Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. (c) 1993 John Wiley & Sons, Inc.     

Bird carcass detection from integrated trials at multiple wind farms

It is often necessary to estimate the number of wind turbine collision fatalities to assess impacts to birds following construction of wind farms. Detection of bird carcasses at wind turbines in the field is affected by carcass persistence and searcher detection rate. Integrated detection trials, which integrate carcass persistence and searcher detection trials into the periodic fatality search, have been proposed as an effective method for estimating these parameters. The purpose of our study was to test whether and how environmental factors affect integrated detection trial outcomes at multiple wind farms. We conducted this study at 10 wind farms in various environments of Japan. Binary data on trial outcomes in open versus forested areas served as our response variable in a generalized additive mixed model informed by days into trial, carcass body mass, season, whether snow covered the ground, and precipitation. For both ground cover types, days into trial and body mass were included in all the top models, suggesting that these factors most influenced bird carcass detection probability in integrated trials. The best model in open areas included days into trial, body mass, snow, and precipitation, and the best model in forested areas included days into trial, body mass, snow, precipitation, and season. Values of area under the curve indicated high accuracy of the best model for both ground cover types. The survey design needs to be appropriate to the size of the target species and to the environment in which the impacts will occur, such as the site's seasonality, its ground cover, and whether snow will cover the ground. Frequency of post-construction fatality monitoring should also be set cautiously, especially at wind farms located on small-bird migration routes, at wind farms in open areas, in areas with snow-covered ground in winter, or in forested areas during spring and summer because detection probabilities decline fastest under such conditions.     

Summer Epidemics of Apple Scab: The Relationship between Measurements and their Implications for the Development of Predictive Models and Threshold Levels under Different Disease Control Regimes

A 2‐year study on epidemic progress of apple scab was conducted at Randwijk, the Netherlands, in 1998 and 1999. The summer epidemic caused by conidia was studied instead of the well‐described spring season epidemic originating from ascospores. The aim was to investigate relationships between disease measurements, i.e. disease incidence and severity measures of apple scab, and their implications for the development of predictive models and threshold levels. The study characterized good relationships between the measurements on cultivar Jonagold using regression analyses in three disease control regimes (untreated, organic and integrated). For fruit quality prediction, the relationship between fruit incidence (I_f) and leaf incidence (I_l) in the organic control regime was given by I_f = 1.966 + 0.402 × (I_l) (R² = 0.92). As a result of low level of disease in the integrated control regime, shoot incidence (I_s), with higher values than leaf incidence, was better suited for prediction. The relationship was given by I_f = ?0.162 + 0.028 × (I_s) (R² = 0.91). For the integrated control regime, disease threshold levels were constructed for timing of the final fungicide application. If an apple grower wants to keep fruit infection under 1% incidence (harvest scab threshold), the timing of the final fungicide application (action threshold) should correspond to 4% shoot scab incidence at the beginning of August. The results are compared with similar studies and their biological interpretation is discussed.     

