AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.One of the major challenges to overcome in the development of hepatitis C virus (HCV)-directed antivirals is the high propensity of the virus to mutate. This is due to the lack of proofreading capacity of the HCV NS5B RNA-dependent RNA polymerase (RdRp), which replicates the HCV RNA strand with an error rate of 10⁻² to 10⁻³ nucleotide substitutions per site per year (17). The diversity of HCV has been recognized as six phylogenetically distinct groups, referred to as genotypes and, within each genotype, as subtypes (a, b, c, d, etc.) (44). HCV subtype 1b is the most prevalent genotype in the world, and subtype 1a is widely distributed in northern Europe and in the United States; subtypes 2a and 2b are common in North America, Europe, and Japan, and subtype 2c is found predominantly in Northern Italy; HCV genotype 3a is more prevalent in the Far East and has recently increased in Europe and in the United States, possibly due to the spread of the virus through intravenous drug use (11, 17, 18, 44, 46). Of the other genotypes, genotype 4 is common in Africa and to a lesser extent in Europe (11, 39), whereas genotypes 5 and 6 are found predominantly in southern Africa and Southeast Asia, respectively. Despite the availability of a standard of care for the treatment of hepatitis C, a combination of pegylated alpha interferon and ribavirin, many HCV-infected patients cannot be cured because of the frequent failure of the treatment, particularly in patients with genotype 1 and 4 infections, and perhaps also in those with genotype 6 infections (12, 35). In addition, tolerability issues associated with the standard of care lead to discontinuation of therapy in many patients (31). Therefore, major efforts have been made toward developing novel oral therapeutics that target a specific step of the HCV life cycle (45), with particular attention to HCV subtypes 1a and 1b, as they are the most common genotypes underserved by the current standard of care. Subtypes 1a and 1b are estimated to account for 57% and 17% of the HCV-infected patients in the United States (1) versus approximately 11% and 45% in Europe (11), respectively.The development of HCV polymerase nonnucleoside inhibitors (NNIs) has been successfully validated in phase II clinical trials (21, 24, 41). From the extensive screening of NS5B inhibitors that has been performed to date, several chemotypes have emerged as promising scaffolds, namely, the indole, thiophene, benzothiadiazine, and benzofuran analogs. Each of these NNIs targets four different binding pockets of the HCV polymerase, thumb-1 NNI-1 (10), thumb-2 NNI-2 (29, 48), palm-1 NNI-3 (9), and palm-2 NNI-4 (19), respectively. Historically, the screen for novel NS5B inhibitors was limited to representatives of genotype 1b only (3, 28) because the tools to target other genotypes were not yet available (16, 38, 40). Further assessment of these analogs, using enzyme isolates and intergenotypic chimeric replicons derived from clinical isolates, revealed that the genotypic coverage of the NNI-1 and -4 analogs extends beyond genotype 1, unlike the NNI-2 and -3 derivatives that typically inhibit genotype 1 only (16, 38). An additional drawback stems from the lower genetic barrier of the NNI-2 and -3 analogs in genotype 1 (25) and the reduced susceptibility in subtype 1a of the NNI-3 series (7, 16, 34, 38, 43). This effect was mostly attributed to the Y415F polymorphism observed in the NNI-3 binding site in subtype 1a (38).Here we report the 1a/1b subtype profiling of 1,5-benzodiazepine (1,5-BZD) HCV polymerase inhibitors that bind to the NNI-3 site (32, 36) using a panel of enzyme and chimeric replicons derived from clinical isolates, X-ray crystallography, and surface plasmon resonance (SPR) studies and compare these inhibitors to the four classes of NNIs by thoroughly assessing a representative of each nonnucleoside binding site in subtypes 1a and 1b.     

Synthesis and cancer cell cytotoxicity of substituted xanthenes

A series of substituted xanthenes was synthesized and screened for activity using DU-145, MCF-7, and HeLa cancer cell growth inhibition assays. The most potent compound, 9g ([N,N-diethyl]-9-hydroxy-9-(3-methoxyphenyl)-9H-xanthene-3-carboxamide), was found to inhibit cancer cell growth with IC₅₀ values ranging from 36 to 50 μM across all three cancer cell lines. Structure–activity relationship (SAR) data is presented that indicates additional gains in potency may be realized through further derivatization of the compounds (e.g., the incorporation of a 7-fluoro substituent to 9g). Results are also presented that suggest the compounds function through a unique mechanism of action as compared to that of related acridine and xanthone anticancer agents (which have been shown to intercalate into DNA and inhibit topoisomerase II activity). A structural comparison of these compounds suggests the differences in function may be due to the structure of the xanthene heterocycle which adopts a nonplanar conformation about the pyran ring.     

Are cyanobacteria involved in Ciguatera Fish Poisoning-like outbreaks in New Caledonia?

