Protocols, Toolboxes and Resource papers

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.     

Erratum to: Sinha S,Colbert CL,Becker N,Wei Y,Levine B. Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus 68 Bcl-2 homolog M11. Autophagy 2008; 4:989-97

Table 1 in Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus 68 Bcl-2 homolog M11 (Autophagy 2008; 4:989-97) contained an error in the heading of the last column. "BAD" should have appeared as "BAX." This error has been corrected and the corrected paper can now be downloaded at: http://www.landesbioscience.com/journals/3/article/6803.     

A reagent forum

My lab works with the yeast Saccharomyces cerevisiae, but I frequently get asked questions regarding autophagy reagents for use in higher eukaryotic systems. For example, "Which anti-LC3 antiserum works well in HEK293 cells"? Occasionally I know the answer, or an answer, but not always. Sometimes the information is available in the literature, but it can be difficult to find. With the ever-growing community of autophagy researchers, however, I suspect that many more reagents have been tested, and in many cases not reported. Therefore, I think it would be useful to establish a list of reagents (in particular, antisera and inhibitors) with company-specific information (name and catalog number) that can save other researchers valuable time and money. Autophagy will post the information in the form of a WIKI to allow updates and comments from users.

For example:

Reagent: anti-LC3

Company: Count On Us

Catalog number: 01234

Comments:

Dan Klionsky: I used this antibody and had a very good result (good sensitivity with low background) on protein extracts from mouse.

Danny Klionsky: I also had a good result with this antibody, and found that a higher dilution (1:3,000) worked just as well with rat liver protein extract.

Daniel J. Klionsky: This antibody did not work for yeast Atg8.

We plan to start the WIKI in August 2008, but its success depends entirely on your participation. Please check for the start of this site, and upload your entries. I am open to suggestions for improvements of the site that will make it more useful for autophagy researchers.

http://www.landesbioscience.com/reagent_blog/     

2012–13 Upcoming meetings

The Winter 2012–13 conference season provides ample opportunities to attend meetings that feature topics relevant to antibody research and development (R&D). Meetings such as these serve critical informational and educational functions and are key networking opportunities. Locations are spread throughout the world, reflecting the global nature of antibody R&D.

Please note that Upcoming meetings lists will no longer be included in the print version of mAbs starting with the January/February 2013 issue. Please visit the mAbs home page to find an online meeting list: www.landesbioscience.com/journals/mabs/     

2012–13 Upcoming meetings

The Winter 2012–13 conference season provides ample opportunities to attend meetings that feature topics relevant to antibody research and development (R&D). Meetings such as these serve critical informational and educational functions and are key networking opportunities. Locations are spread throughout the world, reflecting the global nature of antibody R&D. Please note that Upcoming meetings lists will no longer be included in the print version of mAbs starting with the January/February 2013 issue. Please visit the mAbs home page to find an online meeting list: www.landesbioscience.com/journals/mabs/     

The current status of ethnobiological research in Latin America: gaps and perspectives

Background

Recent reviews have demonstrated an increase in the number of papers on ethnobiology in Latin America. Among factors that have influenced this increase are the biological and cultural diversity of these countries and the general scientific situation in some countries. This study aims to assess the panorama of ethnobiological research in Latin America by analyzing its evolution, trends, and future prospects.

Methods

To conduct this study, we searched for papers in the Scopus (http://www.scopus.com) and Web of Science (http://www.isiknowledge.com) databases. The search was performed using combinations of keywords and the name of each Latin American country. The following countries were included in this study: Argentina, Bolivia, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Guatemala, Haiti, Honduras, Mexico, Panama, Paraguay, Peru, Venezuela, and Uruguay.

Results and conclusions

According to our inclusion criteria, 679 ethnobiological studies conducted in Latin America were found for the period between 1963 and 2012. Of these studies, 289 (41%) were conducted in Brazil, 153 in Mexico (22%), 61 in Peru (9%), 58 in Argentina (8%), 45 in Bolivia (6%), and 97 (14%) in other Latin American countries. The increased number of publications related to this area of knowledge in recent years demonstrates the remarkable growth of ethnobiology as a science. Ethnobiological research may be stimulated by an increase in the number of scientific events and journals for study dissemination and by the creation of undergraduate courses and graduate programs to train ethnoscientists who will produce high-quality studies, especially in certain countries.

