Circular RNAs (circRNAs) are covalently closed, single-stranded RNAs that play critical roles in various biological processes and diseases, including cancers. However, the functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) need further clarification. Here, we identified and confirmed that circATF6 is downregulated in HCC tissues and negatively associated with the overall survival of HCC patients. Ectopic overexpression of circATF6 inhibits malignant phenotypes of HCC cells in vitro and in vivo, while knockdown of circATF6 had opposite effects. Mechanistically, we found that circATF6 bound to calreticulin (CALR) protein and acted as a scaffold to enhance the interaction of CALR with calpain2 (CAPN2), which promoted the degradation of CALR by its enzymatic activity. Moreover, we found that circATF6 inhibited HCC cells by suppressing CALR-mediated wnt/β-catenin signaling pathway. Taken together, our findings suggest that circATF6 is a potential prognostic biomarker and therapeutic target for HCC.

This study aims to investigate the role of anoikis-related genes in diabetic foot (DF) by utilizing bioinformatics analysis to identify key genes associated with anoikis in DF. We selected the GEO datasets GSE7014, GSE80178 and GSE68183 for the extraction and analysis of differentially expressed anoikis-related genes (DE-ARGs). GO analysis and KEGG analysis indicated that DE-ARGs in DF were primarily enriched in apoptosis, positive regulation of MAPK cascade, anoikis, focal adhesion and the PI3K-Akt signalling pathway. Based on the LASSO and SVM-RFE algorithms, we identified six characteristic genes. ROC curve analysis revealed that these six characteristic genes had an area under the curve (AUC) greater than 0.7, indicating good diagnostic efficacy. Expression analysis in the validation set revealed downregulation of CALR in DF, consistent with the training set results. GSEA results demonstrated that CALR was mainly enriched in blood vessel morphogenesis, endothelial cell migration, ECM-receptor interaction and focal adhesion. The HPA database revealed that CALR was moderately enriched in endothelial cells, and CALR was found to interact with 63 protein-coding genes. Functional analysis with DAVID suggested that CALR and associated genes were enriched in the phagosome component. CALR shows promise as a potential marker for the development and treatment of DF.

As a promising immune checkpoint of immunogenic cell death (ICD) and multifunctional calcium-binding molecular chaperone, calreticulin (CALR) has been attracting increasing attention. CALR mainly locates in cellular endoplasmic reticulum and significantly affects cell proliferation, invasion, induction of apoptosis, and angiogenesis in breast invasive carcinoma (BRCA). CALR overexpression might be correlated with a worse outcome. Nonetheless, it remains obscure how CALR correlates with immune infiltration and survival prognosis of BRCA. In this study, we investigated CALR expression utilizing RNAseq data from the cancer genome atlas (TCGA) and genotype-tissue expression (GTEx) database. The prognostic value of CALR was analyzed using clinical survival data. Enrichment analysis was conducted using the R package \"clusterProfiler.\" We downloaded the immune cell infiltration score of TCGA samples from published articles and online databases and performed a correlation analysis between immune cell infiltration levels and CALR expression. We further assessed the association between CALR and immunomodulators. Moreover, we also evaluated the expression of CALR in 100 formalin-fixed and paraffin-embedded breast cancer and adjacent normal breast tissue specimens. Our results found that CALR was highly expressed in BRCA, and CALR expression levels differed in pathological stages, T stages, and N stages. Besides, these results suggested that CALR overexpression may have adverse effects on the progression-free interval (PFI) and disease-free interval (DFI), which may be related to tumor proliferation, invasion, and metastasis, leading to tumor deterioration. Meanwhile, immune cell infiltration analysis revealed a correlation between the expression of CALR and the number of neutrophils and dendritic cells, suggesting that CALR was highly correlated with many immunomodulators in BRCA. Our results provide potential biomarkers of CALR in BRCA. CALR may interact synergistically with other immunomodulators to regulate the immune microenvironment, which could be utilized to develop new immunotherapy drugs.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.

