A worldwide perspective on large carnivore attacks on humans.

Large carnivores have long fascinated human societies and have profound influences on ecosystems. However, their conservation represents one of the greatest challenges of our time, particularly where attacks on humans occur. Where human recreational and/or livelihood activities overlap with large carnivore ranges, conflicts can become particularly serious. Two different scenarios are responsible for such overlap: In some regions of the world, increasing human populations lead to extended encroachment into large carnivore ranges, which are subject to increasing contraction, fragmentation, and degradation. In other regions, human and large carnivore populations are expanding, thus exacerbating conflicts, especially in those areas where these species were extirpated and are now returning. We thus face the problem of learning how to live with species that can pose serious threats to humans. We collected a total of 5,440 large carnivore (Felidae, Canidae, and Ursidae; 12 species) attacks worldwide between 1950 and 2019. The number of reported attacks increased over time, especially in lower-income countries. Most attacks (68%) resulted in human injuries, whereas 32% were fatal. Although attack scenarios varied greatly within and among species, as well as in different areas of the world, factors triggering large carnivore attacks on humans largely depend on the socioeconomic context, with people being at risk mainly during recreational activities in high-income countries and during livelihood activities in low-income countries. The specific combination of local socioeconomic and ecological factors is thus a risky mix triggering large carnivore attacks on humans, whose circumstances and frequencies cannot only be ascribed to the animal species. This also implies that effective measures to reduce large carnivore attacks must also consider the diverse local ecological and social contexts.

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.

Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans.

This study describes two novel regulators, GalX and GalR, that control d-galactose utilization in Aspergillus nidulans. This system is unique for A. nidulans since no GalR homologs were found in other ascomycetes. GalR shares significant sequence identity with the arabinanolytic and xylanolytic regulators AraR and XlnR, but GalX is more distantly related.

Regulation of pentose utilisation by AraR, but not XlnR, differs in Aspergillus nidulans and Aspergillus niger.

Filamentous fungi are important producers of plant polysaccharide degrading enzymes that are used in many industrial applications. These enzymes are produced by the fungus to liberate monomeric sugars that are used as carbon source. Two of the main components of plant polysaccharides are L-arabinose and D-xylose, which are metabolized through the pentose catabolic pathway (PCP) in these fungi. In Aspergillus niger, the regulation of pentose release from polysaccharides and the PCP involves the transcriptional activators AraR and XlnR, which are also present in other Aspergilli such as Aspergillus nidulans. The comparative analysis revealed that the regulation of the PCP by AraR differs in A. nidulans and A. niger, whereas the regulation of the PCP by XlnR was similar in both species. This was demonstrated by the growth differences on L-arabinose between disruptant strains for araR and xlnR in A. nidulans and A. niger. In addition, the expression profiles of genes encoding L-arabinose reductase (larA), L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA) differed in these strains. This data suggests evolutionary changes in these two species that affect pentose utilisation. This study also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.

Corrigendum to "Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate" [Animal Nutrition 6/1 (2020) 24-30].

[This corrects the article DOI: 10.1016/j.aninu.2019.11.003.].

HacA-dependent transcriptional switch releases hacA mRNA from a translational block upon endoplasmic reticulum stress.

Activation of the unfolded protein response (UPR) in eukaryotes involves the splicing of an unconventional intron from the mRNA encoding the transcriptional activator of the pathway. In Saccharomyces cerevisiae a 252-nucleotide (nt) unconventional intron is spliced out of the transcript of HAC1, changing the 3' end of the HAC1 open reading frame and relieving the transcript from a translational block in a single step. The translational block is caused by the base pairing of part of the unconventional intron with the 5'-untranslated region (5'UTR). In Aspergillus niger and other aspergilli, the unconventional intron in hacA mRNA is only 20 nt long. Since this intron is part of a stable stem-loop structure, base pairing with the 5'UTR, in contrast to the case with yeast HAC1, is not possible. However, analysis of the hacA mRNA revealed a GC-rich inverted repeat (18 base pairings). Upon the activation of the UPR, the 5'UTR of hacA mRNA is truncated by 230 nt, removing the left part of this inverted repeat. This implies a similar release of a translational block as in the case of S. cerevisiae HAC1 but in two steps. The mechanism behind the 5' truncation, which does not take place in either yeast HAC1 or mammalian xbp1 mRNA, has been hitherto unknown. Here we show that during secretion stress in A. niger, hacA transcription starts from a new start site closer to the ATG, relieving the transcript from translational attenuation. This transcriptional switch is mediated by HacA itself and the unfolded protein response element 2 (UPRE2) in the hacA promoter.

Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer.

BACKGROUND: Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. RESULTS: We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. CONCLUSIONS: This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate.

The utility of a next generation biosynthetic bacterial 6-phytase (PhyG) in restoring bone ash, bone phosphorus (P) content and performance in piglets depleted in P was evaluated. A total of 9 treatments were tested as follows. Treatment 1, a negative control (NC) diet; treatments 2, 3, 4, NC supplemented with 250, 500 or 1,000 FTU/kg of PhyG; treatments 5, 6, NC supplemented with 500 or 1,000 FTU/kg of a commercial Buttiauxella sp phytase (PhyB); treatments 7, 8, 9, NC supplemented with monocalcium phosphate (MCP) to provide 0.7, 1.4 and 1.8 g/kg digestible P, equating to a digestible P content of 1.8, 2.5 and 2.9 g/kg. The latter constituting the positive control (PC) diet with adequate P and calcium (Ca). The NC was formulated without inorganic P (1.1 g digestible P/kg) and reduced in Ca (5.0 g/kg). Additional limestone was added to treatments 7 to 9 to maintain Ca-to-P ratio between 1.2 and 1.3. A total of 162 crossed Pietrain × (Large White × Landrace) 21-d-old piglets (50% males and 50% females) were fed adaptation diets until 42 d old and then assigned to pens with 2 pigs/pen and 9 pens/treatment in a completely randomized block design. Piglets were fed mash diets based on corn and soybean meal ad libitum for 28 d. At the end of the study, one piglet perpen was euthanized and the right feet collected for determination of bone strength, bone ash and mineral content. Compared with the PC, the NC group had reduced average daily gain (ADG) and increased feed conversion ratio (FCR) during all growth phases and overall, and at d 28 (70 d old) NC pigs had bones with reduced ash, Ca and P content (P < 0.05). The PhyG at 250 FTU/kg improved bone ash vs. NC. Increasing PhyG dose linearly or quadratically improved bone ash, ADG and FCR (P < 0.05). At ≥ 500 FTU/kg, both PhyG and PhyB maintained ADG and FCR equivalent to PC. Linear regression analysis was done to compare the measured response parameters to increasing digestible P from MCP. Based on this analysis it was shown that PhyG and PhyB at 1,000 FTU/kg could replace 1.83 and 1.66 g/kg digestible P from MCP in the diet, respectively, on average across metacarpi bone ash, ADG or FCR. These findings suggest that the biosynthetic phytase is highly effective in the tested dietary setting.

Roles of the Aspergillus nidulans homologues of Tup1 and Ssn6 in chromatin structure and cell viability.

For three different carbon catabolite repressible promoters, alcA, alcR and the bidirectional promoter prnD-prnB, a deletion of rcoA, the Aspergillus nidulans homologue of TUP1, does not result in carbon catabolite derepression. Surprisingly, it results in disruption of the chromatin default structure of alcR and prnD-prnB promoters. In these promoters, and at variance with the wild type, repression occurs in the absence of nucleosome positioning. For alcR, repression occurs together with a nucleosome pattern identical to that found under conditions of full expression, and for prnD-prnB it occurs with a novel pattern that does not correspond to the pattern seen under conditions of repression in a wild-type strain. Deletion of the putative RcoA partner, SsnF, is lethal in A. nidulans.