     

Kinetics of ammonium, nitrate and phosphorus uptake by Canna indica and Schoenoplectus validus

Canna indica L. is an upright perennial rhizomatous herb, and Schoenoplectus validus (Vahl) A. Löve and D. Löve is a tall, perennial, herbaceous sedge. The nutrient uptake kinetics of C. indica and S. validus were investigated using the modified depletion method after plants were grown for 4 weeks in simulated secondary-treated wastewater. The maximum uptake rate (I_max) and Michaelis–Menten constant (K_m) were estimated by iterative curve fitting. The I_max for NH₄N (623 μmol g⁻¹ dry root weight h⁻¹) was significantly higher than that for NO₃N (338 μmol g⁻¹ dry root weight h⁻¹) in S. validus. In contrast, no difference was observed in C. indica. The I_max values for NO₃N and NH₄N were higher in S. validus than in C. indica. A significantly lower K_m was detected for NO₃N uptake in C. indica (385 μmol L⁻¹) compared to that in S. validus (1908 μmol L⁻¹). The I_max for PO₄P did not differ between the plant species. The K_m for PO₄P was significantly higher in C. indica (157 μmol L⁻¹) than in S. validus (60 μmol L⁻¹). In conclusion, we found that S. validus preferred NH₄N over NO₃N, had greater capacity for N uptake and higher affinity for PO₄P, but C. indica had greater affinity for NO₃N. Nutrient uptake capacity is likely related to habitat preference, and is influenced by the structure of roots and rhizomes.     

Antihaitianismo: an embodied discourse

ABSTRACT

In this article, I argue that contemporary manifestations of social, political and economic discrimination – antihaitianismo – in the Dominican Republic towards their Haitian neighbours have become embodied responses which are reproduced through everyday actions. Using ethnographic fieldwork and phenomenology, I explore a variety of contexts in which antihaitianismo has become part of a Dominican existential background that is provoked to surface in situations of heightened tension, stress or discomfort. I also show how this embodied discourse has affected and affects a race-centred, essentialist form of governance. This article is supplemented by a short film which can be found at the following link: https://www.youtube.com/watch?v=GlIH4fpwUlU     

Spectroscopic characterization of cytochrome P450 Compound I

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon–hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophillic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O–porphyrin radical with the ferryl iron spin (S = 1) antiferromagnetically coupled to the porphyrin radical spin (S′ = 1/2) yielding a S_tot = 1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.     

There is more to autophagy than induction: regulating the roller coaster

Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic—for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under noninducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death.¹ Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?Key words: Atg1, autophagosome, flux, lysosome, macroautophagy, phagophore, regulation, stress, TOR, Ulk1, vacuoleOne possibility for downregulating macroautophagy is suggested by Tom Neufeld’s lab, which showed that p70S6 kinase is a positive regulatory factor for autophagy in the Drosophila fat body.² Accordingly, inhibition of TOR activity ultimately results in decreased p70S6K function, which in turn downregulates autophagy. An alternate suggestion from Fred Meijer and Patrice Codogno is that p70S6K acts in part by negatively regulating the class I PtdIns 3-kinase.³ In this scenario, when TOR is inhibited the decrease in p70S6K activity results in the eventual reactivation of the class I PtdIns3K, which then stimulates TOR and downregulates autophagy.Further insight into this question is provided by a relatively recent study from Adi Kimchi’s lab. The conserved protein DAP1 is an mTOR substrate that inhibits macroautophagy. In nutrient-rich conditions, active mTORC1 inhibits DAP1 so that the latter has no effect on autophagy. The inhibition of mTORC1 during nutrient starvation results in the dephosphorylation and activation of DAP1, and the subsequent inhibition of macroautophagy, which limits the magnitude of autophagy-dependent degradation.^4,5Another mechanism of regulation is indicated in studies by Li Yu and colleagues who showed that autophagy is downregulated through mTOR reactivation in an autophagy-dependent manner that requires protein degradation in autolysosomes.⁶ This negative feedback mechanism provides another simple means of self-regulation whereby the nutrient levels within the cell dictate whether autophagy needs to be maintained or shut down. A study described in this issue of the journal provides further support for this mechanism, demonstrating that autophagy can be downregulated during starvation in yeast.⁷ Shin and Huh found that TOR activity is recovered during prolonged starvation, and that this again depends on autophagy (see Fig. 1 in the Autophagic Flux section, p. 803). These studies suggest that autophagy may cycle on and off repeatedly during starvation as nutrient supplies are consumed and then resupplied, ensuring that autophagy is maintained at optimal, and not excessive, levels. The latter mechanism, however, cannot explain how autophagy is regulated during other types of stress, suggesting that multiple control systems are involved.In closing, we introduce a new category of papers and a new section to the journal that we are calling Resource and Autophagic Flux, respectively. Resource papers will provide information that may be useful to the autophagy community, but that may not have specific mechanistic information, such as may occur with large-scale screens. For example, in this issue, see the paper from Marja Jäättelä’s lab that describes the use of a human kinome siRNA library to identify new kinases that regulate macroautophagy. Finally, we have chosen the name Autophagic Flux for the new section because it encompasses the full spectrum of the autophagic process. The schematic summary in Figure 1 of that section highlights the paper by Shin and Huh that we mention here. Our intention is to provide schematic highlights of most of the research papers in the Autophagic Flux section, providing readers with a quick overview and summary of the key point(s) of the study. We hope you find this useful; we welcome feedback.     