Background: JAK2, CALR, and MPL gene mutations are recognized as driver mutations of myeloproliferative neoplasms (MPNs). MPNs without these mutations are called triple-negative (TN) MPNs. Recently, novel mutation loci were continuously discovered using next-generation sequencing (NGS), along with continued discussion and modification of the traditional TN MPN. Case presentation: Novel pathogenic mutations were discovered by targeted NGS in 4 patients who were diagnosed as JAK2 unmutated polycythaemia vera (PV) or TN MPN. Cases 1, 2, and 3 were of patients with PV, essential thrombocythemia (ET), and primary myelofibrosis (PMF); NGS detected JAK2 p.H538_K539delinsQL (uncommon), CALR p.E380Rfs*51 (novel), and MPL p.W515_Q516del (novel) mutations. Case 4 involved a patient with PMF; JAK2, CALR, or MPL mutations were not detected by qPCR or NGS, but a novel mutation SH2B3 p.S337Ffs*3, which is associated with the JAK/STAT signal transduction pathway, was found by NGS. Conclusion: NGS, a more multidimensional and comprehensive gene mutation detection, is required for patients suspected of having MPN to detect non-canonical driver variants and avoid the misdiagnosis of TN MPN. SH2B3 p.S337Ffs*3 can drive MPN occurrence, and SH2B3 mutation may also be a driver mutation of MPN.

The increased frequency of marine heat waves (MHWs) caused by global climate change is predicted to threaten the survival of economic bivalves, therefore having severely adverse effects on local ecological communities and aquaculture production. However, the study of scallops facing MHWs is still scarce, particularly in the scallop Argopecten irradians irradians, which has a significant share of \"blue foods\" in northern China. In the present study, bay scallop heart was selected to detect its cardiac performance, oxidative impairment and dynamic molecular responses, accompanied by assessing survival variations of individuals in the simulated scenario of MWHs (32 °C) with different time points (0 h, 6 h, 12 h, 24 h, 3 d, 6 d and 10 d). Notably, cardiac indices heart rate (HR), heart amplitude (HA), rate-amplitude product (RAP) and antioxidant enzyme activities superoxide dismutase (SOD) and catalase (CAT) all peaked at 24 h but sharply dropped on 3 d, coinciding with mortality. Transcriptome analysis revealed that the heart actively defended against heat stress at the acute stage (<24 h) via energy supply, misfolded proteins correction and enhanced signal transduction, whereas regulation of the defense response and apoptotic process combined with twice transcription initiation were the dominant responses at the chronic stage (3-10 d). In particular, HSP70 (heat shock protein 70), HSP90 and CALR (calreticulin) in the endoplasmic reticulum were identified as the hub genes (top 5 %) in the HR-associated module via WGCNA (weighted gene co-expression network analysis) trait-module analysis, followed by characterization of their family members and diverse expression patterns under heat exposure. Furthermore, RNAi-mediated knockdown of CALR expression (after 24 h) significantly weakened the thermotolerance of scallops, as evidenced by a drop of 1.31 °C in ABT (Arrhenius break temperature) between the siRNA-injected group and the control group. Our findings elucidated the dynamic molecular responses at the transcriptome level and verified the cardiac functions of CALR in bay scallops confronted with stimulated MHWs.

Essential thrombocythemia (ET) with double driver mutations is a rare disease. ET patients with both MPL and Type 1 CALR mutations have been reported. Here, we report the first case of an ET patient with both MPL S204P and Type 2 CALR mutations and a summary of our literature review findings. In the patient whose case is reported here, the disease progressed to an accelerated phase 3.5 months after diagnosis. CALR mutation disappeared and new mutations emerged as the disease progressed, such as ASXL1, CBL, ETV6, and PTPN11 mutations. This case highlights that screening for additional mutations using NGS should be considered in patients with ET to assess the prognosis, especially as the disease progresses.

Simultaneous mutations in Janus kinase 2 (JAK2), calreticulin, and myeloproliferative leukemia (MPL) genes are generally not considered for characterizing Philadelphia-negative myeloproliferative neoplasms (MPNs), leading to misdiagnosis. Sanger sequencing and quantitative polymerase chain reaction were used to detect gene mutations in patients with MPN. We retrospectively screened the data of patients with double mutations in our center and from the PubMed database. Two patients tested positive for both JAK2V617F and CALR mutations (2/352 0.57%) in our center, while data of 35 patients from the PubMed database, including 26 patients with essential thrombocythemia (ET), 6 with primary myelofibrosis (PMF), 2 with unexplained thrombosis, and 1 with polycythemia vera were screened for double mutations. Among these mutations, co-mutation of JAKV617F-CALR constituted the majority (80.0%), when compared with JAKV617F-MPL (17.1%) and CALR-MPL (2.9%) mutations. Moreover, patients with concurrent mutational myeloproliferative neoplasm (MPN) were relatively older (P  =  .010) with significantly higher platelet counts than their counterparts with single gene mutations (P < .001). The occurrence of palpable splenomegaly (P < .001) and leukocyte count (P  =  .041) were also significantly different between patients with single and simultaneous gene mutations. These 4 risk factors also showed significant test effectiveness in the ET and PMF cohorts (P < .05). In terms of clinical characteristics of patients with ET, those with JAK2V617F-CALR mutation had higher but normal hemoglobin levels (P  =  .0151) than those carrying JAK2V617F-MPL mutation. From a clinical perspective, patients with multiple mutational MPN are different from those with single gene mutations. The poor treatment response by patients in our center and unfavorable indicators for patients with co-mutations in published literature indicate that customized treatment may be the best choice for patients with MPN carrying co-mutations.

UNASSIGNED: Calreticulin (CALR) mutations have been identified as driver mutations in a quarter of patients with essential thrombocythaemia (ET) and primary myelofibrosis (PMF), which are subgroups of myeloproliferative neoplasms (MPNs). A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent variants. To better understand the impact of different CALR mutant variants, with or without nondriver mutations, on the clinical subtypes of MPN needs further investigation.
UNASSIGNED: The clinical characteristics, laboratory parameters and genetic mutation statuses were analysed in a cohort of 77 MPN patients with CALR mutations (ET = 24, prePMF = 33, and overt PMF = 20). Targeted NGS using a 38-gene panel was performed to evaluate the variant allele frequency (VAF) of CALR type I/type II mutations and assess the molecular landscape of nondriver gene mutations.
UNASSIGNED: A lower VAF of type I vs. type II was observed in CALR-mutant ET, prePMF and overt PMF, and a higher frequency of type I vs. type II was found in CALR-mutant overt PMF. Additional somatic mutations were indicated to be useful in understanding the pathogenesis of MPN. In this study, the mutation landscape was more complex in overt PMF than in ET or in prePMF. Mutations in epigenetic regulators (ASXL1, EZH2 and TET2) were more common in overt PMF.
UNASSIGNED: The two different subtypes of CALR mutations may have different impacts on MPN. A lower VAF of CALR type I indicates a greater contribution to disease progression in MPN, and increased nondriver mutations may be important in myelofibrosis progression.

The aim of this study was to explore and compare routine blood features and pathological characteristics of bone marrow tissues in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis, prefibrotic stage (prePMF) and overt fibrotic stage (overtPMF), and the correlation between common driver gene mutations and clinical manifestations of myeloproliferative neoplasms (MPN). Methods: We analyzed 259 MPN patients treated at Tongji Hospital of Huazhong University of Science and Technology from January 2016 to December 2020. Results: Among ET, PV, prePMF, and overtPMF, the median leukocyte counts of PV and prePMF were significantly higher than those of ET. The average hemoglobin level of overtPMF was significantly lower than that of ET, PV, and prePMF. ET and prePMF had higher platelet counts than PV and overtPMF, whereas ET had the lowest platelet distribution width. Regarding hematopoietic tissues in the bone marrow, enlarged megakaryocytes were easily found in ET, PV, and prePMF, whereas the average diameter of megakaryocytes in prePMF was smaller than in ET, and PV showed various sizes of megakaryocytes. An increased M/E ratio and dilation of sinus were seen more frequently in PMF. Additionally, JAK2-positive patients tended to have significantly higher leukocyte counts than CALR-positive patients in ET and PMF.