A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein.

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

The avidin-biotin interaction is a robust protein-ligand interaction utilized for several applications. We have developed a miniaturized assay protocol using the small molecule dye HABA to quantify the available biotin binding sites in avidin-containing fusion proteins. This method also serves as a quality control assay for different batches of assembled avidin-biotin fusion proteins, reducing technical complexities and time required for batch release.

Methodological assessment for efficient collection of Schistosoma mansoni environmental DNA and improved schistosomiasis surveillance in tropical wetlands.

Schistosomiasis, caused by trematodes of genus Schistosoma, is among the most seriously neglected tropical diseases. Although rapid surveillance of risk areas for Schistosoma transmission is vital to control schistosomiasis, the habitat and infection status of this parasite are difficult to assess. Environmental DNA (eDNA) analysis, involving the detection of extra-organismal DNA in water samples, facilitates cost-efficient and sensitive biomonitoring of aquatic environments and is a promising tool to identify Schistosoma habitat and infection risk areas. However, in tropical wetlands, highly turbid water causes filter clogging, thereby decreasing the filtration volume and increasing the risk of false negatives. Therefore, in this study, we aimed to conduct laboratory experiments and field surveys in Lake Victoria, Mbita, to determine the appropriate filter pore size for S. mansoni eDNA collection in terms of particle size and filtration volume. In the laboratory experiment, aquarium water was sequentially filtered using different pore size filters. Targeting >3 µm size fraction was found to be sufficient to capture S. mansoni eDNA particles, regardless of their life cycle stage (egg, miracidia, and cercaria). In the field surveys, GF/D (2.7 µm nominal pore size) filter yielded 2.5-times the filtration volume obtained with a smaller pore size filter and pre-filtration methods under the same time constraints. Moreover, a site-occupancy model was applied to the field detection results to estimate S. mansoni eDNA occurrence and detection probabilities and assess the number of water samples and PCR replicates necessary for efficient eDNA detection. Overall, this study reveals an effective method for S. mansoni eDNA detection in turbid water, facilitating the rapid and sensitive monitoring of its distribution and cost-effective identification of schistosomiasis transmission risk areas.

Site-occupancy factors in the Debye scattering equation. A theoretical discussion on significance and correctness.

The Debye scattering equation (DSE) [Debye (1915). Ann. Phys. 351, 809-823] is widely used for analyzing total scattering data of nanocrystalline materials in reciprocal space. In its modified form (MDSE) [Cervellino et al. (2010). J. Appl. Cryst. 43, 1543-1547], it includes contributions from uncorrelated thermal agitation terms and, for defective crystalline nanoparticles (NPs), average site-occupancy factors (s.o.f.'s). The s.o.f.'s were introduced heuristically and no theoretical demonstration was provided. This paper presents in detail such a demonstration, corrects a glitch present in the original MDSE, and discusses the s.o.f.'s physical significance. Three new MDSE expressions are given that refer to distinct defective NP ensembles characterized by: (i) vacant sites with uncorrelated constant site-occupancy probability; (ii) vacant sites with a fixed number of randomly distributed atoms; (iii) self-excluding (disordered) positional sites. For all these cases, beneficial aspects and shortcomings of introducing s.o.f.'s as free refinable parameters are demonstrated. The theoretical analysis is supported by numerical simulations performed by comparing the corrected MDSE profiles and the ones based on atomistic modeling of a large number of NPs, satisfying the structural conditions described in (i)-(iii).

Vanadium Electronic Configuration Determination From L2,3 Transition in V-oxide Compounds and Roscoelite.

We report on the electronic structure of vanadium in synthetic V-oxides and in natural roscoelite (V-rich phyllosilicate). This study applied electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM), combined with first-principle calculations, to (1) establish relationships between the V oxidation state and EELS L2,3 features and (2) better constrain the oxidation state and crystallographic siting of V in roscoelite, with implications for other V-bearing phyllosilicates. Both EELS measurements and band structure calculations show that the EELS L2/L3 ratio increases as the oxidation state of V increases. We establish a quantitative relationship between the V L2,3 near-edge structure and the V oxidation state by normalizing the L2 maximum peak intensity to the L3 peak intensity. By applying this method to roscoelite, we find that it hosts a mix of trivalent and tetravalent V distributed between the octahedral and tetrahedral sites with a V4+/ΣV = 0.6 ± 0.1. This relationship is applicable to measurements of V oxidation states in oxide and phyllosilicate minerals, which is useful for constraining the conditions of rock and mineral formation and has potential implications for metal extraction from phyllosilicate ores.

