Explore the vast range of titles available.

Influenza A virus is a major human pathogen, and its replication is widely studied. One important aspect for effective virus propagation is the host cell, since cellular properties can limit or favor virus entry, viral genome and viral protein synthesis and virus release. To establish detailed mathematical models for these processes, quantitative experimental data on the intracellular dynamics of viral compounds together with the number of infectious and non-infectious virus particles released are required. In this study, we report results obtained from an optimized mass spectrometry assay for the quantification of viral proteins that was applied to compare the production of influenza A virus HA, NP, NA, M1, and NS1 proteins for different seed viruses and host cells of batch cultures. With canine MDCK cell-adapted seed virus, a maximum of about 1.0E+08 copies/cell were found for all five viral proteins after infection of avian AGE1.CR and human HEK293 cells. These intracellular levels are about fivefold lower than in MDCK cells. However, after five passages of seed virus adaptation, intracellular protein copy numbers comparable to those in MDCK cells were achieved. Highest levels were found for the NS1 protein with about 1.0E+09 copies/cell. Furthermore, the onset of virus particle release started earlier for both cell lines (about 3–6 h). In contrast, the maximum virus titers did not change for AGE1.CR cells but increased for HEK293 cells. Nevertheless, the highest HA titers were always obtained for MDCK cells. Overall, the experimental data indicate that influenza A virus replication is different due to specifics of innate host cell immune response, viral protein production, precursor consumption, and degradation rates. Key points • Application of absolute quantification for five major proteins of influenza A virus. • NS1 protein most abundant protein with 1.0E+09 copies/cell at the end of infection. • Virus adaptation leads to earlier release and higher virus titers in HEK293 cell.

Suspension MDCK cells are a substrate for producing influenza A virus (IAV) and typically show very high virus yields compared to other animal cells. Due to the significant heterogeneity within cell populations, studying and comparing clonal cell lines with regard to specific properties, such as superior growth or higher productivity, could facilitate process optimization. In this study, we analyzed the expressed proteins of two clonal cell lines to identify intrinsic characteristics of effective IAV producers. We compared proteome changes in two human IAV PR8 (H1N1, A/PR/8/34) infected monoclonal suspension MDCK cell lines: C59, a low-yield IAV producer with fast cell growth and small cell diameter, and C113, a high-yield IAV producer with average cell growth and large cell diameter. We examined growth rate, size, metabolism and IAV production. A total of 5177 host cell proteins were detected in both cell lines using DIA-PASEF mode with a TimsTOFpro mass spectrometer. Analysis of the differentially expressed proteins revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in highly productive cells. In contrast, steroid biosynthesis and DNA replication were more active in faster-growing cells. Following infection, 122 proteins were significantly upregulated (p < 0.05, log 2 -fold change ≥1) in the high-producing cell line. These proteins were associated with membrane trafficking, interactions with the IAV-NS1 protein and virus production. Additionally, 98 proteins associated with antiviral pathways such as the proto-oncogenic receptor tyrosine kinase MET and tumor necrosis factor (TNF) signaling were downregulated (p < 0.05, log 2 -fold change ≤1). In the cell line that produced lower IAV PR8 titers, 77 proteins were downregulated and 57 were upregulated after infection. RNA metabolism appeared to be downregulated, while the tricarboxylic acid (TCA) cycle and the stress response were both upregulated. In the high-yield C113 clone, only proteins associated with apoptosis and the target of rapamycin kinase (TOR) were expressed following infection. This may indicate a more effective release of virus particles. A comparison of intracellular IAV PR8 protein levels demonstrated that M1 and NA levels were 4-fold and 8-fold higher, respectively, for the high-yield C113 cell line. These findings again suggest an improved virus release.

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

Constraint-based modeling (CBM) is increasingly used to analyze the metabolism of complex microbial communities involved in ecology, biomedicine, and various biotechnological processes. While CBM is an established framework for studying the metabolism of single species with linear stoichiometric models, CBM of communities with balanced growth is more complicated, not only due to the larger size of the multi-species metabolic network but also because of the bilinear nature of the resulting community models. Moreover, the solution space of these community models often contains biologically unrealistic solutions, which, even with model linearization and under application of certain objective functions, cannot easily be excluded. Here we present RedCom, a new approach to build reduced community models in which the metabolisms of the participating organisms are represented by net conversions computed from the respective single-species networks. By discarding (single-species) net conversions that violate a minimality criterion in the exchange fluxes, it is ensured that unrealistic solutions in the community model are excluded where a species altruistically synthesizes large amounts of byproducts (instead of biomass) to fulfill the requirements of other species. We employed the RedCom approach for modeling communities of up to nine organisms involved in typical degradation steps of anaerobic digestion in biogas plants. Compared to full (bilinear and linearized) community models, we found that the reduced community models obtained with RedCom are not only much smaller but allow, also in the largest model with nine species, extensive calculations required to fully characterize the solution space and to reveal key properties of communities with maximum methane yield and production rates. Furthermore, the predictive power of the reduced community models is significantly larger because they predict much smaller ranges of feasible community compositions and exchange fluxes still being consistent with measurements obtained from enrichment cultures. For an enrichment culture for growth on ethanol, we also used metaproteomic data to further constrain the solution space of the community models. Both model and proteomic data indicated a dominance of acetoclastic methanogens (Methanosarcinales) and Desulfovibrionales being the least abundant group in this microbial community.

Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1 m NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D-electrophoresis (2-DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry (MS) via electrospray ionization source (LC-ESI-MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid-degrading bacteria by incubation with 2,4-dichlorophenoxy acetic acid (2,4-D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene-contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP “OP7” that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.

Background Power-to-gas is the pivotal link between electricity and gas infrastructure, enabling the broader integration of renewable energy. Yet, enhancements are necessary for its full potential. In the biomethanation process, transferring H 2 into the liquid phase is a rate-limiting step. To address this, we developed a novel tubular foam-bed reactor (TFBR) and investigated its performance at laboratory scale. Results A non-ionic polymeric surfactant (Pluronic ® F-68) at 1.5% w/v was added to the TFBR’s culture medium to generate a stabilized liquid foam structure. This increased both the gas–liquid surface area and the bubble retention time. Within the tubing, cells predominantly traveled evenly suspended in the liquid phase or were entrapped in the thin liquid film of bubbles flowing inside the tube. Phase (I) of the experiment focused primarily on mesophilic (40 °C) operation of the tubular reactor, followed by phase (II), when Pluronic ® F-68 was added. In phase (II), the TFBR exhibited 6.5-fold increase in biomethane production rate ( MPR ) to 15.1 ( L CH 4 /L R /d) , with a CH 4 concentration exceeding 90% (grid quality), suggesting improved H 2 transfer. Transitioning to phase (III) with continuous operation at 55 °C, the MPR reached 29.7 L CH 4 /L R /d while maintaining the grid quality CH 4 . Despite, reduced gas–liquid solubility and gas–liquid mass transfer at higher temperatures, the twofold increase in MPR compared to phase (II) might be attributed to other factors, i.e., higher metabolic activity of the methanogenic archaea. To assess process robustness for phase (II) conditions, a partial H 2 feeding regime (12 h 100% and 12 h 10% of the nominal feeding rate) was implemented. Results demonstrated a resilient MPR of approximately 14.8 L CH 4 /L R /d even with intermittent, low H 2 concentration. Conclusions Overall, the TFBR’s performance plant sets the course for an accelerated introduction of biomethanation technology for the storage of volatile renewable energy. Robust process performance, even under H 2 starvation, underscores its reliability. Further steps towards an optimum operation regime and scale-up should be initiated. Additionally, the use of TFBR systems should be considered for biotechnological processes in which gas–liquid mass transfer is a limiting factor for achieving higher reaction rates.

Phosphonates such as ethylenediaminetetra (methylenephosphonic acid) (EDTMP) and aminotris (methylenephosphonic acid) (ATMP) are used every day in water treatment processes or in household products. Their consumption is still increasing, regardless of the debates on their environmental impact. Here, the microbial characterisation and determination of the biodegradation potential of selected industrially relevant phosphonates for the isolate Delftia sp. UMB14 is reported. The opportunistic strain was isolated from a biofilm that was derived from a conventional washing machine using conventional detergents containing phosphonates. In antimicrobial susceptibility testing, the strain was only susceptible to sulfonamide, tetracycline, and chloramphenicol. Physiological and biochemical characteristics were determined using the BIOLOG EcoPlate assay. Most importantly, the strain was shown to convert D-malic acid and D-mannitol, as confirmed for strains of Delftia lacustris, and thus the new isolate could be closely related. Biodegradation tests with different phosphonates showed that the strain preferentially degrades ATMP and EDTMP but does not degrade glyphosate (GS) and amino (methylphosphonic acid) (AMPA). A specific gene amplification confirmed the presence of phnX (phosphonoacetaldehyde hydrolase) and the absence of PhnJ (the gene for the core component of C–P lyase). The presence of PhnCDE is strongly suggested for the strain, as it is common in Delftia lacustris species.

One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions. Graphical Abstract Word Cloud showing the abundance of keywords in combination with the “Microbiome” in PubMed NCBI. As abundance values, the rounded logarithm with the base of 2 hits were used and submitted to https://wordart.com/create . For microbiome, the number without any combination was used for calculation. The word cloud displays different aspects of microbiome research: (i.) sources of microbiomes (green), (ii.) interactions (purple), (iii.) involved taxa (red), (iv.) applied experimental approaches (blue), and (vi.) societal effects and recent or future applications (gray). FN-L_zSKWXUyi3FV_c3WKh Video Abstract

Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO 2 production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea.