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Research Question: Does the dose of gonadotropin used for superovulation in IVF affect the proportion of euploid blastocysts obtained after fertilization? Study Design: Multicentre randomized controlled trial recruiting 57 women who were treated with ovarian stimulation using either 150 or 300 IU Menopur per day. Both groups received GnRH antagonist from day 5 of ovarian stimulation and final oocyte maturation was induced using a leuprolide GnRH (gonadotropin releasing hormone) agonist trigger when three or more follicles reached 17 mm diameter. Oocyte collection was scheduled 36–38 hours post trigger. In vitro fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI) were performed according to individual unit protocol and embryos were cultured to blastocyst stage. A trophectoderm biopsy was performed on day 5 of embryo culture and used for preimplantation genetic testing for aneuploidy. Euploid embryos were transferred in subsequent frozen embryo transfer cycles with appropriate endometrial preparation. Results: The number of oocytes obtained from women randomized to 150 IU Menopur was between 3 and 17 (mean = 9), whereas the number of oocytes obtained from women randomized to 300 IU Menopur was between 3 and 24 (mean = 11). There was a positive linear relationship between serum AMH concentration and oocyte yield in both the 150 and 300 IU Menopur groups (R = 0.3359, R2 = 0.1129 and R = 0.3741, R2 = 0.1399). The percentage of euploid to aneuploid embryos in the 150 IU Menopur group was 63% and in the 300 IU Menopur group, the proportion was 75%, which was not significantly different (p = 0.17). Conclusion: The higher dose ovarian stimulation protocol did not significantly increase the number of oocytes retrieved, nor did the higher dose protocol reduce the proportion of euploid embryos created. This study does not support the hypothesis that use of higher doses of gonadotropin for ovarian stimulation results in a reduction in the proportion of euploid embryos obtained after IVF.

Abstract Background Coronavirus disease 2019 (COVID-19) has become a global pandemic and is posing a serious public health problem for almost all countries. Spontaneous pneumomediastinum, a rare condition, is usually seen in patients with underlying pulmonary pathology, infections, or mechanical ventilation. Spontaneous pneumomediastinum is a rare complication in COVID-19 pneumonia. Case presentation We report a case of spontaneous pneumomediastinum, pneumopericardium, and subcutaneous emphysema in a 62-year-old diabetic patient with COVID-19 infection who presented with cough, fever, and breathlessness, which turned to be a fatal complication. Conclusion Pneumomediastinum/subcutaneous emphysema, a not so common complication associated with COVID-19 infection, should be considered as a bad prognostic indicator of worsening disease and hence requires early recognition and careful monitoring of the patient for any possible unfavorable outcome.

Introduction: Second generation Supraglottic Airway Devices (SADs) contain a gastric drain tube which separates the respiratory and the alimentary tract. This provides a better oropharyngeal seal and reduces the risk of pulmonary aspiration of refluxed gastric contents compared to the first generation SADs. Aim: To compare Ambu® AuraGain™ (AAU) laryngeal mask airway with LMA® ProSeal™ (PLMA) in terms of Oropharyngeal Leak Pressure (OLP) in laparoscopic surgeries. Materials and Methods: This randomised clinical study was conducted from December 2017-September 2019, at Shri Dharmasthala Manjunatheshwara College of Medical Sciences and Hospital, Dharwad, India in 80 patients, aged 18-65 years, of American Society of Anaesthesiologists (ASA) physical status I and II undergoing laparoscopic surgeries. Patients were randomly assigned to one of the two groups: group PLMA and group AAU. After induction of anaesthesia, SADs were inserted by an experienced anaesthesiologist. OLP, pharyngeal mucosal pressure, peak airway pressure and secondary outcome parameters (the number of attempts, time required, ease, and haemodynamic response associated with insertion of LMA) were recorded at set time points. Data was analysed using Statistical Packages for Social Sciences (SPSS) version 22. Results: All patients in both the groups were comparable in terms of demographic data and baseline vital parameters. The Oropharyngeal Leak Pressure of group AAU was comparable to group PLMA at all measured time-points. The two groups were comparable in terms of pharyngeal mucosal pressure immediately after insertion of LMA, but group AAU had lesser pharyngeal mucosal pressure compared to group PLMA immediately after pneumoperitoneum, at 30 and 60 minutes. Mean peak airway pressures were lower in group AAU than group PLMA immediately after insertion of LMA (15.53±1.50 versus 17.06±2.56 cmH2O, p=0.004) and immediately after creation of pneumoperitoneum (23.03±2.96 versus 26.58±10.12 cmH2O, p=0.04). Both the groups were comparable in terms of number of attempts, time taken, haemodynamic response associated with LMA insertion and with passage of gastric tube except that PLMA was easier to insert in the first attempt compared to AAU (26/40 versus 13/40, Grade 1 ease of insertion). Conclusion: Ambu® AuraGain™ could be a useful alternative to LMA® ProSeal™ in patients undergoing laparoscopic surgeries.

