A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis.

CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at providing a mass spectrometry (MS)-based validation of the CRNDEP sequence and an investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli and revealed interactions of this micropeptide with p54, an RNA helicase. Additionally, we proved that high CRNDE(P) expression increases the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve the screening, diagnosis, and treatment of this disease.

Contamination of aquatic environment with anticancer reagents influences Daphnia magna - Ecotoxicogenomics approach.

Pharmaceuticals used in human medicine contaminate freshwater ecosystems. Chemotherapeutics applied in cancer treatment are found in freshwaters at low concentrations (in the range of ng L-1) which, however, can be toxic or mutagenic to aquatic organisms. The aim of this study was to determine the impact of the alkylating/crosslinking anticancer agents, cyclophosphamide (CP) and cisplatin (CDDP), at the concentration detected in water, on Daphnia magna life history, transcriptome, and proteome. This filter feeding cladoceran is an important member of the aquatic food webs controlling algal biomass and forming basic food for planktivorous fish. Here, observations of the D. magna growth rate, age at first reproduction, and the number of eggs produced were performed in the presence of CP or CDDP. The D. magna proteins and RNA were isolated and analysed by mass spectrometry and the mRNA-seq method, respectively. Five generations of contact with the pharmaceuticals in question significantly influenced the D. magna life history parameters with the growth rate and number of laid eggs decreased, whereas age at first reproduction was increased. A decrease in survivorship was observed when daphnids were exposed to CP. These changes are the result of modifications in the gene/transcript expression followed by differences in the proteome profile in comparison to the untreated control. The proteome changes were generally in accordance with the modified transcriptome. The ecotoxicogenomics approach makes it possible to get closer to a complete picture of the influence of CP and CDDP on Daphnia. We have gathered evidence that animals in the presence of anticancer pharmaceuticals attempt to cope with permanent stress by changing their proteome and transcriptome profile. Additionally, our analyses indicate that CDDP showed a stronger effect on tested organisms than CP.

Sugar Starvation Disrupts Lipid Breakdown by Inducing Autophagy in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome-a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions.

Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system.

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.

Proteome Analysis of PC12 Cells Reveals Alterations in Translation Regulation and Actin Signaling Induced by Clozapine.

Although antipsychotics are routinely used in the treatment of schizophrenia for the last decades, their precise mechanism of action is still unclear. In this study, we investigated changes in the PC12 cells' proteome under the influence of clozapine, risperidone, and haloperidol to identify protein pathways regulated by antipsychotics. Analysis of the protein profiles in two time points: after 12 and 24 h of incubation with drugs revealed significant alterations in 510 proteins. Further canonical pathway analysis revealed an inhibition of ciliary trophic factor signaling after treatment with haloperidol and showed a decrease in acute phase response signaling in the risperidone group. Interestingly, all tested drugs have caused changes in PC12 proteome which correspond to inhibition of cytokines: tumor necrosis factor (TNF) and transforming growth factor beta 1 (TGF-ß1). We also found that the 12-h incubation with clozapine caused up-regulation of protein kinase A signaling and translation machinery. After 24 h of treatment with clozapine, the inhibition of the actin cytoskeleton signaling and Rho proteins signaling was revealed. The obtained results suggest that the mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) play a central role in the signal transduction of clozapine.

Palm Oil-Rich Diet Affects Murine Liver Proteome and S-Palmitoylome.

