Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.Despite good remission rates observed in acute myeloid leukemia (AML) patients, the 5-year event-free survival rates reach only 35–40% in adults and 60–70% in children (1, 2). Apoptosis is one of the crucial mechanisms influencing survival of AML cells, and its deregulation can possibly lead to chemotherapy resistance and eventually relapse (3–5). The ability of cells to undergo apoptosis is largely defined by the relative expression of anti- (i.e. BCL-2, BCL-X long isoform - BCL-XL, or MCL-1) and proapoptotic (i.e. BAX, BH3 interacting domain death agonist - BID, caspases) proteins. Several studies have shown that the levels of BCL-2 and BCL-2/BAX ratio are a determinant of apoptosis-resistance in AML blasts and are associated with survival in AML patients (3, 6). We have previously demonstrated that the expression of several apoptosis-related proteins, such as BCL-2, BCL-XL, MCL-1, and BAX, can be reliably measured in AML samples by flow cytometry (6). These four quantitative parameters, which constitute an anti-apoptosis index (AAI)¹, have proven to be a reliable predictor of AML patients'' survival, with a high apoptosis-resistant profile (i.e. higher AAI) of diagnosis leukemic blasts being associated with shorter disease-free survival (7). Accordingly, AAI determined at the time of diagnosis also correlated with the frequency of minimal residual disease (MRD), which is a reflection of drug-resistant leukemic cells that have survived chemotherapy (7). MRD can be detected at a low frequency in bone marrow (BM) at the time of remission and is thought to contain the relapse-initiating cells (8–10). These observations imply that leukemic cells that harbor an apoptosis-resistant protein profile at diagnosis can better survive chemotherapy, thereby eventually causing a relapse. Consequently, we further hypothesized that the AAI of MRD cells would be either elevated or at least similar to the profile of leukemic cells at diagnosis. Surprisingly, in complete remission patients, the AAI decreased in the MRD situation compared with apoptosis-resistant profile as measured in leukemic blasts at diagnosis. The values of the AAI profile increased again at relapse, indicating apoptosis-resistance (11). Based on these unexpected findings, we hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly also in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment.Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer. In this respect, both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment (12–14). Apoptosis in the AML setting can be regulated by several cytokines as well as by EVs, which carry variable cargoes, including multiple proteins (15–18). In line with our hypothesis, apoptosis of BM cells was shown to be inhibited in the presence of secretome derived from AML blasts (19). These observations suggest that factors secreted by apoptosis-resistant leukemic blasts are likely to confer a drug resistance phenotype upon initially sensitive blasts. Therefore, the aim of our current study was to characterize the microenvironment produced by apoptosis-resistant AML blasts in terms of its capacity to influence apoptosis regulation in neighboring cells and protein content.     

CNS thermosensitivity and control of latent heat dissipation in the pigeon

Thermoregulatory responses to heat exposure were studied in 12 hand-reared, acclimated pigeons (Columbia livia). Measurements of body temperature (Tcl), brain temperature (Tbr), cutaneous water evaporation (CWE) and respiratory frequency (fr) were carried out in intact conscious heat exposed birds. In a second group of lightly restrained birds, fr and CWE were taken when temperatures of the trunk, brain and air (Ta) were independently changed. Increasing Tbr to 43.5–43.8°C induced a pronounced polypnea (deep and fast, (300 breaths min⁻¹) when Tcl regulated at 42.4°C. Moreover, when hyperthermia (Tcl = 43.0°C) was combined with increased Tbr (43.0–43.8°C) shallow and fast panting (>500 breaths min⁻¹) was evoked. CWE was probably elicited by inputs generated by the skin warm receptors as a result of increased Ta. Moreover it was demonstrated that warming the brain to 42.5°C elicits cutaneous water evaporation in birds exposed to 26°C. When a high Ta (60°C) is accompanied by a high relative humidity (17%), the combined effect generates inputs eliciting intensive panting. The integration of the present and earlier data allows us to generate a model demonstrating the distinguished significance of the trunk, skin and brain thermosensors in the regulation of both respiratory and cutaneous latent heat dissipation. The present model also emphasizes the fact that the highly thermosensitive pigeon brain responds in a similar pattern to that found in mammals     

Effects of streptomycin on plastids in dividing Euglena

Summary The plastids of dividing Euglena cells growing in the light in the presence of streptomycin decreased in length after a lag period of seven generations. The typical structure of the chloroplast was lost after a similar lag period. This loss of structure did not follow a regular pattern. After 11 generations the plastids resembled normal proplastids of dark-grown cells. Initial chlorophyll loss of treated cells was slow, but after 3 generations the rate of loss was about 0.5/generation, indicating a cessation of synthesis and a dilution among the progeny.     