Conservation and management issues of Prespa National Park

The present article describes the human activities inthe area and their changes and trends, given that thecontemporary nature conservation key issues areclosely related to past and present socio-economic andcultural conditions, both within Prespa itself andoutside of it. The liberation of Prespa, a remote andrather socially isolated borderline area, from Ottoman occupation took place in 1912–13. Recentevents in its environmental history have been forestclearings and human depopulation in the Civil War(1944–49), irrigation system construction in the1960s, its discovery by ornithologists in the late1960s, the high emigration rates of 1960–1980, itsdesignation as a National Park in 1974, thedestructive development works of 1984–86 afterGreeces entry to the EEC and the conversion to intensivebean cultivation in the mid 1980s.The human population of ca 7000 at the start of thiscentury suffered an almost 80% decrease after theCivil War. A resettlement in the mid 1950s increasedthe population by 40% but in the 1970s emigrationreduced it to its previous size of around 1500, stillits present size. The extensive and diverse farmingsystems of previous centuries changed gradually afterthe construction of an irrigation network in the1960s, which twenty years later and along withinternational changes in production and economypatterns, made possible the present day domination ofa bean monoculture within irrigated croplands. Thiswas accompanied by increased energy inputs,mechanisation and the use of chemicals.Intensification of agriculture brought increasedincomes which attracted also livestock keepers,resulting in a 56% decrease in numbers of livestockin 1964–1993. Cattle are presently kept for meatproduction only. They graze on the mountains, and nolonger in the meadows around the lakeshore. A localshorthorn breed accounting for 93% of all cattle in1963 declined to less than 18% in 1993. Fishing aimedmainly at carp and Prespa bleak, continues today as inthe past to be a source of supplementary income.Eighty-six per cent of the – mainly oak and beech –forests are State owned and the rest belong to localmunicipalities. The forest area has not changedessentially in the last 40 years, but forest roadshave expanded. Forests provide mainly fuelwood forlocal needs and small quantities of industrial timber.No specific management is conducted to safeguard theconservation values of the forest, the majority ofwhich consists of even aged coppiced stands withlimited value for wildlife. Manufacturing never playedan important role for the economy of the area. Tourismhas increased in the last 20 years, concentratedmainly in summer and spring. Accommodation in thevillages is not yet satisfactory though it hasimproved enormously in recent years. The NationalParks infrastructure for visitors remains poor.Tourist pressure generally is not yet that high toimpact drastically the social and environmentalfabric. In places, hunting, angling and poaching cannegatively affect the target species. The constructionof the irrigation network in the eastern part of LakeMikri Prespa was the most important change to thelandscape and the habitats of Prespa the last 30years. It resulted in the embankment of free-flowingstreams, drainage of wet meadows and dramaticreduction of trees and hedges. In the last 15 years,the reedbeds, no longer burnt or mowed and assisted byincreased nutrient loadings from agricultural runoff,have expanded landward to occupy formerly open,periodically flooded, areas. The above changes inhabitats and land use have brought an impoverishmentof biodiversity which can be seen in the exterminationor the decrease in the numbers of certain plant andbird species.The problems preventing a better management of theNational Park and the perpetuation of its values areinappropriate legislation, the poor degree ofcoordination of the public services, the lack of botha specific National Park Service in Greece and amanagement authority for the Park and the hostileattitude of local people due to their limitedinformation and involvement. The scenic beauty, thewetlands and a number of rare habitats, the diversityof habitats, flora and fauna, the endemic life forms,the rare mammals and the colonial waterbirds, thelocal architecture and the cultural expressions suchas the Byzantine monuments, are the values of Prespathat must be preserved as a whole. The initialconservation efforts aimed at preserving biodiversitybut later it became apparent that not only are humansan inseparable part of the whole, but additionally,several of their extensive activities have contributedsubstantially to the rich biodiversity of the area.Through the prerequisites of keeping a balance betweenprimary and tertiary sector activities and ofrevitalizing the social fabric, the following are thekey management and conservation issues of today: anunequivocal agreement on the values to be preservedand their limits; the formation of a managementauthority; a new realistic protection and land usezoning; the diversification and extensification of allfarming activities; the restoration of the wet meadowhabitats; the ensuring of a high water level and agood water quality for both lakes through tri-lateralcooperation between Greece, Albania and FYROM; and theestablishment of a permanent environmental monitoringscheme.     

A global invasion by the thrip,Frankliniella occidentalis: Current virus vector status and its management

Western flower thrip, Frankliniella occidentalis (Pergande), is among the most economically important agricultural pests globally, attacking a wide range of vegetable and horticultural crops. In addition to causing extensive crop damage, the species is notorious for vectoring destructive plant viruses, mainly belonging to the genera Orthotospovirus, Ilarvirus, Alphacarmovirus and Machlomovirus. Once infected by orthotospoviruses, thrips can remain virulent throughout their lifespan and continue transmitting viruses to host plants when and wherever they feed. These irruptive viral outbreaks in crops will permanently disrupt functional integrated pest management systems, and typically require a remedial treatment involving insecticides, contributing to further development of insecticide resistance. To mitigate against this continuing cycle, the most effective management is early and comprehensive surveillance of the pest species and recognition of plant viruses in the field. This review provides information on the pest status of F. occidentalis, discusses the current global status of the viruses vectored by this thrip species, examines the mechanisms involved in transmitting virus‐induced diseases by thrips, and reviews different management strategies, highlighting the potential management tactics developed for various cropping systems. The early surveillance and the utilization of potential methods for control of both F. occidentalis and viruses are proposed.     