Sodium chloride impacts glycosylation and N- and O-glycan site occupancy of an Fc-fusion protein.

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.

Challenges of copro-parasitological surveys in wild Iberian ibex (Capra pyrenaica) populations addressed through a combination of molecular and statistical tools.

Copro-parasitological surveys in wildlife face challenges due to the secretive nature of many species and the unknown performance of the diagnostic tests employed. To overcome these issues, we used a combination of hierarchical models (site-occupancy and N-mixture models) applied to copro-parasitological data obtained from fecal samples assigned to the host species by molecular methods in the Iberian ibex in north-western Iberian Peninsula. The aims were to compare the performance of four diagnostic tests (Mini-FLOTAC, McMaster, Willis flotation, and natural sedimentation) and to use this methodological approach (molecular analysis with hierarchical models) to better estimate positivity proportion and shedding intensity in a wild ibex population. Pooled fecal samples were collected, and those confirmed by molecular analyses to be the host species in question were included in the study. Hierarchical models confirmed different performances of each diagnostic test, with Mini-FLOTAC showing higher sensitivity for eimeriid coccidia, Willis flotation (for proportion positive) and McMaster (for shedding intensity) in gastrointestinal Strongylida, and equal performance of MiniFlotac/Willis flotation (for proportion positive) and MiniFlotac/McMaster (for shedding intensity) in Moniezia spp. This study employed a combination of molecular and statistical methods that improved the estimates of prevalence and shedding intensity and allowed us to compare the performance of four diagnostic tests while assessing the effect of covariates. Such improvements are critical to enhancing inference in non-invasive wildlife copro-parasitological studies.

Research on Space Occupancy, Activity Rhythm and Sexual Segregation of White-Lipped Deer (Cervus albirostris) in Forest Habitats of Jiacha Gorge on Yarlung Zangbo River Basin Based on Infrared Camera Technology.

The white-lipped deer (Cervus albirostris) is a rare and endangered species found in the Qinghai-Tibet Plateau in China. To understand the space occupancy, activity rhythm, and sexual segregation of the white-lipped deer, 24,096 effective photos and 827 effective videos were captured using infrared cameras from February 2020 to January 2022. The ecology and behavior of the white-lipped deer in Jiacha Gorge were studied in more detail using site occupancy models, relative abundance index, and other technologies and methods. The results show that The occupancy predicted by the model exceeds or approaches 0.5. The occupancy increases with greater altitude and with larger EVI values, while the detection rate increases with altitude only during spring and decreases with EVI values only in summer. The daily activity peaks for white-lipped deer were observed from 7:00 to 11:00 and 17:00 to 22:00, with annual activity peaks occurring from April to June and from September to November. From July to the following January, white-lipped deer mostly move in mixed-sex groups, while during the remainder of the year, they predominantly associate with individuals of the same sex. Climate, vegetation coverage, food resources, and human disturbance collectively influenced the behavior and habitat utilization of white-lipped deer. The foundational research conducted on white-lipped deer over the past two years is expected to enhance the basic understanding of white-lipped deer in the Qinghai-Tibet Plateau and contribute to future protection and management decisions.

Site occupancy by American martens and fishers in temperate deciduous forests of Québec.

Interspecific interactions can mediate site occupancy of sympatric species and can be a key factor in habitat use patterns. American martens (Martes americana) and Fishers (Pekania pennanti) are two sympatric mesocarnivores in eastern North American forests. Due to their larger size, fishers have a competitive advantage over martens. We investigated site occupancy of martens and fishers in temperate deciduous forests of Québec, an environment modified by forest management and climate change. We formulated hypotheses on the spatial distribution of the studied species based on the knowledge of local trappers and on the scientific literature regarding forest cover composition, habitat fragmentation, and competitive relationships. We used a network of 49 camera traps monitored over two fall seasons to document site occupancy by both species. We used two-species site occupancy models to assess habitat use and the influence of fishers on martens at spatial grains of different sizes. None of the habitat variables that we considered explained site occupancy by fishers. Availability of dense old coniferous stands explained the spatial distribution of martens both at the home range grain size and at the landscape grain size. We identified the characteristics of habitat hotspots based on the knowledge of trappers, which highlighted the importance of stand composition, height, age, and canopy closure. The characteristics of habitat hotspots for martens in temperate deciduous forests refine the habitat suitability model for American martens that was originally developed for boreal forests of Québec.

