Assessment of intestinal status in MPLW515L mutant myeloproliferative neoplasms mice model.

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1ß signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1ß signaling pathway, and the composition of gut microbial communities.

CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.

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.

Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.

RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS.

BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.

Calreticulin Immunohistochemistry in Myeloproliferative Neoplasms - Evolution of a New Cost-Effective Diagnostic Tool: A Retrospective Study with Histological and Molecular Correlation.

OBJECTIVE: Recent WHO 2017 guidelines mandates mutational analysis for the diagnosis of myeloproliferative disorders (MPN). JAK2V617F has been found in only 50-60% of Primary myelofibrosis (PMF) and Essential thrombocythaemia (ET). A recently discovered somatic Calreticulin (CALR) mutation has been linked to MPN. This mutation leads to a common 36 amino acid C-terminus that can be detected accurately by immunohistochemistry (IHC). Limited published literature exists on the utility of CAL2IHC as a diagnostic tool. The study aimed to validate the sensitivity and specificity of CAL2IHC for its use as a cost effective and rapid diagnostic tool. MATERIAL AND METHOD: Subjects included 23 patients of MPN (15 PMF, 6 ET, 2 PV (Polycythaemia Vera)), diagnosed between January 2014 to November 2016 with adequate available tissue for histopathological and mutational analysis. Mutational analysis had been performed with Bidirectional Sanger sequencing. CAL2IHC was performed in all cases and the sensitivity and specificity of CAL2 IHC to identify the Calreticulin mutation was evaluated with respect to comparison with the gold standard mutation analysis. RESULTS: In the 23 MPN patients, CAL2 IHC detected CALR mutation with a sensitivity of 95% and a specificity of 100%. Both cases of PV were negative for CAL2IHC. CAL2IHC showed cytoplasmic positivity in ET (2-3+) and PMF (1-3+) with (62-69%) positive megakaryocyte staining. All 6 ET cases and all 14/15 PMF cases were CAL2IHC positive, and these results were concordant with CALR mutational analysis. CONCLUSION: Anti-CAL2 immunohistochemistry is a specific and a sensitive marker to detect CALR mutation. Its' cost effectiveness and fast results are quite advantageous as compared to molecular analysis.

Integration of Molecular Information in Risk Assessment of Patients with Myeloproliferative Neoplasms.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell, characterized by an abnormal proliferation of largely mature cells driven by mutations in JAK2, CALR, and MPL. All these mutations lead to a constitutive activation of the JAK-STAT signaling, which represents a target for therapy. Beyond driver ones, most patients, especially with myelofibrosis, harbor mutations in an array of "myeloid neoplasm-associated" genes that encode for proteins involved in chromatin modification and DNA methylation, RNA splicing, transcription regulation, and oncogenes. These additional mutations often arise in the context of clonal hematopoiesis of indeterminate potential (CHIP). The extensive characterization of the pathologic genome associated with MPN highlighted selected driver and non-driver mutations for their clinical informativeness. First, driver mutations are enlisted in the WHO classification as major diagnostic criteria and may be used for monitoring of residual disease after transplantation and response to treatment. Second, mutation profile can be used, eventually in combination with cytogenetic, histopathologic, hematologic, and clinical variables, to risk stratify patients regarding thrombosis, overall survival, and rate of transformation to secondary leukemia. This review outlines the molecular landscape of MPN and critically interprets current information for their potential impact on patient management.

Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo.

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Mutant specific anti calreticulin antibody (CAL2) immunohistochemistry as a screening test for calreticulin (CALR) mutation testing.

BACKGROUND: About 50 different CALR frameshift mutations have been identified in BCR-ABL1 negative MPN, all leading to the development of common new protein C terminus. Antibody targeting this terminal epitope can be useful to identify this driver mutation using immunohistochemistry. MATERIALS AND METHODS: CALR mutation analysis was carried out in 51 JAK2V617F negative cases, PMF (n = 22) and ET (n = 29). PCR followed by fragment analysis was performed for molecular detection of CALR mutation. Bone marrow biopsy specimens of corresponding patients were subjected to IHC using mutation specific antibody CAL2. Staining pattern and intensity were observed. Staining of <2% of background nonmegakaryocytic (non- MK) cells were regarded as Pattern A, while staining of more than 2% of background nonmegakaryocytic (non-MK) was regarded as pattern B. RESULTS: CALR mutation was noted in 40.9% (9/22) and 41.4% (12/29) of JAK2V617F negative PMF and ET, respectively. All CALR mutated cases, irrespective of the mutation type, showed a positive IHC staining in the megakaryocytes with moderate to bright intensity. All CALR wild-type cases were negative on IHC. (Concordance rate- 100%). Pattern A was noted in 40% cases, while pattern B was noted in 60% cases. Pattern A staining had significantly higher chances of having type 1 mutation as compared to pattern B. In contrast, pattern B had a nonsignificant trend toward higher bone marrow cellularity and marrow fibrosis. CONCLUSION: CAL2 IHC detects all types of CALR mutation. This can act as a sensitive, specific, rapid, and cost-effective screening test for CALR mutation analysis.

Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

Rapid, low cost and sensitive detection of Calreticulin mutations by a PCR based amplicon length differentiation assay for diagnosis of myeloproliferative neoplasms.

BACKGROUND: Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes. METHODS: One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established. RESULTS: PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method. CONCLUSION: PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients.

Quantitative assessment of JAK2 V617F and CALR mutations in Philadelphia negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. PATIENTS AND METHODS: Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. RESULTS: Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). CONCLUSION: Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis.

BACKGROUND: Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. METHODS: After choosing MeSH keywords, including essential thrombocythemia, JAK2V617F, calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. RESULTS: The frequency of JAK2V617F and CALR shows heterogeneity in Caucasian population [JAK2V617 I 2% = 84.3, P < 0.001, 95% CI 0.56 (0.51-0.61)], [CALR I 2% = 96.1, P < 0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2V617F and CALR was 0.57 (95% CI 0.53-0.61), I 2% = 79.3 and 0.22 (95% CI 0.16-0.27), I 2% = 94, respectively. JAK2V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P < 0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. CONCLUSIONS: Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2V617F. Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2V617F mutation. Therefore, detection of mutation in CALR and JAK2V617F may contribute to diagnosis and prognosis of ET patients.

Detección de mutaciones del gen de calreticulina para neoplasias mieloproliferativas / Calreticulin gene mutation testing in myeloproliferative neoplasms

CONTEXTO CLÍNICO: Las neoplasias mieloproliferativas (NMP) son un grupo de patologías de las células madres hematopoyéticas clonales caracterizadas por la proliferación de una o más líneas celulares. Existen muchos tipos de NMP, siendo las más prevalentes la leucemia mieloide crónica (LMC), policitemia vera (PV), trombocitemia esencial (TE) y mielofibrosis primaria (MFP), razón por la cual son consideradas NMP clásicas. Entre las NMP clásicas, sólo la LMC está genéticamente caracterizada por la translocación entre el cromosoma 9 y 22 produciendo en 95% de los casos un cromosoma 22 acortado (Cromosoma Filadelfia) que expresa el oncogen BCR-ABL. La proliferación de células hematopoyéticas que se produce en las NMP Filadelfia negativas (PV, TE y MFP) está relacionada, entre otras, con mutaciones somáticas en diferentes genes como JAK2, MPL, IZF1, TET2 y las recientemente descriptas en el gen de calreticulina (CALR). El 90% de NMP filadelfia negativas tiene mutaciones en los genes JAK2, MPL o CALR, por lo que son las más estudiadas ayudando a diferenciar la enfermedad de un proceso reactivo. A diferencia de la LMC, estas mutaciones se han detectado en diferentes frecuencias como mutaciones singulares o combinadas entre las NMP Filadelfia negativas. Por ejemplo, la mutación JAK2 ha sido descripta en la mayoría de casos de PV, en la mitad de casos de TE y en un tercio de los casos de MFP. Recientes estudios demuestran que la detección de estas mutaciones podría tener utilidad pronóstica de las NMP Filadelfia negativas, además de ayudar en la precisión diagnóstica.7 Se postula que la detección de mutaciones del gen de CALR podría tener utilidad pronóstica y eventualmente a informar decisiones terapéuticas en pacientes con NMP Filadelfia negativas. TECNOLOGÍA: En el año 2013, se identificó la mutación del gen de CALR en pacientes con NMP Filadelfia negativas y sin mutación JAK2 y MPL. A partir de entonces se comenzó a investigar acerca de las implicancias de esta mutación en las NMP. NMP.8-10La calreticulina está involucrada en el mantenimiento de niveles adecuados de calcio y también actúa ayudando a otras proteínas a plegarse correctamente. Se han descripto dos tipos de mutaciones en el exón 9 del gen de la CALR, una de deleción (tipo 1) y otra de inserción (tipo 2). Las mutaciones somáticas en el gen CALR se detectan en sangre periférica en el 65-85% de los pacientes con TE y MFP que son negativos para mutaciones en los genes JAK2 y MPL. OBJETIVO: Evaluar la evidencia disponible acerca de la utilidad pronóstica y las consecuentes decisiones terapéuticas, así como aspectos relacionados a las políticas de cobertura de la detección de mutaciones del gen de calreticulina en pacientes con neoplasias mieloproliferativas. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas (incluyendo Medline, Cochrane y CRD), en buscadores genéricos de Internet, agencias de evaluación de tecnologías sanitarias y financiadores de salud utilizando la siguiente estrategia: ( ̈myeloproliferative disorders ̈ OR myeloproliferative neoplas*[tiab] OR ̈polycythemia vera ̈ OR ̈primary myelofibrosis ̈ OR ̈thrombocythemia, essential ̈ OR ̈Leukemia, Myelogenous, Chronic, BCR-ABL Positive ̈) AND (calreticulin OR calreticulin mutation[tiab] OR CALR[All]). Se priorizó la inclusión de revisiones sistemáticas, ensayos clínicos controlados aleatorizados, estudios observacionales, evaluaciones de tecnologías sanitarias y económicas, guías de práctica clínica y políticas de cobertura de otros sistemas de salud cuando estaban disponibles.RESULTADOS: Para el siguiente informe los resultados de los estudios fueron agrupados según enfermedad. En el caso de LMC no se encontraron estudios que investigaran mutaciones en CALR. Para el caso de la TE, se encontraron una revisión sistemática y un estudios de cohorte. Para el caso de MFP se encontraron dos estudios de cohorte. En PV se encontró sólo un estudio de corte transversal. No se encontraron guías de práctica clínica ni evaluaciones de tecnologías sanitarias que mencionaran a la tecnología y se halló una política de cobertura que la menciona. CONCLUSIONES: Evidencia de moderada calidad metodológica muestra que la presencia de mutaciones del gen de calreticulina se asocia a un menor riesgo de eventos trombóticos en trombocitemia esencial y podría asociarse a una mayor sobrevida en mielofibrosis primaria. No se encontró evidencia con respecto al impacto que tendría la detección de estas mutaciones en la conducta terapéutica. No se reportó la presencia de mutaciones del gen de calreticulina en leucemia mieloide crónica y policitemia vera, por lo que no se plantea su uso. No se hallaron guías de práctica clínica ni evaluaciones de tecnologías sanitarias que mencionaran a la detección mutaciones del gen de calreticulina en el manejo de neoplasias mieloproliferativas. Una sola política de cobertura encontrada no brinda cobertura por considerarla experimental.

