MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

Evaluation of Early Concrete Damage Caused by Chloride-Induced Steel Corrosion Using a Deep Learning Approach Based on RNN for Ultrasonic Pulse Waves.

The objective of this study is to explore the feasibility of using ultrasonic pulse wave measurements as an early detection method for corrosion-induced concrete damages. A series of experiments are conducted using concrete cube specimens, at a size of 200 mm, with a reinforcing steel bar (rebar) embedded in the center. The main variables include the water-to-cement ratio of the concrete (0.4, 0.5, and 0.6), the diameter of the rebar (10 mm, 13 mm, 19 mm, and 22 mm), and the corrosion level (ranging from 0% to 20% depending on rebar diameter). The impressed current technique is used to accelerate corrosion of rebars in concrete immersed in a 3% NaCl solution. Ultrasonic pulse waves are collected from the concrete specimens using a pair of 50 kHz P-wave transducers in the through-transmission configuration before and after the accelerated corrosion test. Deep learning techniques, specifically three recurrent neural network (RNN) models (long short-term memory, gated recurrent unit, and bidirectional long short-term memory), are utilized to develop a classification model for early detection of concrete damage due to rebar corrosion. The performance of the RNN models is compared to conventional ultrasonic testing parameters, namely ultrasonic pulse velocity and signal consistency. The results demonstrate that the RNN method outperforms the other two methods. Among the RNN methods, the bidirectional long short-term memory RNN model had the best performance, achieving an accuracy of 74% and a Cohen's kappa coefficient of 0.48. This study establishes the potentiality of utilizing deep learning of ultrasonic pulse waves with RNN models for early detection of concrete damage associated with steel corrosion.

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

Copper (I) or (II) Replacement of the Structural Zinc Ion in the Prokaryotic Zinc Finger Ros Does Not Result in a Functional Domain.

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper's interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.

Characterization and cytotoxic activity of ribotoxin-like proteins from the edible mushroom Pleurotus eryngii.

Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar. Complete in vitro digestibility by pepsin-trypsin, and lack of cytotoxicity towards human HUVEC cells suggest low toxicity of eryngitins, if ingested. However, eryngitins exhibit cytotoxic action against insect Sf9 cells, suggesting their possible use in biotechnological applications as bioinsecticides. This cytotoxicity was not enhanced in the presence of cytolytic protein complexes based on aegerolysin proteins from Pleurotus mushrooms.

Ribotoxic Proteins, Known as Inhibitors of Protein Synthesis, from Mushrooms and Other Fungi According to Endo's Fragment Detection.

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant 'ribosome inactivating proteins' (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo's assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the ß-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the ß-fragment experimentally detected by Endo's assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

Electrical Resistivity Measurements for Nondestructive Evaluation of Chloride-Induced Deterioration of Reinforced Concrete-A Review.

The objective of this study is to review, evaluate, and compare the existing research and practices on electrical resistivity as a nondestructive technique in evaluating chloride-induced deterioration of reinforced concrete elements in buildings and civil infrastructure systems. First, this paper summarizes the different measurement techniques for gathering electrical resistivity (ER) values on concrete. Second, comparison analyses are performed to review the correlation of ER to different parameters representing corrosive environment and activity of steel corrosion in concrete, such as degree of water saturation, chloride penetration and diffusivity, and corrosion rate. In addition, this research enumerates and individually discusses the different environmental and interference factors that are not related to the electrochemical process of steel corrosion in concrete but directly affect the ER measurements, including temperature, the presence of steel reinforcement, cracks and delamination defects, specimen geometry, and concrete composition. Lastly and most importantly, discussions are made to determine the current gap of knowledge, to improve the utilization of this method in field and laboratory measurements, and future research.

An Updated Review of Bioactive Peptides from Mushrooms in a Well-Defined Molecular Weight Range.

Here, we report the current status of the bioactive peptides isolated and characterized from mushrooms during the last 20 years, considering 'peptide' a succession from to 2 to 100 amino acid residues. According to this accepted biochemical definition, we adopt ~10 kDa as the upper limit of molecular weight for a peptide. In light of this, a careful revision of data reported in the literature was carried out. The search revealed that in the works describing the characterization of bioactive peptides from mushrooms, not all the peptides have been correctly classified according to their molecular weight, considering that some fungal proteins (>10 kDa MW) have been improperly classified as 'peptides'. Moreover, the biological action of each of these peptides, the principles of their isolation as well as the source/mushroom species were summarized. Finally, this review highlighted that these peptides possess antihypertensive, antifungal, antibiotic and antimicrobial, anticancer, antiviral, antioxidant and ACE inhibitory properties.

