Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Histological evaluation of human intrabony periodontal defects treated with an unsintered nanocrystalline hydroxyapatite paste

OBJECTIVES The aim of the study was to clinically and histologically evaluate the healing of human intrabony defects treated with open flap surgery (OFD) and application of a new, resorbable, fully synthetic, unsintered, nanocrystalline, phase-pure hydroxyapatite (nano-HA). MATERIALS AND METHODS Six patients, each of them displaying very advanced intrabony defects around teeth scheduled for extraction due to advanced chronic periodontitis and further prosthodontic considerations, were included in the study. Following local anaesthesia, mucoperiosteal flaps were reflected; the granulation tissue was removed, and the roots were meticulously debrided by hand and ultrasonic instruments. A notch was placed at the most apical extent of the calculus present on the root surface or at the most apical part of the defect (if no calculus was present) in order to serve as a reference for the histological evaluation. Following defect fill with nano-HA, the flaps were sutured by means of mattress sutures to allow primary intention healing. At 7 months after regenerative surgery, the teeth were extracted together with some of their surrounding soft and hard tissues and processed for histological analysis. RESULTS The postoperative healing was uneventful in all cases. At 7 months following surgery, mean PPD reduction and mean CAL gain measured 4.0 ± 0.8 and 2.5 ± 0.8 mm, respectively. The histological analysis revealed a healing predominantly characterized by epithelial downgrowth. Limited formation of new cementum with inserting connective tissue fibers and bone regeneration occurred in three out of the six biopsies (i.e. 0-0.86 and 0-1.33 mm, respectively). Complete resorption of the nano-HA was found in four out of the six biopsies. A few remnants of the graft particles (either surrounded by newly formed mineralized tissue or encapsulated in connective tissue) were found in two out of the six biopsies. CONCLUSION Within their limits, the present results indicate that nano-HA has limited potential to promote periodontal regeneration in human intrabony defects. CLINICAL RELEVANCE The clinical outcomes obtained following surgery with OFD + nano-HA may not reflect true periodontal regeneration.

Mosaic arthroplasty of the medial femoral condyle in horses - an experimental study.

One Arabian and 5 Hungarian half-bred horses were used to study the macroscopic and microscopic survival of autologous osteochondral grafts in the weight-bearing surface of the medial femoral condyle (MFC). Grafts were harvested from the cranial surface of the medial femoral trochlea (MFT) under arthroscopic control. Three of them were transplanted into the weight-bearing surface of the contralateral MFC using an arthrotomy approach. Three months later this transplantation procedure was repeated on the opposite stifle joints in the same animals, but at that time transplantation was performed arthroscopically. Follow-up arthroscopy was carried out 12 months after the first operations, and biopsies were taken from both the recipient and the donor sites for histological examination. During follow-up arthroscopy, the transplanted areas looked congruent and smooth. Microscopically, the characteristics of hyaline cartilage were present in 5 out of the 10 biopsies examined; however, in the other half of biopsies glycosaminoglycan (GAG) loss and change in the architecture of the transplanted cartilage was observed. In a 16-year-old horse, all grafts broke during harvesting, and thus transplantation was not performed. No radiological signs of osteoarthritic changes were detected 9 to 12 months after the operations in the donor and recipient joints. Clinically, no lameness or effusion was present three months after the transplantations.

Recovery of large carnivores in Europe's modern human-dominated landscapes.

The conservation of large carnivores is a formidable challenge for biodiversity conservation. Using a data set on the past and current status of brown bears (Ursus arctos), Eurasian lynx (Lynx lynx), gray wolves (Canis lupus), and wolverines (Gulo gulo) in European countries, we show that roughly one-third of mainland Europe hosts at least one large carnivore species, with stable or increasing abundance in most cases in 21st-century records. The reasons for this overall conservation success include protective legislation, supportive public opinion, and a variety of practices making coexistence between large carnivores and people possible. The European situation reveals that large carnivores and people can share the same landscape.

Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation

MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBL-associated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

A computer-assisted microscopic analysis of bone tissue developed inside a polyactive plymer implanted into an equine articular surface

One of the most promising applications for the restoration of small or moderately sized focal articular lesions is mosaicplasty (MP). Although recurrent hemarthrosis is a rare complication after MP, recently, various strategies have been designed to find an effective filling material to prevent postoperative bleeding from the donor site. The porous biodegradable polymer Polyactive (PA; a polyethylene glycol terephthalate - polybutylene terephthalate copolymer) represents a promising solution in this respect. A histological evaluation of the longterm PA-filled donor sites obtained from 10 experimental horses was performed. In this study, attention was primarily focused on the bone tissue developed in the plug. A computer-assisted image analysis and quantitative polarized light microscopic measurements of decalcified, longitudinally sectioned, dimethylmethylene blue (DMMB)- and picrosirius red (PS) stained sections revealed that the coverage area of the bone trabecules in the PA-filled donor tunnels was substantially (25%) enlarged compared to the neighboring cancellous bone. For this quantification, identical ROIs (regions of interest) were used and compared. The birefringence retardation values were also measured with a polarized light microscope using monochromatic light. Identical retardation values could be recorded from the bone trabeculae developed in the PA and in the neighboring bone, which indicates that the collagen orientation pattern does not differ significantly among these bone trabecules. Based on our new data, we speculate that PA promotes bone formation, and some of the currently identified degradation products of PA may enhance osteo-conduction and osteoinduction inside the donor canal.