RNA self-cleavage activated by ultraviolet light-induced oxidation

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5′-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C 79 and CSFV A45 in some molecules and at the immediately adjacent 5′-positions HCV U 78 and CSFV U 44 in others. Among other reaction products, the majority of biochemically active products detected contained 3′-phosphate and 5′-phosphate-end groups at the newly generated termini, along with a much lower amount of 3′-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (∼33nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures. © 2012 The Author(s).http://dx.doi.org/10.1093/nar/gkr82

A system dynamics model to predict the human monocyte response to endotoxins

System dynamics is a powerful tool that allows modeling of complex and highly networked systems such as those found in the human immune system. We have developed a model that reproduces how the exposure of human monocytes to lipopolysaccharides (LPSs) induces an inflammatory state characterized by high production of tumor necrosis factor alpha (TNFα), which is rapidly modulated to enter into a tolerant state, known as endotoxin tolerance (ET). The model contains two subsystems with a total of six states, seven flows, two auxiliary variables, and 14 parameters that interact through six differential and nine algebraic equations. The parameters were estimated and optimized to obtain a model that fits the experimental data obtained from human monocytes treated with various LPS doses. In contrast to publications on other animal models, stimulation of human monocytes with super-low-dose LPSs did not alter the response to a second LPSs challenge, neither inducing ET, nor enhancing the inflammatory response. Moreover, the model confirms the low production of TNFα and increased levels of C-C motif ligand 2 when monocytes exhibit a tolerant state similar to that of patients with sepsis. At present, the model can help us better understand the ET response and might offer new insights on sepsis diagnostics and prognosis by examining the monocyte response to endotoxins in patients with sepsisThis work was supported by grants from the “Instituto de Salud Carlos III” (ISCiii), “Fondo de Investigación Sanitaria” (FIS), and Fondos FEDER (PI14/01234, PIE15/00065) to EL-C. EA work contract is supported by the Torres Quevedo program from “Ministerio de Economía y Competitividad” (SPTQ1300X006175XV0). VT work contract is supported by the “Ministerio de Economía y Competitividad” (PTA2013-8265-I

Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type

8 Pág.Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.This study was funded by grant PID2020-113288RB-100/AEI/10.13039/501100011033.Peer reviewe

Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year

10 Pág. Centro de Investigación en Sanidad Animal (CISA) / Departamento de Reproducción animalThe roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P<0.05) values for post-thaw acrosome integrity (43.3% vs. 25.0%) and straight-line velocity (19 μm/s vs. 4 μm/s). For both freezing methods, sperm heads were smaller post-thawing than pre-freezing (P<0.001). In conclusion, both the pre-rutting and rutting season are suitable periods for freezing roe deer sperm. Ultra-rapid freezing did not provide suitable results.This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. P. Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was the recipient of a research fellowship from the PASPA-DGAPA-UNAM (México). V.N. Flores-Gil was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).Peer reviewe

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation

9 Pág. Departamento de Reproducción animal. (INIA)Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.This research was funded by MCINN/AEI/FEDER grant AGL2017-85753-R and by the Fundación Parques Reunidos INIA agreement CC19-096.Peer reviewe

Developing energy efficient lignin biomass processing: towards understanding mediator behaviour in ionic liquids

Environmental concerns have brought attention to the requirement for more efficient and renewable processes for chemicals production. Lignin is the second most abundant natural polymer, and might serve as a sustainable resource for manufacturing fuels and aromatic derivatives for the chemicals industry after being depolymerised. In this work, the mediator 2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) diammonium salt (ABTS), commonly used with enzyme degradation systems, has been evaluated by means of cyclic voltammetry (CV) for enhancing the oxidation of the non-phenolic lignin model compound veratryl alcohol and three types of lignin (organosolv, Kraft and lignosulfonate) in the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate, ([C2mim][C2SO4]). The presence of either veratryl alcohol or organosolv lignin increased the second oxidation peak of ABTS under select conditions, indicating the ABTS-mediated oxidation of these molecules at high potentials in [C2mim][C2SO4]. Furthermore, CV was applied as a quick and efficient way to explore the impact of water in the ABTS-mediated oxidation of both organosolv and lignosulfonate lignin. Higher catalytic efficiencies of ABTS were observed for lignosulfonate solutions either in sodium acetate buffer or when [C2mim][C2SO4] (15 v/v%) was present in the buffer solution, whilst there was no change found in the catalytic efficiency of ABTS in [C2mim][C2SO4]–lignosulfonate mixtures relative to ABTS alone. In contrast, organosolv showed an initial increase in oxidation, followed by a significant decrease on increasing the water content of a [C2mim][C2SO4] solution