The impact of a "short-term" basic intensive care training program on the knowledge of nonintensivist doctors during the COVID-19 pandemic: An experience from a population-dense low- and middle-income country.

BACKGROUND: The utility of basic intensive care unit (ICU) training comprising a "1-day course" has been scientifically evaluated and reported in very few studies, with almost no such study from resource-limited settings. AIM: The study assessed the utility of basic ICU training comprising of a "1-day course" in increasing the knowledge of nonintensivist doctors. MATERIALS AND METHODS: This is an observational study conducted at a medical university in North India in 2020. The participants were nonintensivist doctors attending the course. The course was designed by intensivists, and it had four domains. The participants were categorised on the basis of their duration of ICU experience and broad speciality. Pretest and posttest was administered, which was analysed to ascertain the gain in the knowledge score. RESULTS: A total of 252 participants were included, of which the majority were from the clinical medicine speciality (85.3%) and had ICU experience of 1-6 months (47.6%). There was a significant improvement in the mean total score of the participants after training from 14/25 to 19/25, with a mean difference (MD) of 5.02 (p < 0.001). Based on ICU experience, in groups I (<1 month), II (1-6 months), and III (>6 months), there was a significant improvement in the total score of the participants after training with MD with 95% confidence interval (CI) limits of 5.27 (4.65-5.90), 4.70 (4.38-5.02), and 5.33 (4.89-5.78), respectively. In the clinical surgery specialty (n = 37), there was a significant improvement in the total score after training from 11/25 to 16.4/25 with an MD (95% CI limits) of 5.38 (4.4-6.3). Similarly, in the clinical medicine group (n = 215), the MD (95% CI limits) score after training was 4.95 (4.71-5.20), from 14.5/25 to 19.5/25. In feedback, more than half of the participants showed interest in joining ICU after training. CONCLUSIONS: Training nonintensivist doctors for 1 day can be useful in improving their knowledge, regardless of their prior ICU experience and speciality.

Use of methylene blue dye for sentinel lymph node mapping in early-stage gynecological cancers - An option for low resource settings.

Context: Sentinel lymph node (SLN) mapping is a standard of care in gynecological cancers but with limited resources and equipment, intraoperative use of methylene blue for SLN mapping may be more useful. Aims: To authenticate the use of methylene blue dye for intraoperative SLN mapping in cases of early-stage gynecological cancers. Settings and Design: This pilot study was conducted in a tertiary care teaching hospital. Subjects and Methods: The cases included 14 cervical, 4 endometrial, 1 vulvar, and 1 synchronous cervical-vulvar cancer wherein 21 SLN mappings were done. Four ml of methylene blue was injected submucosally in the cervix in cervical and endometrial cancer. It was injected around the tumor in vulvar cancer. The pelvic lymphatic drainage area was examined in cervical and endometrial cancer while inguinofemoral area was examined in vulvar cancer for any blue LNs. Histopathological examination results were compared for the presence of metastasis in stained and unstained LNs. Statistical Analysis Used: The observations were presented as numbers and percentages. Results: The SLN mappings showed a detection rate of 76%. The mean number of LNs removed in each case was 10.3 with an average of 2.4 stained and 7.6 unstained LNs. SLN was most commonly found among right external iliac nodes. All stained and unstained nodes among the 16 SLN detections did not show any histological evidence of metastasis suggesting a negative predictive value of 100%. Conclusions: Methylene blue is an efficient, feasible, and safe dye for SLN mapping in early-stage gynecological cancer.

Increased HDAC1 deposition at hematopoietic promoters in AML and its association with patient survival.

Epigenetic changes play a crucial role in leukemogenesis. HDACs are frequently recruited to target gene promoters by balanced translocation derived oncogenic fusion proteins. As important epigenetic effector mechanisms, histone deacetylases (HDAC) have emerged as potential therapeutic targets. However, the patterns of HDAC1 localization and the role of HDACs in leukemia pathogenesis remain to be elucidated. Using ChIP-Chip analyses we analyzed HDAC1 deposition patterns at more than 10,000 gene promoters in a large cohort of leukemia patients and CD34+ controls. HDAC1 binding was significantly increased in AML blasts compared to CD34+ progenitor cells at 130 gene promoters whereas decreased binding was observed at 66 gene promoters. Distinct HDAC1 binding patterns occurred in AML subtypes with balanced translocations t(15;17), t(8;21) and inv(16). In addition, a more generalized signature was established, that revealed an AML specific pattern of HDAC1 distribution. Many of the HDAC1-binding altered promoters regulate genes involved in hematopoiesis, transcriptional regulation and signal transduction. HDAC1 binding patterns were associated with patients' event free survival. This is the first study to determine HDAC1 modification patterns in a large number of AML and ALL specimens. Our findings suggest that dyslocalization of HDAC1 is a common feature in AML. Importantly, HDAC1 modifications possess prognostic power for patient survival. Our findings suggest that altered HDAC1 localization is an explanation for the observed benefit of HDAC inhibitors in AML therapy.

Chromatin modifications induced by PML-RARalpha repress critical targets in leukemogenesis as analyzed by ChIP-Chip.

The translocation t(15;17) generates the chimeric PML-RARalpha transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARalpha transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)-chip experiments identified 372 direct genomic PML-RARalpha targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARalpha targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARalpha binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARalpha to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARalpha regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

DNA methylation of tumor suppressor genes in clinical remission predicts the relapse risk in acute myeloid leukemia.