Les interactions interspécifiques peuvent affecter l'occupation de sites par des espèces sympatriques et jouer un rôle clé dans leur utilisation des habitats. La martre d'Amerique (Martes americana) et le pékan (Pekania pennanti) sont deux mésocarnivores sympatriques des forêts de l'est de l'Amérique du Nord. En raison de sa grande taille, le pékan est un compétiteur dominant de la martre. Nous avons étudié l'occupation des sites par la martre et le pékan dans la forêt tempérée feuillue du Québec, un environnement modifié par l'aménagement forestier et les changements climatiques. Nous avons formulé des hypothèses sur la répartition spatiale des espèces étudiées en nous basant sur les connaissances des trappeurs locaux et sur la littérature scientifique en ce qui a trait à la composition du couvert forestier, à la fragmentation de l'habitat, et aux relations de compétition. Nous avons utilisé un réseau de 49 appareils photo à déclenchement automatique pendant deux automnes pour documenter l'occupation des sites par les deux espèces. Nous avons utilisé des modèles d'occupation de sites à deux espèces afin d'évaluer l'effet de la présence du pékan sur l'utilisation de l'habitat par la martre à des échelles spatiales de résolutions variables. Aucune des variables d'habitat que nous avons prises en compte n'explique l'occupation des sites par les pékans. La disponibilité de vieux peuplements denses de conifères explique la répartition spatiale de la martre aux échelles spatiales du domaine vital et du paysage. Nous avons développé un indice d'habitat potentiel basé sur les connaissances des trappeurs, qui a mis en évidence l'importance de la composition, de la hauteur, de l'âge et de la densité des peuplements. Cet indice affine, pour les forêts tempérées feuillues du Québec, le modèle de qualité de l'habitat de la martre d'Amérique originellement élaboré pour la forêt boréale.

A manager's guide to using eDNA metabarcoding in marine ecosystems.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes, providing more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model.

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In-depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5,608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where a ∼2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

Using machine learning to model nontraditional spatial dependence in occupancy data.

Spatial models for occupancy data are used to estimate and map the true presence of a species, which may depend on biotic and abiotic factors as well as spatial autocorrelation. Traditionally researchers have accounted for spatial autocorrelation in occupancy data by using a correlated normally distributed site-level random effect, which might be incapable of modeling nontraditional spatial dependence such as discontinuities and abrupt transitions. Machine learning approaches have the potential to model nontraditional spatial dependence, but these approaches do not account for observer errors such as false absences. By combining the flexibility of Bayesian hierarchal modeling and machine learning approaches, we present a general framework to model occupancy data that accounts for both traditional and nontraditional spatial dependence as well as false absences. We demonstrate our framework using six synthetic occupancy data sets and two real data sets. Our results demonstrate how to model both traditional and nontraditional spatial dependence in occupancy data, which enables a broader class of spatial occupancy models that can be used to improve predictive accuracy and model adequacy.

Streamlining Peptide Mapping LC-MS Approach for Studying Fusion Peptide-Conjugated Vaccine Immunogens.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MSE fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation.

Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglBC. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

Differential Analysis of N-glycopeptide Abundance and N-glycosylation Site Occupancy for Studying Protein N-glycosylation Dysregulation in Human Disease.

Protein N-glycosylation plays a vital role in diverse cellular processes, and dysregulated N-glycosylation is implicated in a variety of human diseases including neurodegenerative disorders and cancer. With recent advances in high-resolution mass spectrometry-based glycoproteomics technologies enabling large-scale N-glycoproteome profiling of disease and control samples, analysis of the large datasets has become a challenge. Here, we provide a protocol for the systems-level analysis of in vivo N-glycosylation sites on N-glycosylated proteins and their changes in human disease, such as Alzheimer's disease. The protocol includes quantitation and differential analysis of N-glycopeptide abundance, in addition to integrative N-glycoproteome and proteome data analyses, to determine disease-associated changes in N-glycosylation site occupancy and identify differentially N-glycosylated proteins in human disease versus control samples. This protocol can be modified and applied to study proteome-wide N-glycosylation alterations in response to different cellular stresses or pathophysiological states in other organisms or model systems.

Site-occupancy scheme in disordered Ca3RE2(BO3)4: a dependence on rare-earth (RE) ionic radius.

