Background: Bariatric surgery, especially Roux-en-Y gastric bypass (RYGB), is the most effective and durable treatment option for severe obesity. The mechanisms involving adipose tissue may be important to explain the effects of surgery.

Methods: We aimed to identify the genetic signatures of adipose tissue in patients undergoing RYGB. We evaluated 13 obese, non-diabetic patients (mean age 37 years, 100% women, Body mass index (BMI) 42.2 kg/m²) one day before surgery, 3 and 6 months (M) after RYGB.

Results: Analysis of gene expression in adipose tissue collected at surgery compared with samples collected at 3 M and 6 M Post-RYGB showed that interleukins [Interleukin 6, Tumor necrosis factor-α (TNF-α), and Monocyte chemoattractant protein-1(MCP1)] and endoplasmic reticulum stress (ERS) genes [Eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) and Calreticulin (CALR)] decreased during the follow-up (P ≤ 0.01 for all). Otherwise, genes involved in energy homeostasis [Adiponectin and AMP-activated protein kinase (AMPK)], cellular response to oxidative stress [Sirtuin 1, Sirtuin 3, and Nuclear factor erythroid 2-related factor 2 (NRF2)], mitochondrial biogenesis [Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)] and amino acids metabolism [General control nonderepressible 2 (GCN2)] increased from baseline to all other time points evaluated (P ≤ 0.01 for all). Also, expression of Peroxisome proliferator-activated receptor gamma (PPARϒ) (adipogenesis regulation) was significantly decreased after RYGB (P < 0.05). Additionally, we observed that PGC1α, SIRT1 and AMPK strongly correlated to BMI at 3 M (P ≤ 0.01 for all), as well as ADIPOQ and SIRT1 to BMI at 6 M (P ≤ 0.01 for all).

Conclusions: Our findings demonstrate that weight loss is associated with amelioration of inflammation and ERS and increased protection against oxidative stress in adipose tissue. These observations are strongly correlated with a decrease in BMI and essential genes that control cellular energy homeostasis, suggesting an adaptive process on a gene expression level during the caloric restriction and weight loss period after RYGB. Trial registration CAAE: 73,585,317.0.0000.5440.

Keywords: Bariatric surgery; Endoplasmic reticulum stress; Human adipose tissue; Inflammation; Obesity; Oxidative stress; Roux-en-Y gastric bypass.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (<i>JAK</i>2), Calreticulin (<i><em>CALR</em></i>) and <i>MPL</i> (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase <i>TET2</i>, polycomb group protein <i>ASXL1</i> and histone-lysine N-methyltransferase <i>EZH2</i> genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in <i>JAK2</i>V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of <i>NANOG</i> and <i>E2F4</i> transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase <i>SUV39H1</i>, SWI/SNF complex subunit <i>SMARCC2, SMARCE2</i>, chromatin remodeling complex subunit <i>SS18</i>, tubulin β (<i>TUBB</i>) and cyclin dependent kinase <i>CDK1</i>. Among the upregulated epigenetic markers, there was a ~10-fold increase in <i>MYB</i> expression in <i>JAK2</i>V617F negative samples. A significant increase in total CD34 counts in <i>JAK</i>2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in <i>JAK</i>2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

Keywords: CD34; JAK; MYB; cell cycle; chromatin; epigenetics; microarray; myeloproliferative neoplasm.

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides - 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) - as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6-21%, p < 0.001) and 6% (95%CI: 0.4-11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12-2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49-110%, p < 0.001) and 105% (95%CI: 80-133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

Keywords: 8-oxoGuo; 8-oxodG; CALR; JAK2V617F; Myeloproliferative neoplasms; Oxidative stress; Oxidized nucleosides.

Mutations in the calreticulin (CALR) gene are found in the majority of Janus kinase 2-negative myeloproliferative neoplasms MPN and, thus far, have exclusively been reported as acquired, somatic mutations. We assessed the mutational status of exon 9 of the CALR gene in 2000 blood samples submitted to our centre and identified 12 subjects (0.6%) harbouring distinctive CALR mutations, all with an allelic frequency of 50% and all involving indels occurring as multiples of 3 bp. Buccal cell samples obtained from these patients confirmed the germline nature of the mutations. Importantly, these germline mutations were not diagnostic of MPN. We thus report for the first time the identification and confirmation of germline mutations in CALR distinct from those somatic mutations that define classical MPN. The finding of a non-standard CALR mutation with an allelic frequency of 50% should raise suspicion of the possibility of a germline CALR mutation and these cases investigated further.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

OBJECTIVE

To analyze the mutation rate and clinical characteristics of CALR, MPL W515K and JAK2 V617F genes in patients with primary thrombocythemia (PT).

