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Background: Renewed interest in the effects of intensity on treatment has led to development of short-term, intensive treatment protocols, such as Constraint-Induced Language Therapy (CILT), in which participants with chronic aphasia begin to show statistically significant language improvements in as little as 2 weeks. Given its relatively short treatment cycle, CILT is also a good choice of treatment methodology for studying brain/behaviour plasticity in post-stroke aphasia. Aims: This study aimed to examine differences between two short, intensive treatment protocols in a participant with chronic Wernicke's aphasia both in terms of treatment outcomes and changes in patterns of BOLD signal activation. Methods & Procedures: The participant (ACL) participated in language testing and an fMRI overt speech confrontation-naming paradigm pre and post 2 weeks of CILT, post 2 weeks of unconstrained language therapy (PACE), and 6 months post-CILT. He named 48 black/white line drawings from each of four conditions: treated (CILT or PACE), untreated, or consistently correctly named pictures. Outcomes & Results: Naming treated pictures improved, even in the scanner, while naming untreated pictures did not. About one third of PACE and three-fourths of CILT gains were maintained. Rather than a distinct pattern of activation distinguishing treated from untreated or CILT from PACE pictures, ACL recruited a frontal network during naming of all pictures that included left middle and inferior frontal cortex, SMA and pre-SMA, and that varied in spatial extent and degree of activation according to accuracy and performance expectation. In post-hoc analyses of accuracy, this frontal network was most active during incorrect trials. At 6 months post-CILT, compared to controls, incorrect naming recruited a large and significant bilateral network including right Wernicke's area homologue. Conclusions: Results suggest that short, intensive therapy can improve naming and jumpstart language recovery in chronic aphasia, whether responses are constrained to the speech modality or not. Modulation of a left frontal network was associated with accuracy in naming and may represent compensatory adaptation to improve response selection, self-monitoring, and/or inhibition. [ABSTRACT FROM AUTHOR]

The frequency of mature hybrids, including post-F1 individuals, between loblolly (Pinus taeda L.) and shortleaf pine (Pinus echinata Mill.), detectable with a codominant nuclear marker, was studied in a sympatric population from central Arkansas. The direction of introgression was also examined. The marker revealed 10 putative hybrids from the 80 trees sampled. PCR-RFLP analysis of their rbcL gene showed two of the putative hybrids (HL) share loblolly pine chloroplast DNA, and eight (HS) share the shortleaf pine chloroplast DNA. The two putative HL hybrids were morphologically similar to loblolly pine, and the eight putative HS hybrids were morphologically similar to shortleaf pine. Utilizing microsatellite data, Nei's measure of genetic identity showed the putative HL hybrids to be similar to loblolly pine, and the putative HS hybrids as being similar to shortleaf pine. An inferred tree of the individuals, using simple sequence repeat data and the neighbor-joining method, also suggested that some of the putative hybrids were not F1 individuals. Principle component analysis of the morphological characters groups the HL trees with loblolly pine and the HS trees with shortleaf pine. These results suggest bidirectional introgression occurred within the study population, and some of the putative hybrids were likely derived from early-generation backcross(es) with either shortleaf or loblolly pine. [ABSTRACT FROM AUTHOR]

A high-density restriction fragment length polymorphism (RFLP) linkage map is being constructed for loblolly pine ( Pinus taeda L.). Loblolly pine cDNA and genomic DNA clones were used as probes in hybridizations to genomic DNAs prepared from grandparents, parents, and progeny of a three-generation outbred pedigree. Approximately 200 probes were evaluated for their ability to detect polymorphic loci between DNAs prepared from the two parent trees, 20-1010 and 11-1060, and cut with four different restriction enzymes: BamHI , DraI, EcoRI, and HindIII. More than half of the probes detecting single- or low-copy number sequences (56%) revealed polymorphisms between the two parents with at least one restriction enzyme. If necessary, an additional hybridization to DNAs prepared from the four grandparent trees was conducted to determine the zygosity of parent trees. Ten of these probes were hybridized to progeny DNAs from this cross and, as expected, the markers were inherited as simple codominant Mendelian alleles. Four of the ten probes detected segregation of three alleles at one locus, and four probes detected more than one independently segregating locus. RFLPs can be used immediately to assess genetic diversity in conifer populations and to 'efingerprint' genotypes in tree improvement programs. [ABSTRACT FROM AUTHOR]