From 2001 to 2005, numerous cases of seafood poisonings were reported in a tribe from Lifou (Loyalty Islands Province, New Caledonia) of which 35 were thoroughly examined. Observations outlined by the epidemiological and clinical data (including severity and rapid onset of certain symptoms following consumption of either giant clams (Tridacna spp.) or grazing and molluscivorous fish together with the apparent inefficacy of traditional remedies, were not in favour of a classical Ciguatera Fish Poisoning (CFP) outbreak. From 2005 onwards, an environmental offshore survey of the affected area was conducted. Screening of the damaged coral area revealed the presence of large populations of cyanobacteria identified as Hydrocoleum Kützing, but the absence of Gambierdiscus spp., the well-known dinoflagellate causative agent of CFP. In vivo and in vitro toxicological studies of extracts obtained from cyanobacteria and giant clams, strongly suggested the co-occurrence of ciguatoxin-like, anatoxin-like and paralytic shellfish toxins in these samples.These new findings shed new light on the complexity of the CFP symptomatology and treatment and also on the diversity and origin of the CFP toxins. Furthermore they provide new evidence of the overall variability of seafood poisonings following the ingestion of different sea products living in a marine environment where significant harmful populations of microalgae and cyanobacteria coexist.This is the first report on the involvement of cyanobacteria in CFP-like outbreaks following the consumption of giant clams or fish specimens. Consequently, it is recommended that CFP risk assessment programs now include monitoring of cyanobacteria besides the obvious screening of CFP-promoting dinoflagellates.     

Pedigree‐free quantitative genetic approach provides evidence for heritability of movement tactics in wild roe deer

Assessing the evolutionary potential of animal populations in the wild is crucial to understanding how they may respond to selection mediated by rapid environmental change (e.g. habitat loss and fragmentation). A growing number of studies have investigated the adaptive role of behaviour, but assessments of its genetic basis in a natural setting remain scarce. We combined intensive biologging technology with genome‐wide data and a pedigree‐free quantitative genetic approach to quantify repeatability, heritability and evolvability for a suite of behaviours related to the risk avoidance‐resource acquisition trade‐off in a wild roe deer (Capreolus capreolus) population inhabiting a heterogeneous, human‐dominated landscape. These traits, linked to the stress response, movement and space‐use behaviour, were all moderately to highly repeatable. Furthermore, the repeatable among‐individual component of variation in these traits was partly due to additive genetic variance, with heritability estimates ranging from 0.21 ± 0.08 to 0.70 ± 0.11 and evolvability ranging from 1.1% to 4.3%. Changes in the trait mean can therefore occur under hypothetical directional selection over just a few generations. To the best of our knowledge, this is the first empirical demonstration of additive genetic variation in space‐use behaviour in a free‐ranging population based on genomic relatedness data. We conclude that wild animal populations may have the potential to adjust their spatial behaviour to human‐driven environmental modifications through microevolutionary change.     

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore‐forming toxin is detrimental for the host

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase‐1, which in turn triggers macrophage pyroptosis and IL‐1β/IL‐18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore‐forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase‐11 activation. Surprisingly, previous studies indicated that a T3SS‐induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS‐negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore‐forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll‐like receptors, and thus enhanced the expression of inflammatory proteins including pro‐IL‐1β and TNF‐α. However, mature‐IL‐1β and IL‐18 were undetectable in wild‐type mice, suggesting that ExlA failed to effectively activate caspase‐1. Nevertheless, caspase‐1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA‐induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome‐dependent process.     

三种被动式采集方法对甲虫收集效果的比较研究: 以香港城门样地为例

标本标准化采集是昆虫多样性研究的根本。昆虫种类繁多、习性复杂、分布广泛, 基于不同的研究目标, 昆虫学家会选用不同的采集方法。由于主动式采集方法存在较多干扰因素和重现性差等问题, 以飞行阻隔器(flight interception trap, FIT)、马氏网(Malaise trap, MT)和罐诱(pitfall trap, PT)为代表的被动式采集方法被广泛应用, 并在昆虫多样性研究中展现独特的优势。然而关于这些被动式采集方法的收集特点和采集效果等还缺乏系统性研究。本研究选取香港城门13个样点, 利用上述3种被动式采集方法共156个采集装备开展为期24天的鞘翅目昆虫采集工作, 并通过多样性指数分析、多度分析、体型与食性相关分析、相似性分析以及物种累积曲线分析评估了不同采集方法对甲虫的收集效果。本研究共采集甲虫6,380头, 涉及40科197种, 分析结果显示: (1)采用不同采集方法获得的物种数量和组成存在差异。从科级和种级的数量来看, FIT (36科, 149种) > MT (24科, 79种) > PT (17科, 60种); 在物种组成方面, FIT与PT之间、MT与PT之间区别较大, FIT与MT对应的物种相似度稍高于前两组。(2)多样性指数和物种多度分布分析显示: 丰富度指数为FIT > MT > PT, 优势度指数为FIT > PT > MT, 多样性指数为MT > FIT > PT, 均匀度指数为MT > PT > FIT。3种方法采集到的甲虫个体数为1头的种较多, 个体数超过1头的种在时间和空间方面的分布较广, 优势科的种类较少, 但其个体数占总个体数的比例较高。(3) FIT和PT均采集到了6类食性的甲虫, 其中藻食性的缨甲科甲虫仅见于FIT和PT采集方法。(4)物种累积曲线的结果表明3种采集方法效果均较好。3种采集方法各有特点, 但FIT采集的综合效果最优。FIT和MT两种方法的结合提升了采集甲虫的种类、食性和体型等方面的覆盖度, 更利于对甲虫多样性及类群与生态环境功能互作的研究。3种方法所收集到的甲虫存在一定差异, 因此可以针对不同研究目的选取适宜的采集方式。