Efeito de diferentes intensidades do exercício antagonista no desempenho de repetições dos agonistas em séries pareadas / Effect of different intensities of antagonist exercise on agonist repetitions performance in paired series

O objetivo do presente estudo foi verificar o efeito agudo de diferentes intensidades em séries pareadas agonistas e antagonistas sobre o desempenho de repetições máximas. Participaram de estudo, 12 homens recreacionalmente treinados (idade: 25.83 ± 3.43 anos; peso: 76.33 ± 5.58 kg; altura 1.72 ± 0.04 metros; IMC: 25.95 ± 1.34 kg/m²). Os procedimentos foram separados em cinco sessões: o primeiro encontro constou de anamnese, medidas antropométricas e teste de 10 repetições máximas (RM) nos exercícios rosca bíceps supinada com barra (RB) e tríceps no pulley (TP). Nos demais encontros, os participantes executaram o máximo de repetições no RB com 80% da carga de 10RM após realizarem, de maneira aleatória e em dias separados, um dos 4 protocolos de diferentes intensidades na musculatura antagonista (TP): Protocolo controle (PC), 10 repetições com 40% de 10RM (P40), 10 repetições com 60% de 10RM (P60) e 10 repetições com 80% de 10RM (P80). Para a análise estatística foi realizada uma ANOVA de medidas repetidas e o nível de significância adotado foi de p < 0.05. Os resultados demonstraram que maiores intensidades (P60 = 15.83 ± 0.94 repetições e P80 = 16.42 ± 0.79 repetições) possibilitaram um aumento significativo (p < 0.05) no desempenho de repetições quando comparado ao PC (14.83 ± 0.72 repetições). Além disso, P80 foi também superior ao P40 (15.25 ± 0.97 repetições, p = 0.00), mostrando existir uma dosagem mínima (60% de 10RM) para a melhora de desempenho no método pareado agonista e antagonista. (AU)

The aim of the present study was to verify the acute effect of different intensities of paired agonist and antagonist series on the performance of maximum repetitions. Twelve recreational trained men participated in the study (age: 25.83 ± 3.43 years; weigth: 76.33 ± 5.58 kg; height 1.72 ± 0.04 meters; IMC: 25.95 ± 1.34 kg/m²). The experimental procedure took place over five sessions: the first consisted of anamnesis, anthropometric measurements and a test of 10 maximum repetitions (RM) in the barbell curved supine (RB) and triceps pulley (TP) exercises. nother meetings, the participants performed maximum repetitions in the RB with 80% of the 10RM load after performing, in random order and on separate days, one of the 4 protocols of different intensities in the antagonist musculature (TP): Control protocol (PC), 10 repetitions with 40% of 10RM (P40), 10 repetitions with 60% of 10RM (P60) and 10 repetitions with 80% of 10RM (P80). For the statistical analysis, an ANOVA was performed and the level of significance adopted was p <0.05. The results showed that higher intensities (P60 = 15.83 ± 0.94 repetitions e P80 = 16.42 ± 0.79 repetitions) enabled a significant increase in repetition performance when compared to CP (14.83 ± 0.72 repetitions). In addition, P80 was also superior to P40 (15.25 ± 0.97 repetitions, p = 0.00), showing that there is a minimum dosage (60% of 10RM) to improve performance in the paired agonist and antagonist method. (AU)

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Effect of the Geometrical Constraints to the Wenner Four-Point Electrical Resistivity Test of Reinforced Concrete Slabs.

The main objectives of this study are to evaluate the effect of geometrical constraints of plain concrete and reinforced concrete slabs on the Wenner four-point concrete electrical resistivity (ER) test through numerical and experimental investigation and to propose measurement recommendations for laboratory and field specimens. First, a series of numerical simulations was performed using a 3D finite element model to investigate the effects of geometrical constraints (the dimension of concrete slabs, the electrode spacing and configuration, and the distance of the electrode to the edges of concrete slabs) on ER measurements of concrete. Next, a reinforced concrete slab specimen (1500 mm (width) by 1500 mm (length) by 300 mm (thickness)) was used for experimental investigation and validation of the numerical simulation results. Based on the analytical and experimental results, it is concluded that measured ER values of regularly shaped concrete elements are strongly dependent on the distance-to-spacing ratio of ER probes (i.e., distance of the electrode in ER probes to the edges and/or the bottom of the concrete slabs normalized by the electrode spacing). For the plain concrete, it is inferred that the thickness of the concrete member should be at least three times the electrode spacing. In addition, the distance should be more than twice the electrode spacing to make the edge effect almost negligible. It is observed that the findings from the plain concrete are also valid for the reinforced concrete. However, for the reinforced concrete, the ER values are also affected by the presence of reinforcing steel and saturation of concrete, which could cause disruptions in ER measurements.