METHODS

Fifty-six patients with PT were selected as the research objects in our hospital. The CALR and MPL W515K gene mutations were determined by genomic DNA-PCR direct sequencing of the PCR products, and the JAK2 V617F gene mutation was detected by allele specific PCR method.

RESULTS

Among the 56 patients with PT there were 14 cases of CALR gene mutation with the incidence rate of 25%, including 6 cases of type I, 5 cases of type II and 3 cases of type III. The sex, age, platelet(Plt) count, white blood cell (WBC) count and hemoglobin (Hb) level in the type I case of CALR gene mutation all were not significantly different from that in type II and III(all P>0.05); the WBC level in type III group significantly increased in comparison of type II group (P<0.05), while the sex, age, Hb and Plt levels showed no significant difference between the type III and type II groups (P>0.05). There were 3 cases of MPL W515K gene mutation with the incidence rate of 5.36%; 21 cases of JAK2 V617F gene mutation with the incidence rate of 37.50%. There were 13 cases of CALR gene mutation in negative patients with MPL W515K and JAK2 V617F (18 cases) with 72.22% incidence rate (13/18), and there was no cases of 1 or 2 gene mutations coexisted. The levels of Hb and WBC in peripheral blood of patients with CALR mutation were significantly lower than those of JAK2 V617F mutation (both P<0.05). In 56 cases, there were 3 cases of abnormal karyotype, with the incidence rate of 5.36%. The mutation rate of CALR gene in abnormal karyotypes (66.67%) was significantly higher than that of normal karyotypes (20.75%) (P<0.01).

CONCLUSIONS

The incidence of JAK2 V617F gene mutation increases in the patients with primary thrombocythemia; CALR mutation rate is higher in the patients with negative MPL W515K and JAK2 V617F gene mutation, which may closely correlate with abnormal karyotype; the levels of peripheral Hb and WBC in PT the patients with CALR gene mutation are significantly lower than those in patients with JAK2 V617F mutation.

OBJECTIVE

To explore the proteomic changes in thyroid tissue from GD patients and find new biomarkers for the prevention, diagnosis as well as the treatment of GD.

METHODS

Group1 included five thyroid specimens of GD cases and 5 normal thyroid tissue samples which were removed surgically and collected. The proteins were extracted from these thyroid tissues and then the differentially expressed protein spots were identified by MALDI-TOF-MS. The interested proteins were further validated in more specimens (group2: 11 pathological thyroid specimens and 7 normal thyroid tissue samples).

RESULTS

A total of 34 differentially expressed proteins were observed, and the majority of these proteins were involved in endoplasmic reticulum stress (ER-stress), oxidative stress, energy metabolism, cytoskeleton and movement. The overexpression of calreticulin(CALR) and heat shock 70kDa protein 5(HSPA5) was further validated.

CONCLUSIONS

Alltogether, abundant new candidate molecules, especially proteins related to ER-stress, were found to be involved in the pathogenesis of GD.

Background: The picture of Vietnamese patients with essential thrombocythemia (ET) remains mostly undetermined.Our study intended to determine the frequency of JAK2V617F, CALR exon 9, and MPL exon 10 mutations as well asto analyze clinical characteristics associated with different mutational status in Vietnamese ET patients. Methods: Weexplored mutations of JAK2V617F, MPL, and CALR from 395 patients using allele specific oligonucleotide – polymerasechain reaction and Sanger sequencing techniques; then, the clinical and hematological features were compared accordingto mutation patterns. Results: We found that JAK2V617F, CALR exon 9, and MPL exon 10 mutations were present in56.2%, 27.6%, and 1% of the 395 patients with ET, respectively. Twelve different types of CALR mutation were detectedin 109 patients, with the CALR type 1 mutation (c.1099_1150del; L367fs*46) was the most common, followed byCALR type 2 mutation (c.1154_1155insTTGTC; K385fs*47). The JAK2V617F-positive patients had older age, higherwhite blood cell counts and higher hemoglobin levels but lower platelet counts than patients with CALR mutationsor patients negative for triple tests. There was no significant difference regarding sex ratio, white blood cell counts,platelet counts and hemoglobin levels among CALR mutation subtypes. Conclusion: we reported high frequency ofJAK2V617F, CALR, and MPL mutations in Vietnamese patients with ET and underscored the importance of combinedgenetic tests for diagnosis and classification of ET into different subtypes.

