and to efficiently triage cases for full genomic sequencing.

Detection of Pathogenic Isoforms of IKZF1 in Leukemic Cell Lines and Acute Lymphoblastic Leukemia Samples: Identification of a Novel Truncated IKZF1 Transcript in SUP-B15.

Leukemia-associated alternative splicing of IKZF1 can result in proteins with loss of one to four copies of its N-terminal zinc finger domains (N-ZnF). The best characterized pathogenic splice isoforms, Ik-6 and Ik-8, have been commonly found in BCR-ABL1+ acute lymphoblastic leukemia (ALL) and a subset of BCR-ABL1-like ALL. Infantile and childhood ALL that express these pathogenic IKZF1 isoforms have shown inferior clinical outcomes and can be resistant to tyrosine kinase inhibitors. Using ALL cell lines, we designed and validated a method to detect abnormal IKZF1 transcripts. In the SUP-B15 leukemia cell line, we noted novel IKZF1 transcripts that include both an Ik-6 splice and a transcript with a 14 base pair insertion at the C-terminus. There was also increased IKZF2 protein in SUP-B15 as compared to other ALL lines. Expression of Ik-6 could be suppressed by treatment with the pro-apoptotic type II histone deacetylase inhibitor givinostat. In 17 adult ALL samples, we noted the Ik-6 isoforms in 6 of 15 BCR-ABL1-, and 1 of 2 BCR-ABL1+ cases, with Ik-8 also expressed in one case. Cases with Ik-6 expression showed inferior survival as well as older age at presentation, lower expression of CD10 and more commonly a diploid karyotype.

Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.

Interleukin-10 regulated gene expression in cells of hypothalamic-pituitary-adrenal axis origin.

AIM: The hypothalamic-pituitary-adrenal (HPA) axis is a mediator for interactions between the immune and neuroendocrine systems. Pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) have been shown to activate the HPA axis. Recently, interleukin-10, an important anti-inflammatory cytokine in the immune system, has been shown to be expressed in the central nervous system and neuroendocrine system. Little is known, however, about IL-10's functions in the HPA axis. METHODS: The Affymetrix DNA microarray (mouse genome U74Av2 Probe Array) was conducted to determine the gene expression profile regulated by IL-10 in cells of HPA axis origin. RESULTS: In this study, we analyzed gene expression regulated by IL-10 in cells derived from the HPA axis. The results showed that quorums of genes are modulated by IL-10 in these neuroendocrine cells. CONCLUSIONS: These findings will provide a valuable repository to aid in understanding IL-10's functions in the HPA axis at the molecular level.

Regulation of adrenal glucocorticoid synthesis by interleukin-10: a preponderance of IL-10 receptor in the adrenal zona fasciculata.

Several lines of evidence indicate that cytokines can affect adrenal function. To date most of these cytokines have been shown to be pro-inflammatory, such as interleukin (IL)-1, tumor necrosis factor (TNFalpha), and IL-6. However, we have previously shown that IL-10-/- (IL-10 knockout) mice have higher serum corticosterone levels than IL-10+/+ (wild type) mice following acute immune and physiologic stress, implying that IL-10, an anti-inflammatory cytokine, regulates glucocorticoid synthesis in a negative manner. Here, we show that IL-10 knockout mice produce more corticosterone under basal conditions as well (shown by ELISA). We further support this contention by showing that in Y-1 adrenocortical cells IL-10 inhibits steroid production (StAR) (measured by the production of the corticosterone precursor, progesterone), the expression of steroidogenic acute regulatory protein (semi-quantitative RT-PCR), as well as the activity of the proximal steroidogenic enzymes P450scc and/or 3beta-hydroxysteroid dehydrogenase (3beta-HSD) (measured by progesterone production in 22(R)-hydroxycholesterol-treated cells). Interestingly, all of the above-mentioned effects of IL-10 occur through its inhibition of ACTH effects, but not by IL-10 alone. Furthermore, immunocytochemistry data shows that the region of the adrenal gland responsible for the vast majority of corticosterone synthesis, the zona fasciculata, predominantly expresses the IL-10 receptor 1 (IL-10R1), with little expression in the zona glomerulosa and reticularis. These data demonstrate that IL-10 could play an important role in the regulation of glucocorticoid biosynthesis and in maintenance of homeostasis and immunity during periods of stress.