A false sense of security

Some journals are using ineffective software to screen images for manipulation. In doing so, they are creating a false sense of security in the research community about the integrity of the image data they publish.“There must be an easier way!” It''s the mantra of anyone performing a labor-intensive task, and the motivation behind the human desire for automation. Apparently, it also holds true for image screening.Open in a separate windowFigure 1©cartoonbank.com. All Rights Reserved.At the Rockefeller University Press, we screen all images in all accepted papers for evidence of manipulation (1). We do this by visually inspecting every image using basic adjustments in Photoshop. When editors from other publishers see a demonstration of our process, they often assert, “There must be an easier way!”The possibility of automating the image screening process was described in a Nature news article more than two years ago (2). About a year ago, one of the largest publisher services providers, Cadmus Communications, started offering an automated image screening service using a program called Rigour, which they publicize as “the world''s first automated Image Manipulation Analysis Software” (www.suprocktech.com).Cadmus demonstrated an early version of this software at the Press, but we found that it could not detect blatant examples of band deletions, band intensity adjustments, large regions of duplication, or composite images. In an e-mail to Cadmus dated September 11, 2007, I expressed my concern, “I am worried about causing a setback in the publishing community if editors think the current Rigour software is effective at detecting problems in biomedical images (specifically gel images). I have already heard of editors saying they will not initiate visual screening because they will just use the Cadmus software. This is creating a false sense of security in the community, because the software is not yet an effective screening tool.” I received no response to this e-mail.I was surprised to learn that, within a couple of months, Cadmus had started to sell an image screening service to publishers using this software. But given the availability of such a service, I was not surprised to learn that editors at two very prominent journals were using it. Publishers were clearly looking for a less labor-intensive solution to an image problem, in two senses of the word—image data, and public image. They wanted to be seen by the public to be actively addressing the problem of image manipulation.I asked these publishers if they had tested the service before they started to use it. Both had done so, but one of them declined to send the results of their tests; the other indicated that the Cadmus service had a 20% success rate. It seems that these publishers were not really concerned if the screening process they used actually worked.Problems with the service were still evident recently when I was consulted by a third party about a case of image manipulation in a paper published in one of these journals. The paper made a surprising claim with important clinical implications. Given that journal''s policy of only screening a fraction of papers for image manipulation, one might expect that they would at least select those with important clinical implications. In fact, the papers are selected at random, and this one had not been screened. After questions were raised, the figures were screened by Cadmus using their software, but they did not detect problems with the images that were easily revealed with visual screening.In personal communications, publishers have argued that using the Cadmus service must be better than doing nothing. In fact, it is worse than doing nothing. These publishers are creating a false sense of security in the community about the integrity of the image data they publish.A recent test of the Cadmus image screening service showed some improvement, with the software detecting manipulation in 10 out of 22 images (45%) in which image manipulation had previously been detected by visual inspection. However, when multiplied by the small fraction of images being screened by these journals, the percentage of images that are effectively screened is dramatically lower. At the very least, these journals should fully disclose their screening practices (and their efficacy) to their readers.Although complete protection against manipulated images cannot be guaranteed, it is incumbent on journal editors to screen the images they publish using the best available method, not just to some known (and low) percentage of efficacy. The issue of data integrity should not be left to chance and probability. This is scholarly publishing, not blackjack.There are others developing software to detect image manipulation, and it is possible that these applications may eventually prove to be useful and effective tools for editors. But journal editors should not rely on an automated method for image screening unless they know it is as effective as the visual method. Otherwise, readers are left to hedge their bets.     

