Treatment outcome of atypical EGFR mutations in the German National Network Genomic Medicine Lung Cancer (nNGM).

BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.

Glacial history of the European marine mussels Mytilus, inferred from distribution of mitochondrial DNA lineages.

Mussels of the genus Mytilus have been used to assess the circumglacial phylogeography of the intertidal zone. These mussels are representative components of the intertidal zone and have rapidly evolving mitochondrial DNA, suitable for high resolution phylogeographic analyses. In Europe, the three Mytilus species currently share mitochondrial haplotypes, owing to the cases of extensive genetic introgression. Genetic diversity of Mytilus edulis, Mytilus trossulus and Mytilus galloprovincialis was studied using a 900-bp long part of the most variable fragment of the control region from one of their two mitochondrial genomes. To this end, 985 specimens were sampled along the European coasts, at sites ranging from the Black Sea to the White Sea. The relevant DNA fragments were amplified, sequenced and analyzed. Contrary to the earlier findings, our coalescence and nested cladistics results show that only a single M. edulis glacial refugium existed in the Atlantic. Despite that, the species survived the glaciation retaining much of its diversity. Unsurprisingly, M. galloprovincialis survived in the Mediterranean Sea. In a relatively short time period, around the climatic optimum at 10 ky ago, the species underwent rapid expansion coupled with population differentiation. Following the expansion, further contemporary gene flow between populations was limited.

NEEM: UNUSUALLY VERSATILE PLANT GENUS AZADIRACHTA WITH MANY USEFUL AND SO FAR INSUFFICIENTLY EXPLOITED PROPERTIES FOR AGRICULTURE, MEDICINE, AND INDUSTRY.

Neem plants (Rutales: Meliaceae) are well known for their multitude of human benefits in various fields. Specifically well investigated are the Indian neem tree Azadirachta indica A. Juss., the Thai neem A. siamensis Val., the originally Malaysian/Philippinean neem A. excelsa (Jack) and, as a close relative, the Persian lilac, Melia azedarach. The major and most active natural products are azadirachtin, salannin, nimbin and marrangin from Azadirachta species, and azadirachtin analogues like meliantriol from Melia species. Neem fruits, leaves, bark, and roots have specific virtues. They have been traditionally exploited for a considerable part of human history and are documented in Sanskrit texts. Due to human activity in trade and travel both at land and sea, the plant species has been distributed around the globe and is cultivated in many tropical, and subtropical regions. A multitude of natural products of neem have been isolated, chemically characterized or identified, and investigated for their properties in the management of insects, Acarina, Crustacea, nematodes, bacteria, fungi, viruses and soil fertility (for reviews see Kraus, 2002; Schmutterer, 2002A; Rembold, 2002; Koul, 2004; Schmutterer and Huber, 2005; Kleeberg and Strang, 2009; Hummel et al., 2008, 2011, 2012). Neem products are virtually nontoxic, compatible with beneficial insects, pollinators and bees. They are environmentally benign, sustainable, renewable, and of a price affordable for developed countries. In conclusion, neem is a prime example of a natural resource with many beneficial applications in agriculture, human and veterinary medicine. So far, its use is practically free of resistance problems which are frustratingly prevalent in many areas of synthetic insecticide and drug development. Investigating more neem applications will increase future human welfare and health while being of general ecological benefit to the planet.

EXPLOITING CHEMICAL ECOLOGY FOR LIVELIHOOD IMPROVEMENT OF SMALL HOLDER FARMERS IN KENYA.

