The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Anti-HIV Activity of Snake Venom Phospholipase A2s: Updates for New Enzymes and Different Virus Strains.

Since the beginning of the HIV epidemic, lasting more than 30 years, the main goal of scientists was to develop effective methods for the prevention and treatment of HIV infection. Modern medicines have reduced the death rate from AIDS by 80%. However, they still have side effects and are very expensive, dictating the need to search for new drugs. Earlier, it was shown that phospholipases A2 (PLA2s) from bee and snake venoms block HIV replication, the effect being independent on catalytic PLA2 activity. However, the antiviral activity of human PLA2s against Lentiviruses depended on catalytic function and was mediated through the destruction of the viral membrane. To clarify the role of phospholipolytic activity in antiviral effects, we analyzed the anti-HIV activity of several snake PLA2s and found that the mechanisms of their antiviral activity were similar to that of mammalian PLA2. Our results indicate that snake PLA2s are capable of inhibiting syncytium formation between chronically HIV-infected cells and healthy CD4-positive cells and block HIV binding to cells. However, only dimeric PLA2s had pronounced virucidal and anti-HIV activity, which depended on their catalytic activity. The ability of snake PLA2s to inactivate the virus may provide an additional barrier to HIV infection. Thus, snake PLA2s might be considered as candidates for lead molecules in anti-HIV drug development.

SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

Serglycin induces osteoclastogenesis and promotes tumor growth in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

Mini-TrpRS is essential for IFNγ-induced monocyte-derived giant cell formation.

Truncated tryptophanyl-tRNA synthetase (mini-TrpRS), like any other aminoacyl-tRNA synthetases, canonically functions as a protein synthesis enzyme. Here we provide evidence for an additional signaling role of mini-TrpRS in the formation of monocyte-derived multinuclear giant cells (MGCs). Interferon-gamma (IFNγ) readily induced monocyte aggregation leading to MGC formation with paralleled marked upregulation of mini-TrpRS. Small interfering (si)RNA, targeting mini-TrpRS in the presence of IFNγ prevented monocyte aggregation. Moreover, blockade of mini-TrpRS, either by siRNA, or the cognate amino acid and decoy substrate D-Tryptophan to prevent mini-TrpRS signaling, resulted in a marked reduction in expression of the purinergic receptor P2X 7 (P2RX7) in monocytes activated by IFNγ. Our findings identify mini-TrpRS as a critical signaling molecule in a mechanism by which IFNγ initiates monocyte-derived giant cell formation.

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis.

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.

Comparison of bone wax and chitosan usage on post-sternotomy bone healing.

BACKGROUND: Sternotomy is a standard approach performed in almost every surgical procedure on the heart and mediastinum. Effective hemostasis of the sternum is required to keep the operative field dry, avoid excessive blood transfusions during surgery, and prevent reoperation due to massive postoperative bleeding, which can further increase morbidity and mortality in patients. Bone wax is a mechanical hemostat commonly used after sternotomy and has been known to affect bone healing, trigger chronic inflammatory reactions, and increase the rate of infection. The application of chitosan, which has intrinsic hemostat ability, as hemostatic material is believed to improve bone healing following sternotomy. This study aimed to compare the effectiveness of bone wax and chitosan on bone healing after sternotomy. METHODS: Median sternotomies were performed on 2 groups of New Zealand White rabbits. Each group of 16 animals received either bone wax or chitosan powder as hemostatic material. The degree of bone healing, the number of foreign-body giant cells, and the number of osteoblasts were evaluated after 6 weeks. RESULTS: Radiographs showed that significantly more animals in the chitosan group had total sternal healing (p = 0.033). Histopathology revealed that the number of foreign-body giant cells was significantly less (p = 0.036) and the number of osteoblasts was significantly greater (p < 0.0001) in the group of animals that received chitosan. CONCLUSION: The use of chitosan as hemostatic material can promote better bone healing compared to bone wax.

Loss of Aurora Kinase Signaling Allows Lung Cancer Cells to Adopt Endoreplication and Form Polyploid Giant Cancer Cells That Resist Antimitotic Drugs.

Polyploid giant cancer cells (PGCC) are common in tumors and have been associated with resistance to cancer therapy, tumor relapse, malignancy, immunosuppression, metastasis, cancer stem cell production, and modulation of the tumor microenvironment. However, the molecular mechanisms that cause these cells to form are not yet known. In this study, we discover that Aurora kinases are synergistic determinants of a switch from the proliferative cell cycle to polyploid growth and multinucleation in lung cancer cell lines. When Aurora kinases were inhibited together, lung cancer cells uniformly grew into multinucleated PGCCs. These cells adopted an endoreplication in which the genome replicates, mitosis is omitted, and cells grow in size. Consequently, such cells continued to safely grow in the presence of antimitotic agents. These PGCC re-entered the proliferative cell cycle and grew in cell number when treatment was terminated. Thus, PGCC formation might represent a fundamental cellular response to Aurora kinase inhibitors and contributes to therapy resistance or tumor relapse. SIGNIFICANCE: These findings provide a novel insight about how cancer cells respond to Aurora kinase inhibitors and identify a new mechanism responsible for resistance to these agents and other antimitotic drugs.

Cisplatin-Induced Giant Cells Formation Is Involved in Chemoresistance of Melanoma Cells.

