Pulmonary Tumor Embolism and Pulmonary Tumor Thrombotic Microangiopathy Causing Rapidly Progressive Respiratory Failure: A Case Series.

Pulmonary tumor embolism (PTE) and pulmonary tumor thrombotic microangiopathy (PTTM) are rare etiologies for rapidly progressive dyspnea in the setting of undiagnosed metastatic cancer. They occur most frequently in association with adenocarcinomas, with PTE being most frequently associated with hepatocellular carcinoma and PTTM being most commonly reported with gastric adenocarcinoma. Pulmonary tumor embolism and PTTM appear to be a disease spectrum where PTTM represents an advanced form of PTE. Pulmonary tumor embolism and PTTM are mostly identified postmortem during autopsy as the antemortem diagnosis remains a clinical challenge due to the rapidly progressive nature of these rare diseases. We report 2 cases of rapidly progressive respiratory failure leading to death, due to tumoral pulmonary hypertension resulting from PTE and PTTM, diagnosed postmortem. Both of the patients were middle-aged females, nonsmokers, and had a gastrointestinal source of their primary malignancy.

Pulmonary Barotrauma in COVID-19 Patients With ARDS on Invasive and Non-Invasive Positive Pressure Ventilation.

BACKGROUND: We experienced a high incidence of pulmonary barotrauma among patients with coronavirus disease-2019 (COVID-19) associated acute respiratory distress syndrome (ARDS) at our institution. In current study, we sought to evaluate the incidence, clinical outcomes, and characteristics of barotrauma among COVID-19 patients receiving invasive and non-invasive positive pressure ventilation. METHODOLOGY: This retrospective cohort study included adult patients diagnosed with COVID-19 pneumonia and requiring oxygen support or positive airway pressure for ARDS who presented to our tertiary-care center from March through November, 2020. RESULTS: A total of 353 patients met our inclusion criteria, of which 232 patients who required heated high-flow nasal cannula, continuous or bilevel positive airway pressure were assigned to non-invasive group. The remaining 121 patients required invasive mechanical ventilation and were assigned to invasive group. Of the total 353 patients, 32 patients (65.6% males) with a mean age of 63 ± 11 years developed barotrauma in the form of subcutaneous emphysema, pneumothorax, or pneumomediastinum. The incidence of barotrauma was 4.74% (11/232) and 17.35% (21/121) in the non-invasive group and invasive group, respectively. The median length of hospital stay was 22 (15.7 -33.0) days with an overall mortality of 62.5% (n = 20). CONCLUSIONS: Patients with COVID-19 ARDS have a high incidence of barotrauma. Pulmonary barotrauma should be considered in patients with COVID-19 pneumonia who exhibit worsening of their respiratory disease as it is likely associated with a high mortality risk. Utilizing lung-protective ventilation strategies may reduce the risk of barotrauma.

A Case of Extracorporeal Membrane Oxygenation as a Salvage Therapy for COVID-19-Associated Severe Acute Respiratory Distress Syndrome: Mounting Evidence.

Coronavirus disease 2019 (COVID-19) caused by a novel human coronavirus has led to a tsunami of viral illness across the globe, originating from Wuhan, China. Although the value and effectiveness of extracorporeal membrane oxygenation (ECMO) in severe respiratory illness from COVID-19 remains unclear at this time, there is emerging evidence suggesting that it could be utilized as an ultimate treatment in appropriately selected patients not responding to conventional care. We present a case of a 32-year-old COVID-19 positive male with a history of diabetes mellitus who was intubated for severe acute respiratory distress syndrome (ARDS). The patient's hypoxemia failed to improve despite positive pressure ventilation, prone positioning, and use of neuromuscular blockade for ventilator asynchrony. He was evaluated by a multidisciplinary team for considering ECMO for refractory ARDS. He was initiated on venovenous ECMO via dual-site cannulation performed at the bedside. Although his ECMO course was complicated by bleeding, he showed a remarkable improvement in his lung function. ECMO was successfully decannulated after 17 days of initiation. The patient was discharged home after 47 days of hospitalization without any supplemental oxygen and was able to undergo active physical rehabilitation. A multidisciplinary approach is imperative in the initiation and management of ECMO in COVID-19 patients with severe ARDS. While ECMO is labor-intensive, using it in the right phenotype and in specialized centers may lead to positive results. Patients who are young, with fewer comorbidities and single organ dysfunction portray a better prognosis for patients in which ECMO is utilized.

Regulation of the Nanog gene by both positive and negative cis-regulatory elements in embryonal carcinoma cells and embryonic stem cells.

The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis-regulatory elements that influence the activity of the Nanog promoter, including adjacent cis-regulatory elements that bind Sox2 and Oct-3/4. Given the importance of Nanog during mammalian development, we examined the cis-regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis-regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis-regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis-regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis-regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis-regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds.