Abstract Background COVID-19 has caused a global pandemic of unprecedented proportions. Elective cardiac surgery has been universally postponed with only urgent and emergency cardiac operations being performed. The National Health Service in the United Kingdom introduced national measures to conserve intensive care beds and significantly limit elective activity shortly after lockdown. Case presentation We report two cases of early post-operative mortality secondary to COVID-19 infection immediately prior to the implementation of these widespread measures. Conclusion The role of cardiac surgery in the presence of COVID-19 is still very unpredictable and further studies on both short term and long term outcomes are warranted.

Abstract Background Despite declining incidence over the past decade, malaria remains an important health burden in India. This study aimed to assess the village-level temporal patterns of Plasmodium infection in two districts of the north-eastern state of Meghalaya and evaluate risk factors that might explain these patterns. Methods Primary Health Centre passive malaria case data from 2014 to 2018 were analysed to characterize village-specific annual incidence and temporal trends. Active malaria case detection was undertaken in 2018 and 2019 to detect Plasmodium infections using PCR. A questionnaire collected socio-demographic, environmental, and behavioural data, and households were spatially mapped via GPS. Adult mosquitoes were sampled at a subset of subjects' houses, and Anopheles were identified by PCR and sequencing. Risk factors for Plasmodium infection were evaluated using bivariate and multivariate logistic regression analysis, and spatial cluster analysis was undertaken. Results The annual malaria incidence from PHC-based passive surveillance datasets in 2014–2018 was heterogenous but declining across villages in both districts. Active surveillance in 2018 enrolled 1468 individuals from 468 households (West Jaintia Hills) and 1274 individuals from 359 households (West Khasi Hills). Plasmodium falciparum prevalence per 100 people varied from 0 to 4.1% in the nine villages of West Jaintia Hills, and from 0 to 10.6% in the 12 villages of West Khasi Hills. Significant clustering of P. falciparum infections [observed = 11, expected = 2.15, Relative Risk (RR) = 12.65; p

During the times of the ongoing COVID pandemic, aerosol-generating procedures such as bronchoscopy have the potential of transmission of severe acute respiratory syndrome coronavirus 2 to the healthcare workers. The decision to perform bronchoscopy during the COVID pandemic should be taken judiciously. Over the years, the indications for bronchoscopy in the clinical practice have expanded. Experts at the Indian Association for Bronchology perceived the need to develop a concise statement that would assist a bronchoscopist in performing bronchoscopy during the COVID pandemic safely. The current Indian Association for Bronchology Consensus Statement provides specific guidelines including triaging, indications, bronchoscopy area, use of personal protective equipment, patient preparation, sedation and anesthesia, patient monitoring, bronchoscopy technique, sample collection and handling, bronchoscope disinfection, and environmental disinfection concerning the coronavirus disease-2019 situation. The suggestions provided herewith should be adopted in addition to the national bronchoscopy guidelines that were published recently. This statement summarizes the essential aspects to be considered for the performance of bronchoscopy in COVID pandemic, to ensure safety for both for patients and healthcare personnel.

