Immune pathway upregulation and lower genomic instability distinguish EBV-positive nodal T/NK-cell lymphoma from ENKTL and PTCL-NOS

Primary EBV+ nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of PTCL-NOS. Herein, we analyzed copy-number aberrations (n=77) with focus on global measures of genomic instability (GI) and homologous recombination deficiency (HRD) and performed gene expression (n=84) and EBV miRNA expression profiling (n=24) and targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed a significantly worse outcome of PTCL-EBV compared to PTCL-NOS (P=0.002) but not ENKTL. Remarkably, PTCL-EBV exhibited significantly lower GI and HRD scores compared to ENKTL and PTCL-NOS. Gene Set Enrichment Analysis revealed many immune-related pathways, interferon alpha/gamma response, and IL6_JAK_STAT3 signaling to be significantly upregulated in PTCL-EBV and correlated with lower GI-scores. We also identified NFκB-associated genes, BIRC3, NFκB1 (p50) and CD27, and their proteins to be upregulated in PTCLEBV. PTCL-EBV demonstrated mostly type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNAs compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are microsatellite stable. Overall, the poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNAs are distinctive features of PTCL-EBV. Our data support the consideration of PTCL-EBV as a distinct entity, provide novel insights into the disease pathogenesis and offer potential new therapeutic targets for this tumor.

Mutational Analysis of Compound Heterozygous Mutation P.q6x/p.h232r in Srd5a2 Causing 46,xy Disorder of Sex Development

Background: Over 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration. Results: To clarify the cause of 46,XY DSD in the affected family focused on here, SRD5A2 sequencing was performed. Heterozygous p.H232R mutation was identified in the proband’s father, so we concluded that this mutation originated from the paternal side of the family and did not cause 46,XY DSD. Meanwhile, heterozygous p.Q6X mutation was identified in the proband’s mother, maternal uncle, and maternal grandfather, indicating that this mutation originated from maternal side of the family and did not cause 46,XY DSD. To clarify the effect of the p.H232R mutation in SRD5A2 on dihydrotestosterone (DHT) production, p.H232R mutant SRD5A2 plasmids were transfected into HEK293 cells. LC-MS indicated that DHT production decreased compared with that in cells transfected with wild-type SRD5A2.Conclusions: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage

Intramembrane metalloproteases regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σ K during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σ K cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired Pro-σ K interaction with SpoIVFB in co-purification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the Proregion of Pro-σ K . Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σ K cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis . We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined interaction of the IMMP SpoIVFB with its substrate Pro-σ K , whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σ K binding and cleavage. Corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

Mutational Analysis of a Wheat O-methyltransferase Involved in Flavonoid Metabolism

Methylated flavones, and tricin in particular, have been implicated in protecting wheat plants against a variety of biotic and abiotic stresses. Methylated flavones are produced via O-methylation of the hydroxyl groups in flavones, which is catalyzed by O-methyltransferases (OMTs). To examine the role of wheat OMT2 in methylated flavone biosynthesis and facilitate interrogation of tricin functions in wheat-environment interactions, loss-of-function mutants of OMT2 homoeologs, omt-A2 and omt-B2, were identified from a tetraploid wheat Targeting Induced Local Lesions in Genomes (TILLING) mutant population and crossed to generate the omt-A2omt-B2 double mutant. Although tricin and most other soluble phenolics did not differ in leaves and glumes of TILLING control and the omt-A2, omt-B2, and omt-A2 omt-B2 mutants, chlorogenic acid was increased in glumes of omt-A2 omt-B2 relative to TILLING control, suggesting that it might serve as a substrate for OMT2. The omt2 mutant lines showed similar growth phenotypes as well as comparable lignin deposition in cell walls of stems compared to TILLING control. These results collectively suggest that OMT2 and its close homolog OMT1 may possess overlapping activities in tricin production, with OMT1 compensating for the missing OMT2 activities in the omt2 mutant lines.