I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.

We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site.

Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed ß-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

Identifying ecological corridors for Amur tigers (Panthera tigris altaica) and Amur leopards (Panthera pardus orientalis).

The rapid explosion of human populations and the associated development of human-dominated landscapes have drastically reduced and fragmented habitat for tigers (Panthera tigris) and leopards (Panthera pardus) across Asia, resulting in multiple small populations. However, Amur tiger (Panthera tigris altaica) habitat in Russia has remained largely interconnected, except for a break between tigers in southwest Primorye and the southern Sikhote-Alin Mountains. This habitat patch in southwest Primorye also retains the last population of Amur leopards (Panthera pardus orientalis). Genetic differentiation of tigers in southwest Primorye and the Sikhote-Alin Mountains along with survey data suggest that habitat fragmentation is limiting movement of tigers and leopards across the Razdolnaya River basin. We looked at historical and recent survey data on tigers and leopards and mapped existing cover types to examine land-use patterns of both large felids and humans in the development strip along the Razdolnaya River. We then used least-cost distance analyses to identify the most effective potential corridor to retain connectivity for large felids between Land of the Leopard National Park and Ussuriskii Zapovednik (Reserve). We identified a single potential corridor that still exists with a total distance of 62.5 km from Land of the Leopard National Park to Ussuriskii Zapovednik, mostly (93%) through forested habitat. We recommend formal recognition of a Razdolnaya ecological corridor and provide specific recommendations for each of 3 proposed management sections.

Indications and results of sleeve carinal resection.

OBJECTIVE: Carinal resection is the most complicated procedure in tracheobronchial surgery. The main aspects of the technique are still debated at the present time. We present our experience of 231 carinal resections with analysis of operative techniques, complications and long-term survival. METHODS: Since 1979 we have performed 231 carinal resections. Indications for surgery included lung cancer in 151 cases (65.4%), non-bronchogenic carcinoma in 45 (19.4%), main bronchus fistula with short stump in 25 (10.8%), stenosis of tuberculous and nonspecific etiology in nine (4%), and trauma in one case (0.4%). We have performed 156 right-sided resections (67.5%) and 75 left-sided (32.5%). In 162 cases carinal pneumonectomy was undertaken, carinal resection following pneumonectomy was performed in 28 cases, isolated resection of bronchial bifurcation was performed in 25 cases, and in 15 cases we combined lobectomy and resection of bifurcation. The length of resection extended from one to nine tracheal rings. The operative approach was lateral thoracotomy in 102 cases (44.2%), and sternotomy in 129 (55.8%). RESULTS: Thirty-seven patients died postoperatively (16%). Complications were observed in 82 patients (35.4%), dominated by anastomotic problems which occurred in 58 cases (25.1%). The most frequent causes of death were respiratory distress syndrome and anastomotic dehiscence (P < 0.05). Mortality and the incidence of complications were significantly correlated to length of resection, laryngeal nerves injury, and mode of intraoperative ventilation. CONCLUSIONS: The feasibility of carinal resection is limited by the patient's functional status and extension of tumor growth. Thorough selection of patients may improve immediate and long-term results.

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production.

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated by UV mutagenesis in an industrial strain improvement program. Using enzyme assays, gene expression, sequencing of the ladA locus in the parent and mutant, and complementation of the mutation, we were able to show that improved xylanase production was mainly caused by a chromosomal translocation that occurred between a subtilisin-like protease pepD gene and the L-arabitol dehydrogenase encoding gene (ladA), which is part of the L-arabinose catabolic pathway. This genomic rearrangement resulted in disruption of both genes and, as a consequence, the inability of the mutant to use L-arabinose as a carbon source, while growth on D-xylose was unaffected. Complementation with constitutively expressed ladA confirmed that the xylanase overproducing phenotype was mainly caused by loss of ladA function, while a knockout of xlnR in the UV mutant demonstrated that improved xylanase production was mediated by XlnR. This study demonstrates the potential of metabolic manipulation for increased production of fungal enzymes.