Seed from 126 trees from populations representing 15 geographic locations covering much of the natural range of shortleaf pine (Pinus echinata Mill.) were analyzed using 23 enzyme systems covering 39 loci to determine patterns of genetic variation and structure. Populations were polymorphic (p) at 87.2% of the loci, had 2.18 alleles (A) per locus, and had 2.35 alleles per polymorphic locus (Ap). Mean expected heterozygosity (He) was 0.194 and mean observedheterozygosity (Ho) was 0.174. Western populations had a higher p, higher A, similar Ap, and a higher Hoand He than eastern populations, in part because of six private alleles (alleles seen only in one population) in the west but only one in the east. Genetic structure analysis revealed interpopulation genetic variation at 9%, meaning 91% of the genetic variation inshortleaf pine resides within populations. Interpopulation gene flowwas 2.56, indicating two or three allele migrations per generation, which is relatively high and explains the low interpopulation geneticvariation in the species. There was no apparent relationship betweengeographic distance among populations and their genetic distance. Shortleaf pine populations exist in naturally outcrossing random-matingpopulations and have a relatively large amount of natural variability. Western populations are more diverse than their eastern counterparts. [ABSTRACT FROM AUTHOR]

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N 6 -[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli. We used the orthogonal pyrrolysyl-tRNA synthetase/suppressor tRNA CUA Pyl pair from Methanosarcina mazei to incorporate Azk site-specifically. We characterized Azk uptake and Fab production at bench-scale under different fermentation conditions, varying timing and mode of Azk addition, Azk-to-cell ratio and induction time. Our results indicate that Azk uptake is comparatively efficient in the batch phase. We discovered that the time between Azk uptake and inducing its incorporation into the Fab must be kept short, which suggests that intracellular Azk is consumed and/or degraded. The results obtained in this study are an important step towards the development of efficient production methods for Azk-incorporated proteins in E. coli. The developed process is scalable and provides excellent yields of 2.95 mg functionalized Fab per g CDM, which corresponds to 80% of yield obtained with the wild type Fab. We also identified the cellular uptake of Azk being dependent on the physiological state of the cell as a potential bottleneck in production., (© 2024. The Author(s).)

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation., Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L -1 , and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds., Conclusion: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis., (© 2024. The Author(s).)

Background: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations., Results: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg 2+ , indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising., Conclusions: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production., (© 2024. The Author(s).)

