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This paper aims to explain recent empirical findings on subjunctive II formation in the Bavarian dialects of Austria from both functional and formal perspectives. For this purpose, the explanatory power of the functional principles of natural morphology (NM) is compared with the formal framework of constructional morphology (CxM). It is argued that the two approaches complement each other. Thus, it is shown that the key concepts of NM (constructional iconicity, uniformity and transparency) can easily be adapted in terms of CxM. These adjustments are needed to explain the ongoing changes in subjunctive II formation in the Bavarian dialects of Austria. This is due to the well-documented shift towards the use of periphrastic constructions (with the täte- and würde-auxiliary) that are located at the interface between morphology and syntax.

This paper investigates the geographical and structural diffusion of “Viennese Monophthongization” (VM). By means of a new numerical measure to assess and compare formant movement in 18 lexical items, we provide evidence that VM is an ongoing, regular sound change transforming [aɛ̯] and [aɔ̯] gradually into [æː] and [ɒː]. Data are based on direct dialect recordings of 76 speakers in two age groups in 19 eastern and central Austrian rural locations. Results indicate that VM is diffusing from Vienna in a wave-like fashion. Even though VM is reported to have been established in other bigger cities for more than 30 years, the data show no evidence of diffusion from these cities. There are also other factors affecting the degree of formant movement: The phonetic-phonological environment (stress and the following consonant) explains most of the variance in the data, whereas no frequency effects could be found. Social identity, cultural space, and gender-related network structures are discussed as additional social factors.

The Alpine region stands out in the German-speaking world for its well-preserved traditional dialects, which continue to play a significant role in daily life. However, the vast geographical range of the Alpine region and the limitations imposed by national and regional borders have hindered comprehensive investigations of the entire Alpine area. To overcome these obstacles, this study utilizes crowdsourcing data from the VerbaAlpina project to investigate phonological and morpho-phonological variation in the German-speaking Alpine region. Although the data were collected in a written form and with a focus on lexical variation, it is shown that the data contain detailed phonological information. By using multivariate dialectometric measurements (i.e. factor analysis [FA]) based on 19 (morpho-)phonological variables (comprising 8,582 tokens), the study explores the geolinguistic structures of the German dialects in the Alpine region. The results confirm, on a general level, the validity of the traditionally established dialect classification. However, the findings also reveal previously underrated border effects, in particular for Bavarian dialects along the German-Austrian border, which are argued to be the outcome of divergence processes. Hence, the study highlights the importance of cross-dialectal and cross-national perspectives in understanding dialect variation and emphasizes the value of crowdsourcing data for dialectological research.

Previously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 μl samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10-50 m°C for concentrations of 0.05-0.30 M glucose or sucrose present in the original non-diluted samples. The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant. On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance. [ABSTRACT FROM AUTHOR]

Background The AthaMap database generates a genome-wide map for putative transcription factor binding sites for A. thaliana. When analyzing transcriptional regulation using AthaMap it may be important to learn which genes are also post-transcriptionally regulated by inhibitory RNAs. Therefore, a unified database for transcriptional and post-transcriptional regulation will be highly useful for the analysis of gene expression regulation. Methods To identify putative microRNA target sites in the genome of A. thaliana, processed mature miRNAs from 243 annotated miRNA genes were used for screening with the psRNATarget web server. Positional information, target genes and the psRNATarget score for each target site were annotated to the AthaMap database. Furthermore, putative target sites for small RNAs from seven small RNA transcriptome datasets were used to determine small RNA target sites within the A. thaliana genome. Results Putative 41,965 genome wide miRNA target sites and 10,442 miRNA target genes were identified in the A. thaliana genome. Taken together with genes targeted by small RNAs from small RNA transcriptome datasets, a total of 16,600 A. thaliana genes are putatively regulated by inhibitory RNAs. A novel web-tool, ‘MicroRNA Targets’, was integrated into AthaMap which permits the identification of genes predicted to be regulated by selected miRNAs. The predicted target genes are displayed with positional information and the psRNATarget score of the target site. Furthermore, putative target sites of small RNAs from selected tissue datasets can be identified with the new ‘Small RNA Targets’ web-tool. Conclusions The integration of predicted miRNA and small RNA target sites with transcription factor binding sites will be useful for AthaMap-assisted gene expression analysis. URL: http://www.athamap.de/

