Scanning the solutions for the sustainable supply of forest ecosystem services in Europe.

Forests are key components of European multifunctional landscapes and supply numerous forest ecosystem services (FES) fundamental to human well-being. The sustainable provision of FES has the potential to provide responses to major societal challenges, such as climate change, biodiversity loss, or rural development. To identify suitable strategies for the future sustenance of FES, we performed a solution scanning exercise with a group of transdisciplinary forest and FES experts from different European regions. We identified and prioritized fifteen major challenges hindering the balanced provision of multiple FES and identified a series of potential solutions to tackle each of them. The most prominent challenges referred to the increased frequency and impacts of extreme weather events and the normative mindset regarding forest management. The respective solutions pointed to the promotion of forest resilience via climate-smart forestry and mainstreaming FES-oriented management through a threefold strategy focusing on education, awareness raising, and networking. In a subsequent survey, most solutions were assessed as highly effective, transferable, monitorable, and with potential for being economically efficient. The implementation of the solutions could have synergistic effects when applying the notion of leverage points. Seven emerging pathways towards the sustainable supply of FES have been identified. These pathways build on each other and are organized based on their potential for transformation: (1) shifting forest management paradigms towards pluralistic ecosystem valuation; (2) using integrated landscape approaches; (3) increasing forest resilience; (4) coordinating actions between forest-related actors; (5) increasing participation in forest planning and management; (6) continuous, open, and transparent knowledge integration; and (7) using incentive-based instruments to support regulating and cultural FES. These pathways can contribute to the implementation of the new EU Forestry Strategy to support the balanced supply of multiple FES. Supplementary Information: The online version contains supplementary material available at 10.1007/s11625-022-01111-4.

Metastasizing Esthesioneuroblastoma in a Dog.

A 7-year-old Afghan hound presented with a history of disorientation, loss of vision, and seizures. Magnetic resonance imaging helped identify a mass at the level of the main olfactory bulb that compressed and displaced adjacent tissues in the cribriform plate into the nasal cavity and nasopharynx. Bony structures were osteolytic. After removing almost 80% of the mass, the tumor recurred a few months later. Due to severe respiratory distress and subsequent to an ultrasound diagnosis of a liver tumor, the dog was euthanized. In addition to the nasal mass, a single nodule in the liver and multiple nodules in the lung were present. All masses had similar cell morphology and were diagnosed as metastasizing esthesioneuroblastoma. The neoplastic cells expressed neuron-specific enolase and chromogranin A, and a few cells within the nasal mass were positive for cytokeratin. This is the first description of a canine esthesioneuroblastoma with distant metastases.

Predictors for the development of temporomandibular disorders in scuba divers.

The aim was to determine predictors for the development of complaints of temporomandibular disorders (TMD) in a large sample of Dutch scuba divers who were free of any TMD complaints before they started diving actively. Five-hundred and thirty-six scuba divers (mean ± SD age = 40.4 ± 11.9 years; 34.1% women) completed a specifically developed questionnaire, either online or on paper. Stepwise forward logistic regression analysis was performed to predict the presence of TMD pain, with several potential risk factors as predictors. Four hundred and eighty-five of the 536 respondents were free of any TMD pain before they started diving actively. In this sample, TMD pain was present in 214 persons (44.1%). Four predictors contributed significantly to the presence of TMD pain, viz., clenching (OR = 2.466), warm water (OR = 1.685), biting on the mouthpiece (OR = 1.598), and the quality rating of the mouthpiece (OR = 0.887, that is, a higher rating means a smaller odds of having TMD pain). TMD pain is a common complaint among scuba divers who were free of such complaints before they started diving actively. Clenching, biting on the mouthpiece, and a low rating of the mouthpiece are predictors for the presence of TMD pain in scuba divers, while diving in cold water serves as a protective factor for TMD pain.

Genetic variations in polyunsaturated fatty acid metabolism--implications for child health?