It is time to change thrombosis risk assessment for PV and ET?

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms to be diagnosed according to the WHO classification. Molecular profiling must include the analysis of JAK2 (looking for the V617F point-mutation in PV and ET, screening exon 12 for mutations only in V617F-negative PV), CALR and MPL mutations (both in V617F-negative ET). The current risk stratification to predict thrombosis requires two parameters: age over 60 years and prior history of thrombosis. On the basis of these two risk factors patients can be stratified in low-risk and high-risk and receive a proper treatment. However, a modern stratification of thrombotic risk might consider "new" low-risk patients: conventional low-risk plus absence of leukocytosis from diagnosis onwards and a hematocrit level below 45% during the course of disease for PV; conventional low-risk plus absence of leukocytosis from diagnosis onwards, JAK2 negativity, CALR positivity, and absence of cardiovascular risk factors for ET.

Proteomic assessment shows that many endoplasmic reticulum (ER)-resident proteins are targeted by N(epsilon)-lysine acetylation in the lumen of the organelle and predicts broad biological impact.

In addition to the nucleus, cytosol, and mitochondrial lumen, N(ε)-lysine acetylation also occurs in the lumen of the endoplasmic reticulum (ER). However, the impact of such an event on ER functions is still unknown. Here, we analyzed the "ER acetyl-lysine proteome" by nano-LC-MS/MS and discovered that a large number of ER-resident and -transiting proteins undergo N(ε)-lysine acetylation in the lumen of the organelle. The list of ER-resident proteins includes chaperones and enzymes involved with post-translational modification and folding. Grouping of all acetylated proteins into major functional categories suggests that the ER-based acetylation machinery regulates very diverse biological events. As such, it is predicted to play a fundamental role in human physiology as well as human pathology.

Multilevel ecotoxicity assessment of polycyclic musk in the earthworm Eisenia fetida using traditional and molecular endpoints.

The ecotoxicity assessment of galaxolide (HHCB) and tonalide (AHTN) was investigated in the earthworm Eisenia fetida using traditional and novel molecular endpoints. The median lethal concentration (LC(50)) for 7-day and 14-day exposures was 573.2 and 436.3 µg g(-1) for AHTN, and 489.0 and 392.4 µg g(-1) for HHCB, respectively. There was no observed significant effect on the growth rate of E. fetida after a 28-day exposure except that at the highest concentration (100 µg g(-1)) of AHTN and HHCB, whereas a significant decrease of cocoon production was found in earthworms exposed to 50 and 100 µg g(-1). To assess molecular-level effect, the expression of encoding antioxidant enzymes and stress protein genes were investigated upon sublethal exposures using the quantitative real time PCR assay. The expression level of SOD, CAT and calreticulin genes was up-regulated significantly, while the level of annetocin (ANN) and Hsp70 gene expression was down-regulated in E. fetida. Importantly, the level of ANN expression had a significant positive correlation with the reproduction rate of earthworms. Furthermore, the lowest observed effect concentration (LOECs) of ANN expression level was 3 µg g(-1) for AHTN and 10 µg g(-1) for HHCB, suggesting that ANN gene expression can serve as a more sensitive indicator of exposure to AHTN and HHCB than traditional endpoints such as cocoon production. The transcriptional responses of these genes may provide early warning molecular biomarkers for identifying contaminant exposure, and the data obtained from this study will contribute to better understand the toxicological effect of AHTN and HHCB.