Melanoma is notoriously resistant to current cancer therapy. However, the chemoresistance mechanism of melanoma remains unclear. The present study unveiled that chemotherapy drug cisplatin induced the formation of giant cells, which exhibited enlargement in cell diameter and nucleus in mice and human melanoma cells. Giant cells were positive with melanoma maker S100 and cancer stem cell markers including ABCB5 and CD133 in vitro and in vivo. Moreover, giant cells retained the mitotic ability with expression of proliferation marker Ki-67 and exhibited multiple drug resistance to doxorubicin and actinomycin D. The mitochondria genesis/activities and cellular ATP level were significantly elevated in giant cells, implicating the demand for energy supply. Application of metabolic blockers such as sodium azide or 2-deoxy glucose abolished the cisplatin-induced giant cells formation and expression of cancer stemness markers. The present study unveils a novel chemoresistance mechanism of melanoma cells via size alteration and the anti-neoplastic strategy by targeting giant cells.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

Vimentin filaments drive migratory persistence in polyploidal cancer cells.

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.

Delta-9-tetrahydrocannabinol disrupts mitochondrial function and attenuates syncytialization in human placental BeWo cells.

The psychoactive component in cannabis, delta-9-tetrahydrocannabinol, can restrict fetal growth and development. Delta-9-tetrahydrocannabinol has been shown to negatively impact cellular proliferation and target organelles like the mitochondria resulting in reduced cellular respiration. In the placenta, mitochondrial dysfunction leading to oxidative stress prevents proper placental development and function. A key element of placental development is the proliferation and fusion of cytotrophoblasts to form the syncytium that comprises the materno-fetal interface. The impact of delta-9-tetrahydrocannabinol on this process is not well understood. To elucidate the nature of the mitochondrial dysfunction and its consequences on trophoblast fusion, we treated undifferentiated and differentiated BeWo human trophoblast cells, with 20 µM delta-9-tetrahydrocannabinol for 48 hr. At this concentration, delta-9-tetrahydrocannabinol on BeWo cells reduced the expression of markers involved in syncytialization and mitochondrial dynamics, but had no effect on cell viability. Delta-9-tetrahydrocannabinol significantly attenuated the process of syncytialization and induced oxidative stress responses in BeWo cells. Importantly, delta-9-tetrahydrocannabinol also caused a reduction in the secretion of human chorionic gonadotropin and the production of human placental lactogen and insulin growth factor 2, three hormones known to be important in facilitating fetal growth. Furthermore, we also demonstrate that delta-9-tetrahydrocannabinol attenuated mitochondrial respiration, depleted adenosine triphosphate, and reduced mitochondrial membrane potential. These changes were also associated with an increase in cellular reactive oxygen species, and the expression of stress responsive chaperones, HSP60 and HSP70. These findings have important implications for understanding the role of delta-9-tetrahydrocannabinol-induced mitochondrial injury and the role this might play in compromising human pregnancies.

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections.

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-µM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Histone deacetylase 1 and 2 drive differentiation and fusion of progenitor cells in human placental trophoblasts.

Cell fusion occurs when several cells combine to form a multinuclear aggregate (syncytium). In human placenta, a syncytialized trophoblast (syncytiotrophoblast) layer forms the primary interface between maternal and fetal tissue, facilitates nutrient and gas exchange, and produces hormones vital for pregnancy. Syncytiotrophoblast development occurs by differentiation of underlying progenitor cells called cytotrophoblasts, which then fuse into the syncytiotrophoblast layer. Differentiation is associated with chromatin remodeling and specific changes in gene expression mediated, at least in part, by histone acetylation. However, the epigenetic regulation of human cytotrophoblast differentiation and fusion is poorly understood. In this study, we found that human syncytiotrophoblast development was associated with deacetylation of multiple core histone residues. Chromatin immunoprecipitation sequencing revealed chromosomal regions that exhibit dynamic alterations in histone H3 acetylation during differentiation. These include regions containing genes classically associated with cytotrophoblast differentiation (TEAD4, TP63, OVOL1, CGB), as well as near genes with novel regulatory roles in trophoblast development and function, such as LHX4 and SYDE1. Prevention of histone deacetylation using both pharmacological and genetic approaches inhibited trophoblast fusion, supporting a critical role of this process for trophoblast differentiation. Finally, we identified the histone deacetylases (HDACs) HDAC1 and HDAC2 as the critical mediators driving cytotrophoblast differentiation. Collectively, these findings provide novel insights into the epigenetic mechanisms underlying trophoblast fusion during human placental development.

Mechanical Circulatory Support Combined With Immunosuppression for the Treatment of Giant Cell Myocarditis　- A Single-Center Experience in Japan.

BACKGROUND: The therapeutic strategy for giant cell myocarditis (GCM) remains controversial, so we reviewed the clinical status of Japanese patients with GCM.Methods and Results:We retrospectively reviewed 6 consecutive patients with GCM requiring percutaneous mechanical circulatory support (p-MCS), with 3 further requiring ventricular assist devices. One patient died during p-MCS. Cardiac function improved in the other 5 with immunosuppressive therapy, but only 3 patients treated with dual immunosuppressants, including cyclosporine (CyA), achieved >1-year survival. CONCLUSIONS: The prognosis of patients with fulminant GCM is poor, but a treatment that combines MCS and early administration of CyA-based immunosuppressants will be useful.

Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function.

INTRODUCTION: Placental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined. METHODS: An ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions. RESULTS: In the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01). CONCLUSION: The current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.