Optimising cell-based bioassays via integrated design of experiments (ixDoE) - A practical guide.

For process optimisation Design of Experiments (DoE) has long been established as a more powerful strategy than a One Factor at a Time approach. Nevertheless, DoE is not widely used especially in the field of cell-based bioassay development although it is known that complex interactions often exist. We believe that biopharmaceutical manufacturers are reluctant to move beyond standard practices due to the perceived costs, efforts, and complexity. We therefore introduce the integrated DoE (ixDoE) approach to target a smarter use of DoEs in the bioassay setting, specifically in optimising resources and time. Where in a standard practice 3 to 4 separate DoEs would be performed, our ixDoE approach includes the necessary statistical inference from only a single experimental set. Hence, we advocate for an innovative, ixDoE approach accompanied by a suitable statistical analysis strategy and present this as a practical guide for a typical bioassay development from basic research to biopharmaceutical industry.

Measurement and assessment of grief in a large international sample.

BACKGROUND: In 2022, the International Classification of Diseases (ICD-11) and an update of the Diagnostic Statistical Manual of Mental Disorders (DSM 5 TR) were released for implementation worldwide and now include the new Prolonged Grief Disorder (PGD). The newest definition of PGD is based on robust clinical research from the Global North yet until now has not been tested for global applicability. METHODS: The current study assesses the new PGD ICD-11 criteria in a large international sample of 1393 bereaved adults. The majority of the sample was included from the USΑ. Additionally, we conduct a sub-sample analysis to evaluate the psychometric properties, probable caseness of PGD, and differences in network structure across three regions of residency (USA, Greece-Cyprus, Turkey-Iran). RESULTS: The psychometric validity and reliability of the 33-item International Prolonged Grief Disorder Scale (IPGDS) were confirmed across the whole sample and for each regional group. Using the strict diagnostic algorithm, the probable caseness for PGD for the whole sample was 3.6 %. Probable caseness was highest for the Greece-Cyprus group (6.9 %) followed by Turkey-Iran (3.2 %) and the USA (2.8 %). Finally, the network structure of the IPGDS standard items and cultural supplement items (total of 33 items) confirmed the strong connection between central items of PGD, and revealed unique network connections within the regional groups. LIMITATIONS: Future research is encouraged to include larger sample sizes and a more systematic assessment of culture. CONCLUSION: Overall, our findings confirm the global applicability of the new ICD-11 PGD disorder definition as evaluated through the newly developed IPGDS. This scale includes culturally sensitive grief symptoms that may improve clinical precision and decision-making.

MH2c: Characterization of major histocompatibility α-helices - an information criterion approach.

Major histocompatibility proteins share a common overall structure or peptide binding groove. Two binding groove domains, on the same chain for major histocompatibility class I or on two different chains for major histocompatibility class II, contribute to that structure that consists of two α-helices ("wall") and a sheet of eight anti-parallel beta strands ("floor"). Apart from the peptide presented in the groove, the major histocompatibility α-helices play a central role for the interaction with the T cell receptor. This study presents a generalized mathematical approach for the characterization of these helices. We employed polynomials of degree 1 to 7 and splines with 1 to 2 nodes based on polynomials of degree 1 to 7 on the α-helices projected on their principal components. We evaluated all models with a corrected Akaike Information Criterion to determine which model represents the α-helices in the best way without overfitting the data. This method is applicable for both the stationary and the dynamic characterization of α-helices. By deriving differential geometric parameters from these models one obtains a reliable method to characterize and compare α-helices for a broad range of applications. PROGRAM SUMMARY: Program title: MH2c (MH helix curves) Catalogue identifier: AELX_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AELX_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 327 565 No. of bytes in distributed program, including test data, etc.: 17 433 656 Distribution format: tar.gz Programming language: Matlab Computer: Personal computer architectures Operating system: Windows, Linux, Mac (all systems on which Matlab can be installed) RAM: Depends on the trajectory size, min. 1 GB (Matlab) Classification: 2.1, 4.9, 4.14 External routines: Curve Fitting Toolbox and Statistic Toolbox of Matlab Nature of problem: Major histocompatibility (MH) proteins share a similar overall structure. However, identical MH alleles which present different peptides differ by subtle conformational alterations. One hypothesis is that such conformational differences could be another level of T cell regulation. By this software package we present a reliable and systematic way to compare different MH structures to each other. Solution method: We tested several fitting approaches on all available experimental crystal structures of MH to obtain an overall picture of how to describe MH helices. For this purpose we transformed all complexes into the same space and applied splines and polynomials of several degrees to them. To draw a general conclusion which method fits them best we employed the "corrected Akaike Information Criterion". The software is applicable for all kinds of helices of biomolecules. Running time: Depends on the data, for a single stationary structure the runtime should not exceed a few seconds.

Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes.

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Conservation, localization and expression of HopZ, a protein involved in adhesion of Helicobacter pylori.

From a sarkosyl-insoluble outer membrane fraction prepared from the Helicobacter pylori strain ATCC 43504, 19 proteins could be sequenced N-terminally by Edman degradation. Oligonucleotides were deduced and used for screening of a genomic library. From the isolated genes, five code for different members of a H.pylori outer membrane protein (Hop) family. Among these, the hopZ gene was characterized in more detail. It encodes a protein which was shown to be located at the bacterial surface by immunofluorescence studies. Sequence analysis of the hopZ gene from 15 different H.pylori strains revealed the existence of two alleles and the possible regulation of hopZ expression by slipped-strand mispairing within a CT dinucleotide repeat motif located in the signal-peptide coding region. Among the different strains, the influence of this region on the expression of HopZ was analyzed on a translational level by western blot analysis of bacterial extracts and immunofluorescence studies on intact cells. The protein is expressed only in those strains in which the number of the CT dinucleotide repeats allow for an open reading frame encoding the complete protein. Addionally the function of HopZ was investigated in an adhesion assay. The wild-type strain ATCC 43504 adhered to human gastric epithel cells whereas a knockout mutant strain showed significantly reduced binding to the cells.

Interleukin-4 and interferon-γ orchestrate an epithelial polarization in the airways.

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are key effector cytokines for the differentiation of T helper type 1 and 2 (Th1 and Th2) cells. Both cytokines induce fate-decisive transcription factors such as GATA3 and TBX21 that antagonize the polarized development of opposite phenotypes by direct regulation of each other's expression along with many other target genes. Although it is well established that mesenchymal cells directly respond to Th1 and Th2 cytokines, the nature of antagonistic differentiation programs in airway epithelial cells is only partially understood. In this study, primary normal human bronchial epithelial cells (NHBEs) were exposed to IL-4, IFN-γ, or both and genome-wide transcriptome analysis was performed. The study uncovers an antagonistic regulation pattern of IL-4 and IFN-γ in NHBEs, translating the Th1/Th2 antagonism directly in epithelial gene regulation. IL-4- and IFN-γ-induced transcription factor hubs form clusters, present in antagonistically and polarized gene regulation networks. Furthermore, the IL-4-dependent induction of IL-24 observed in rhinitis patients was downregulated by IFN-γ, and therefore IL-24 represents a potential biomarker of allergic inflammation and a Th2 polarized condition of the epithelium.

From evil to good: a cytolysin in vaccine development.

Current vaccination strategies mainly target antigens into the phagosomal, major histocompatibility complex class II antigen-processing pathway and thus lead predominantly to humoral immune responses. The elicitation of cytotoxic T-cell responses instead requires introduction of antigens into the cytosol of professional antigen-presenting cells (APCs). The intracellular bacterium Listeria monocytogenes gains access to the host cell cytosol by means of a cytolysin, listeriolysin O. Vaccine researchers have successfully employed listeriolysin in novel vaccination approaches to provide access to the cytosol of professional APCs for purified protein antigens, attenuated bacterial vaccine strains, DNA vaccines and liposome contents.

Characterization of four members of a multigene family encoding outer membrane proteins of Helicobacter pylori and their potential for vaccination.