"Push-Pull" is an inexpensive and eminently practical strategy designed for developing countries in order to exploit sound principles of chemical ecology for agricultural pest management. This strategy is specifically suitable for small holder farmers. Their experience can easily be integrated into existing farming practices in their immediate environment. "Push-pull" within one and a half decades became widely established and meanwhile is greatly beneficial to practitioners in East Africa, mainly Kenya. The classical push-pull approach used for applied plant-insect management was pioneered by Khan and Pickett (2000) and subsequent papers of Pickett (2003) and Khan et al. (2006, 2008). Relevant plant species explored so far were maize or sorghum intercropped with other East African plants (Desmodium spp. resp. Melinis minutiflora) possessing natural chemicals repellent resp. attractive for stem borer moths Chilo partellus (Lepidoptera), whereby Desmodium spp. was grown inside the maize rows while M. minutiflora surrounded it. Both simultaneous actions combined resulted in a significant decrease of stem borers in the area to be protected. A benefit to cost ratio of 2.5 was realized. Within a period of 15 years the number of subscribing farmers substantially increased from a few dozen to more than 80,000 in 2014. Two experiments along the paths of chemical ecology were undertaken between Sept 2012 and Feb 2013: One was designed to investigate if the legume D. intortum known to produce repellent volatiles against stem borer moths induces defence in Zea mays varieties. We looked at two open-pollinated farmers' varieties and two commercial hybrid varieties suspecting the farmers' varieties to be responsive rather than the hybrids. However, no defence induction was detected in this study so far. This could be explained by an insufficient production of defence inducing volatiles in leaves of D. intortum whereas flowers might produce a sufficient response. More detailed study is needed. A second approach made use of species-specific insect monitoring traps baited with highly specific female sex pheromones for attracting and monitoring destructive insect pests. The female sex pheromone (8-methyl-decane-2-ol propanoate) of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (Western Corn Rootworm) is readily available as bait in the "Metcalf sticky cup trap" for trapping males, an extraordinarily sensitive technique for monitoring the presence or absence of male beetles in a given area. Li et al. (2006) had argued for the likelihood of easy immigration of this cosmopolitan maize pest into East Africa. Our results, however, so far indicate the absence of a local population in the area of Mbita, while not excluding its presence at Nairobi or Mombasa. Both investigations contribute to different aspects of Kenyan economic development and may be seen as two independent but complementary contributions towards livelihood improvement of small holder farmers in Kenya.

Diabrotica virgifera virgifera on South-Northern expansion drive?

As Wudtke and colleagues predicted and Hummel and colleagues later confirmed, the Western corn rootworm Diabrotica v. virgifera LeConte (Coleoptera: Chrysomelidae) (WCR) had shown the ability to expand northward from South and Eastern into Central Europe. Decades earlier WCR had done the same in North America. Yet, European entomologists erroneously assumed they would be somehow magically protected from such aggressive moves of WCR. Meanwhile we know better: Southern Germany is infested, and also many East European countries are victims of these Northern and also Eastern advances. In Germany, neither speed nor extent of such advances is well defined. Jumping advances similar to earlier spreading episodes may be the rule, but they are not well predictable. In this report, we argue for a persistent northbound trend of WCR.

New dispenser types for integrated pest management of agriculturally significant insect pests: an algorithm with specialized searching capacity in electronic data bases.

Pheromone effects discovered some 130 years, but scientifically defined just half a century ago, are a great bonus for basic and applied biology. Specifically, pest management efforts have been advanced in many insect orders, either for purposes or monitoring, mass trapping, or for mating disruption. Finding and applying a new search algorithm, nearly 20,000 entries in the pheromone literature have been counted, a number much higher than originally anticipated. This compilation contains identified and thus synthesizable structures for all major orders of insects. Among them are hundreds of agriculturally significant insect pests whose aggregated damages and costly control measures range in the multibillions of dollars annually. Unfortunately, and despite a lot of effort within the international entomological scene, the number of efficient and cheap engineering solutions for dispensing pheromones under variable field conditions is uncomfortably lagging behind. Some innovative approaches are cited from the relevant literature in an attempt to rectify this situation. Recently, specifically designed electrospun organic nanofibers offer a lot of promise. With their use, the mating communication of vineyard insects like Lobesia botrana (Lep.: Tortricidae) can be disrupted for periods of seven weeks.

Sex pheromone monitoring as a versatile tool for determining presence and abundance of Cydia pomonella (Lep.: Tortricidae) in German apple orchards.

Cydia pomonella (Lep.: Tortricidae), the codling moth, is an apple, pear, quince and walnut pest with considerable impact on horticultural production systems in many parts of the world. In commercial apple production, it is responsible for a yearly damage level of 40 billion dollars. In response to the need of tight codling moth control there are several options for intervention by pest managers in commercially operated orchards. Spray and count methods have been used for decades with success, but at considerable external costs for the integrity of ecological cycles. Also, problems with pesticide residues and with resistant strains are an issue of concern. For environmental reasons, toxicological means are discounted here. Instead, flight curves based on sex pheromone trapping and monitoring are preferred means towards determining the optimal timing of interventions by biotechnical and biological control methods. Finally, ecological reasons are discussed for vastly different population levels of C. pomonella developing in closely neighboring field sections which operated under different environmental management.

Organic nanofibers containing insect pheromone disruptants: a novel technical approach to controlled release dispensers with potential for process mechanization.