Cytokine regulation of tryptophan metabolism in the hypothalamic-pituitary-adrenal (HPA) axis: implications for protective and toxic consequences in neuroendocrine regulation.

AIM: Indoleamine 2,3-dioxygenase (IDO) catalyzation of tryptophan is the first rate-limiting step of the kynurenine pathway in the majority of tissues. The kynurenine pathway produces neurotoxic metabolites such as 3-hydroxykinurenine and quinolinic acid. IDO is inducible by the cytokine interferon-gamma (IFN-gamma) and has been proposed to mediate the sickness behavior of patients with infectious or other inflammatory diseases.To better understand the neuroendocrine component of cytokine induced sickness behavior we determined the effects of the pro-inflammatory cytokine IFN-gamma and the anti-inflammatory cytokine IL-10 on IDO expression in cells derived from the hypothalamic-pituitary-adrenal axis (HPA): GT1-7 hypothalamic, AtT-20 pituitary, and Y-1 adrenal cells. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to check the IDO expression from IFN-gamma and IL-10 treated cells such as GT1-7, AtT-20 and Y-1 cells. RESULTS: We found that IFN-gamma induces IDO expression after 4 h treatment in GT1-7 and AtT-20 cells. IL-10 was also able to suppress IFN-gamma induced IDO expression in these cells. In Y-1 adrenal cells, IFN-gamma treatment had no effect on IDO expression. CONCLUSIONS: Our results indicate that cytokines such as IFN-gamma and IL-10 are able to regulate IDO expression in cells of hypothalamic and pituitary origin. The ability of IL-10 to suppress IFN-gamma induced IDO expression implies that IL-10 has a putative neuroprotective role in the HPA axis. It can act at two levels, systemically by inhibiting sickness behavior-related Th1 cytokine synthesis and more centrally by inhibiting the kynurenine pathway.

Cloning and characterization of an outer membrane protein (Lip18) from Helicobacter bizzozeronii.

A recombinant lambda-Zap II phage was selected by screening a genomic library of Helicobacter bizzozeronii (Hb) using antibodies from a naturally infected cat. DNA sequencing resulted an open reading frame containing 172 codons with a predicted molecular mass of 18 kDa (Lip18). The amino acid sequence showed 22.1, 55.2, 56.7 and 57.1% identity to peptidoglycan-associated lipoprotein of Helicobacter pylori, Agrobacterium tumefaciens, Bartonella bacilliformis, respectively. A peptidoglycan associating alpha-helical motif (LALGQRRSVAVRDYLVS) was located in the C-terminal region. H. bizzozeronii contains a potential lipoprotein signal peptide cleavage site (Val-Val-Gly-Cys), and yields a predicted mature protein with 148 amino acids. The Lip18 was localized into the outer membrane of the bacteria. Immunoblot analysis of serum samples from a dog and cat naturally infected with Helicobacter spp was able to recognize the purified recombinant Lip18.

Evidence for endogenous interleukin-10 during nociception.

Cytokines such as IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) have been shown to contribute directly to central and peripheral neuropathic pain. Recently, exogenous interleukin-10 (IL-10) was shown to impede development of dynorphin-induced allodynia presumably by inhibiting IL-1beta. We therefore wanted to determine whether endogenous IL-10 had a role in pain perception. By measuring the latency of the paw licking response, we show in IL-10 knockout mice and in normal mice treated with anti-IL-10 that latency times are increased, suggesting that endogenous IL-10 increases nociception. This does not appear to be directly correlated with IL-10's regulation of DREAM, a transcriptional regulator of prodynorphin synthesis.