BCR/ABL1-negative myeloproliferative neoplasms (MPNs) are characterized by recurrent mutations in JAK2, CALR, and MPL, each of which has been reported to alter JAK/STAT signaling pathways. This report characterizes JAK/STAT signaling patterns in molecularly defined subsets of MPN utilizing immunohistochemistry for pSTAT3 and pSTAT5. Analysis of 30 BCR/ABL1-negative, nonpolycythemia vera MPN identified 15 (50%) with JAK2 V617F, 2 with MPL mutations (7%), and 8 with CALR mutations (27%). All mutations were mutually exclusive, except for 1 case with concurrent JAK2 V617F and CALR mutations. pSTAT3 staining in megakaryocyte nuclei was found in 4 cases (13%) and was not significantly associated with mutation status. pSTAT5 staining in megakaryocyte nuclei was found in 16 cases (53%), as was significantly associated with JAK2 V617F versus CALR mutation (P=0.009). Erythroid staining for pSTAT5 was seen exclusively in "triple-negative (TN)" cases lacking JAK2 V617F, MPL, and CALR mutations (P=0.006, TN vs. other genotypes), and pSTAT5 staining in megakaryocyte nuclei was seen in 2 TN cases. pSTAT5 staining in TN MPN suggests that other unknown abnormalities in this pathway may contribute to the pathogenesis of these cases. Furthermore, the demonstration of distinct STAT staining patterns in molecularly defined MPN suggests that these mutations result in divergent signaling events that may contribute to the biological and prognostic differences in these molecular subsets of MPN.

The aim of this study was to explore the distinct protein profiles of different subtype of acute myeloid leukemia (AML), including M(1), M(2), M(3) and acute lymphoid leukemia (ALL) by differential proteomic expression analysis. The proteins of bone marrow leukemia cells from AML and ALL patients were separated by two-dimensional electrophoresis (2-DE). 2-DE patterns were analyzed by PDQuest 7.4 software and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and bioinformatics. The results indicated that 21 differentially expressed proteins were found by 2-DE and 15 were identified by MS to be significantly differentially expressed. In AML, seven proteins were highly expressed such as MPO, PRDX3, CALR and ECH1 and so on, and eight proteins were highly expressed in ALL, including ARHGDIB, PFN1 and ACTG1 and so on. It is concluded that the distinct protein profiles between AML and ALL have been proved. It may be helpful for the identification of new targets for specific treatment approaches and the molecular markers for the early diagnosis of leukemia.

The aim of this study is to investigate the effect of miR-148b on cell proliferation and migration of Schwann cells and explore its mechanism. The miR-148b group, miR-con group and the anti-miR-148b group, anti-miR-con group, si-con group, si-CALR group, Ctrl group, CALR group were transfected into Schwann cells by liposome method; the expression of miR-148b was detected by qRT-PCR; the cell viability was detected by MTT assay; the migration of cells was detected by Transwell method; WB assay was used to detect the protein expression of CALR. Firstly, we found that compared with miR-con group and si-con group, the proliferation and migration of miR-148b group and si-CALR group were significantly down-regulated (P < .05). Moreover, compared with anti-miR-con group and Ctrl group, anti-miR-148b group and CALR group cells proliferation and migration were significantly up-regulated (P < .05). In addition, miR-148b was targeted to CALR, and silencing CALR could reverse the inhibitory effect of miR-148b on Schwann cell proliferation and migration. In conclusion, miR-148b can regulate the proliferation and migration of Schwann cells. The mechanism may be related to the targeted negative regulation of CALR, which will provide a basis for targeted therapy of peripheral nerve injury.

Ultra high thread-level parallelism in modern GPUs usually introduces numerous memory requests simultaneously. So there are always plenty of memory requests waiting at each bank of the shared LLC (L2 in this paper) and global memory. For global memory, various schedulers have already been developed to adjust the request sequence. But we find few work has ever focused on the service sequence on the shared LLC. We measured that a big number of GPU applications always queue at LLC bank for services, which provide opportunity to optimize the service order on LLC. Through adjusting the GPU memory request service order, we can improve the schedulability of SM. So we proposed a critical-aware shared LLC request scheduling algorithm (CaLRS) in this paper. The priority representative of memory request is critical for CaLRS. We use the number of memory requests that originate from the same warp but have not been serviced when they arrive at the shared LLC bank to represent the criticality of each warp. Experiments show that the proposed scheme can boost the SM schedulability effectively by promoting the scheduling priority of the memory requests with high criticality and improves the performance of GPU indirectly.