Sulfate-Reducing Bacteria and Their Activities in Cyanobacterial Mats of Solar Lake (Sinai, Egypt)

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10⁶ and 10⁷ cultivable sulfate-reducing bacteria ml⁻¹ and showed sulfate reduction rates between 1,000 and 2,200 nmol ml⁻¹ day⁻¹, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 10⁴ to 10⁶ cells ml⁻¹. A Desulfonema-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO₂ from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO₂ demand of the mat.     

Composition and dynamics of the gill microbiota of an invasive Indo-Pacific oyster in the eastern Mediterranean Sea

Gill bacterial communities of Chama pacifica, an Indo-Pacific invasive oyster to the eastern Mediterranean Sea, were compared with those of Chama savignyi, its northern Red Sea congeneric species. Summer and winter bacterial populations were characterized and compared using 16S rDNA clone libraries, and seasonal population dynamics were monitored by automated ribosomal intergenic spacer analysis (ARISA). Clone libraries revealed a specific clade of bacteria, closely related to marine endosymbionts from the Indo-Pacific, found in both ecosystems, of which one taxon was conserved in oysters from both sites. This taxon was dominant in summer libraries and was weakly present in winter ones, where other members of this group were dominant. ARISA results revealed significant seasonal variation in bacterial populations of Mediterranean Sea oysters, as opposed to Red Sea ones that were stable throughout the year. We suggest that this conserved association between bacteria and oyster reflects either a symbiosis between the oyster host and some of its bacteria, a co-invasion of both parties, or both.     

First evidence of maternal transmission of algal endosymbionts at an oocyte stage in a triploblastic host, with observations on reproduction in Waminoa brickneri (Acoelomorpha)

Abstract. Examination of sexual reproduction in a symbiotic acoelomorph worm, Waminoa brickneri from Eilat (Red Sea), presents the first definitive evidence for maternal transmission of dinoflagellate algal symbionts in a triploblastic organism. Sexually mature worms were removed from the stony coral Plesiastrea laxa and raised in the laboratory. Eggs were detected 18 d after the collection of the worms and hatched 4 d later. Histological sections performed on sexually mature worms showed an ovary with oocytes containing two distinct types of algal endosymbionts within their ooplasm. Transmission electron microscopy corroborated the presence of algal symbionts within the developing embryos. Our findings regarding maternal transmission of symbionts shed new light on the diversity of modes of algal symbiont acquisition known in triploblastic organisms.     

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.     

The effects of MIF-I, beta-endorphin and alpha-MSH on d-amphetamine induced paradoxical behavioral thermoregulation: possible involvement of the dopaminergic system

The neuropharmacological basis for d-amphetamine induced paradoxical behavioral thermoregulation remains unclear. This study examined thermoregulatory behavior of rats in a runway device that housed a heat source at one end and in which locomotion along the length of the runway could be observed. Sprague Dawley rats were pretreated with IP injections of saline, beta-endorphin, MIF-1, or alpha-MSH, with a repeat injection after 30 min. In a second experiment, d-amphetamine was administered as the repeat drug for all Ss. The results showed clear differences for heat-source-on vs. heat-source off. All peptides induced hypermotility, although no differentiated effects for the peptides on d-amphetamine induced paradoxical behavioral thermoregulation were found. These findings are discussed in light of the theoretical possibilities that: (a) a ceiling effect exists; (b) there are separate control systems for maintaining body temperature and another for behavioral thermoregulatory responses, and (c) other neurotransmitters may be involved in such induced paradoxical behavioral thermoregulation.