Sufficient nutritional supply with polyunsaturated fatty acids (PUFAs) has long been considered as beneficial for child health, especially in regard to neuronal development and allergic diseases. In recent years, genetic association studies showed that in addition to nutritional influences, the genetic background is highly important for PUFA composition in human tissues. Specifically, polymorphisms in the fatty acid desaturase genes or FADS determine the efficiency of how PUFAs are processed endogenously. Recent gene-nutrition interaction studies suggest that these polymorphisms modulate the effect of nutritional fatty acid intake on complex phenotypes such as cognitive outcomes and asthma risk in children. These early results may provide the basis for future well-specified dietary recommendations to achieve optimal health benefit for all children. This article presents results from recent gene-nutrition interaction studies, discusses its implications for child health, and gives an outlook how this association might translate into clinical practice in the future.

piggyBac-mediated germline transformation in the beetle Tribolium castaneum.

The lepidopteran transposable element piggyBac can mediate germline insertions in at least four insect orders. It therefore shows promise as a broad-spectrum transformation vector, but applications such as enhancer trapping and transposon-tag mutagenesis are still lacking. We created, cloned, sequenced and genetically mapped a set of piggyBac insertions in the red flour beetle, Tribolium castaneum. Transpositions were precise, and specifically targeted the canonical TTAA recognition sequence. We detected several novel reporter-expression domains, indicating that piggyBac could be used to identify enhancer regions. We also demonstrated that a primary insertion of a non-autonomous element can be efficiently remobilized to non-homologous chromosomes by injection of an immobile helper element into embryos harbouring the primary insertion. These developments suggest potential for more sophisticated methods of piggyBac-mediated genome manipulation.

Analysis of FA contents in individual lipid fractions from human placental tissue.

Data on FA contents in the human placenta are limited. Different methods have been used for the FA analysis, and only percentage results have been presented. We developed and evaluated a method for the determination of FA concentrations in placental tissue. Lipids were extracted from placental tissue with a chloroform/methanol mixture; and phospholipids (PL), nonesterified FA (NEFA), TG, and cholesterol esters (CE) were isolated by TLC. Individual lipid fractions were derivatized with methanolic hydrochloric acid, and the FAME were quantified by GC with FID. The CV of intra-assay (n = 8) of absolute concentrations were evaluated for FA showing a tissue content > 0.01 mg/g. CV ranges were 4.6-11.0% for PL, 6.4-9.3% for NEFA, 6.1-8.9% for TG, and 11.4-16.3% for CE. The relative FA composition across a term placenta indicated no differences between samples of central and peripheral locations of maternal and fetal site (CV 0.5-9.9%), whereas the absolute FA concentrations were only reproducible in the PL fraction (CV 7.0-12.8%). The method shows a reasonably high precision that is well suited for physiological and nutritional studies.

Expression of dachshund in wild-type and Distal-less mutant Tribolium corroborates serial homologies in insect appendages.

We isolated the homologue of the Drosophila gene dachshund (dac) from the beetle Tribolium castaneum. Tc'dac is expressed in all appendages except urogomphi and pleuropodia. Tc'dac is also active in the head lobes, in the ventral nervous system, in the primordia of the Malpighian tubules and in bilateral stripes corresponding to the presumptive dorsal midline. Expression of Tc'dac in the labrum lends support to the interpretation that the insect labrum is derived from a metameric appendage. The legs of Tribolium accommodate two Tc'dac domains, of which the more distal one corresponds to the single dac domain described for Drosophila leg discs. In contrast to Drosophila, where this domain is thought to intercalate between the homothorax (hth) and the Distal-less (Dll) domains, in Tribolium it arises from within the Dll domain. In embryos mutant for the Tc'Dll gene we find that the distal Tc'dac domain in the legs, as well as the expression in the labrum, are deleted while the proximal leg domain and the mandibular expression are unaffected. Based on Tc'dac expression in wild-type and mutant embryos, we demonstrate serial homology of the complete mandible with the coxa of the thoracic legs, which affirms the gnathobasic nature of the insect mandible.

The influence of airway pressure changes on intracranial pressure (ICP) and the blood flow velocity in the middle cerebral artery (VMCA).