In search of protective antigens which can be used in a vaccine to prevent Helicobacter pylori infection, we report on the identification of four genes, hopV, hopW, hopX and hopY, and the characterization of the corresponding proteins which belong to the H. pylori outer membrane protein (Hop) family containing 32 homologous members, some of which were shown to function as porins. Sequence analysis of 16 different H. pylori strains revealed that the proteins HopV, HopW, HopX and HopY are highly conserved. Localization of HopV, HopW, HopX and HopY at the surface of the bacteria was investigated by immunofluorescence. Using a planar lipid bilayer system the proteins HopV and HopX were shown to form pores with single-channel conductances of 1.4 and 3.0 nS, respectively.

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.

A new blood stage antigen of Plasmodium falciparum transported to the erythrocyte surface.

On screening a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum against the 41-kDa protein band, which confers protective immunity to monkeys, two strongly reacting clones were isolated. One of the clones codes for parts of the P. falciparum 41-kDa aldolase, while the other (41-2) codes for parts of an unknown antigen; this was analyzed further. The 41-2 insert was used to identify two genomic fragments which carry the entire gene. The 41-2 gene codes for 184 amino acids specifying a 29-kDa schizont protein as estimated by Western blot analysis. The protein contains no repetitive sequences, and is characterized by a signal sequence and by two additional hydrophobic segments which could function as membrane anchor sequences. Computer analysis of the protein sequence predicted a dominant epitope in the N-terminal part which was confirmed by immunoreactivity data. The 41-2 protein could be localized in the schizont membrane, associated with membranous structures in the erythrocytic cytoplasm and with the erythrocyte membrane.

Plasmodium falciparum aldolase: gene structure and localization.

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.

A new blood stage antigen of Plasmodium falciparum highly homologous to the serine-stretch protein SERP.

We have isolated the gene coding for a new protein (SERP H), highly homologous to the 113-kDa serine-stretch protein SERP which confers protective immunity to monkeys. The gene consists of four exons interrupted by three short introns located at positions corresponding to those of the SERP gene. Both genes were shown to be linked on chromosome 2 of Plasmodium falciparum suggesting that both originate from a common ancestral gene. Both genes are transcribed in the blood-stage form as 3.8-kb mRNAs with high yield. The deduced amino acid sequence of SERP H is highly homologous to SERP, although it does not contain a serine stretch. A highly hydrophilic region specific for the protein which was shown to be identical among different P. falciparum isolates was expressed in Escherichia coli for preparation of SERP H specific antisera. A schizont polypeptide of 130 kDa within the parasitophorous vacuole was detected by Western blot analysis and immunoelectron microscopy. Like SERP, the 130-kDa protein exhibits a region homologous to cysteine proteinases, suggesting that these proteins, or their processing products, may play a role as proteinases at the time of merozoite release from the infected erythrocyte.

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch.

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.

In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum.

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.

Responses of T cells from sensitized donors to recombinant and synthetic peptides corresponding to sequences of the Plasmodium falciparum SERP antigen.

In the present work, we intend to determine the capacity of human lymphocytes to recognize subfragments of the serine-stretch protein SERP, a blood-stage antigen from Plasmodium falciparum. Individuals sensitized by a previous P. falciparum infection were studied. Some recombinant proteins (RP) including RP7 and RP10 (amino acids 631-684 and 631-892 of SERP, respectively), were recognized in proliferation assays by lymphocytes from 28 sensitized individuals and not by lymphocytes from control, non-sensitized, donors. Synthetic peptides covering predefined zones of particular interest were tested and appeared to induce proliferative responses of lymphocytes from sensitized donors, allowing identification of putative T cell epitopes.

Serine-stretch protein (SERP) of Plasmodium falciparum corresponds to the exoantigen Ag2, a target of antibodies associated with protection against malaria.

A mixture of Plasmodium falciparum exoantigens inducing lymphocyte activation and cytokine production was shown to contain the malaria vaccine candidate, the serine-stretch protein. This protein was shown serologically to correspond to Ag2, an exoantigen recognized by antibodies linked with protection against malaria. The glycophorin-binding protein, the histidine-rich protein II, the S-antigen, the heat shock protein 70, the ring-infected erythrocyte surface antigen, and the apical membrane antigen-1 were also shown serologically to be present in the mixture of exoantigens.

Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions.

Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.