Abstract Background Proteins can be used in drug delivery systems to improve pharmacological properties of an active substance. Differences in pH between tissues can be utilized in order to achieve a targeted drug release at a specific location or tissue, such as a tumor. The enamel matrix protein amelogenin has a pH dependent solubility profile and self-assemble to form aggregates at neutral pH. This could make amelogenin useful in the design of pH responsive drug delivery systems. Results In this study amelogenin was evaluated as a pH responsive component in drug delivery applications. This was achieved by testing the ability of amelogenin to entrap/release other proteins upon changes in pH, and by testing if amelogenin could confer pH responsiveness to an existing and versatile drug delivery system, such as gelatin microparticles. Amelogenin was able to encapsulate bovine serum albumin and insulin, whichwere used as model target proteins. The composite aggregates of amelogenin and target protein were formed at neutral pH and could be reversibly solubilized at weakly acidic pH. Gelatin microparticles prepared in the presence of amelogenin, showed a modulated structure in response to pH change, when studied by scanning electron microscopy, compared to particles without amelogenin. At neutral pH amelogenin induced formation of pores in the particle surface, which were not present at acidic pH, or in particles lacking amelogenin. Conclusions The results from this study demonstrate that amelogenin can be a useful component in drug delivery systems in order to achieve a pH dependent response.

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation. The γL96Y mutation presented the best profile of oxidative protection unaccompanied by loss of protein stability and function. N-terminal deletions were constructed to facilitate the production of recombinant hemoglobin by fermentation and phenylalanine insertions in the heme pocket to decrease the rate of NO dioxygenation. The resultant mutant (αV1del. αL29F, γG1del. γV67F, γL96Y) significantly decreased NO scavenging and lipid peroxidation in vitro. Unlike native hemoglobin or a recombinant control (αV1del, γG1del), this mutation showed no increase in blood pressure immediately following infusion in a rat model of reperfusion injury, suggesting that it was also able to prevent NO scavenging in vivo. Infusion of the mutant also resulted in no meaningful adverse physiological effects apart from diuresis, and no increase in oxidative stress, as measured by urinary isoprostane levels. Following PEGylation via the Euro-PEG-Hb method to increase vascular retention, this novel protein construct was compared with saline in a severe rat reperfusion injury model (45% blood volume removal for 90 minutes followed by reinfusion to twice the volume of shed blood). Blood pressure and survival were followed for 4 h post-reperfusion. While there was no difference in blood pressure, the PEGylated Hb mutant significantly increased survival., (© 2024. The Author(s).)

Development in hemoglobin-based oxygen carriers (HBOCs) that may be used as alternatives to donated blood requires an extensive supply of highly pure hemoglobin (Hb) preparations. Therefore, it is essential to fabricate inexpensive, stable and highly selective absorbents for Hb purification. Molecular imprinting is an attractive technology for preparing such materials for targeted molecular recognition and rapid separations. In this case study, we developed human fetal hemoglobin (HbF)-imprinted polymer beads through the fusion of surface imprinting and Pickering emulsion polymerization. HbF was firstly covalently coupled to silica nanoparticles through its surface-exposed amino groups. The particle-supported HbF molecules were subsequently employed as templates for the synthesis of molecularly imprinted polymers (MIPs) with high selectivity for Hb. After removing the silica support and HbF, the resulting MIPs underwent equilibrium and kinetic binding experiments with both adult Hb (HbA) and HbF. These surface-imprinted MIPs exhibited excellent selectivity for both HbA and HbF, facilitating the one-step isolation of recombinant Hb from crude biological samples. The saturation capacities of HbA and HbF were found to be 15.4 and 17.1 mg/g polymer, respectively. The present study opens new possibilities for designed resins for tailored protein purification, separation and analysis.

Hemozoin (Hz) is a heme crystal produced during malaria infection that stimulates immune cells, leading to the production of cytokines and chemokines. The immunostimulatory action of Hz has previously been applied in the development of alternative adjuvants. Crystallization of hemin is a chemical approach for producing Hz. Here, we focused on an enzymatic production method for Hz using the heme detoxification protein (HDP), which catalyzes heme dimer formation from hemin in Plasmodium . We examined the immunostimulatory effects of an enzymatically synthesized analog of Hz (esHz) produced by recombinant Plasmodium falciparum HDP. Enzymatically synthesized Hz stimulates a macrophage cell line and human peripheral mononuclear cells, leading to the production of interleukin (IL)-6 and IL-12p40. In mice, subcutaneous administration of esHz together with an antigen, ovalbumin (OVA), increased the OVA-specific immunoglobulin (Ig) G2c isotype level in the serum, whereas OVA-specific IgG1 was not induced. Our findings suggest that esHz is a useful Th-1 cell adjuvant.