Palmitic acid (C16:0) is the most abundant saturated fatty acid in animals serving as a substrate in synthesis and ß-oxidation of other lipids, and in the modification of proteins called palmitoylation. The influence of dietary palmitic acid on protein S-palmitoylation remains largely unknown. In this study we performed high-throughput proteomic analyses of a membrane-enriched fraction of murine liver to examine the influence of a palm oil-rich diet (HPD) on S-palmitoylation of proteins. HPD feeding for 4 weeks led to an accumulation of C16:0 and C18:1 fatty acids in livers which disappeared after 12-week feeding, in contrast to an accumulation of C16:0 in peritoneal macrophages. Parallel proteomic studies revealed that HPD feeding induced a sequence of changes of the level and/or S-palmitoylation of diverse liver proteins involved in fatty acid, cholesterol and amino acid metabolism, hemostasis, and neutrophil degranulation. The HPD diet did not lead to liver damage, however, it caused progressing obesity, hypercholesterolemia and hyperglycemia. We conclude that the relatively mild negative impact of such diet on liver functioning can be attributed to a lower bioavailability of palm oil-derived C16:0 vs. that of C18:1 and the efficiency of mechanisms preventing liver injury, possibly including dynamic protein S-palmitoylation.

Clozapine influences cytoskeleton structure and calcium homeostasis in rat cerebral cortex and has a different proteomic profile than risperidone.

For over the last 50 years, the molecular mechanism of anti-psychotic drugs' action has been far from clear. While risperidone is very often used in clinical practice, the most efficient known anti-psychotic drug is clozapine (CLO). However, the biochemical background of CLO's action still remains elusive. In this study, we performed comparative proteomic analysis of rat cerebral cortex following chronic administration of these two drugs. We observed significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins caused by both antipsychotics. Among other proteins, alterations in collapsin response mediator proteins, CRMP2 and CRMP4, were the most spectacular consequences of treatment with both drugs. Moreover, risperidone increased the level of proteins involved in cell proliferation such as fatty acid-binding protein-7 and translin-associated factor X. CLO significantly up-regulated the expression of visinin-like protein 1, neurocalcin δ and mitochondrial, stomatin-like protein 2, the calcium-binding proteins regulating calcium homeostasis, and the functioning of ion channels and receptors. Using two-dimensional differential electrophoresis, we demonstrate that chronic treatment the healthy rats with anti-psychotics, clozapine and risperidone, induce changes in expression of cytoskeletal, synaptic, and regulatory proteins in the cerebral cortex. While risperidone increases the level of proteins regulating cell proliferation, namely, fatty acid-binding protein-7 and translin-associated factor X, the clozapine significantly up-regulates calcium sensors, i.e., visinin-like protein 1 and neurocalcin δ. 2D DIGE, Differential in Gel Electrophoresis; Cy2, Cy3, and Cy5 are cyanine dyes.

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination.

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SIGNIFICANCE: The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation.

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Exercise-Induced Proteomic Profile Changes in Patients with Advanced Heart Failure.

BACKGROUND/OBJECTIVES: The pathophysiological background of the processes activated by physical activity in patients with heart failure (HF) is not fully understood. Proteomic studies can help to preliminarily identify new protein markers for unknown or poorly defined physiological processes. We aimed to analyse the changes in the plasma proteomic profile of HF patients after a cardiopulmonary exercise test (CPET) to define pathways involved in the response to exercise. METHODS: The study prospectively enrolled 20 male patients with advanced HF (aged 53.3 ± 8.3 years). Blood samples were taken from the patients before and immediately after the CPET to obtain plasma proteomic profiles. Two-sample t-tests (paired or non-paired) were performed with and without false discovery rate (FDR) correction for multiple testing. Enrichment analysis was performed to associate biological processes and pathways with the study results. RESULTS: A total of 968 plasma proteins were identified, of which 722 underwent further statistical analysis. Of these, 236 proteins showed differential expression when comparing all plasma samples collected before and after CPT (p < 0.05), and for 86 of these the difference remained statistically significant after FDR correction. Proteins whose expression changed after exercise are mostly involved in immune response and inflammatory processes, coagulation, cell adhesion, regulation of cellular response to stimulus and regulation of programmed cell death. There were no differences in resting proteomics according to HF etiology (ischemic vs. non-ischemic). CONCLUSIONS: Changes in the proteomic profile revealed a complexity of exercise-induced processes in patients with HF, suggesting that few major physiological pathways are involved. Further studies focusing on specific pathways are needed.

