Identification of an ATP-sensitive potassium channel in mitochondria.

Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular energy requirements, but the mechanisms that link organelle performance to ATP levels are poorly understood. Here we confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents (that is, mitoKATP). We show that-similar to their plasma membrane counterparts-mitoKATP channels are composed of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution of MITOK together with MITOSUR recapitulates the main properties of mitoKATP. Overexpression of MITOK triggers marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our results indicate that mitoKATP channels respond to the cellular energetic status by regulating organelle volume and function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.

Histomorphologic, Biomechanical, and Functional Analysis in Mandibular Osteogenesis Induced by External Distraction.

The induced bone generation procedure (osteogenic distraction [OD]) is frequently used in cases of jaw deformities in patients with, hemifacial microsomia, mandibular hypoplasia, and so on. This study was undertaken to provide criteria to perform this procedure. The authors evaluated the effects of various parameters using OD to determine the optimal variables. Sixty distractors were placed in 30 canine subjects who then underwent OD after appropriate osteotomy. The procedures were performed during varying latency periods, distraction periods, distraction rate distance, and the consolidation period. Bone biopsies and radiographic studies were performed at 0, 7, 14, 21, 45, and 55 days after the distraction process. The observed changes and the most efficient mandibular OD parameters are reported.

Determination of mandibular distraction osteogenesis most suitable parameters. An experimental study.

BACKGROUND: Distraction osteogenesis is a process of induced bone generation. Various protocols have been described for the management of the latency period, distraction speed and consolidation period, with greater or lesser success. OBJECTIVE: To better understand the process of mandibular distraction and establish the determining factors and their optimal times. MATERIAL AND METHODS: Twenty-seven dogs were studied, which had 54 distractors placed and that underwent unidirectional, bilateral mandibular distraction osteogenesis. The distraction processes were applied using six variants, two for each factor: latency period, distraction period and distraction speed. The changes were examined by means of bone biopsies and X-rays of the area at 0, 7, 14, 21, 45 and 55 days of the process. RESULTS: The most efficient osteogenic distraction parameters were a latency period of five days, a consolidation period of six weeks, distraction speed of 1 mm/day for distances of less than 20 mm, and 3 mm/day for longer distances. CONCLUSIONS: The sequential histological study allowed to observe the appearance of cellular elements (osteocytes, osteoclasts, osteoid matrix, trabeculate, etc.) and their participation in granulation tissue, newly-formed bone and compact mature bone.

ANTECEDENTES: Respecto a la distracción osteogénica (generación ósea inducida), con mayor o menor éxito han sido descritos diversos protocolos para el manejo del período de latencia, velocidad de distracción y período de consolidación. ­. OBJETIVO: Entender mejor el proceso de la distracción mandibular y establecer los factores determinantes y sus tiempos óptimos. MATERIAL Y MÉTODOS: Se estudiaron 27 perros sometidos a distracción osteogénica unidireccional, bilateral de la mandíbula. Los procesos de distracción se aplicaron con seis variantes, dos por cada factor (período de latencia, período de distracción y velocidad de distracción). Se estudiaron los cambios mediante biopsias del hueso y radiografías de la zona a los 0, 7, 14, 21, 45 y 55 días del proceso. RESULTADOS: Los parámetros de distracción osteogénica más eficientes fueron período de latencia de cinco días, período de consolidación de seis semanas, 1 mm diario de velocidad de distracción para distancias menores a 20 mm y 3 mm diarios para distancias mayores. CONCLUSIONES: El estudio histológico secuencial permitió observar la aparición de los elementos celulares (osteocitos, osteoclastos, matriz osteoide, trabeculado, etcétera) y su participación en el tejido de granulación, el hueso neoformado y el hueso maduro compacto.

Beyond genetics: Deciphering the impact of missense variants in CAD deficiency.

CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.

A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD.

CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion.

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1-3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators.

Three-Flap Mastopexy with Reduction or Augmentation.

BACKGROUND: Many authors have studied breast ptosis and made contributions to the search for a procedure that allows its correction with long-lasting results and minimal scars. Collective evolution has allowed us to reach the point where we are today and will allow us to continue improving techniques in the future. OBJECTIVES: The mastopexy procedure that we have used for the last 11 years, resulting from our surgical practice and countless observations at conferences, is a versatile procedure, applicable to different types of breasts and mastopexies with and without reduction or augmentation. METHODS: Analysis and the clinical review of the patients and the records of the mastopexy cases that we operated on using the technique from January 2009 to March, 2020, are presented here. Using a periareolar approach for grade I ptosis and an inverted "T" approach for more severe ptosis, the excess skin is removed, and three flaps of breast tissue, superior, medial and lateral, are carved. A retromammary dissection is performed to anchor the end of the flaps, medial and lateral, to the pectoral aponeurosis, followed by invagination of these flaps to concentrate the breast tissue in the upper and central area of the breast. Closure of the wound is then performed, transforming its excess length into a short horizontal one, into the inframammary grove. RESULTS: Sixty-seven female patients, between 36 and 59 years old, underwent this procedure. Complications included hematoma (4%), overcorrection (4%), dehiscence (3.3%), residual ptosis (2.7%), pathological scarring (2.7%) and rotation folds (2%). Maximum follow-up was 8 years 2 months, mean follow-up 2 years 9 months, with satisfactory results for the patients and surgeon. CONCLUSIONS: The mastopexy procedure that we present here is a versatile technique, which can be applied to most cases requiring correction of ptosis. It provides the breast with proper shape, size and location with fewer scars. It is a logical, safe, efficient, reproducible procedure, which is easy to learn. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Interaction of Intestinal Bacteria with Human Rotavirus during Infection in Children.