Beginning fifty years ago, the search for suitable dispensers containing insect pheromones grew with the availability of these synthetic biotechnical tools. Many economic entomologists and application engineers dearly wish they had the "smart, intelligent and ideal dispenser". More or less suitable approximations are available commercially, but none so far meets all demands. Under economic strictures, novel inexpensive systems would be advantageous with release characteristics tailored to the specific life histories of pest insects, the plants considered and the numerous requirements of growers alike. Simultaneously, their field distribution should be mechanizable and be accomplished by one (or very few) application runs. The dispensers should be biodegradable, biocompatible, sustainably applicable, and they should be based on renewable resources. This report presents first results of a novel organic, electrospun nanofiber dispenser with dimensions in the upper nanometer range. Its load of pheromone can be adjusted to be sufficient for 7 weeks of constant disruptive action in vineyards and can be directed against the European Grape Vine Moth Lobesia botrana (Lepidoptera: Tortricidae) which here serves as a readily available model. Mating disruption in L. botrana and the related Eupoecilia ambiguella is a well studied and developed engineering process. Equally, nanofiber production by electrospinning (for a comprehensive review see Greiner and Wendorff, 2007A, B) is well known and already has numerous applications in filtration technology, air conditioning, and medical wound dressing. Our goal was to bring together and successfully mate these (partly incompatible) technologies via technical tricks of a proprietary nature. Even though the lifetime and effectiveness of currently available nanofibers still must be doubled, the rather complicated system of their production and analysis is known well enough to identify the parameters that need future adjustment. Another challenge is the mechanical distribution of the fibers in the vineyards by suitable machinery. Also, in this respect, certain technical leads are available for future development.

Organic electrospun nanofibers as vehicles toward intelligent pheromone dispensers: characterization by laboratory investigations.

Organic nanofibers have a history of technical application in various independent fields, including medical technology, filtration technology, and applications of pharmaceuticals via inhalation into the lungs. Very recently, in a joint effort with polymer chemists, agricultural applications have been added to this list of priorities. The aim is finding novel approaches to insect control. Pheromones, dispensed in a quantifiable way, are being used here in disrupting the mating communication between male and female pest insects, e.g. the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae), where current dispenser technology does not fully meet the high expectations of growers and environmentalists with respect to longevity of constant release, self decomposition, mechanical distribution, renewability as well as sustainability of resources. The methodology of electrospinning is exhaustively covered by Greiner and Wendorff (2007), with technical details reported by Hellmann et al. (2009), Hein et al. (2011), and Hummel et al. (2010). Wind tunnel studies were run within a tunnel with adjustable laminar flow and 0.5 m/sec air velocity. Mass losses of the electrospun fiber bundles were determined with a sensitive analytical balance 2-3 times per week and recorded as time vs. mass change. CLSA experiments were performed with a self developed glass apparatus (Lindner, 2010) based on various suggestions of previous authors. Microgram quantities of volatile pheromone (E,Z)-7,9-Dodecadienylacetate were absorbed on a filter of rigorously purified charcoal and desorbed by repeated micro extraction with a suitable solvent mixture. Aliquots of the solution were subjected to temperature programmed capillary GLC. Retention times were used for identification, whereas the area covered by the pheromone peak originating from a FID detector signal was integrated and compared with a carefully calibrated standard peak. Since these signals were usually in the low nanogram range, several replications were averaged for statistical improvement. - Thermogravimetric analysis between ambient temperature and 500 degrees C provided a series of degradation curves where the diagram contained information on the evaporation of pheromone alone, polymer fiber alone and pheromone included in the fiber.- Microscopic investigations resulted in pictures of nanofibers from which the overall morphology and the fiber dimensions could be quantified. Organic nanofibers loaded with the grapevine moth pheromone have been well characterized by 5 different lab methods, followed by field bioassays reported elsewhere in these communications volumes (HUMMEL et al., 2011). This comprehensive analytical approach to fiber characterization is new and will be further refined. The federal agency JKI Berlin subjected the pheromone loaded organic fibers to various independent toxicological and ecotoxicological tests and found no adverse side effects.

Western corn rootworm Diabrotica v. virgifera in Europe: status, and options for future management.

Diabrotica virgifero virgifera (Col.: Chrysomelidae) (western corn rootworm, WCR) unfortunately is now a firmly established European maize pest without indigenous natural enemies. Twenty years after its first introduction into Europe by traffic and trade, eradication is wishful thinking and not longer a viable option. Instead, European legislation calls for toxicological emergency measures on a case by case basis where and when new infestations occur either by new invasions from the native Americas or by introductions from other European countries. It is unclear at which point in time such treatment systems will break down because of development of resistant strains or by other complications of this seemingly unstable situation. Rather than proceeding as usual, sustainable biological, biotechnical and cultural methods should be practiced. Prominent among them are trapping and survey with attractants in conjunction with traditional crop rotation and phytosanitation.