OBJECTIVE: Due to the exponential shape of the intracranial volume-pressure relation, simple measurement of epidural, parenchymal or intraventricular intracranial pressure (ICP) in traumatic brain injury (TBI) often fails to early recognize patients with a fulminant development of intracranial hypertension even during recently available methods of tissue PO2 and microdialysis measurements. One approach to this problem could be repetitive intracranial volume provocations to evaluate a trend of the intracranial elastance. Several previously published methods use invasive volume challenge through access to the cerebrospinal fluid (CSF). This pilot study describes changes in intracranial pressure due to variations of airway pressure with BIPAP ventilation maneuvers. PATIENTS AND METHODS: Ten patients with severe TBI were enrolled and completed the study. The inclusion was based on radiologic signs due to TBI in the first CT-scan and the clinical indication for insertion of an ICP monitoring device. Patients with elevated ICP above 20 mm Hg were excluded. The epidural ICP response together with haemodynamic parameters in relation to defined airway pressure changes (delta PAW) was detected. The influence of the duration of delta PAW was evaluated additionally. Data of central venous pressure (CVP), ICP, mean arterial pressure (MAP), cerebral perfusion pressure (CPP), airway pressure (PAW) and blood flow velocity of the middle cerebral artery (VMCA) were analyzed on the basis of differences between the maximum (inspiration) and minimum PAW values (expiration). RESULTS: Elevations of PAW in the range of 20 to 35 cm H2O resulted in changes of the ICPmean from 4.1 to 6.0 mm Hg (r = 0.9, p < 0.05). A correlation was estimated for the changes of systolic arterial pressure (Part) and CPPmean due to PAW variations which ranged between 4.5 and 11.6 mm Hg (r = 0.99, p < 0.05). Concerning the transcranial doppler measurements the data of changes of the blood flow velocity of the middle cerebral artery (VMCA) revealed a positive correlation to PAW with a r = 0.99, p < 0.05. CONCLUSIONS: Elevation of the venous outflow resistance and a transient increase in cardiac output have to be considered as mechanisms for transduction of transthoracic pressure changes to intracranial pressure variations. We conclude, that trends of changes in elastance can be derived from intermittent airway pressure variations. This can be useful in easy and on line dynamic monitoring of ICP in traumatic brain injury.

Quantitative analysis of gene function in the Drosophila embryo.

The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells.

The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.

A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum.

Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species.

Structure and evolution of a pair-rule interaction element: runt regulatory sequences in D. melanogaster and D. virilis.

Pair-rule genes serve two important functions during Drosophila development: they first initiate periodic patterns, and subsequently interact with each other to refine these patterns to the precision required for definition of segmental compartments. Previously, we described a pair-rule input region of the runt gene. Here we further characterize this region through the use of reporter gene constructs and by comparison with corresponding sequences from Drosophila virilis. We find that many but not all regulatory properties of this '7-stripe region' are functionally conserved. Moreover, the similarity between these homologous sequences is surprisingly low. When compared to similar data for gap gene input element, our data suggest that pair-rule target sequences are less constrained during evolution, and that functional elements mediating pair-rule interactions can be dispersed over many kilobases.

Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum.

Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes.

Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo.

In short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how an anterior gradient like that of bicoid in Drosophila could in these embryos be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homologue from any species outside the diptera, it has been speculated that the anterior bicoid gradient could be a late addition during insect evolution. We addressed this question by analysing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. We demonstrate that homologues of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal caudal RNA is translationally repressed by bicoid. We find that Tribolium caudal RNA is also translationally repressed by bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homologue must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, we show that caudal activates the posterior of these domains via a specific promoter. Our experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by caudal to direct activation by bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggest how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal.

Disperse versus compact elements for the regulation of runt stripes in Drosophila.

The segmented body pattern of the Drosophila embryo is established through a hierarchical network of interacting genes. At each successive step in this pathway, transcriptional regulation is used to convert coarse positional information into finer patterns of gene expression. Central to this process are the cis-regulatory regions that drive the dynamic spatial expression of the different segmentation genes. Here we describe the cis-regulatory region of the runt gene. As found for both other primary pair-rule genes, hairy and even-skipped, there are stripe-specific elements which mediate the initial regulation of runt stripes by gap genes. We did not find autoregulatory elements as described for even-skipped and fushi tarazu. The regulation of runt by other pair-rule genes is mediated by a large region, extending over 5 kb upstream and downstream of the transcription start site. This "disperse" element cannot be subdivided into functionally independent subelements or minimal elements. Such disperse elements mediating pair-rule gene interactions may have escaped detection in other segmentation genes and may involve molecular mechanisms different from those mediating regulation by gap genes.

