The Cytokinin-Activating LOG-Family Proteins Are Not Lysine Decarboxylases.

A conserved PGGxGTxxE motif misleads the cytokinin (CK) converting LONELY GUY enzymes to be wrongly annotated as lysine decarboxylases (LDCs). However, so far PGGxGTxxE motif-containing LDCs do not show any LDC activity. Instead, they show phosphoribohydrolase activity by converting inactive CK nucleotides into active free-base forms to invoke CK responses.

Population-Predicted MHC Class II Epitope Presentation of SARS-CoV-2 Structural Proteins Correlates to the Case Fatality Rates of COVID-19 in Different Countries.

We observed substantial differences in predicted Major Histocompatibility Complex II (MHCII) epitope presentation of SARS-CoV-2 proteins for different populations but only minor differences in predicted MHCI epitope presentation. A comparison of this predicted epitope MHC-coverage revealed for the early phase of infection spread (till day 15 after reaching 128 observed infection cases) highly significant negative correlations with the case fatality rate. Specifically, this was observed in different populations for MHC class II presentation of the viral spike protein (p-value: 0.0733 for linear regression), the envelope protein (p-value: 0.023), and the membrane protein (p-value: 0.00053), indicating that the high case fatality rates of COVID-19 observed in some countries seem to be related with poor MHC class II presentation and hence weak adaptive immune response against these viral envelope proteins. Our results highlight the general importance of the SARS-CoV-2 structural proteins in immunological control in early infection spread looking at a global census in various countries and taking case fatality rate into account. Other factors such as health system and control measures become more important after the early spread. Our study should encourage further studies on MHCII alleles as potential risk factors in COVID-19 including assessment of local populations and specific allele distributions.

Modeling of shotgun sequencing of DNA plasmids using experimental and theoretical approaches.

BACKGROUND: Processing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments. RESULTS: In silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction. CONCLUSIONS: We applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis.

BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry. CONCLUSION: A proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

Detection of West Nile virus and tick-borne encephalitis virus in birds in Slovakia, using a universal primer set.

West Nile virus (WNV) is a mosquito-borne neurotropic pathogen that presents a major public health concern. Information on WNV prevalence and circulation in Slovakia is insufficient. Oral and cloacal swabs and bird brain samples were tested for flavivirus RNA by RT-PCR using newly designed generic primers. The species designation was confirmed by sequencing. WNV was detected in swab and brain samples, whereas one brain sample was positive for tick-borne encephalitis virus (TBEV). The WNV sequences clustered with lineages 1 and 2. These results confirm the circulation of WNV in birds in Slovakia and emphasize the risk of infection of humans and horses.

Identification of B-cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage-displayed gene fragment library.

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B-cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti-OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N-terminus (OspC E1 aa19-27, OspC E2 aa38-53, OspC E3 aa62-66) and three at the C-terminal end (OspC E4 aa155-163, OspC E5 aa184-190 and OspC E6 aa201-207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B-cell epitopes may provide fundamental data for the development of multi-epitope-based diagnostic tools for Lyme disease.

Deciphering the protein interaction in adhesion of Francisella tularensis subsp. holarctica to the endothelial cells.

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica (Fth) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth. Pull down and affinity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 significantly reduced (500-fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers.

Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

How to make DNA data storage more applicable.

The storage of digital data is becoming a worldwide problem. DNA has been recognized as a biological solution due to its ability to store genetic information without alteration over long periods. The first demonstrations of high-capacity long-lasting DNA digital data storage have been shown. However, high storage costs and slow retrieval of the data must be overcome to make DNA data storage more applicable and marketable. Herein, we discuss the issues and recent advances in DNA data storage methods and highlight pathways to make this technology more applicable to real-world digital data storage. We envision that a combination of molecular biology, nanotechnology, novel polymers, electronics, and automation with systematic development will allow DNA data storage sufficient for everyday use.

Experimental approaches for induction of diabetes mellitus and assessment of antidiabetic activity: An in vitro and in vivo methodological review.