Daphnia stress response to environmental concentrations of chloramphenicol-multi-omics approach.

Commonly used medicines, when discarded or improperly disposed of, are known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic and mutagenic, and can modify freshwater organisms, even at environmentally relevant concentrations. Chloramphenicol (CAP) is an antibiotic banned in Europe. However, it is still found in surface waters around the world. The aim of this study was to evaluate the impact of chloramphenicol contamination in freshwater on the model organism Daphnia magna. Specific life history parameters, proteome, and host-associated microbiome of four D. magna clones were analyzed during a three-generation exposure to CAP at environmental concentrations (32 ng L-1). In the first generation, no statistically significant CAP effect at the individual level was detected. After three generations, exposed animals were smaller at first reproduction and on average produced fewer offspring. The differences in D. magna's life history after CAP treatment were in accordance with proteome changes. D. magna's response to CAP presence indicates the high stress that the tested organisms are under, e.g., male production, upregulation of ubiquitin-conjugating enzyme E2 and calcium-binding protein, and downregulation of glutathione transferase. The CAP-exposed D. magna proteome profile confirms that CAP, being reactive oxygen species (ROS)-inducing compounds, contributes to structural changes in mitochondria. Microbiome analysis showed a significant difference in the Shannon index between control and CAP-exposed animals, the latter having a more diverse microbiome. Multilevel analyses, together with long exposure in the laboratory imitating conditions in a polluted environment, allow us to obtain a more complete picture of the impact of CAP on D. magna.

Visfatin impact on the proteome of porcine luteal cells during implantation.

Visfatin (VIS) is a hormone belonging to the adipokines' group secreted mainly by the adipose tissue. VIS plays a crucial role in the control of energy homeostasis, inflammation, cell differentiation, and angiogenesis. VIS expression was confirmed in the hypothalamic-pituitary-gonadal (HPG) axis structures, as well as in the uterus, placenta, and conceptuses. We hypothesised that VIS may affect the abundance of proteins involved in the regulation of key processes occurring in the corpus luteum (CL) during the implantation process in pigs. In the present study, we performed the high-throughput proteomic analysis (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the in vitro influence of VIS (100 ng/mL) on differentially regulated proteins (DRPs) in the porcine luteal cells (LCs) on days 15-16 of pregnancy (implantation period). We have identified 511 DRPs, 276 of them were up-regulated, and 235 down-regulated in the presence of VIS. Revealed DRPs were assigned to 162 gene ontology terms. Western blot analysis of five chosen DRPs, ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), lanosterol 14-α demethylase (CYP51A1), inhibin subunit beta A (INHBA), notch receptor 3 (NOTCH3), and prostaglandin E synthase 2 (mPGES2) confirmed the veracity and accuracy of LC-MS/MS method. We indicated that VIS modulates the expression of proteins connected with the regulation of lipogenesis and cholesterologenesis, and, in consequence, may be involved in the synthesis of steroid hormones, as well as prostaglandins' metabolism. Moreover, we revealed that VIS affects the abundance of protein associated with ovarian cell proliferation, differentiation, and apoptosis, as well as CL new vessel formation and tissue remodelling. Our results suggest important roles for VIS in the regulation of ovarian functions during the peri-implantation period.

The proteomic profile is altered but not repaired after bariatric surgery in type 2 diabetes pigs.