Identification of DPPA4 and other genes as putative Sox2:Oct-3/4 target genes using a combination of in silico analysis and transcription-based assays.

Sox2 and Oct-3/4 function as master regulators during mammalian embryogenesis, where they are believed to regulate a critical gene regulatory network by cooperatively binding to DNA regulatory regions composed of adjacent HMG and POU motifs (HMG/POU cassettes). Previous studies have identified seven genes that contain highly active HMG/POU cassettes (referred to as Sox2:Oct-3/4 target genes). Importantly, nearly all known Sox2:Oct-3/4 target genes appear to be essential for embryogenesis. Recent genome-wide ChIP-chip studies identified over 300 genes that are co-occupied by Sox2 and Oct-3/4, which suggests that most Sox2:Oct-3/4 target genes remain to be identified. The work described here used a 3-step strategy for identifying additional Sox2:Oct-3/4 target genes. First, we employed in silico analysis to search for putative HMG/POU cassettes in 50 genes reported to be co-occupied by Sox2 and Oct-3/4 in embryonic stem cells. We identified 39 genes that contain putative HMG/POU cassettes. Next, we tested the activity of seven of the putative HMG/POU cassettes in a transcription-based assay and determined that nearly all are functional. Finally, as a proof-of-principle, we tested one of the seven cassettes (DPPA4) in the context of its endogenous promoter using a promoter/reporter gene construct. DPPA4 was tested in part because it was shown recently to play an important role in ES cell self-renewal. We determined that the 5' flanking region of the DPPA4 gene contains a functional HMG/POU cassette and behaves as a Sox2:Oct-3/4 target gene. Finally, we used a transcription-based assay to help develop a refined consensus sequence for HMG/POU cassettes.

Differential regulation of the Oct-3/4 gene in cell culture model systems that parallel different stages of mammalian development.

Oct-3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct-3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis-elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH-1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct-3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH-1 site functions as a positive and a negative cis-regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct-3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct-3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct-3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct-3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct-3/4 gene in EC cells is due to differential action of Sox2 and its associated co-factors.

Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes.

Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma cells that are associated with the transcription factors Sox2 and Oct-3/4. Other studies have shown that Sox2 and Oct-3/4 work together cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.

Differential activity of the FGF-4 enhancer in F9 and P19 embryonal carcinoma cells.

Transcription factors Oct-3/4 and Sox2 behave as global regulators during mammalian embryogenesis. They work together by binding co-operatively to closely spaced HMG and POU motifs (HMG/POU cassettes). Recently, it was suggested that a critical Sox2:Oct-3/4 target gene, FGF-4, is expressed at lower levels in P19 than in F9 embryonal carcinoma (EC) cells, due to lower levels of Sox2 in P19 than in F9 cells. We tested this possibility to better understand how FGF-4 expression is modulated during development. Although we found that P19 EC cells express approximately 10-fold less FGF-4 mRNA than F9 EC cells, we determined that Sox2 levels do not differ markedly in F9 and P19 EC cells. We also determined that Sox2 and Oct-3/4 work together equally well in both EC cell lines. Moreover, in contrast to an earlier prediction based on in vitro binding studies, we demonstrate that the function of the HMG/POU cassettes of the FGF-4 and UTF1 genes does not differ significantly in these EC cell lines when tested in the context of a natural enhancer. Importantly, we determined that the FGF-4 promoter is highly responsive to a heterologous enhancer in both EC cell lines; whereas, the FGF-4 enhancer is 7- to 10-fold less active in P19 than in F9 EC cells. Because F9 and P19 EC cells are likely to represent cells at different stages of mammalian development, we suggest that this difference in FGF-4 enhancer activity may reflect a mechanism used to decrease, but not abolish, FGF-4 expression as the early embryo develops.

Distal enhancer of the mouse FGF-4 gene and its human counterpart exhibit differential activity: critical role of a GT box.

Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells.

Transcriptional regulation of the murine Elf3 gene in embryonal carcinoma cells and their differentiated counterparts: requirement for a novel upstream regulatory region.

The transcription factor Elf3, which is one of over 25 Ets family members, is expressed in a wide variety of carcinomas and has been shown to promote the transcription of many genes implicated in cancer. To understand how the Elf3 gene is regulated at the transcriptional level, we probed its 5'-flanking region, and we report here the identification of both proximal and distal regions that regulate murine Elf3 promoter activity. In addition to mapping the transcription start site of the Elf3 gene, the work described in this study identifies four cis-regulatory elements in the proximal promoter region of the gene. These include a cis-regulatory element previously designated ESE, a kappaB site, a POU motif, and a CCAAT box. In addition, we demonstrate that a novel 94 bp region 2 kb upstream of the transcription start site significantly elevates Elf3 promoter activity in F9-differentiated cells, but not in the parental F9 embryonal carcinoma (EC) cells. This region appears to be largely responsible for the increase in Elf3 promoter activity that accompanies the differentiation of embryonal carcinoma cells.