Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Unexpected Regeneration in Middle-Aged Mice

Complete regeneration of damaged extremities, including both the epithelium and the underlying tissues, is thought to occur mainly in embryos, fetuses, and juvenile mammals, but only very rarely in adult mammals. Surprisingly, we found that common strains of mice are able to regenerate all of the tissues necessary to completely fill experimentally punched ear holes, but only if punched at middle age. Although young postweaning mice regrew the epithelium without typical pre-scar granulation tissue, they showed only minimal regeneration of connective tissues. In contrast, mice punched at 5–11 months of age showed true amphibian-like blastema formation and regrowth of cartilage, fat, and dermis, with blood vessels, sebaceous glands, hair follicles, and, in black mice, melanocytes. These data suggest that at least partial appendage regeneration may be more common in adult mammals than previously thought and call into question the common view that regenerative ability is lost with age. The data suggest that the age at which various inbred mouse strains become capable of epimorphic regeneration may be correlated with adult body weight

Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that degrade the extracellular matrix and other extracellular proteins. Studies in experimental animals demonstrate that MMPs play a number of roles in the detrimental as well as in the beneficial events after spinal cord injury (SCI). In the present correlative investigation, the expression pattern of several MMPs and their inhibitors has been investigated in the human spinal cord. METHODS: An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type. RESULTS: In the unlesioned human spinal cord, MMP and TIMP immunoreactivity was scarce. After traumatic SCI, a lesion-induced bi-phasic pattern of raised MMP-1 levels could be found with an early up-regulation in macrophages within the lesion epicentre and a later induction in peri-lesional activated astrocytes. There was an early and brief induction of MMP-2 at the lesion core in macrophages. MMP-9 and -12 expression peaked at 24 days after injury and both molecules were mostly expressed in macrophages at the lesion epicentre. Whereas MMP-9 levels rose progressively from 1 week to 3 weeks, there was an isolated peak of MMP-12 expression at 24 days. The post-traumatic distribution of the MMP inhibitors TIMP-1, -2 and -3 was limited. Only occasional TIMP immuno-positive macrophages could be detected at short survival times. The only clear induction was detected for TIMP-3 at survival times of 8 months and 1 year in peri-lesional activated astrocytes. CONCLUSION: The involvement of MMP-1, -2, -9 and -12 has been demonstrated in the post-traumatic events after human SCI. With an expression pattern corresponding largely to prior experimental studies, they were mainly expressed during the first weeks after injury and were most likely involved in the destructive inflammatory events of protein breakdown and phagocytosis carried out by infiltrating neutrophils and macrophages, as well as being involved in enhanced permeability of the blood spinal cord barrier. Similar to animal investigations, the strong induction of MMPs was not accompanied by an expression of their inhibitors, allowing these proteins to exert their effects in the lesioned spinal cord

Allografts Use in Nasal Reconstruction

The outcomes of destructive processes of the nasal structure such as infection, chronic inflammation, or resective procedures can lead to the need of complex reconstruction of the nasal framework [1]. Various types of grafts and implants have been employed [2]. When dealing with a nasal reconstruction of whatever kind, the surgeon is faced by a clinical dilemma: which is the best material for reconstructive purposes in that patient? The materials used for augmentation are therefore an important issue among reconstructive rhinoplasty surgeons [3, 4]. A basic differentiation must be outlined between the terms of grafts and implants: a graft is made of tissue either from the same patient (autograft) or from a member of the same species (homograft). Implants are synthetic and if implantable are called alloplasts [5]. Alloplastic material is deemed desirable if noncarcinogenic, nonallergenic, readily available, resistant to mechanical strain, and entirely reabsorbable and still reliable. It is commonly perceived that autologous grafts are the first choice for augmenting the nose; unfortunately, this material is not always available or sufficient in cases of atrophic changes of the nose of whatever cause to fulfill the needs. However, limited availability, unpredictable resorption rates, difficulty of handling, and donor-site morbidity are possible drawbacks. In such instances, other choices must be considered, and alloplastic materials can represent an attractive alternative tool to take into account [6]. On the other hand, their efficacy complications and limited usage are 192 debated, such not uniform feelings and disputed possibilities have given rise to the development of different technologies to possibly reach ideal grafting substance (Fig. 16.1). In Western countries, surgeons prefer costal or auricular cartilage when septal cartilage is not available or insufficient, whereas alloplastic materials are more widely used in Asia [7]. Since the very beginning of the rhinoplasty history, many efforts have been made over time to use implants such as gold, iron, ivory, paraffin, celluloid, glass, and cork, eventually discorded due to unsurpassable troubles [8] (Figs. 16.2 and 16.3). Today, commonly used alloplastic materials are silicon, Gore-Tex® (Surgiform Technology, SC, USA), Medpor® (Stryker Corporate, a b c d Fig. 16.1 (a) CT scan and pathology specimen showing foreign body cystic reaction. Fibrous capsule with implant inside. (b) Latex implant of the dorsum (implanted 10 years previously), at moment inflamed, cistic and mobile, removed. (c) The removing of latex implant. (d) A fibrous capsule with implant inside P. G. Giacomini et al. 193 a b Fig. 16.2 (a) Kirschner steel wire and preserved costal cartilage implant of dorsum (10 years previously), for cocaine abuse outcomes. (b) Picture 6 months after removal a b Fig. 16.3 (a) CT scan, showing implant and the infected. (b) Mobile dorsal implant 16 Allografts Use in Nasal Reconstruction 194 MI, USA), and polydioxanone plate (PDS Flexible Plate, Johnson & Johnson Company, Langhorne, Pennsylvania, USA) [9]. An overview of their pros and cons will be conducted on the basis of the literature data and personal experience to highlight their possible use in case of atrophic nose outcomes that require surgical correction. Some exemplificative clinical cases of patients treated at the ENT Dept., School of Medicine, University of Rome Tor Vergata, at Nose Plastic Surgery Clinic in the past 10 years for complications associated with alloplastic materials used in atrophic rhinitis of various etiologies are reported. Clinical profiles: eight cocaine abuse, one purulent chronic infection, two outcomes of facial trauma, and one previous nasal surgery. M/F ratio: 1:4. The patients’ age ranged from 42 to 81 years (mean: 49 years). The follow-up period was 3–15 years (mean: 4.2 years). All had been treated elsewhere for augmentation rhinoplasty with alloplastic materials end eventually revised for complications occurred. Type of alloplastic materials used, complications developed, and results obtained were revised by medical charts, photo documentation, and histopathologic data examined. Literature data were considered in order to define alloplastic materials possibilities in this kind of nasal reconstruction