The intense gravitational attraction of autophagy

In the course of my work as Autophagy editor, I try to gauge the overall patterns of interest in autophagy research. Not surprisingly, the number of papers associated with this topic has increased steadily. However, that trend provides only one glimpse into the way interest in this field has been changing—that the number of people working on autophagy has expanded. Perhaps not surprisingly, the number of different research areas that now include autophagy studies is also increasing. Thus, I decided to carry out an informal, imprecise analysis of the number of different journals (presumably reflecting in part the number of topics) that include papers on autophagy.     

Demographic and phylogeographic histories of two venomous North American snakes of the genus Agkistrodon

Many studies have revealed that lineages currently inhabiting formerly glaciated areas were pushed into southern glacial refugia and have expanded into their modern range since the last glacial maximum. There have been few studies that compare the effects of glacial cycles on lineage diversification and historical demography in closely related species with overlapping ranges. In this study we compare phylogeographic structure, historical demography, and approximate lineage age in two closely related and broadly co-occurring venomous snakes in eastern North America, the cottonmouth (Agkistrodon piscivorus) and copperhead (A. contortrix) using sequences from the mtDNA gene cytochrome b. We inferred three geographic lineages of A. contortrix and two of A. piscivorus with no common geographic or temporal pattern of lineage diversification identified for these species. Lineage diversification occurred in the Late Pliocene for A. piscivorus (2.5 mya) and in the Early Pleistocene for A. contortrix (1.5 mya). Demographic estimates revealed population expansion following the last glacial maximum (20,000 years ago) in two lineages of A. contortrix (the Central clade and Eastern clade) and one lineage of A. piscivorus (the Continental clade). The Florida clade of A. piscivorus is the only lineage for which constant population size through time was inferred, possibly due to stable populations persisting in areas unaffected by glacial advances. Our data suggest that unique habitat preferences may have shaped both the phylogeographic and demographic histories of each species.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

Nitrogen dioxide solubility and permeation in lipid membranes

Nitrogen dioxide (NO₂) is an important oxidant molecule in biology that is produced by several biological processes, and it is also an important air pollutant. It can oxidize proteins and lipids with important consequences on their biological functions. Despite its relevance, the interaction of NO₂ with the cell barrier, the lipid membrane, is poorly understood. For instance, can lipid membranes limit NO₂ diffusion? To estimate the permeability of lipid membranes to NO₂ it is necessary to learn more about its solubility in the lipid phase. However, experimental data on NO₂ solubility is very limited. To improve our knowledge on this matter, we used a mixed approach consisting in calculating the solubility of NO₂ and related diatomic and triatomic gases (NO, O₂, CO₂, etc.) in different solvents using quantum calculations and Tomasi’s Polarizable Continuum Model and validating and correcting these results using experimental data available for the related gases. This approach led to an estimated partition coefficient for NO₂ of 2.7 between n-octanol and water, and 1.5 between lipid membranes and water, meaning that NO₂ is a moderately hydrophobic molecule (less than NO, more than CO₂). Based on the solubility-diffusion permeability theory, the permeability coefficient was estimated to be 5 cm s⁻¹, up to 4000 times higher than that of peroxynitrous acid. It is concluded that lipid membranes are not significant barriers to NO₂ transport.     