The structures of polycrystalline Ca3RE2(BO3)4 (RE = La, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Y; space group Pnma) orthoborates were determined using powder X-ray diffraction. Trends in the unit-cell dimensions and yet unreported trends in other structural properties (interatomic distances and the fractional occupation of three Ca/RE sites) for these compounds are demonstrated as a function of RE ionic radius. The unit-cell volume and a unit-cell parameter present a linear dependence, while the b and c unit-cell parameters change in a nonlinear manner. For the whole series, the RE atoms are present at all three cationic sites (labelled as M1, M2 and M3), but the fractional occupancies depend on the RE ionic radius. The small rare-earth atoms tend to enter mainly the M3 site; for the larger rare earths, the occupancy of this site decreases sharply. The occupancy of the M1 site by RE atoms is around 0.5 and tends to increase with increasing RE ionic radius. The M2 site is the least preferentially occupied by RE ions, but the occupancy discernibly increases with rising radius as well. These findings are assembled with properties of isostructural strontium and barium borates, allowing prediction of occupancy schemes for not yet investigated compounds from the A3RE2(BO3)4 (A = Ca, Ba, Sr).

Targeted Quantification of Phosphorylation Sites Identifies STRIPAK-Dependent Phosphorylation of the Hippo Pathway-Related Kinase SmKIN3.

We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation.IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip.

Elemental Sub-Lattice Occupation and Microstructural Evolution in γ/γ' Co-12Ti-4Mo-Cr Alloys.

We report on comparative atom probe tomography investigations of γ/γ'-forming Co­12Ti­4Mo­Cr alloys. Moderate additions of Cr (2 and 4 at%) reduced the γ/γ' lattice misfit and increased the γ' volume fraction of a Co­12Ti­4Mo alloy significantly. These microstructural changes were accompanied by changes in the elemental partitioning between γ and γ' and site-occupancy in γ'. Spatial distribution maps revealed that Mo occupied both Co and Ti sub-lattice sites in γ'. In agreement with the experimental data, thermodynamic calculations predicted a stronger tendency for Mo to occupy the Co-sites than for Cr and an increase in Cr fraction on the Ti-sites with increasing Cr content.

Accommodating the role of site memory in dynamic species distribution models.

First-order dynamic occupancy models (FODOMs) are a class of state-space model in which the true state (occurrence) is observed imperfectly. An important assumption of FODOMs is that site dynamics only depend on the current state and that variations in dynamic processes are adequately captured with covariates or random effects. However, it is often difficult to understand and/or measure the covariates that generate ecological data, which are typically spatiotemporally correlated. Consequently, the non-independent error structure of correlated data causes underestimation of parameter uncertainty and poor ecological inference. Here, we extend the FODOM framework with a second-order Markov process to accommodate site memory when covariates are not available. Our modeling framework can be used to make reliable inference about site occupancy, colonization, extinction, turnover, and detection probabilities. We present a series of simulations to illustrate the data requirements and model performance. We then applied our modeling framework to 13 yr of data from an amphibian community in southern Arizona, USA. In this analysis, we found residual temporal autocorrelation of population processes for most species, even after accounting for long-term drought dynamics. Our approach represents a valuable advance in obtaining inference on population dynamics, especially as they relate to metapopulations.

Multispecies models reveal that eDNA metabarcoding is more sensitive than backpack electrofishing for conducting fish surveys in freshwater streams.

Environmental DNA (eDNA) sampling can provide accurate, cost-effective, landscape-level data on species distributions. Previous studies have compared the sensitivity of eDNA sampling to traditional sampling methods for single species, but similar comparative studies on multispecies eDNA metabarcoding are rare. Using hierarchical site occupancy detection models, we examined whether key choices associated with eDNA metabarcoding (primer selection, low-abundance read filtering and the number of positive water samples used to classify a species as present at a site) affect the sensitivity of metabarcoding, relative to backpack electrofishing for fish in freshwater streams. Under all scenarios (teleostei and vertebrate primers; 0%, 0.1% and 1% read filtering thresholds; one or two positive samples required to classify species as present), we found that eDNA metabarcoding is, on average, more sensitive than electrofishing. Combining vertebrate and teleostei markers resulted in higher detection probabilities relative to the use of either marker in isolation. Increasing the threshold used to filter low-abundance reads decreased species detection probabilities but did not change our overall finding that eDNA metabarcoding was more sensitive than electrofishing. Using a threshold of two positive water samples (out of five) to classify a species as present typically had negligible effects on detection probabilities compared to using one positive water sample. Our findings demonstrate that eDNA metabarcoding is generally more sensitive than electrofishing for conducting fish surveys in freshwater streams, and that this outcome is not sensitive to methodological decisions associated with metabarcoding.