Regulation of runt transcription by Drosophila segmentation genes.

The runt gene plays an important role in the genetic hierarchy that generates the segmented body pattern during the early stages of Drosophila embryogenesis. We studied mRNA expression in mutant embryos in order to investigate the regulation of runt transcription during these stages. We used sensitive whole-mount in situ hybridization procedures to identify the earliest, and therefore most likely direct regulatory effects. There are several distinct phases of runt expression in the early embryo. We find that each phase depends on a different set of regulators. The first phase of expression is a broad-field of mRNA accumulation in the central regions of syncytial blastoderm stage embryos. This pattern is due to terminal repression by the anterior and terminal maternal systems. The effect of the terminal system, even at this early stage, is mediated by two zygotic gap genes, tailless and huckebein. A 7 stripe pattern of runt mRNA accumulation emerges during the process of cellularization. The initial formation of this pattern depends on position-specific repression by zygotic gap genes. Examination of the early RNA patterns of the pair-rule genes even-skipped, hairy, and fushi tarazu indicate that they are also regulated in a similar manner. Three pair-rule genes, hairy, even-skipped, and runt itself, also affect runt's 7 stripe pattern. The effects of runt are stripe specific; the effects of hairy are more uniform; and the patterns obtained in even-skipped mutant embryos show a combination of both stripe specific and uniform regulatory effects. A third distinct phase of expression occurs at the onset of gastrulation when runt becomes expressed in 14 stripes. fushi tarazu plays a negative regulatory role in generating this pattern, whereas the pair-rule genes paired and odd-paired are required for activating or maintaining runt expression during these stages.

Long-term low-dose cyclosporin A in steroid dependent nephrotic syndrome of childhood.

Therapy of steroid-dependent idiopathic nephrotic syndrome is often unsatisfactory. Since 1986 we have treated nine children (six male and three female), aged 3-16 years, with cyclosporin A (CsA) during 2.0-5.2 (median 3.1) years. All had minimal change disease on renal biopsy and had previously received cyclophosphamide. Mean daily dosage of CsA was 4.1 mg/kg (range 2.7-5.8) and mean whole blood trough level was 220 ng/ml (range 141-271). The relapse rate decreased from 3.4/patient year before CsA to 0.55 on CsA. Discontinuation of CsA or reduction below 2 mg/kg daily was always followed by a relapse. The overall relapse rate, including the period with very low-dose CsA, was 0.95/patient year. Four patients required additional low-dose alternate-day prednisone. Repeat renal biopsy showed minimal change disease in eight patients and focal segmental glomerulosclerosis in one; CsA-toxicity was mild in two and moderate in one. The latter was the only patient with slightly reduced glomerular filtration rate. Two boys with delayed puberty spontaneously matured and reached expected final height. We conclude that long-term low-dose CsA is very effective and steroid-sparing. Its use is justified in selected patients, particularly in those with numerous relapses and in male patients before and during puberty, as long as renal function and CsA-toxicity are carefully monitored.

Localized requirement for torso-like expression in follicle cells for development of terminal anlagen of the Drosophila embryo.

The torso (tor) gene, one of six identified maternal genes essential for the development of the anterior and posterior terminal structures in the Drosophila embryo, is likely to function as a transmembrane receptor tyrosine kinase. Although tor protein is uniformly distributed in the membrane of the egg cell and syncytial embryo, genetic and molecular data suggest that tor is locally activated at the ends of the embryo by a ligand present in the perivitelline space. Local activation of tor could be achieved if the ligand were expressed by a subpopulation of the follicle cells that surround the developing oocyte. Here we describe torso-like (tsl), the sixth member of the terminal gene class, and show that it is unique among these genes in that its expression is required in the somatic follicle cells rather than in the germ line. Moreover, mosaic analysis demonstrates that tsl expression is necessary only in subpopulations of follicle cells located at the poles of the oocyte. Thus, the spatially regulated expression of tsl in the follicle cell layer may generate a localized signal that is transduced by tor, ultimately resulting in the formation of the terminal structures of the embryo.