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies.

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy.

The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to ß-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of ß-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with ß-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy.

Surgical Planning for Mandibular Distraction Osteogenesis Using Low-Cost Three-Dimensional-Printed Anatomic Models.

Mandibular distraction osteogenesis (MDO) is a fairly common procedure in specialized craniofacial surgery centers. The MDO is a minimally invasive technique that is able to generate new bone in patients with a severe hypoplastic ascending ramus, while also expanding the overlying soft tissues; therefore, it has become the treatment of choice for the surgical correction of mandibular hypoplasias. One of the most common postoperative complications involves misplacement of the distractor and/or corticotomy, which can result in the creation of an erroneous distraction vector. The introduction of 3-dimensional (3D) printing has revolutionized preoperative planning in several surgical fields; however, cost can hinder its application in developing nations, specially in public institutions. The aim of this work is present our experience using low-cost 3D-printed anatomic models for preoperative planning and surgical simulation in patients requiring MDO.

Restoration of the Age Elongated Auricular Lobe.

BACKGROUND: The ears increase in all dimensions over the years (eg, length, helix diameter, lobe length), but the most obvious change is the elongation or ptosis of the lobe. Its correction should be considered a part of facial rejuvenation. OBJECTIVES: The authors presented the surgical procedure they have been utilizing to correct elongated earlobes. METHODS: The authors described a simple and effective procedure consisting of the curved resection of the excess tissue of the lobe at its lower end and its reconstruction through the utilization of 2 triangular flaps of skin, taken from the lateral surface of the lobe, rotated backwards, and sutured to the medial surface, leaving a zigzag hidden scar. RESULTS: The authors have utilized this surgical procedure over the last 16 years to correct ptosis of the earlobe as well as some of its other alterations due to aging. The results obtained are presented as well as the classification that the authors have utilized to qualify their degree of ptosis. CONCLUSIONS: This procedure has yielded excellent results, with imperceptible scars, and is therefore very pleasing to patients and surgeon alike.

Managing low productive forests at catchment scale: Considering water, biomass and fire risk to achieve economic feasibility.

Semi-arid forests are water limited environments considered as low-productive. As a result, these forests usually end up unmanaged and abandoned, with the subsequent wild fire risk increasing, water yield decreasing and a general diminishing of the forest resilience. Hydrological-oriented silviculture could be a useful alternative that increases management possibilities by combining forest productivity and water yield. However, the slight water yield increase after forest management together with the low forest productivity, could make this option insufficient for semi-arid forests, and other goods and services should be included and quantified. In this sense, the present study analyzes to what extent semi-arid forest management for water yield results effective and profitable at catchment scale, and how does it improve when it is combined with other benefits such as biomass production and fire risk diminishing. To that end, the effects of forest management of semi-arid Aleppo pine post-fire regeneration stands are analyzed in terms of water yield (TETIS-VEG model), fire risk (KDBY index and FARSITE) and biomass production, at catchment scale. Regarding to water yield, the results confirmed the slight effect of forest management on its increase (average increase of 0.27 ±â€¯0.29 mm yr-1), at the same time that highlighted the role of the upper catchment area as an important water contributor. The management produced 4161.6 Mg of biomass, and decreased in 27±17% and 25.6 ±â€¯14.1% the fire risk and fire propagation, respectively. Finally, a simple economic estimation of the management profitability is carried out by means of comparing the Benefit/Cost ratio of the managed and unmanaged scenarios. Both scenarios were always above the unity when just considering water as benefit, although the unmanaged scenario produced a higher ratio, as no management costs are expended. Contrarily, when wildfire was also included into the evaluation, the situation is overturned for wildfires equal or higher than 1.5 day duration, where the forest management is shown as the most convenient alternative.

Distinctive physiological and molecular responses to cold stress among cold-tolerant and cold-sensitive Pinus halepensis seed sources.