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

Screening for Mitochondrial tRNA Mutations in 318 Patients with Dilated Cardiomyopathy

Objectives: Dilated cardiomyopathy (DCM) is a complex cardiovascular disease with unknown etiology. Although nuclear genes play active roles in DCM, mitochondrial dysfunction was believed to be involved in the pathogenesis of DCM. The objective of this study is to analysis the association between mitochondrial tRNA (mt-tRNA) mutations and DCM. Material and Methods: We performed a mutational analysis of mt-tRNA genes in a cohort of 318 patients with DCM and 200 age- and gender-matched control subjects. To further assess their pathogenicity, phylogenetic analysis and mitochondrial functions including mtDNA copy number, ATP and ROS were analyzed. Results: 7 possible pathogenic mutations: MT-TL1 3302A>G, MT-TI 4295A>G, MT-TM 4435A>G, MT-TA 5655T>C, MT-TH 12201T>C, MT-TE 14692A>G and MT-TT 15927G>A were identified in DCM group but absent in controls. These mutations occurred at extremely conserved nucleotides of corresponding tRNAs, and led to the failure in tRNAs metabolism. Moreover, a significant reduction in ATP and mtDNA copy number, whereas a markedly increased in ROS level were observed in polymononuclear leukocytes (PMNs) derived from the DCM patients carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for DCM. Conclusions: Our data indicated that mt-tRNA mutations may be the molecular basis for DCM, which shaded novel insight into the pathophysiology of DCM that was manifestated by mitochondrial dysfunction.

Identification of CHEK2 germline mutations in BRCA1/2 and PALB2 negative breast and ovarian cancer patients

Introduction: The CHEK2 gene is known to be an important signal transducer involved in DNA repair, apoptosis, or cell cycle arrest in response to DNA damage. The mutations in this gene have been associated with a wide range of cancers, both sporadic and hereditary. Germline CHEK2 mutations are linked to an increased risk of breast cancer. Therefore, the aim of this study was to identify the prevalence of CHEK2 variants in BRCA1/2 and PALB2 negative early-onset patients with breast cancer and/or ovarian cancer in a Turkish population for the first time. Methods: The study included 95 patients with BRCA1/2 and PALB2 negative early-onset breast cancer and/or ovarian cancer and also 60 unaffected women. All the intron/exon boundaries and coding exons of CHEK2 were subjected to mutational analysis by heteroduplex analysis and DNA sequencing. Results: A total of 16 CHEK2 variants were found in breast cancer patients within the Turkish population. CHEK2 c.1100delC mutation studied in the CHEK2 gene most frequently was not detected in our study. The prevalence of variants of uncertain significance in CHEK2 was found to be 7.3% (n= 7) in BRCA1/2 and PALB2 mutation negative Turkish patients with early-onset breast and/or ovarian cancer. Discussion/Conclusion: The present study may shed light on alternative variations that could be significant for understanding the prevalence and clinical suitability of the CHEK2 gene.

The Stroma Liquid Biopsy Panel Contains a Stromal-Epithelial Gene Signature Ratio That Is Associated with the Histologic Tumor-Stroma Ratio and Predicts Survival in Colon Cancer

Liquid biopsy has emerged as a novel approach to tumor characterization, offering advantages in sample accessibility and tissue heterogeneity. However, as mutational analysis predominates, the tumor microenvironment has largely remained unacknowledged in liquid biopsy research. The current work provides an explorative transcriptomic characterization of the Stroma Liquid BiopsyTM (SLB) proteomics panel in colon carcinoma by integrating single-cell and bulk transcriptomics data from publicly available repositories. Expression of SLB genes was significantly enriched in tumors with high histologic stromal content in comparison to tumors with low stromal content (median enrichment score 0.308 vs. 0.222, p = 0.036). In addition, we identified stromal-specific and epithelial-specific expression of the SLB genes, that was subsequently integrated into a gene signature ratio. The stromal-epithelial signature ratio was found to have prognostic significance in a discovery cohort of 359 colon adenocarcinoma patients (OS HR 2.581, 95%CI 1.567–4.251, p < 0.001) and a validation cohort of 229 patients (OS HR 2.590, 95%CI 1.659–4.043, p < 0.001). The framework described here provides transcriptomic evidence for the prognostic significance of the SLB panel constituents in colon carcinoma. Plasma protein levels of the SLB panel may reflect histologic intratumoral stromal content, a poor prognostic tumor characteristic, and hence provide valuable prognostic information in liquid biopsy.

Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera of 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. However, some Delta patients have relatively preserved neutralization efficacy, comparable to 3-month double BNT162b2 vaccination. Domain-wise analysis using chimeric spike revealed that this efficient evasion was, at least in part, caused by multiple mutations in the N-terminal domain. Omicron escapes the therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective against Omicron. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from mutational escape. Deep mutational analysis demonstrated that, indeed, the engineered ACE2 overcomes every single-residue mutation in the receptor-binding domain, similar to immunized sera. Like previous SARS-CoV-2 variants, Omicron and some other sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.