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N 6 -[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2 Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2 Azk variants were efficiently produced in fed-batch like μ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2 Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups., Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification., Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus., Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms., Funding: WWTF, Project No. COV20-016; BOKU, LBI/LBG., Competing Interests: Declaration of Competing Interest Dr. Klausberger has nothing to disclose. Dr. Duerkop has nothing to disclose. Dr. Haslacher has nothing to disclose. Dr. Wozniak-Knopp has nothing to disclose. Dr. Cserjan-Puschmann has nothing to disclose. Dr. Perkmann has nothing to disclose. Dr. Lingg has nothing to disclose. Dr. Pereira Aguilar has nothing to disclose. Dr. Laurent has nothing to disclose. Dr. De Vos has nothing to disclose. Mag.rer.nat. Hofner has nothing to disclose. Dr. Holzer has nothing to disclose. Mrs. Stadler has nothing to disclose. Dipl.-Ing. Manhart has nothing to disclose. DI Vierlinger has nothing to disclose. Dr. Egger has nothing to disclose. Dipl. Ing. Milchram has nothing to disclose. Dr. Gludovacz has nothing to disclose. Dr. Marx has nothing to disclose. Dipl.-Ing. Köppl has nothing to disclose. Christopher Tauer, BSc has nothing to disclose. Jürgen Beck, MSc reports nothing to disclose. Daniel Maresch has nothing to disclose. Dr. Grünwald-Gruber has nothing to disclose. Mr. Strobl has nothing to disclose. Dr. Satzer has nothing to disclose. Dr. Stadlmayr has nothing to disclose. Ing. Vavra has nothing to disclose. Ms. Huber BSc has nothing to disclose. Dr. Wahrmann has nothing to disclose. Dr. Eskandary has nothing to disclose. Dr. Breyer has nothing to disclose. Dr. Sieghart has nothing to disclose. Dr. Quehenberger reports other from Roche Austria, personal fees from Takeda, outside the submitted work; . Dr. Leitner has nothing to disclose. Dr. Strassl has nothing to disclose. Dr. Egger has nothing to disclose. Dr. IRSARA has nothing to disclose. Dr. Griesmacher has nothing to disclose. Dr. Hoermann has nothing to disclose. Dr. Weiss has nothing to disclose. Dr. Bellmann-Weiler has nothing to disclose. Dr. Löffler-Ragg has nothing to disclose. Dr. Borth has nothing to disclose. Dr. Strasser has nothing to disclose. Dr. Jungbauer has nothing to disclose. Dr. Hahn has nothing to disclose. Dr. Mairhofer reports other from enGenes Biotech GmbH, outside the submitted work; In addition, Dr. Mairhofer has a patent PCT/EP2016/059597-Uncoupling growth and protein production issued. Dr. Hartmann has nothing to disclose. Dr. Binder reports grants from Vienna Business Agency, during the conduct of the study; and Employee of Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH. Dr. Striedner reports other from enGenes GmbH, outside the submitted work; In addition, Dr. Striedner has a patent. US20180282737A1 issued to enGenes GmbH. Dr. Mach has nothing to disclose. Dr. Weinhaeusel has nothing to disclose. Dr. Dieplinger has nothing to disclose. Dr. Grebien has nothing to disclose. Dr. Gerner has nothing to disclose. Dr. Christoph Binder is board member of Technoclone GmbH. Dr. Grabherr has nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges' λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based "All-In-One" genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

Chronic recalcitrant dermatophytoses, due to Trichophyton ( T .) mentagrophytes Type VIII are on the rise in India and are noteworthy for their predominance. It would not be wrong to assume that travel and migration would be responsible for the spread of T . mentagrophytes Type VIII from India, with many strains resistant to terbinafine, to other parts of the world. From September 2016 until March 2020, a total of 29 strains of T . mentagrophytes Type VIII (India) were isolated. All patients were residents of Germany: 12 females, 15 males and the gender of the remaining two was not assignable. Patients originated from India (11), Pakistan (two), Bangladesh (one), Iraq (two), Bahrain (one), Libya (one) and other unspecified countries (10). At least two patients were German-born residents. Most samples (21) were collected in 2019 and 2020. All 29 T . mentagrophytes isolates were sequenced (internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1-α)). All were identified as genotype VIII (India) of T . mentagrophytes . In vitro resistance testing revealed 13/29 strains (45%) to be terbinafine-resistant with minimum inhibitory concentration (MIC) breakpoints ≥0.2 µg/mL. The remaining 16 strains (55%) were terbinafine-sensitive. Point mutation analysis revealed that 10/13 resistant strains exhibited Phe 397 Leu amino acid substitution of squalene epoxidase (SQLE), indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe 397 Leu and Ala 448 Thr amino acid substitutions, and one strain a single Leu 393 Phe amino acid substitution. Out of 16 terbinafine-sensitive strains, in eight Ala 448 Thr, and in one Ala 448 Thr +, new Val 444 Ile amino acid substitutions were detected. Resistance to both itraconazole and voriconazole was observed in three out of 13 analyzed strains. Treatment included topical ciclopirox olamine plus topical miconazole or sertaconazole. Oral itraconazole 200 mg twice daily for four to eight weeks was found to be adequate. Terbinafine-resistant T . mentagrophytes Type VIII are being increasingly isolated. In Germany, transmission of T . mentagrophytes Type VIII from the Indian subcontinent to Europe should be viewed as a significant public health issue.

In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high-pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins. Our main conclusion was that although we identified industrially scalable lysis conditions that significantly increased the starting purity for further purification, none of the tested conditions were selective for periplasmic protein over cytoplasmic protein. Furthermore, we demonstrated that efficient extraction of the expressed recombinant proteins was largely dependent on the overall protein concentration in the cell., (© 2020 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)