BACKGROUND: Diabetes mellitus poses a global health challenge, driving the need for innovative therapeutic solutions. Experimental methods play a crucial role in evaluating the efficacy of potential antidiabetic drugs, both in vitro and in vivo. Yet concerns about reproducibility persist, necessitating comprehensive reviews. OBJECTIVES: This review aims to outline experimental approaches for inducing diabetes and evaluating antidiabetic activity, synthesizing data from authoritative sources and academic literature. METHODS: We conducted a systematic search of prominent databases, including PubMed, ScienceDirect, and Scopus, to identify relevant articles spanning from 1943 to the present. A total of 132 articles were selected for inclusion in this review, focusing on in vitro and in vivo experimental validations of antidiabetic treatments. RESULTS: Our review highlights the diverse array of experimental methods employed for inducing diabetes mellitus and evaluating antidiabetic interventions. From cell culture assays to animal models, researchers have employed various techniques to study the effectiveness of novel therapeutic agents. CONCLUSION: This review provides a comprehensive guide to experimental approaches for assessing antidiabetic activity. By synthesizing data from a range of sources, we offer valuable insights into the current methodologies used in diabetes research. Standardizing protocols and enhancing reproducibility are critical for advancing effective antidiabetic treatments.

Cat-E: A comprehensive web tool for exploring cancer targeting strategies.

Identifying potential cancer-associated genes and drug targets from omics data is challenging due to its diverse sources and analyses, requiring advanced skills and large amounts of time. To facilitate such analysis, we developed Cat-E (Cancer Target Explorer), a novel R/Shiny web tool designed for comprehensive analysis with evaluation according to cancer-related omics data. Cat-E is accessible at https://cat-e.bioinfo-wuerz.eu/. Cat-E compiles information on oncolytic viruses, cell lines, gene markers, and clinical studies by integrating molecular datasets from key databases such as OvirusTB, TCGA, DrugBANK, and PubChem. Users can use all datasets and upload their data to perform multiple analyses, such as differential gene expression analysis, metabolic pathway exploration, metabolic flux analysis, GO and KEGG enrichment analysis, survival analysis, immune signature analysis, single nucleotide variation analysis, dynamic analysis of gene expression changes and gene regulatory network changes, and protein structure prediction. Cancer target evaluation by Cat-E is demonstrated here on lung adenocarcinoma (LUAD) datasets. By offering a user-friendly interface and detailed user manual, Cat-E eliminates the need for advanced computational expertise, making it accessible to experimental biologists, undergraduate and graduate students, and oncology clinicians. It serves as a valuable tool for investigating genetic variations across diverse cancer types, facilitating the identification of novel diagnostic markers and potential therapeutic targets.

DNA storage-from natural biology to synthetic biology.

Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades).

PRO-Simat: Protein network simulation and design tool.

PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.

Genome-wide scan for potential CD4+ T-cell vaccine candidates in Candida auris by exploiting reverse vaccinology and evolutionary information.

Candida auris is a globally emerging fungal pathogen responsible for causing nosocomial outbreaks in healthcare associated settings. It is known to cause infection in all age groups and exhibits multi-drug resistance with high potential for horizontal transmission. Because of this reason combined with limited therapeutic choices available, C. auris infection has been acknowledged as a potential risk for causing a future pandemic, and thus seeking a promising strategy for its treatment is imperative. Here, we combined evolutionary information with reverse vaccinology approach to identify novel epitopes for vaccine design that could elicit CD4+ T-cell responses against C. auris. To this end, we extensively scanned the family of proteins encoded by C. auris genome. In addition, a pathogen may acquire substitutions in epitopes over a period of time which could cause its escape from the immune response thus rendering the vaccine ineffective. To lower this possibility in our design, we eliminated all rapidly evolving genes of C. auris with positive selection. We further employed highly conserved regions of multiple C. auris strains and identified two immunogenic and antigenic T-cell epitopes that could generate the most effective immune response against C. auris. The antigenicity scores of our predicted vaccine candidates were calculated as 0.85 and 1.88 where 0.5 is the threshold for prediction of fungal antigenic sequences. Based on our results, we conclude that our vaccine candidates have the potential to be successfully employed for the treatment of C. auris infection. However, in vivo experiments are imperative to further demonstrate the efficacy of our design.