Toxicity of Stemona root against onion cutworm by using different contacting methods and solvents.

Roots of Stemona tuberoso have been extracted by using fixed-bed contacting method with serial and parallel technique. In order to extract secondary metabolites from roots of Stemona sp., various organic solvents with different polarities were employed. Optimal solvents for the extraction of effective ingredients from root were determined. The results showed that in most cases the yields of crude extract were highest by using parallel technique rather than serial technique. The highest yields of crude extract by using both techniques were obtained with acetone, followed by water, ethanol, methanol, dichloromethane and hexane, respectively. Furthermore it was found that the L-a-b value and the total soluble solid (TSS) value in terms of degree Brix of crude extracts were different depending on choice of organic solvents. In particular, dichloromethane extracted more total soluble solid from plant material with both techniques, followed by hexane, ethanol, acetone, water and methanol, respectively. The biological activities of different crude extracts from S. tuberosa against Spodoptera exigua were experimentally determined. It was observed that the crude extract with highest toxicity to Larvae was obtained with high polarity solvents such as water, methanol and ethanol. The results suggested that water extract tested under high concentration could promote higher mortality. On the other hand, hexane extract caused approximately 30 % mortality with highest concentration at 15% w/w. Dichloromethane and acetone extract could be considered of similar efficiency.

Ergography: a method for analyzing and understanding temporal and thematic working patterns of prolific entomologists.

Ergographic analysis is a new approach for qualitative assessment and quantitative depiction of publication records of prolific authors. For proving the merits of ergographic analysis, the method is applied to the work of 3 scientists who are well known for their individual contributions to basic and applied entomology, insect toxicology, insect biochemistry, and chemical ecology including integrated pest management. Authors thus considered are R.L. Metcalf (1916-1998), H. Schmutterer (born 1926) and P. Karlson (1918-2001). Ergograms obtained through ergographic analysis are graphic presentations of when, which, and how many papers appeared within the author's lifetime (two-dimensional ergogram), and how parallel or subsequent strands of distinct thematic interests and subject areas intertwine or follow each other over time (three-dimensional ergograms). Unlike conventional printed bibliographies which have a tendency to be SUMMARY, rather unwieldy and boring, ergograms provide easy graphic access to information. They can be understood and interpreted at first glance and with a conceptual clarity so far unknown from conventionally printed reference lists.

The western corn rootworm (Diabrotica virgifera virgifera) and its first appearance in Germany 2007.

Diabrotica v. virgifera (WCR) is one of the most successful invasive insect species and owes this success to its close association with mankind, to worldwide trade and commerce connections, and to widely practiced, monoculturally oriented production patterns which are characteristic for contemporary agriculture in several parts of the world. Without a drastic change in attitudes and approaches of a globalizing agriculture, WCR will in the foreseeable future have invaded all maize growing areas. WCR is continuing its spreading within Europe with its first documented appearance in Germany 2007, where two major and three minor sites of invasion are identified. Unfortunately, WCR already has secondary hosts and, through accelerated microevolution, soon may acquire more of them. The beetle may be seen as a clever follower in the footsteps of Homo sapiens and may thrive on worldwide ecological imbalances. Without major new paradigms in control and management approaches, WCR will be one of the big winners of globalization and mercilessly occupy the niches opening up. Some biotechnical and cultural alternatives are discussed which, in combination with biocontrol, may assist in evironmentally compatible management of WCR.

Monitoring Diabrotica v. virgifera (Col.: Chrysomelidae) in southeastern Slovenia: increasing population trend and host spectrum expansion.

Ever since the western corn rootworm (WCR) (Diabrotica virgifera virgifera), an alien invasive species from North America, has been introduced into Europe on at least 3 separate occasions, it spread within 15 years over the entire area of south-eastern and central Europe (except Denmark). Until quite recently, Zea mays L. was the only known host plant whereas in North America WCR also attacks members of the plant family Cucurbitaceae. In August of 2006, we were able to validate these findings also in the Old World by observing WCR visiting blossoms of oil pumpkin (Cucurbita pepo L.). Beside this first report of WCR on this regionally and economically important crop, a population increase in Gaberje near Lendava, Eastern Slovenia, was observed. Some future consequences of multiple hosts for integrated pest management (IPM) of WCR are being discussed.

A comparative assessment of heavy metal accumulation in soft parts and byssus of mussels from subarctic, temperate, subtropical and tropical marine environments.