Missing piece of top predator-based conservation: Demographic analysis of an owl population on a remote subtropical island

Top predators are frequently the target of conservation programs. Owls are such predators. However, previous studies of owls are biased to species occurring in temperate regions, whereas most owl species occur in tropical or subtropical regions and are understudied. Furthermore, owls are often endemic to islands and of unknown conservation status. Demographic data for such species are especially scarce although they are essential for initiating and promoting their conservation. As a case study of demographic analysis of owls in a tropical or subtropical area and on islands, we applied an integrated population model to 7-year monitoring data (2012–2018) of the Ryukyu Scops Owl population on Minami-daito Island, Japan. We used survival history data from 903 individuals, reproduction and sex ratio data from 213 broods, and count data of 2,526 individuals in total. Long-term averages of annual survival rates were 0.73 for adult females and 0.74 for adult males, although the sexual difference was not significant. Sex ratio estimates fluctuated annually and long-term averages were slightly skewed to males: 0.51 among fledglings, 0.54 among yearlings, and 0.52 among adults. Long-term averages of population size were estimated to be 273.4 females and 296.8 males. The long-term average of population growth rate was 0.98, suggesting a slightly declining trend. It was fortunate to recognize the declining trend during the early phase. Considering the general lack of fundamental ecological data on owls of tropical or subtropical areas and on islands, it seems likely that many endangered owl populations await conservation efforts.     

Molecular phylogeny and diversity of penaeid shrimps (Crustacea: Decapoda) from South-East Asian waters

The decapod family Penaeidae comprises most of the economically important marine shrimp species. Its members are widespread throughout the world, with its highest species diversity centred in the Indo-West Pacific region. Despite this importance, their taxonomy, classification and phylogeny are not yet settled due in part to incongruence among hypotheses proposed from molecular versus morphological studies. In this study, using a thorough taxonomic sampling of especially the South-East Asian species, we aim to (a) utilize a reconstructed phylogeny to test the monophyly of the Penaeidae and its currently recognized genera and (b) explore its species diversity in South-East Asian waters. To infer the phylogeny, a combined gene data set (including 109 ingroup and six outgroup taxa) of mitochondrial genes, COI and 16S rRNA, and two nuclear genes, NaK and PEPCK, was utilized. To explore its diversity, another data set that included 371 COI gene sequences (231 newly generated and 140 retrieved from public sources) was compiled and subsequently analysed with two different tools (ABGD and bPTP) for species delimitation. Other than supporting the non-monophyly of the Penaeidae with the Sicyoniidae nested within the penaeid tribe Trachypenaeini, the genera Penaeus, Mierspenaeopsis and Parapenaeopsis were also revealed to be polyphyletic. Our species delimitation analysis inferred that 94 putative species actually existed within the 71 morphospecies reviewed, indicating an underestimated biodiversity in this family and the potential presence of new species within the following morphospecies: Kishinouyepenaeopsis cornuta, K. incisa, Mierspenaeopsis sculptilis, M hardwicki, Parapenaeopsis coromandelica and Penaeus monodon.     

Comprehensive analysis of mRNA‐level and miRNA‐level subpathway activities for identifying robust ovarian cancer prognostic signatures

Ovarian cancer (OvCa) causes the highest mortality among all gynaecologic cancers. A large number of mRNA‐ or miRNA‐based signatures were identified for OvCa patient prognosis. However, the comprehensive analysis of function‐level prognostic signatures is currently not considered in OvCa. In the present study, we respectively inferred subpathway activities from mRNA and miRNA levels based on high‐throughput expression profiles and reconstructed subpathways. Firstly, the activities of two tumour pathways were calculated and the difference between normal and tumour samples were analysed using multiple tumour types. Then, we calculated subpathway activities for OvCa based on the expression profiles from both mRNA and miRNA levels. Furthermore, based on these subpathway activity matrices, we performed bootstrap analysis to obtain sub‐training sets and utilized univariate method to identify robust OvCa prognostic subpathways. A comprehensive comparison of subpathway results between these two levels was performed. As a result, we observed subpathway mutual exclusion trend between the levels of mRNA and miRNA, which indicated the necessary of combining mRNA‐miRNA levels. Finally, by using ICGC data as testing sets, we utilized two strategies to verify survival predictive power of the mRNA‐miRNA combined subpathway signatures and performed comparisons with results from individual levels. It was confirmed that our framework displayed application to identify robust and efficient prognostic signatures for OvCa, and the combined signatures indeed exhibited advantages over individual ones. In the study, we took a step forward in relevant novel integrated functional signatures for OvCa prognosis.     

Afforestation for climate change mitigation: Potentials,risks and trade‐offs

Afforestation is considered a cost‐effective and readily available climate change mitigation option. In recent studies afforestation is presented as a major solution to limit climate change. However, estimates of afforestation potential vary widely. Moreover, the risks in global mitigation policy and the negative trade‐offs with food security are often not considered. Here we present a new approach to assess the economic potential of afforestation with the IMAGE 3.0 integrated assessment model framework. In addition, we discuss the role of afforestation in mitigation pathways and the effects of afforestation on the food system under increasingly ambitious climate targets. We show that afforestation has a mitigation potential of 4.9 GtCO₂/year at 200 US$/tCO₂ in 2050 leading to large‐scale application in an SSP2 scenario aiming for 2°C (410 GtCO₂ cumulative up to 2100). Afforestation reduces the overall costs of mitigation policy. However, it may lead to lower mitigation ambition and lock‐in situations in other sectors. Moreover, it bears risks to implementation and permanence as the negative emissions are increasingly located in regions with high investment risks and weak governance, for example in Sub‐Saharan Africa. Afforestation also requires large amounts of land (up to 1,100 Mha) leading to large reductions in agricultural land. The increased competition for land could lead to higher food prices and an increased population at risk of hunger. Our results confirm that afforestation has substantial potential for mitigation. At the same time, we highlight that major risks and trade‐offs are involved. Pathways aiming to limit climate change to 2°C or even 1.5°C need to minimize these risks and trade‐offs in order to achieve mitigation sustainably.     

Genomic and phenotypic effects of inbreeding across two different hatchery management regimes in Chinook salmon

Genomic approaches permit direct estimation of inbreeding and its effect on fitness. We used genomic‐based estimates of inbreeding to investigate their relationship with eight adult traits in a captive‐reared Pacific salmonid that is released into the wild. Estimates were also used to determine whether alternative broodstock management approaches reduced risks of inbreeding. Specifically, 1,100 unlinked restriction‐site associated (RAD) loci were used to compare pairwise relatedness, derived from a relationship matrix, and individual inbreeding, estimated by comparing observed and expected homozygosity, across four generations in two hatchery lines of Chinook salmon that were derived from the same source. The lines are managed as “integrated” with the founding wild stock, with ongoing gene flow, and as “segregated” with no gene flow. While relatedness and inbreeding increased in the first generation of both lines, possibly due to population subdivision caused by hatchery initiation, the integrated line had significantly lower levels in some subsequent generations (relatedness: F₂–F₄; inbreeding F₂). Generally, inbreeding was similar between the lines despite large differences in effective numbers of breeders. Inbreeding did not affect fecundity, reproductive effort, return timing, fork length, weight, condition factor, and daily growth coefficient. However, it delayed spawn timing by 1.75 days per one standard deviation increase in F (~0.16). The results indicate that integrated management may reduce inbreeding but also suggest that it is relatively low in a small, segregated hatchery population that maximized number of breeders. Our findings demonstrate the utility of genomics to monitor inbreeding under alternative management strategies in captive breeding programs.