Background: Masticatory forces generate various degrees of stress and strain in the periodontium of teeth which determine the clinical functions and load-bearing capacity of the teeth. There are few in vitro studies that have analyzed stress generated due to combined forces acting on the teeth. Thus, the objective of the present study was to do a comparative analysis of the influence of various stresses on the periodontal ligament and alveolar bone of maxillary central incisor with normal bone height and reduced bone height under simulated standard masticatory using finite element stress analysis. Methodology: A 3D model of the tooth was obtained with the help of ANSYS software. These models were subjected to various oblique forces, i.e., 100N and 235.9N, applied at 45° angle on the lingual surface of the maxillary central incisor and stress values were recorded in three dimensions. The results from FE analysis were analyzed using 3D Von Mises Criteria. Results: It was observed that in healthy periodontium; it was observed that among the periodontal structure studied, the maximum stress levels were exerted on root followed by cortical bone, cancellous bone, and PDL, irrespective of the force, as compared to the diseased periodontium, in which the bone height was reduced, the maximum stresses were on root followed by cortical bone, PDL, and cancellous bone. Conclusion: The main factor governing the success of any periodontal procedure depends on the height of the remaining bone and the amount of force exerted on to the tooth and the stress generated within the tooth. The finite element method could be of substantial importance in this respect as it can assess the stresses of various occlusal forces on the periodontal ligament, root, cortical bone, and cancellous bone of teeth in a periodontally healthy and diseased state.

Background: High dose gonadotropin stimulation protocols may increase costs, patient discomfort, likelihood of ovarian hyper stimulation syndrome (OHSS), ovarian torsion and increased bleeding after multiple ovarian punctures. In addition, it is possible that high dose stimulation may result in impaired oocyte “quality” with adverse effect of embryonic ploidy. This may negate any benefits seen from a higher oocyte yield after high dose stimulation. Aim: To determine the proportion of euploid blastocysts resulting from higher versus lower dose ovarian stimulation protocols. Method: Multicentre randomised controlled trial recruiting 57 women who were treated with ovarian stimulation using either 150 or 300 IU Menopur per day. In Vitro Fertilisation (IVF) or Intracytoplasmic Sperm Injection (ICSI) were performed according to individual unit protocol and embryos were cultured to blastocyst stage. A trophectoderm biopsy was performed for preimplant action genetic testing for aneuploidy. Results: The number of oocytes obtained from women randomised to 150 IU Menopur was between 3–17 (mean=9), whereas the number of oocytes obtained from women randomised to 300 IU Menopur was between 3–24 (mean=11). There was a positive linear relationship between serum AMH concentration and oocyte yield in both the 150 & 300 IU Menopur groups (R=0.3359, R2=0.1129 & R=0.3741, R2=0.1399). 63% of blastocysts were euploid in the 150 IU Menopur group and 75% in the 300 IU Menopur group (p=0.17). Conclusion: The higher dose ovarian stimulation protocol did not significantly increase the number of oocytes retrieved, nor did the higher dose protocol reduce the proportion of euploid embryos created. This study does not support the hypothesis that use of higher doses of gonadotropin for ovarian stimulation results in a reduction in the proportion of euploid embryos obtained after IVF.

Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 TC cell and 40 TH cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by TC and TH cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro and in vivo with insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells.

Liposarcoma, the most common soft tissue sarcoma, is a group of fat cell mesenchymal tumors with different histological subtypes. The dysregulation of long non-coding RNAs (lncRNAs) has been observed in human cancers including a few studies in sarcoma. However, the global transcriptome analysis and potential role of lncRNAs remain unexplored in liposarcoma. The present investigation uncovers the transcriptomic profile of liposarcoma by RNA sequencing to gain insight into the global transcriptional changes in liposarcoma. Our RNA sequencing analysis has identified that many oncogenic lncRNAs are differentially expressed in different subtypes of liposarcoma including MALAT1, PVT1, SNHG15, LINC00152, and MIR210HG. Importantly, we identified a highly overexpressed, unannotated, and novel lncRNA in dedifferentiated liposarcomas. We have named it TODL, transcript overexpressed in dedifferentiated liposarcoma. TODL lncRNA displayed significantly higher expression in dedifferentiated liposarcoma cell lines and patient samples. Interestingly, functional studies revealed that TODL lncRNA has an oncogenic function in liposarcoma cells by regulating proliferation, cell cycle, apoptosis, differentiation, and tumorigenesis in the murine model. Silencing of TODL lncRNA highlighted the enrichment of several key oncogenic signaling pathways including cell cycle, transcriptional misregulation, FOXM1 network, p53 signaling, PLK1 signaling, FoxO, and signaling Aurora signaling pathways. RNA pull-down assay revealed the binding of TODL lncRNA with FOXM1, an oncogenic transcription factor, and the key regulator of the cell cycle. Silencing of TODL lncRNA also induces adipogenesis in dedifferentiated liposarcomas. Altogether, our finding indicates that TODL could be utilized as a novel, specific diagnostic biomarker, and a pharmacological target for therapeutic development in controlling aggressive and metastatic dedifferentiated liposarcomas., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.

Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12-THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome caused by mutation in FA pathway proteins, involved in Interstrand Cross Link (ICL) repair. FA cells exhibit in vitro proliferation arrest due to accumulated DNA damage, hence understanding the rescue mechanism that renders proliferation advantage is required. Gene expression profiling performed in FA patients Peripheral Blood Mononuclear Cells (PBMCs) revealed a wide array of dysregulated biological processes. Functional enrichment and gene clustering analysis showed crippled autophagy process and escalated Notch signalling pathway in FA clinical samples and cell lines. Notch pathway mediators overexpression were reverted in FANCA mutant cells when treated with Rapamycin, an autophagy inducer. Additionally, Rapamycin stabilized cell viability after treatment with the DNA damaging agent, MitomycinC (MMC) and enhanced cell proliferation genes expression in FANCA mutant cells. Inherently FANCA mutant cells express impaired autophagy; thus activation of autophagy channelizes Notch signalling cascade and sustains cell viability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro and in vivo with insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells., Competing Interests: HPK received funding from Morgan Stanley Inc and the Department of Defense (DoD). There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved adenylate-uridylate-rich elements (ARE) located in 3'untranslated regions (UTR) to mediate mRNA decay. We hypothesized that ZFP36L1 is a negative regulator of a posttranscriptional hub involved in mRNA half-life regulation of cancer-related transcripts. Analysis of in silico data revealed that ZFP36L1 was significantly mutated, epigenetically silenced, and downregulated in a variety of cancers. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo , whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wild-type and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells {plus minus} tet-on ZFP36L1 was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1. These targets included a number of key oncogenic transcripts such as HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3'UTRs of these targets for mRNA degradation, thus suppressing their expression. Dual luciferase reporter assays and RNA electrophoretic mobility shift assays showed that wild-type, but not zinc-finger mutant ZFP36L1, bound to HIF1A 3'UTR and mediated HIF1A mRNA degradation, leading to reduced expression of HIF1A and its downstream targets. Collectively, our findings reveal an indispensable role of ZFP36L1 as a posttranscriptional safeguard against aberrant hypoxic signaling and abnormal cell-cycle progression. SIGNIFICANCE: RNA-binding protein ZFP36L1 functions as a tumor suppressor by regulating the mRNA stability of a number of mRNAs involved in hypoxia and cell-cycle signaling., (©2019 American Association for Cancer Research.)

Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients., (Copyright© 2019 Ferrata Storti Foundation.)

Maturation of granulocytes is dependent on controlled gene expression by myeloid lineage restricted transcription factors. CEBPE is one of the essential transcription factors required for granulocytic differentiation. Identification of downstream targets of CEBPE is vital to understand better its role in terminal granulopoiesis. In this study, we have identified Card10 as a novel target of CEBPE. We show that CEBPE binds to regulatory elements upstream of the murine Card10 locus, and expression of CARD10 is significantly reduced in Cebpe knock-out mice. Silencing Card10 in a human cell line and in murine primary cells impaired granulopoiesis, affecting expression of genes involved in myeloid cell development and function. Taken together, our data demonstrate for the first time that Card10 is expressed in granulocytes and is a direct target of CEBPE with functions extending to myeloid differentiation., (Copyright© 2018 Ferrata Storti Foundation.)