Estimation of R0 for the spread of SARS-CoV-2 in Germany from excess mortality.

For SARS-CoV-2, R0 calculations in the range of 2-3 dominate the literature, but much higher estimates have also been published. Because capacity for RT-PCR testing increased greatly in the early phase of the Covid-19 pandemic, R0 determinations based on these incidence values are subject to strong bias. We propose to use Covid-19-induced excess mortality to determine R0 regardless of RT-PCR testing capacity. We used data from the Robert Koch Institute (RKI) on the incidence of Covid cases, Covid-related deaths, number of RT-PCR tests performed, and excess mortality calculated from data from the Federal Statistical Office in Germany. We determined R0 using exponential growth estimates with a serial interval of 4.7 days. We used only datasets that were not yet under the influence of policy measures (e.g., lockdowns or school closures). The uncorrected R0 value for the spread of SARS-CoV-2 based on RT-PCR incidence data was 2.56 (95% CI 2.52-2.60) for Covid-19 cases and 2.03 (95% CI 1.96-2.10) for Covid-19-related deaths. However, because the number of RT-PCR tests increased by a growth factor of 1.381 during the same period, these R0 values must be corrected accordingly (R0corrected = R0uncorrected/1.381), yielding 1.86 for Covid-19 cases and 1.47 for Covid-19 deaths. The R0 value based on excess deaths was calculated to be 1.34 (95% CI 1.32-1.37). A sine-function-based adjustment for seasonal effects of 40% corresponds to a maximum value of R0January = 1.68 and a minimum value of R0July = 1.01. Our calculations show an R0 that is much lower than previously thought. This relatively low range of R0 fits very well with the observed seasonal pattern of infection across Europe in 2020 and 2021, including the emergence of more contagious escape variants such as delta or omicron. In general, our study shows that excess mortality can be used as a reliable surrogate to determine the R0 in pandemic situations.

Prediction and validation of host-pathogen interactions by a versatile inference approach using Aspergillus fumigatus as a case study.

Biological networks are characterized by diverse interactions and dynamics in time and space. Many regulatory modules operate in parallel and are interconnected with each other. Some pathways are functionally known and annotated accordingly, e.g., endocytosis, migration, or cytoskeletal rearrangement. However, many interactions are not so well characterized. For reconstructing the biological complexity in cellular networks, we combine here existing experimentally confirmed and analyzed interactions with a protein-interaction inference framework using as basis experimentally confirmed interactions from other organisms. Prediction scoring includes sequence similarity, evolutionary conservation of interactions, the coexistence of interactions in the same pathway, orthology as well as structure similarity to rank and compare inferred interactions. We exemplify our inference method by studying host-pathogen interactions during infection of Mus musculus (phagolysosomes in alveolar macrophages) with Aspergillus fumigatus (conidia, airborne, asexual spores). Three of nine predicted critical host-pathogen interactions could even be confirmed by direct experiments. Moreover, we suggest drugs that manipulate the host-pathogen interaction.

Nanocellulose Composites as Smart Devices With Chassis, Light-Directed DNA Storage, Engineered Electronic Properties, and Chip Integration.