To reveal the sources of obesity and type 2 diabetes (T2D) in humans, animal models, mainly rodents, have been used. Here, we propose a pig model of T2D. Weaned piglets were fed high fat/high sugar diet suppling 150% of metabolizable energy. Measurements of weight gain, blood morphology, glucose plasma levels, cholesterol, and triglycerides, as well as glucose tolerance (oral glucose tolerance test, OGTT) were employed to observe T2D development. The histology and mass spectrometry analyses were made post mortem. Within 6 months, the high fat-high sugar (HFHS) fed pigs showed gradual and significant increase in plasma triglycerides and glucose levels in comparison to the controls. Using OGTT test, we found stable glucose intolerance in 10 out of 14 HFHS pigs. Mass spectrometry analysis indicated significant changes in 330 proteins in the intestine, liver, and pancreas of the HFHS pigs. These pigs showed also an increase in DNA base modifications and elevated level of the ALKBH proteins in the tissues. Six diabetic HFHS pigs underwent Scopinaro bariatric surgery restoring glycaemia one month after surgery. In conclusion, a high energy diet applied to piglets resulted in the development of hyperlipidaemia, hyperglycaemia, and type 2 diabetes being reversed by a bariatric procedure, excluding the proteomic profile utill one month after the surgery.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H2S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.

Chemerin Effect on the Endometrial Proteome of the Domestic Pig during Implantation Obtained by LC-MS/MS Analysis.

Chemerin (CHEM) is a hormone mainly expressed in adipocytes involved in the regulation of energy homeostasis and inflammatory response. CHEM expression has been demonstrated in the structures of the porcine hypothalamic-pituitary-gonadal axis, as well as in the uterus, trophoblasts and conceptuses of pigs. In this study, we performed high-throughput proteomic analyses (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the influence of CHEM (400 ng/mL) on differentially regulated proteins (DRPs) in the porcine endometrial tissue explants during implantation (15 to 16 days of gestation). Among all 352 DRPs, 164 were up-regulated and 188 were down-regulated in CHEM-treated group. DRPs were assigned to 47 gene ontology (GO) terms (p-adjusted < 0.05). Validation of four DRPs (IFIT5, TGFß1, ACO1 and PGRMC1) by Western blot analysis confirmed the veracity and accuracy of the LC-MS/MS method used in the present study. We suggest that CHEM, by modulating various protein expressions, takes part in the endometrial cell proliferation, migration and invasion at the time of implantation. It also regulates the endometrial immune response, sensitivity to P4 and the formation of new blood vessels. Additionally, CHEM appears to be an important factor involved in endothelial cell dysfunction during the pathogenesis of preeclampsia. The identification of a large number of DRPs under the influence of CHEM provides a valuable resource for understanding the molecular mechanisms of this hormone action during implantation, which is a prerequisite for better control of pig reproduction.

Proteomic Profile of Saliva in Parkinson's Disease Patients: A Proof of Concept Study.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. It affects many organs. Lewy bodies-a histopathological "hallmark" of PD-are detected in about 75% of PD submandibular gland samples. We hypothesize that saliva can be a source of biomarkers of PD. The aim of the study was to evaluate and compare the salivary proteome of PD patients and healthy controls (HC). Salivary samples from 39 subjects (24 PD patients, mean age 61.6 ± 8.2; 15 HC, mean age 60.9 ± 6.7) were collected. Saliva was collected using RNA-Pro-Sal kits. Label-free LC-MS/MS mass spectrometry was performed to characterize the proteome of the saliva. IPA analysis of upstream inhibitors was performed. A total of 530 proteins and peptides were identified. We observed lower concentrations of S100-A16, ARP2/3, and VPS4B in PD group when compared to HC. We conclude that the salivary proteome composition of PD patients is different than that of healthy controls. We observed a lower concentration of proteins involved in inflammatory processes, exosome formation, and adipose tissue formation. The variability of expression of proteins between the two groups needs to be considered.

Mitochondrial Processes during Early Development of Dictyostelium discoideum: From Bioenergetic to Proteomic Studies.

The slime mold Dictyostelium discoideum's life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria's structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

Composition and function of the C1b/C1f region in the ciliary central apparatus.

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.

PD-L1 Overexpression, SWI/SNF Complex Deregulation, and Profound Transcriptomic Changes Characterize Cancer-Dependent Exhaustion of Persistently Activated CD4+ T Cells.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.