HACA, the transcriptional activator of the unfolded protein response (UPR) in Aspergillus niger, binds to partly palindromic UPR elements of the consensus sequence 5'-CAN(G/A)NTGT/GCCT-3'.

The promoters of UPR target genes contain an unfolded protein response element (UPRE), which confers the stress inducibility to the gene, via an interaction with the transcription activator HACA. In the promoters of the Aspergillus ER-stress responsive genes bipA, cypB, pdiA, prpA, tigA, and hacA, a consensus sequence was identified, which was located close to the transcription start site of the gene (<81 bp), and corresponds to the sequence CAN(G/A)NTGT/GCCT. The UPRE is a partly palindromic sequence around a dispensable spacer nucleotide, followed by four highly conserved bases. By an in vitro selection procedure, an optimal binding site for HACA was isolated. This sequence, ACACGTGTCCT, resembles the UPRE but lacks the spacer nucleotide. It has a much higher binding affinity than the identified UPREs, and in vivo it behaves as a more powerful cis-acting element.

Patterns of nucleosomal organization in the alc regulon of Aspergillus nidulans: roles of the AlcR transcriptional activator and the CreA global repressor.

We have studied the chromatin organization of three promoters of the alc regulon of Aspergillus nidulans. No positioned nucleosomes are seen in the aldA (aldehyde dehydrogenase) promoter under any physiological condition tested by us. In the alcA (alcohol dehydrogenase I) and alcR (coding for the pathway-specific transcription factor) promoters, a pattern of positioned nucleosomes is seen under non-induced and non-induced repressed conditions. While each of these promoters shows a specific pattern of chromatin restructuring, in both cases induction results in loss of nucleosome positioning. Glucose repression in the presence of inducer results in a specific pattern of partial positioning in the alcA and alcR promoters. Loss of nucleosome positioning depends absolutely on the AlcR protein and it is very unlikely to be a passive result of the induction of transcription. In an alcR loss-of-function background and in strains carrying mutations of the respective AlcR binding sites of the alcA and alcR promoters, nucleosomes are fully positioned under all growth conditions. Analysis of mutant AlcR proteins establishes that all domains needed for transcriptional activation and chromatin restructuring are included within the first 241 residues. The results suggest a two-step process, one step resulting in chromatin restructuring, a second one in transcriptional activation. Partial positioning upon glucose repression shows a specific pattern that depends on the CreA global repressor. An alcR loss-of-function mutation is epistatic to a creA loss-of-function mutation, showing that AlcR does not act by negating a nucleosome positioning activity of CreA.

Nuclear import of zinc binuclear cluster proteins proceeds through multiple, overlapping transport pathways.

In Aspergillus nidulans, the high transcriptional level of the ethanol utilization pathway genes (alc) is regulated by the specific activator AlcR. Here we have analyzed the mechanism of the nuclear import of AlcR, as well as that of other proteins belonging to the Zn(2)Cys(6) binuclear cluster family. The nuclear localization signal of AlcR maps within the N-terminal 75 amino acid residues and overlaps with its DNA-binding domain. It consists of five clusters rich in basic residues. Four of them are necessary and sufficient for nuclear targeting. The first two basic regions are crucial for both nuclear localization and recognition of AlcR-specific DNA targets. This nuclear localization signal (NLS) motif is recognized by the nuclear transport machinery of Saccharomyces cerevisiae and requires both Ran/Gsp1p activity and specific transport receptors. AlcR can be imported into nuclei via multiple transport pathways mediated by a distinct set of karyopherins composed of Kap104p, Sxm1p, and Nmd5p transport receptors. The two former karyopherins interact with the NLS of AlcR directly. Other Zn binuclear cluster proteins from S. cerevisiae, such as Gal4p and Pdr3p, also appear to be transported to the nuclei in a nonclassical, importin-alpha-independent manner and can share common importin beta receptors.