The rapid development of green and sustainable materials opens up new possibilities in the field of applied research. Such materials include nanocellulose composites that can integrate many components into composites and provide a good chassis for smart devices. In our study, we evaluate four approaches for turning a nanocellulose composite into an information storage or processing device: 1) nanocellulose can be a suitable carrier material and protect information stored in DNA. 2) Nucleotide-processing enzymes (polymerase and exonuclease) can be controlled by light after fusing them with light-gating domains; nucleotide substrate specificity can be changed by mutation or pH change (read-in and read-out of the information). 3) Semiconductors and electronic capabilities can be achieved: we show that nanocellulose is rendered electronic by iodine treatment replacing silicon including microstructures. Nanocellulose semiconductor properties are measured, and the resulting potential including single-electron transistors (SET) and their properties are modeled. Electric current can also be transported by DNA through G-quadruplex DNA molecules; these as well as classical silicon semiconductors can easily be integrated into the nanocellulose composite. 4) To elaborate upon miniaturization and integration for a smart nanocellulose chip device, we demonstrate pH-sensitive dyes in nanocellulose, nanopore creation, and kinase micropatterning on bacterial membranes as well as digital PCR micro-wells. Future application potential includes nano-3D printing and fast molecular processors (e.g., SETs) integrated with DNA storage and conventional electronics. This would also lead to environment-friendly nanocellulose chips for information processing as well as smart nanocellulose composites for biomedical applications and nano-factories.

An Ebola, Neisseria and Trypanosoma human protein interaction census reveals a conserved human protein cluster targeted by various human pathogens.

Filovirus ebolavirus (ZE; Zaire ebolavirus, Bundibugyo ebolavirus), Neisseria meningitidis (NM), and Trypanosoma brucei (Tb) are serious infectious pathogens, spanning viruses, bacteria and protists and all may target the blood and central nervous system during their life cycle. NM and Tb are extracellular pathogens while ZE is obligatory intracellular, targetting immune privileged sites. By using interactomics and comparative evolutionary analysis we studied whether conserved human proteins are targeted by these pathogens. We examined 2797 unique pathogen-targeted human proteins. The information derived from orthology searches of experimentally validated protein-protein interactions (PPIs) resulted both in unique and shared PPIs for each pathogen. Comparing and analyzing conserved and pathogen-specific infection pathways for NM, TB and ZE, we identified human proteins predicted to be targeted in at least two of the compared host-pathogen networks. However, four proteins were common to all three host-pathogen interactomes: the elongation factor 1-alpha 1 (EEF1A1), the SWI/SNF complex subunit SMARCC2 (matrix-associated actin-dependent regulator of chromatin subfamily C), the dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 (RPN1), and the tubulin beta-5 chain (TUBB). These four human proteins all are also involved in cytoskeleton and its regulation and are often addressed by various human pathogens. Specifically, we found (i) 56 human pathogenic bacteria and viruses that target these four proteins, (ii) the well researched new pandemic pathogen SARS-CoV-2 targets two of these four human proteins and (iii) nine human pathogenic fungi (yet another evolutionary distant organism group) target three of the conserved proteins by 130 high confidence interactions.

Computational Enzyme Engineering Pipelines for Optimized Production of Renewable Chemicals.

To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.

Topological Analysis of the Carbon-Concentrating CETCH Cycle and a Photorespiratory Bypass Reveals Boosted CO2-Sequestration by Plants.

Synthetically designed alternative photorespiratory pathways increase the biomass of tobacco and rice plants. Likewise, some in planta-tested synthetic carbon-concentrating cycles (CCCs) hold promise to increase plant biomass while diminishing atmospheric carbon dioxide burden. Taking these individual contributions into account, we hypothesize that the integration of bypasses and CCCs will further increase plant productivity. To test this in silico, we reconstructed a metabolic model by integrating photorespiration and photosynthesis with the synthetically designed alternative pathway 3 (AP3) enzymes and transporters. We calculated fluxes of the native plant system and those of AP3 combined with the inhibition of the glycolate/glycerate transporter by using the YANAsquare package. The activity values corresponding to each enzyme in photosynthesis, photorespiration, and for synthetically designed alternative pathways were estimated. Next, we modeled the effect of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (CETCH), which is a set of natural and synthetically designed enzymes that fix CO2 manifold more than the native Calvin-Benson-Bassham (CBB) cycle. We compared estimated fluxes across various pathways in the native model and under an introduced CETCH cycle. Moreover, we combined CETCH and AP3-w/plgg1RNAi, and calculated the fluxes. We anticipate higher carbon dioxide-harvesting potential in plants with an AP3 bypass and CETCH-AP3 combination. We discuss the in vivo implementation of these strategies for the improvement of C3 plants and in natural high carbon harvesters.