Gene expression analysis mainly helps to study gene quantification methods by using various downstream detection approaches like imaging, amplification, probe hybridization, or sequencing. With respect to DNA, which is less static, mRNA levels vary over time, between cell types under divergent conditions. Gene expression analysis is principally focused on determination of mRNA levels transcribed from DNA. DNA microarrays are one of the robust and powerful tools to detect changes in multiple transcripts in larger cohorts in parallel. The basic principle of DNA microarray hybridization is complementary base pairing of single-stranded nucleic-acid sequences. On a microarray platform (also called a chip), known sequences called targets are attached at fixed locations (spots) to a solid surface such as glass using robotic spotting. Since a large number of samples (variables) are used in a typical hybridization experiment, which often leads to impreciseness for example, target mRNA transcribed from the same source should be identical every time. In such cases, developing an optimized protocol for microarray platform to study the expression profiling of differentially regulated genes is a challenging task. Thus genome-wide expression array analysis yields data about candidate genes that may be involved in disease acquisition progression, and helps in better understanding the pathophysiology of the disease. In this chapter we describe in detail the microarray technique, a well-accepted method for understanding the development and progression of Fanconi anemia (FA), a genetic disorder which is characterized by progressive bone marrow failure and a predisposition to cancer.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria., Competing Interests: CONFLICTS OF INTERESTS None.

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. FA cells have been characterized by an overproduction of cytokines, MAPKs, and Interleukins. Through this study we have identified the overexpression of additional secretory factors such as IL-6, IL-8, MMP-2, and MMP-9 in FA cells and in cells depleted of FANCA or FANCC and proved that their expression is under the control of NF-κB/TNF-α signaling pathways. We also demonstrated that these overexpressed secretory factors were effective in promoting the proliferation, migration, and invasion of surrounding tumor cells a fundamental event in the process of epithelial mesenchymal transition (EMT) and that they also modulated the expression of EMT markers such as E-cadherin and SNAIL. Overall our data suggest that the upregulation of EMT promoting factors in FA may contribute to predisposing FA patients to cancer, thereby providing new insights into possible therapeutic interventions., (© 2014 Wiley Periodicals, Inc.)

The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage-neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.

Fanconi anemia (FA) is a rare genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. Besides the well-established role of FA genes in DNA damage and repair pathways, recent reports have revealed an overproduction of epithelial to mesenchymal transition (EMT) factors via a NF-κB-dependent mechanism that results in the proliferation of neighboring tumor cells and FA cells have also been shown to possess damaged mitochondria, accompanied by altered RedOx pathways. This study has focused on reactive oxygen species Modulator-1 (ROMO1), an oncomarker and mitochondrial membrane protein, which is known to be associated with cancer growth and in the modulation of RedOx states in some cancer models. Here, we reveal the role of ROMO1 and demonstrate its link in regulating RedOx states and in the activation of NF-κB-dependent EMT factors in FA., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Fanconi anemia (FA) is known as an inherited bone marrow failure syndrome associated with cancer predisposition and susceptibility to a number of DNA damaging stimuli, along with a number of clinical features such as upper limb malformations, increased diabetes incidence and typical anomalies in skin pigmentation. The proteins encoded by FA-defective genes (FANC proteins) display well-established roles in DNA damage and repair pathways. Moreover, some independent studies have revealed that mitochondrial dysfunction (MDF) is also involved in FA phenotype. Unconfined to FA, we have shown that other syndromes featuring DNA damage and repair (such as ataxia-telangiectasia, AT, and Werner syndrome, WS) display MDF-related phenotypes, along with oxidative stress (OS) that, altogether, may play major roles in these diseases. Experimental and clinical studies are warranted in the prospect of future therapies to be focused on compounds scavenging reactive oxygen species (ROS) as well as protecting mitochondrial functions.

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype., (© 2013 Elsevier B.V. All rights reserved.)

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers like acute myeloid leukemia, lung and squamous cell carcinomas. DNA damage in a healthy cell activates the FA pathway where 15 individual FA proteins interact and function together to maintain genomic stability. The disruption of this pathway results in the characteristic cellular phenotype and clinical outcome of the disease. The diverse clinical symptoms of FA such as impaired immunity and predisposition to cancers may not be explained exclusively by a non-functional FA pathway. These symptoms could then be attributed to defects in other functions of the individual FA proteins. To identify the effects of a mutant FA protein, FANCC, a transcriptome analysis was carried out on a FANCC mutant cell line (EUFA 450) and its revertant isogenic control cell line (EUFA 450Rev). Microarray data revealed dysregulation of genes involved in regulation of cell death and immune response. This study reports for the first time, the lowered expression of a tumor suppressor gene - caveolin-1, in FANCC mutant cells. The downregulation of caveolin-1 can be significant as Fanconi anemia patients have an elevated predisposition to develop cancer., (© 2013 Elsevier B.V. All rights reserved.)