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The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

Eukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

Generation of Chimeric Antigen Receptors (CARs) presented a significant advance in the field of immunotherapy, allowing the targeting of cell-surface expressed molecules in an MHC-independent manner. Arming NK cells with CARs merges their innate natural cytotoxicity with the refined precision of targeted antigen recognition. The success of these therapies hinges on selecting the right tumor-specific targets to ensure effective activation and avoid self-reactivity. Optimization of CAR design and targeting is based on NK cell intrinsic properties (CAR modules and sources of NK cells), as well as on NK-tumor cell interactions (multi-antigen, multi-step, multi-switch). Additionally, the dynamics of tumor infiltration and adaptation to the tumor microenvironment play a critical role in CAR-NK cell efficacy. Combining CAR-NK cell therapies with chemotherapy, radiotherapy, checkpoint inhibitors, and emerging approaches like epigenetic modulators and oncolytic viruses, may address some of these challenges. The development of CAR-NK cell strategies for metastatic disease is especially promising, though the complexities of metastasis require refined targeted designs. As immunomics and multi-omics continue to evolve, the potential for designing more effective CAR-NK cell therapies expands. As results from preclinical and clinical trials unfold, a multidisciplinary approach integrating all those aspects will be key to unlock the full potential of CAR-NK cell-based adoptive transfers.

Immunoepigenetics is a growing field, as there is mounting evidence on the key role played by epigenetic mechanisms in the regulation of tumor immune cell recognition and control of immune cell anti-tumor responses. Moreover, it is increasingly acknowledgeable a tie between epigenetic regulation and prostate cancer (PCa) development and progression. PCa is intrinsically a cold tumor, with scarce immune cell infiltration and low inflammatory tumor microenvironment. However, Natural Killer (NK) cells, main anti-tumor effector immune cells, have been frequently linked to improved PCa prognosis. The role that epigenetic-related mechanisms might have in regulating both NK cell recognition of PCa tumor cells and NK cell functions in PCa is still mainly unknown. Epigenetic modulating drugs have been showing boundless therapeutic potential as anti-tumor agents, however their role in immune cell regulation and recognition is scarce. In this review, we focused on studies addressing modulation of epigenetic mechanisms involved in NK cell-mediated responses, including both the epigenetic modulation of tumor cell NK ligand expression and NK cell receptor expression and function in different tumor models, highlighting studies in PCa. The integrated knowledge from diverse epigenetic modulation mechanisms promoting NK cell-mediated immunity in various tumor models might open doors for the development of novel epigenetic-based therapeutic options for PCa management.

Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.

Sea urchin population harvest in the North Atlantic coast of Portugal was characterized in terms of gonad yield, nutritional composition and important market-related traits, over one reproductive cycle (March 2016 to March 2017). Most of the quality attributes showed a seasonal variation strongly dependent on sea urchin sex. Maximum gonad yield (18%) was observed in March 2017. A single spawning event occurred between May and July. Gonads are rich sources of protein (12–18% WW) with low fat content (≤6% WW), that increase during the gametogenic stages of recovery and growing (November-December). Polyunsaturated fatty acids were the dominant class in both sexes (4.2–14.7 mg.g −1 WW), being preferentially accumulated in females. Total gonads carotenoid varied seasonally, with the highest level being observed in males during spawning season. Echinenone was the main pigment present in gonads, showing highest concentrations in males during spawning and gonad recovering. During the growing and early maturation period gonads were more reddish, yellowish and brighter, as well as more firm, irrespectively of the sex. Based on all seasonal changes affecting gonad yield and quality, the period between November and February seems the most suitable to harvest high quality gonads in the Atlantic coast of Portugal.