Gentamicin affects the bioenergetics of isolated mitochondria and collapses the mitochondrial membrane potential in cochlear sensory hair cells

Aminoglycoside antibiotics are widely prescribed to treat a variety of serious bacterial infections. They are extremely useful clinical tools, but have adverse side effects such as oto- and nephrotoxicity. Once inside a cell they are thought to cause mitochondrial dysfunction, subsequently leading to apoptotic cell death due to an increase in reactive oxygen species (ROS) production. Here we present evidence of a direct effect of gentamicin (the most commonly prescribed aminoglycoside) on the respiratory activities of isolated rat liver and kidney mitochondria. We show that gentamicin stimulates state 4 and inhibits state 3u respiratory rates, thereby reducing the respiratory control ratio (RCR) whilst simultaneously causing a collapse of the mitochondrial membrane potential (MtMP). We propose that gentamicin behaves as an uncoupler of the electron transport chain (ETC) – a hypothesis supported by our evidence that it reduces the production of mitochondrial ROS (MtROS). We also show that gentamicin collapses the MtMP in the sensory hair cells (HCs) of organotypic mouse cochlear cultures

Cisplatin increases PD-L1 expression and optimizes immune check-point blockade in non-small cell lung cancer

The number of clinical protocols testing combined therapies including immune check-point inhibitors and platinum salts is currently increasing in lung cancer treatment, however preclinical studies and rationale are often lacking. Here, we evaluated the impact of cisplatin treatment on PD-L1 expression analyzing the clinicopathological characteristics of patients who received cisplatin-based neoadjuvant chemotherapy followed by surgery and showed that cisplatin-based induction treatment significantly increased PD-L1 staining in both tumor and immune cells from the microenvironment. Twenty-two patients exhibited positive PD-L1 staining variation after neoadjuvant chemotherapy; including 9 (23.1%) patients switching from <50% to 6550% of stained tumor-cells. We also confirmed the up-regulation of PD-L1 by cisplatin, at both RNA and protein levels, in nude and immunocompetent mice bearing tumors grafted with A549, LNM-R, or LLC1 lung cancer cell lines. The combined administration of anti-PD-L1 antibodies (3 mg/kg) and cisplatin (1 mg/kg) to mice harboring lung carcinoma significantly reduced tumor growth compared to single agent treatments and controls. Overall, these results suggest that cisplatin treatment could synergize with PD-1/PD-L1 blockade to increase the clinical response, in particular through early and sustainable enhancement of PD-L1 expression

Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis ▿ †

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells.

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34(+) progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA

The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences