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The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. The direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiac myocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in an improved intracellular Ca2+ handling as well as an enhanced contractility and relaxation. The PKC-mediated inhibition of PP2A also leads to a modulation of the LTCC activation and inactivation kinetics. This study highlights the importance of exploring regulatory subunits of PP2A.

Phthalocyanines (Pcs) are promising candidates for photodynamic therapy (PDT) due to their absorption in the phototherapeutic window. However, the highly aromatic Pc core leads to undesired aggregation and decreased reactive oxygen species (ROS) production. Therefore, short PEG chain functionalized A3B type asymmetric Pc photosensitizers (PSs) were designed in order to decrease aggregation and increase the aqueous solubility. Here we report the synthesis, characterization, optical properties, cellular localization, and cytotoxicity of three novel Pc-based agents (LC31, MLC31, and DMLC31Pt). The stepwise functionalization of the peripheral moieties has a strong effect on the distribution coefficient (logP), cellular uptake, and localization, as well as photocytotoxicity. Additional experiments have revealed that the presence of the malonic ester moiety in the reported agent series is indispensable in order to induce photocytotoxicity. The best-performing agent, MLC31, showed mitochondrial targeting and an impressive phototoxic index (p.i.) of 748 in the cisplatin-resistant A2780/CP70 cell line, after a low-dose irradiation of 6.95 J/cm2. This is the result of a high photocytotoxicity (IC50 = 157 nM) upon irradiation with near-infrared (NIR) light, and virtually no toxicity in the dark (IC50 = 117 μM). Photocytotoxicity was subsequently determined under hypoxic conditions. Additionally, a preliminarily pathway investigation of the mitochondrial membrane potential (MMP) disruption and induction of apoptosis by MLC31 was carried out. Our results underline how agent design involving both hydrophilic and lipophilic peripheral groups may serve as an effective way to improve the PDT efficiency of highly aromatic PSs for NIR light-mediated cancer therapy.

We report the first exocyclically metallated tetrapyridinoporphyrazine, [tetrakis-(trans-Pt(NH3)2Cl)-tetra(3,4-pyrido)porphyrazine-zinc(II)](NO3)4 (4), synthesized in a multistep synthesis starting from 3,4-pyridinedicarbonitrile (1). The synthetic procedure involved a platination reaction of the intermediate tetra(3,4-pyrido)porphyrazine-zinc(II) (2), whereby the zinc(II) enhanced the solubility of the intermediate enabling the platination reaction. A similar approach to synthesize [tetrakis-(trans-Pt(NH3)2Cl)-tetra(3,4-pyrido)porphyrazine](NO3)4 (5) failed due to the unsuitable solubility properties of the intermediate tetra(3,4-pyrido)porphyrazine (3). The final product 4 and the intermediates were characterized, the photochemical and photophysical properties were determined and the photocytotoxicities were investigated. We demonstrate that the platinated tetra-pyridinoporphyrazine 4 is a potential photosensitizer for photodynamic therapy (PDT).

Error-free repair of DNA double-strand break is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM execute extensive resection of DNA ends to produce 3’-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification of EXO1 followed by Orbitrap mass spectrometry led to the identification of novel partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Depletion of a selected subset of EXO1 interacting proteins led to reduction of the DNA damage response. Among those, we examined the RRP5-homologue and NFκB-interacting protein PDCD11/ALG-4, which has roles in apoptosis and is a putative driver gene in cutaneous T-cell lymphoma. We provide evidence that depletion of PDCD11 decreased the formation of γH2AX foci and the phosphorylation of DNA damage response signaling intermediates in response to camptothecin or bleomycin, resulting in increased cellular resistance to DNA damage. Furthermore, extensive coverage of EXO1 sequence (>85%) by mass spectrometry allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.As a whole, these results provide the basis for future in-depth studies on novel roles of EXO1 in genome stability and indicate targets for pharmacological inhibition of pathways of cancer development.HIGHLIGHTSProteome-wide analysis of Exonuclease-1 (EXO1) interacting proteins revealed novel partners involved in RNA processing or that are the causative agents of rare X-linked disorders.We provide evidence for a role of PDCD11 in the DNA Damage Response.We conducted a comprehensive identification of EXO1 phosphorylation sites.

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model. Funding Agencies|Promedica-Stiftung-UBS; Stiftung fur Krebsbekampfung and Stiftung fur wissenschaftliche Forschung of the University of Zurich; Swiss National Science Foundation; Forschungskredit of the University of Zurich; AIRC [IG 14180]; University of Padua [COZZ_SID18_01]; Swedish Society for Medical Research (SSMF); Swedish Research Council (VR); Linkoping University (LiU); H2020-MSCA-RISE network 3D-NEONET; foundation Jeanssons Stiftelser; Magnus Bergvall Foundation

We report the synthesis of novel tetraplatinated metalloporphyrin-based photosensitizers (PSs) for photodynamic therapy (PDT), their characterization, cellular uptake and localization, as well as the determination of their in vitro light-induced anticancer properties. The PSs show excellent phototoxic indexes up to 5800 against HeLa cells, which is, to the best of our knowledge, the highest value reported for any porphyrin so far. Furthermore, isotopic labelling of the porphyrin with a highly enriched 67Zn isotope was performed in order to determine the distribution ratio of zinc to platinum by ICP-MS, allowing to differentiate between naturally occurring zinc and 67Zn that was introduced into the cells by the PS. We conclude that the platinum units within the platinum-PS conjugates help to solubilize the PS and, at the same time, act as cell-penetrating vectors, enhancing the efficiency of the PS without causing a significant dark toxicity.

Im Uberblickskapitel zur Weimarer Republik werden die juristischen, technischen, asthetischen, diskursiven und performativen Besonderheiten von Film und Kino von 1918 bis ca. 1933 besprochen. Dazu zahlt die Besonderheit der nicht vorhandenen Zensur 1918/19. Es entstanden die sogenannten Aufklarungs- und Sensationsfilme. Dazu zahlt selbstredend der expressionistische Film. Ebenso relevant ist die sezierende, geradezu praskriptive und dynamisierte Kamera ab ca. 1924. Der entsprechende Bildmodus dichtet das Sichtbare zugleich mit einem luminiszenten Uberzug (glow) ab. Durch diese Aufmerksamkeitsstrategie thematisiert das Medium den Realitatsbezug der Themen. Das Verhaltnis der Geschlechterordnung zum Apparat brachte im Zuge dessen die moderne Figur der femme fatale als Resultat einer spezifischen differenzialen Relationalitat von ‚authentischer Identitat‘ und (projektivem) ‚Schein‘ hervor. Diese auserte sich fur mannliche Subjektivitat in einer Spaltung von ‚psychischen Innenraumen‘ und ‚sozialer Realitat‘, sprich in Dissoziationen und Dopplungen. Diese Aufteilungen lassen sich als spezifische Reorganisation eines nicht (mehr) exklusiv aufeinander bezogenen Geschlechterverhaltnisses schliesen. Die Implementierung des Tons ins Medium Film sowie in die Kinosale ist ebenfalls von groser Bedeutung. Mit ihm reorganisiert sich die Konstituierungsweise von (geschlechtlicher und sexueller) Identitat erneut. Auch im Musik- und Sangerfilm wird dies in aller Komplexitat ins Verhaltnis zu einer vermehrt durch Medien vermittelten sozialen Realitat gesetzt.

Dieses Uberblickskapitel stellt zunachst den Umbau des Kulturbetriebs unter der Agide der Asthetisierung des Politischen und vice versa durch die Nazis vor. Hierbei spielt das Konzept der Metaphysik der Prasenz eine zentrale Rolle, womit das volkische Ideal einer harmonisierten Gemeinschaft unter radikalem Ausschluss alles Nicht-Genehmen legitimiert wurde. Hieraus wird ersichtlich, warum es einerseits keine nationalsozialistische Asthetik geben konnte. Andererseits sollte durch eine bestimmte Raumzeitkonstruktion alles Partikulare auf ein noch nicht eingelostes Ideal bezogen werden konnen. Sowohl das Gemeinschaftliche als auch das Individuelle, inklusive Geschlecht und Sexualitat, wurden daher in einer success story raumzeitlich angeordnet, worin sie schlussendlich stets larger than life als uber-menschlich, uber-zeitlich in Erscheinung traten.

In diesem Kapitel wird der Fokus auf die Konstituierung von (geschlechtlicher, sexueller und race- sowie class-bedingter) Identitat durch das Medium Film von 1896 bis 1910 in der Methodik medialer differenzialer Relationalitat, inklusive filminterner Differenzen vorgestellt. Konkret werden die zeitgenossischen Phanomene von Nummernprogrammen bis hin zum Langfilm und deren Einfluss auf die Herausbildung von Identitaten erlautert. Deutlich wird hierbei, dass der filmische Apparat Identitat eben nie ‚realitatsgetreu‘ abbildete, sondern als stets in Differenz zu bereits bestehenden Konzepten von Identitat oder anderen Formen der Identitat existierende konstituierte. Zugleich wurde daran arbeitete, die geschlechtliche und sexuelle Identitat als heterosexuelle Geschlechterbinaritat in den Apparat zu installieren. Speziell in Bezug auf queere Mannlichkeit konnte dies aufgrund des in jeglicher Konstituierung von Mannlichkeit wirksamen double bind von ‚gleich‘ und ‚anders‘ sowie ‚homo‘ und ‚hetero‘ bei den Wieder-auffuhrungen nie vollstandig gelingen.

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase λ (Polλ) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Polλ is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Polλ induced by oxidative stress is dependent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersensitivity of cells lacking WRN and/or Polλ to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Polλ and specifically stimulates DNA gap filling by Polλ over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Polλ during MUTYH-initiated repair of 8-oxo-G:A mispairs.

Genera of flowering plants that are endemic to oceanic islands are often of great biological interest. These groups represent adaptive complexes that confer distinction to the islands or archipelagos in which they are found, and this often results in a focus on their conservation. In recent decades, numerous molecular phylogenetic (and other evolutionary) studies have been done on island genera, hence providing much valuable new information on relationships and evolution of island groups. Genera restricted to oceanic islands derive evolutionarily from parental stocks usually in continental regions. These parental genera are often themselves evolutionarily successful, being particularly adept at dispersal, adaptation, and speciation. These immigrants to isolated oceanic islands derive from common ancestors of large and diverse parents or directly from within the lineages themselves. If in the latter case the island derivatives are treated at the generic level, then the parental genus becomes parap...

This article is about the potentiality of Queer Theory not as a tool to analyze and criticize power structures and their mechanisms with respect to sexualities, desire, sexual difference, binarized gender identities, and, finally heteronormativity. This kind of Theory is not to be seen as a way to ref lect critically on something which is represented or discursively constructed either. Queer Theory, here, is a way of embodied thinking that has internalized the principles of queer as traversing the traditional ways of coherent thinking, of analysis, of representation, of linear narratives and causalities. In the course of its unfolding it actualizes two entities, biodigital composits and nanobots. Those two entities will already have modified on an abstract level the objects of its outcome: sexuality, gender, desire in an unprecedented way beyond oedipal constellations, heteronormative sexualities and gender identities. I call this process techno-ontogenetic queer becoming.

It is attractive to use vitamin B12 as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B12 by fast proliferating cells. The basic {B12–CN–PtII} conjugates are recognized by intracellular enzymes and converted to coenzyme B12 in an enzymatic adenosylation assay. The reductive adenosylation of {B12–CN–PtII} conjugates leads to the release of the PtII complexes; thus, {B12–CN–PtII} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin–B12 conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B12–CN–PtII} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding PtII species. Kurnakov tests and coordination of 2′-deoxyguanosine or GMP to the released PtII complexes allowed isolation and characterization of PtII complexes as released during enzymatic adenosylation. The biological activity of these PtII complexes was evaluated. Since the cleaved PtII complexes show cytotoxicity, the {B12–CN–PtII} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC50 between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B12–CN–PtII} concentration, comparison with pure cisplatin is of limited use. We could show that the PtII complexes cleaved from B12 exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B12 free media showed a dependence on the addition of transcobalamin II for B12–Pt(II) conjugates.

The monophyletic Anemone section Anemone (Ranunculaceae) includes predominantly diploid and outbreeding geophytic perennials. A revised taxonomy of the section with 16 species (and some infraspecific taxa) is proposed on the basis of a critical morphological analysis of living populations and extensive herbarium material, together with karyological, cytogenetical and DNA-analytical data. A key, descriptions, figures illustrating some type specimens and differential characters, examples of seedling development and pollen grain micromorphology (scanning electron microscopy) and distribution maps are presented. The position of A. section Anemone within the family is illustrated by a plastid DNA phylogram from sequences of the atpB-rbcL intergenic region. A penalized likelihood approach permitted the approximate dating of the origin and major differentiation phases of the section. The analysis of 20 morphological characters from all species of A. section Anemone with A. blanda (A. section Tuberosa) as an outgroup resulted in a morphology-based phylogram which supports the recognition of four subsections, i.e. Somalienses (one species, northern Somalia), Anemone (three species, Mediterranean area), Biflorae (five species, South-West and Central Asia) and Carolinianae (seven species, North and South America). These data allow a discussion of the phylogenetic diversification and stepwise expansion of the section since the late Miocene (c. 9 Mya). Partly by long distance dispersion, section Anemone has developed from a palaeo-Mediterranean ancestor to its present transcontinental distribution. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 312–354.

Genetic variation in 42 populations throughout the range of Biscutella laevigata L. (Brassicaceae), a morphologically variable central European species, has been investigated by enzyme electrophoresis with three loci (Amy1, Amy2, and Gpi2). Genetic identities and the Fitch-Margoliash tree suggest differentiation into four regional groups: 1) a northwestern diploid group (northern France and northern Germany), 2) a northeastern diploid group (southern Germany, Upper Austria, northern Lower Austria, Poland, and Romania), 3) a central diploid group in southern Lower Austria corresponding to subspecies austriaca, and 4) a southern tetraploid group in Alpine areas of France, Germany, Switzerland, Austria, Italy, and Slovenia corresponding to subspecies laevigata. Geographically isolated diploid relic populations that are genetically depauperate are found in the NW and NE diploid groups. On the other hand, the diploid relic subspecies austriaca from the NE Prealps and Alps is highly variable. Subspecies laevigata appears to be a genetical autotetraploid with multiple origins involving several diploid progenitors (the NW diploids, subspecies austriaca and B. prealpina).

SUMMARYA program system for microcomputer assisted karyotype analysis of banded chromosomes is presented. Measurement is done with a digitizer pad (graphics tablett), the quantification and statistical calculation is processed by a computer program with a microcomputer DEC PDP11/23. The significant karyotype parameters can be calculated and plotted in an idiogram: length of the chromosome arms, centromeric index, position of secondary constriction, position and size of bands. The program system is exemplified in C-banded Hyacinthoides italica s.l. (Hyacinthaceae), which shows a rather complex banding pattern.

Since standard susceptibility tests reflect the physiological rather than the pathological conditions prevailing within an infected abdomen, as recently documented, the effect of reduced pH and pO2 and increased pCO2 on the activity of antibiotics in vitro was studied. MICs were determined in vitro under standard culture conditions (MICstandard) and modified conditions (MICmodification) simulating the previously determined pathological values. Various classes of antibiotics were affected differently by the modified conditions. However, within an antibiotic class similar results were obtained for gram-negative and gram-positive pathogens. Median MICmodification/MICstandard ratios were 4 for aminoglycosides, 2 for quinolones and clindamycin, 1 for cephalosporins, and 0.5 for penicillins and vancomycin. Anaerobic conditions and a pH of 6.4 further increased the ratio of aminoglycosides to 8. Ratios were similar within an antibiotic class at inocula of 105 or 107 cfu/ml. All MICs determined in tests with imipenem against gram-negative and gram-positive bacteria and with vancomycin against gram-positive organisms were below the susceptibility breakpoint, whatever conditions and inocula were employed. In contrast, the percentage of MICs in susceptibility range using high inocula and modified conditions decreased to 78 % for penicillins, 73 % for cephalosporins, 22 % for aminoglycosides, 11 % for quinolones and 0 % for clindamycin. In conclusion, routine susceptibility testing may overestimate the activity of aminoglycosides and underestimate the activity of beta-lactams under the conditions prevailing during abdominal infection.

It is attractive to use vitamin B₁₂ as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B₁₂ by fast proliferating cells. The basic {B₁₂-CN-Pt(II)} conjugates are recognized by intracellular enzymes and converted to coenzyme B₁₂ in an enzymatic adenosylation assay. The reductive adenosylation of {B₁₂-CN-Pt(II)} conjugates leads to the release of the Pt(II) complexes; thus, {B₁₂-CN-Pt(II)} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin-B₁₂ conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B₁₂-CN-Pt(II)} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding Pt(II) species. Kurnakov tests and coordination of 2'-deoxyguanosine or GMP to the released Pt(II) complexes allowed isolation and characterization of Pt(II) complexes as released during enzymatic adenosylation. The biological activity of these Pt(II) complexes was evaluated. Since the cleaved Pt(II) complexes show cytotoxicity, the {B₁₂-CN-Pt(II)} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC(50) between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B₁₂-CN-Pt(II)} concentration, comparison with pure cisplatin is of limited use. We could show that the Pt(II) complexes cleaved from B₁₂ exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B₁₂ free media showed a dependence on the addition of transcobalamin II for B₁₂-Pt(II) conjugates.

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity.

We report the phylogeographic pattern of the Patagonian and Subantarctic plant Hypochaeris incana endemic to southeastern South America. We applied amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) analysis to 28 and 32 populations, respectively, throughout its distributional range and assessed ploidy levels using flow cytometry. While cpDNA data suggest repeated or simultaneous parallel colonization of Patagonia and Tierra del Fuego by several haplotypes and/or hybridization, AFLPs reveal three clusters corresponding to geographic regions. The central and northern Patagonian clusters (approximately 38-51 degrees S), which are closer to the outgroup, contain mainly tetraploid, isolated and highly differentiated populations with low genetic diversity. To the contrary, the southern Patagonian and Fuegian cluster (approximately 51-55 degrees S) contains mainly diploid populations with high genetic diversity and connected by high levels of gene flow. The data suggest that H. incana originated at the diploid level in central or northern Patagonia, from where it migrated south. All three areas, northern, central and southern, have similar levels of rare and private AFLP bands, suggesting that all three served as refugia for H. incana during glacial times. In southern Patagonia and Tierra del Fuego, the species seems to have expanded its populational system in postglacial times, when the climate became warmer and more humid. In central and northern Patagonia, the populations seem to have become restricted to favourable sites with increasing temperature and decreasing moisture and there was a parallel replacement of diploids by tetraploids in local populations.