Existing data on metal concentrations in mussels from subarctic, temperate, subtropical and tropical waters were analyzed using multivariate statistics in order to assess regional variations in metal contamination. Potential errors were reduced by only analyzing data from surveys that employed the same protocols, analytical methodologies and analysts. Factor analysis demonstrated that mussels inhabiting extremely contaminated areas (e.g. from Japanese and Swedish metallurgy sources) could be separated from mussels from other contaminated areas, and that metals such as Cd, Pb, Cu and Zn could be used to identify heavily contaminated samples while Co, Fe, Cr and Ni concentrations were good markers for exposure to inputs from different industrial sources. Furthermore byssus, like soft tissue, selectively and sensitively reflects variations of certain metal concentrations in ambient waters and thus serves as a reliable biomonitor for these contaminants in a variety of coastal and estuarine areas.

Diabrotica virgifera virgifera in Switzerland: future invasion trend into Germany via transalpine, terrestrial routes.

The western corn rootworm Diabrotica v.virgifera (D.v.v.) (Coleoptera:Chrysomelidae) is endemic to the New World. On at least 3 occasions, it invaded Europe (Miller et al. 2005). The first of these invasions took place in or before 1992 (Baca 1993) to Belgrade, Serbia, and gave rise to a rapidly expanding beetle population in SE Europe. Its larvae are causing economic damage to Zea mays for the last ten years, with increasing severity both in acreage and density. Meanwhile Germany is still without proven infestations as of 2005, but is surrounded by countries invaded by D.v.v. It will be a matter of time and careful search efforts with efficient traps to detect D.v.v. on German territory as well. Three main terrestrial routes of future introduction are likely (Wudtke et al. 2005, Hummel et al. 2005 a and b; Baufeld and Enzian, 2005), one of them leading from Northern Italy through Southern and Northern Switzerland to Southwestern and Southern Germany.

Diabrotica virgifera virgifera in eastern Slovenia: increasing population trend 2005.

The first invasion of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) into Slovenia was recorded in 2003. A few beetles were found in the western province of Slovenia near Italy. More beetles were detected in the eastern district of Prekmurje bordering Austria, Hungary and Croatia. While the western population remained the same in 2005, the eastern population increased considerably. By means of the very sensitive Metcalf sticky trap baited with the female sex pheromone, early infestations could be recorded. At corn fields, Zea mays L., near the villages of Dolina, Gornji Lakos and Pince, Diabrotica flights were observed in 2005 which had their maxima during the first week of August and yielded, respectively, 57, 112, and 45 beetles/trap per 3-4 day trapping interval. The increase from 2004 to 2005 in this eastern most district of Slovenia is noteworthy. Generally in the western and central part of Slovenia, most maize is grown in rotation, typically every second year. It is estimated that about 10 % of the surface of maize is grown as monoculture, typically on farms with intensive cattle production. In eastern Slovenia, a higher proportion of maize is grown in monoculture (about 30 % in the 3-year period from 2002-2004); this percentage decreased in 2005 due to official requirements by the Slovenian Phytosanitarian Administration.

Diabrotica flight in time and space as monitored with a high capacity trap placed in Zea mays fields of Illinois, USA.

Monitoring of insects by trapping is one of the prime tools of field entomologists. The leaf beetle Diabrotica virgifera virgifera LeConte (Coleoptera:Chrysomelidae) is no exception. Since its numbers (and consequently its mass) in a field population can be enormous, tools must be adapted to hold this many insects for later counting, sexing, biomass determinations, and additional investigations to follow. Since counting the high numbers during the flight peak may not be feasible at all, weighing and extrapolating to numbers by the correlation factor 1g = 160 to 170 beetles of mixed female and male sex was the method of choice. Around the perimeter of a hybrid maize (Z. mays) field of 0.6 ha, 16 high capacity traps were established at elevations ranging from 0 to 2 m above ground level. Optimal trapping is possible between 0.5 to 2m which field entomologists intuitively knew from experience and tradition. Below and above that level, the number of beetles is not zero but significantly below the optimum. High capacity traps can be left in the field with one loading of lure for four to seven days. Lures were the D. v. virgifera beetle sex pheromone 8-methyl-decane-2-ol propanoate (0.1 mg, collecting mainly male beetles) and the plant kairomone 4-methoxy-trans-cinnamaldehyde (10 mg, collecting both male and female beetles). The specific kairomone action (being much weaker than the pheromone) can be increased by simultaneously offering a feeding arrestant powder (e.g. prepared from ground Cucurbita texana or C. okeechobeensis) which keeps the beetles attracted by the kairomone lure close to the trapping site. There they can be immobilized and knocked down by a tiny source of carbaryl or dichlorvos fixed inside or at the surface of a plastic pellet or sheet. The high capacity traps are commercially available and can be reused for several flight seasons.