Epigenetic changes play an important role in leukemia pathogenesis. DNA methylation is among the most common alterations in leukemia. The potential role of DNA methylation as a biomarker in leukemia is unknown. In addition, the lack of molecular markers precludes minimal residual disease (MRD) estimation for most patients with hematologic malignancies. We analyzed the potential of aberrant DNA promoter methylation as a biomarker for MRD in acute leukemias. Quantitative real-time PCR methods with bisulfite-modified DNA were established to quantify MRD based on estrogen receptor alpha (ERalpha) and/or p15(INK4B) methylation. Methylation analyses were done in >370 DNA specimens from 180 acute leukemia patients and controls. Methylation of ERalpha and/or p15(INK4B) occurred frequently and specifically in acute leukemia but not in healthy controls or in nonmalignant hematologic diseases. Aberrant DNA methylation was detectable in >20% of leukemia patients during clinical remission. In pediatric acute lymphoblastic leukemia, methylation levels during clinical remission correlated closely with T-cell receptor/immunoglobulin MRD levels (r = +0.7, P < 0.01) and were associated with subsequent relapse. In acute myelogenous leukemia patients in clinical remission, increased methylation levels were associated with a high relapse risk and significantly reduced relapse-free survival (P = 0.003). Many patients with acute leukemia in clinical remission harbor increased levels of aberrant DNA methylation. Analysis of methylation MRD might be used as a novel biomarker for leukemia patients' relapse risk.

Decitabine and vitamin D3 differentially affect hematopoietic transcription factors to induce monocytic differentiation.

Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may re-establish functional hematopoiesis in these patients. We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on U937 cells and AML blasts. Preincubation with decitabine (0.1-1 microM) and subsequent exposure to VD3 (3 nM) synergistically induced monocytic differentiation. To elucidate the mechanisms of decitabine- and VD3-induced monocytic differentiation, we investigated the effects of the two drugs on transcription factors implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Using electromobility shift assays, we demonstrated increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis. In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.

The C/EBPdelta tumor suppressor is silenced by hypermethylation in acute myeloid leukemia.

Aberrant DNA methylation is the most frequent molecular alteration in acute myeloid leukemia (AML). To identify methylation-silenced genes in AML, we performed microarray analyses in U937 cells exposed to the demethylating agent 5-aza-deoxy-cytidine. Overall, 274 transcripts were significantly induced. Interestingly, C/EBPdelta expression was significantly induced (more than 10-fold) by demethylation whereas expression of all other C/EBP family members remained unchanged. The C/EBPdelta promoter was strongly methylated in different leukemic cell lines and showed signs of a repressed chromatin state. Analyses of the promoter regions of the entire C/EBP family (alpha, beta, gamma, delta, epsilon, zeta) in bone marrow samples from AML patients (n = 80) and controls (n = 15) by mass spectrometry revealed that C/EBPdelta is the most commonly hypermethylated C/EBP gene in AML. Hypermethylation occurred in more than 35% of AML patients at primary diagnosis. A significant correlation (P = .016) was observed between hypermethylation of the C/EBPdelta promoter and low expression of C/EBPdelta in AML patients. C/EBPdelta promoter activity was strongly repressed by methylation in vitro, and transcriptional repression partially depended on MeCP2 activity. C/EBPdelta exhibited growth-inhibitory properties in primary progenitor cells as well as in Flt3-ITD-transformed cells. Taken together, C/EBPdelta is a novel tumor suppressor gene in AML that is silenced by promoter methylation.

Cyclin A1, the alternative A-type cyclin, contributes to G1/S cell cycle progression in somatic cells.

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1-/- cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1-/- mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.

Specific protein redirection as a transcriptional therapy approach for t(8;21) leukemia.

Important progress has been achieved in the knowledge about the pathogenesis of cancer. However, despite these advances, the therapeutic strategies are still limited. Leukemias are often characterized by specific balanced translocations, with the t(8;21) balanced translocation being the most frequent chromosomal aberration in acute myeloid leukemia (AML). This translocation produces the AML1-ETO fusion protein, which binds to AML1 target promoter sequences. Transcriptional repression of AML1-dependent genes by AML1-ETO and associated corepressors represents the pathogenetic mechanisms of t(8;21). Here, we show that targeting of AML1-ETO to essential, MYB-dependent gene promoters induces t(8;21)-restricted cell death. We constructed a chimeric protein that contained the MYB DNA-binding domain and the AML1-binding domain of myeloid Elf-1-like factor (MEF). This protein associated with AML1-ETO and directed the complex to MYB-responsive promoters in vitro and in vivo. In the presence of AML1-ETO, the chimeric protein repressed the activity of MYB-responsive promoters, rapidly induced apoptosis, and specifically inhibited colony growth. All these effects occurred only in AML1-ETO-positive cells, whereas no adverse effects were observed in cells not expressing AML1-ETO. Taken together, this study demonstrates that redirection of oncogenic proteins can be used as a strategy to dramatically influence their cellular effects, with the ultimate goal to design highly specific therapies for cancer.

Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations.

The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT- and FLT3-ITD-expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD-expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPalpha, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3-independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.