Antibiotic resistance has become a serious threat to global public health and economic development. Rapid and accurate identification of a patient status for antimicrobial resistance (AMR) are urgently needed in clinical diagnosis. Here we describe the development of an assay method for activity fingerprinting of AMR β-lactamases using panels of 7 β-lactam antibiotics in 35 min. New Deli Metallo β-lactamase-1 (NDM-1) and penicillinase were demonstrated as two different classes of β-lactamases. The panel consisted of three classes of antibiotics, including: penicillins (penicillin G, piperacillin), cephalosporins (cefepime, ceftriaxone, cefazolin) and carbapenems (meropenem and imipenem). The assay employed a scheme combines the catalytic reaction of AMR β-lactamases on antibiotic substrates with a flow-injected thermometric biosensor that allows the direct detection of the heat generated from the enzymatic catalysis, and eliminates the need for custom substrates and multiple detection schemes. In order to differentiate classes of β-lactamases, characterization of the enzyme activity under different catalytic condition, such as, buffer composition, ion strength and pH were investigated. This assay could provide a tool for fast diagnosis of patient AMR status which makes possible for the future accurate treatment with selected antibiotics., Competing Interests: Author MM was employed by the company Omik Bioscience AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Song, Sun, Wang, Bülow, Mecklenburg, Wu, Meng and Xie.)

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli . In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli , we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H 2 O 2 -mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kettisen, Nyblom, Smeds, Fago and Bülow.)

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled ( 2 H, 13 C and 15 N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1 H- 15 N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins' roles in planta.

Oat ( Avena sativa ) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum . The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea -inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea -treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea -treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.

Albumin-based cryogels for capturing haemin were synthesised by crosslinking different biomolecules, bovine serum albumin (BSA) and ovalbumin (OVA). The impact of the protein and coupling agent concentrations on cryogel's mechanical properties, swelling ratios and polymerisation yields, as well as autoclaving as a post-treatment on the cryogel, were studied. We found that BSA (50 mg/ml) and the crosslinker ( N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride, 46 mg/ml) formed a cryogel with optimum physical characteristics at a comparatively low protein concentration. The cryogel's mechanical stability was increased using a double-layer cryogel approach by crosslinking the BSA proteins at subzero temperature inside an acrylamide and hydroxyethyl methacrylate premade cryogels. Batch binding and kinetic adsorption isotherms of haemin on the cryogels were assessed to evaluate their binding capacity toward the porphyrin molecule. The results showed that single-layer cryogels (BSA and OVA) had a higher capacity (∼0.68 mg/ml gel) and higher reaction rate constant towards haemin adsorption than double-layer gels. In contrast, the double-layer cryogels had higher mechanical strength than single-layer gels. The experimental results suggested that the cryogels followed the Freundlich model and the pseudo-second-order isotherm for batch adsorption and kinetics, respectively. The interaction between haemin and the gels was studied by fluorescence quenching. We found between 1.1 and 1.6 binding sites for different cryogels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hajizadeh, Dicko and Bülow.)

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet ( Beta vulgaris ), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum . Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Antimicrobial resistance (AMR) threatens global public health and modern surgical medicine. Expression of β-lactamase genes is the major mechanism by which pathogens become antibiotic resistant. Pathogens expressing extended spectrum β-lactamases (ESBL) and carbapenemases (CP) are especially difficult to treat and are associated with increased hospitalization and mortality rates. Despite considerable effort, identification of ESBLs and CPs in a clinically relevant timeframe remains challenging. In this study, a two-dimensional AMR profiling assay strategy was developed employing panels of antibiotics (penicillins, cephamycins, oximino-cephalosporins and carbapenems) and β-lactamases inhibitors (avibactam and EDTA). The assay required the development of a novel biosensor that employed New Delhi metallo-β-lactamase-1 (NDM-1) as the sensing element. Functionally probing β-lactamase activity using substrates and inhibitors combinatorically increased the informational content that enabled the development of assays capable of simultaneous, differential identification of multiple β-lactamases expressed in a single bacterial isolate. More specifically, the assay enabled the simultaneous identification of ESBL and CP in mock samples, as well as in an engineered construct which co-expressed these β-lactamases. The NDM-1 biosensor assay was 16 times and 8 times more sensitive than the ESBL Nordmann/Dortet/Poirel (NDP) and Carba Nordmann/Poirel (NP) assays, respectively. In a retrospective study, NDM-1 biosensor assays were able to differentially identify ESBLs, metallo-CPs and serine-CPs β-lactamases in 23 clinical isolates with 100% accuracy. An assay algorithm was developed which accelerated data analytics reducing turnaround to <1 h. The assay strategy integrated with AI-based data analytics has the potential to provide physicians with a comprehensive readout of patient AMR status., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hajizadeh, Kettisen, Bülow and Ye.)