Protein kinase C-α negatively regulates EGF-induced PLC- activity through direct phosphorylation

Phospholipase C- is a PLC isozyme that contains a CDC25 homology domain and a pair of RA domains in addition to a conserved PLC catalytic domain. PLC- is activated by both growth factors and GPCR ligands in a distinct manner. Growth factors such as EGF stimulate PLC- in an RA2 domain-dependent manner through Ras and Rap. On the other hand, several GPCR ligands that are linked with Ga₁₂ or Ga₁₃ can activate PLC- by associating with GTP-RhoA. GTP-RhoA binds with the region in the PLC- Y domain. Gs-linked ligands such as PGE1 and adrenaline stimulate PLC- by cAMP-dependent activation of Epac and Rap2B. PLC- is important for cardiac development and function. In addition, several lines of evidence indicate that PLC- promotes cell growth in an activity-dependent or -independent manner. In particular, PLC--dependent suppression of EGF receptor downregulation contributes to its growth promoting activity. Proper regulation of PLC- activity is essential for preventing tumor formation. Our previous report indicated that EGF-dependent ubiquitination of PLC- is required for the control of PLC--dependent cell growth. Recently, we found that PLC- is phosphorylated by growth factor stimulation, and this is another mechanism of the negative regulation. PLC- is phosphorylated by PKC-α upon stimulation with growth factors such as EGF and PDGF. The EGF-induced phosphorylation of PLC- was abolished by PKC inhibitors and by the expression of the dominant negative mutant of PKC-α. Furthermore, PKC-α was found to phosphorylate PLC- directly in vitro, suggesting that PLC- is a substrate of PKC-α in cells. In addition, PLC- was co-immunoprecipitated with PKC-α in an EGF-dependent manner. Immunocytochemical studies showed that PLC- co-localized with PKC-α in the plasma membrane after EGF stimulation. In addition, inhibition of PKC activity enhanced PLC--mediated PIP₂ hydrolysis, suggesting that PKC-α negatively regulates PLC- activity. Taken together, these results suggest for the first time that PLC- is regulated by PKC-α-dependent phosphorylation.     

A review of the Cambrian biostratigraphy of South Australia

Cambrian rocks in South Australia occur in the Stansbury, Arrowie, eastern Officer and Warburton Basins. The succession in the Stansbury and Arrowie Basins can be divided into three sequence sets (supersequences), 1, 2 and 3. Sequence set 1 can be divided into five third-order sequences: 1.0, 1.1A, 1.1B, 1.2 and 1.3. Trilobites from the Stansbury and Arrowie Basins are restricted largely to the lower part of the succession. Four trilobite zones are recognized: Abadiella huoi (latest Atdabanian–earliest Botoman), Pararaia tatei, Pararaia bunyerooensis and Pararaia janeae Zones (all Botoman). Trilobites higher in the succession are known from only a few horizons and in part correlate with the upper Lower Cambrian Lungwangmiaoan Stage of China, equivalent to the top Toyonian. Pagetia sp. has been reported in the Coobowie Formation of the Stansbury Basin, thus suggesting an early Middle Cambrian age.The Cambrian faunas of the Warburton Basin range in age from early Middle Cambrian (Late Templetonian) to very Late Cambrian, although the richest faunal assemblages are late Middle Cambrian (Ptychagnostus punctuosus to Goniagnostus nathorsti Zones). Conodonts, including Cordylodus proavus, occur in a Datsonian fauna.The Arrowie Basin contains the most complete and best studied archaeocyath succession in the Australia–Antarctica region. The Warriootacyathus wilkawillensis, Spirillicyathus tenuis and Jugalicyathus tardus Zones from the lower Wilkawillina Limestone (Arrowie Basin) and equivalents are correlated with the Atdabanian. Botoman archaeocyathids occur higher in the Wilkawillina Limestone. The youngest (Toyonian) archaeocyath fauna in Australia occurs in the Wirrealpa Limestone (Arrowie Basin).Brachiopods and molluscs of the Arrowie and Stansbury Basins can be divided into four biostratigraphic assemblages. Several informal Early Cambrian SSF biostratigraphic assemblages are recognized. Probable tabulate-like corals occur in the Botoman Moorowie Formation. Seven informal acritarch assemblages occur in the Early Cambrian of the Stansbury and Arrowie Basins. Trace fossils may mark the Precambrian–Cambrian boundary. Only two of several tuffaceous horizons from the Stansbury and Arrowie Basins have been dated (i) a date of 522.0 ± 2.1 Ma from the Heatherdale Shale of the Stansbury Basin, about 400 m above latest Atdabanian archaeocyathids and (ii) a date of 522.0 ± 1.8 Ma from the lower part of the Billy Creek Formation in the Arrowie Basin. Neither date is regarded as reliable.