BACKGROUND: Forest species ranges are confined by environmental limitations such as cold stress. The natural range shifts of pine forests due to climate change and proactive-assisted population migration may each be constrained by the ability of pine species to tolerate low temperatures, especially in northern latitudes or in high altitudes. The aim of this study is to characterize the response of cold-tolerant versus cold-sensitive Pinus halepensis (P. halepensis) seedlings at the physiological and the molecular level under controlled cold conditions to identify distinctive features which allow us to explain the phenotypic difference. With this objective gas-exchange and water potential was determined and the photosynthetic pigments, soluble sugars, glutathione and free amino acids content were measured in seedlings of different provenances under control and cold stress conditions. RESULTS: Glucose and fructose content can be highlighted as a potential distinctive trait for cold-tolerant P. halepensis seedlings. At the amino acid level, there was a significant increase and accumulation of glutathione, proline, glutamic acid, histidine, arginine and tryptophan along with a significant decrease of glycine. CONCLUSION: Our results established that the main difference between cold-tolerant and cold-sensitive seedlings of P. halepensis is the ability to accumulate the antioxidant glutathione and osmolytes such as glucose and fructose, proline and arginine.

Analysis and Tools for Improved Management of Connectionless and Connection-Oriented BLE Devices Coexistence.

With the introduction of low-power wireless technologies, like Bluetooth Low Energy (BLE), new applications are approaching the home automation, healthcare, fitness, automotive and consumer electronics markets. BLE devices are designed to maximize the battery life, i.e., to run for long time on a single coin-cell battery. In typical application scenarios of home automation and Ambient Assisted Living (AAL), the sensors that monitor relatively unpredictable and rare events should coexist with other sensors that continuously communicate health or environmental parameter measurements. The former usually work in connectionless mode, acting as advertisers, while the latter need a persistent connection, acting as slave nodes. The coexistence of connectionless and connection-oriented networks, that share the same central node, can be required to reduce the number of handling devices, thus keeping the network complexity low and limiting the packet's traffic congestion. In this paper, the medium access management, operated by the central node, has been modeled, focusing on the scheduling procedure in both connectionless and connection-oriented communication. The models have been merged to provide a tool supporting the configuration design of BLE devices, during the network design phase that precedes the real implementation. The results highlight the suitability of the proposed tool: the ability to set the device parameters to allow us to keep a practical discovery latency for event-driven sensors and avoid undesired overlaps between scheduled scanning and connection phases due to bad management performed by the central node.

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Lactobacillus casei ferments the N-Acetylglucosamine moiety of fucosyl-α-1,3-N-acetylglucosamine and excretes L-fucose.

We have previously characterized from Lactobacillus casei BL23 three α-L-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the L-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the α-L-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-L-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria.

Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine.

UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate by four successive reactions catalyzed by three enzymes: Glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM), and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli.

Carbonic anhydrase IX as a specific biomarker for clear cell renal cell carcinoma: comparative study of Western blot and immunohistochemistry and implications for diagnosis.

OBJECTIVE: This study aimed to evaluate the usefulness of carbonic anhydrase IX (CA-IX) expression in clear cell renal cell carcinoma (CCRCC) using two different techniques to detect protein expression. MATERIAL AND METHODS: An experimental, cross-sectional, analytical study was conducted to analyse proteins in renal tumour and healthy tissue specimens from 38 consecutive patients who underwent nephrectomy for renal cancer. CA-IX protein expression was measured by immunohistochemistry and Western blot analysis and quantified. Statistical analysis was performed with the positive and negative specific agreements and kappa coefficient. The sensitivity and specificity of both techniques were assessed. Statistical tests were conducted to analyse the association between CA-IX expression quantitation and normal prognosis factors (TNM stage and Fuhrman nuclear grade), only in CCRCC. RESULTS: The mean patient age was 65 years, 78.9% of patients were men and 57.9% of tumours were CCRCC. CA-IX protein expression was positive in 63.2% of tumours by immunohistochemistry and in 60.5% by Western blot. Both techniques detected CA-IX expression only in CCRCC and unclassifiable tumours. High concordance indices were observed for CCRCC diagnosis. Western blot and immunohistochemistry had a sensitivity of 95.5% and 100%, respectively; the specificity was 100% in both techniques. CA-IX expression quantitation did not correlate with tumour stage or Fuhrman nuclear grade. CONCLUSIONS: Immunochemistry and Western blot techniques can be used to detect abnormal CA-IX protein expression in CCRCC and to support morphology-based diagnostic techniques.

Limited contribution of post-fire eco-engineering techniques to support post-fire plant diversity.

Eco-engineering techniques are generally effective at reducing soil erosion and restore vegetal cover after wildfire. However, less evidence exists on the effects of the post-fire eco-engineering techniques to restore plant diversity. To fill this knowledge gap, a standardized regional-scale analysis of the influence of post-fire eco-engineering techniques (log erosion barriers, contour felled log debris, mulching, chipping and felling, in some cases with burning) on species richness and diversity is proposed, adopting the Iberian Peninsula as case study. In general, no significant differences in species richness and diversity (Shannon) were found between the forest treated with different post-fire eco-engineering techniques, and the burned and non-treated soils. Only small significant differences were found for some sites treated with log erosion barriers or mulching. The latter technique increased species richness and diversity in some pine species and shrublands. Contour felled log debris with burning slightly increased vegetation diversity, while log erosion barriers, chipping and felling were not successful in supporting plant diversity. This research will help forest managers and agents in Mediterranean forest to decide the best postfire management option for wildfire affected forest, and in the development of more effective post-fire strategies.