Ribotoxin-like proteins from Boletus edulis: structural properties, cytotoxicity and in vitro digestibility.

Porcini are edible mushrooms widely used in cooking due to their extraordinary taste. Despite this, cases of food poisoning have been reported in the recent literature also for ingestion of porcini. Here, we report the isolation from Boletus edulis fruiting bodies of two novel ribotoxin-like proteins (RL-Ps), enzymes already studied in other organisms for their toxicity. These RL-Ps, named Edulitin 1 (16-kDa) and Edulitin 2 (14-kDa), show peculiar structural and enzymatic differences, which probably reflect their different bio-activities and a dose/time dependent toxicity (Edulitin 2) on normal and tumoral human cells. Particularly interesting is the resistance to proteolysis of Edulitin 2, for which it was observed that its toxicity was abolished only after heat treatment (90 °C) followed by proteolysis. As mushroom poisoning is a serious food safety issue, data here presented confirm the existence of toxins also in porcini and the importance of a proper cooking before their consumption.

Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.

This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw.

Electrical Resistivity Measurements of Reinforced Concrete Slabs with Delamination Defects.

The main objectives of this research are to evaluate the effects of delamination defects on the measurement of electrical resistivity of reinforced concrete slabs through analytical and experimental studies in the laboratory, and to propose a practical guide for electrical resistivity measurements on concrete with delamination defects. First, a 3D finite element model was developed to simulate the variation of electric potential field in concrete over delamination defects with various depths and lateral sizes. Second, for experimental studies, two reinforced concrete slab specimens (1500 mm (width) by 1500 mm (length) by 300 mm (thickness)) with artificial delamination defects of various dimensions and depths were fabricated. Third, the electrical resistivity of concrete over delamination defects in the numerical simulation models and the two concrete slab specimens were evaluated by using a 4-point Wenner probe in accordance with AASHTO (American Association of State Highway and Transportation Office) T-358. It was demonstrated from analytical and experimental studies in this study that shallow (50 mm depth) and deep (250 mm depth) delamination defects resulted in higher and lower electrical resistivity (ER) values, respectively, as compared to measurements performed on solid concrete locations. Furthermore, the increase in size of shallow defects resulted in an increase in concrete resistivity, whereas the increase in sizes of deep delamination defects yielded opposite results. In addition, measurements done directly above the steel reinforcements significantly lowered ER values. Lastly, it was observed from experimental studies that the effect of delamination defects on the values of electrical resistivity decreases as the saturation level of concrete increases.

Entropy Multiparticle Correlation Expansion for a Crystal.

As first shown by H. S. Green in 1952, the entropy of a classical fluid of identical particles can be written as a sum of many-particle contributions, each of them being a distinctive functional of all spatial distribution functions up to a given order. By revisiting the combinatorial derivation of the entropy formula, we argue that a similar correlation expansion holds for the entropy of a crystalline system. We discuss how one- and two-body entropies scale with the size of the crystal, and provide fresh numerical data to check the expectation, grounded in theoretical arguments, that both entropies are extensive quantities.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

Zinc Fingers.

Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ßßα architecture consisting in a ß-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a ßßßαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.

Thyroid hormones T3 and T4 regulate human luteinized granulosa cells, counteracting apoptosis and promoting cell survival.

PURPOSE: Fine and balanced regulation of cell proliferation and apoptosis are key to achieve ovarian follicle development from the primordial to the preovulatory stage and therefore assure female reproductive function. While gonadotropins are the major and most recognized regulators of follicle cell growth and function, other factors, both systemic and local, play equally important roles. This work is aimed at evaluating the effects of thyroid hormones (THs) on human granulosa luteinized (hGL) viability. METHODS: Human GL cells derived from assisted reproduction treatments were exposed to T3 or T4. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by the TUNEL assay and active caspase-3 staining. StAR, CYP19A1,Caspase-3, P53 and BAX mRNA were evaluated by real-time PCR. LC3-I/-II, AKT and pAKT were evaluated by western blot. RESULTS: T3 and T4 promoted cell viability in a dose-dependent modality and modulate StAR and CYP19A1 expression. T3 and to a lesser extent T4 mitigated cell death induced by serum starvation by inhibition of caspase-3 activity and expression of P53 and BAX; and attenuate cell death experimentally induced by C2-ceramide. Cell death derived from starvation appeared to be involved in autophagic processes, as the levels of autophagic markers (LC3-II/LC3-I ratio) decreased when starved cells were exposed to T3 and T4. This effect was associated with an increase in pAkt levels. CONCLUSION: From the present study, THs emerge as potent anti-apoptotic agents in hGL cells. This effect is achieved by inhibiting the apoptosis signalling pathway of BAX and caspase-3, while maintaining active the PI3K/AKT pathway.