Cell Rep Methods. 2023 Dec 3:100669. doi: 10.1016/j.crmeth.2023.100669. Online ahead of print.

ABSTRACT

We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory "wild-type" E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.

PMID:38086386 | DOI:10.1016/j.crmeth.2023.100669

Eur J Med Chem. 2023 Dec 11;264:116020. doi: 10.1016/j.ejmech.2023.116020. Online ahead of print.

ABSTRACT

The development of new antiviral agents such as nucleoside analogues or acyclic nucleotide analogues (ANPs) and prodrugs thereof is an ongoing task. We report on the synthesis of three types of lipophilic triphosphate analogues of (R)-PMPA and dialkylated diphosphate analogues of (R)-PMPA. A highly selective release of the different nucleotide analogues ((R)-PMPA-DP, (R)-PMPA-MP, and (R)-PMPA) from these compounds was achieved. All dialkylated (R)-PMPA-prodrugs proved to be very stable in PBS as well as in CEM/0 cell extracts and human plasma. In primer extension assays, both the monoalkylated and the dialkylated (R)-PMPA-DP derivatives acted as (R)-PMPA-DP as a substrate for HIV-RT. In contrast, no incorporation events were observed using human polymerase γ. The dialkylated (R)-PMPA-compounds exhibited significant anti-HIV efficacy in HIV-1/2 infected cells (CEM/0 and CEM/TK-). Remarkably, the dialkylated (R)-PMPA-MP derivative 9a showed a 326-fold improved activity as compared to (R)-PMPA in HIV-2 infected CEM/TK- cells as well as a very high SI of 14,000. We are convinced that this study may significantly contribute to advancing antiviral agents developed based on nucleotide analogues in the future.

PMID:38086193 | DOI:10.1016/j.ejmech.2023.116020

Nat Commun. 2023 Dec 12;14(1):8245. doi: 10.1038/s41467-023-43999-1.

ABSTRACT

Pluripotent stem cell-derived organoids can recapitulate significant features of organ development in vitro. We hypothesized that creating human heart organoids by mimicking aspects of in utero gestation (e.g., addition of metabolic and hormonal factors) would lead to higher physiological and anatomical relevance. We find that heart organoids produced using this self-organization-driven developmental induction strategy are remarkably similar transcriptionally and morphologically to age-matched human embryonic hearts. We also show that they recapitulate several aspects of cardiac development, including large atrial and ventricular chambers, proepicardial organ formation, and retinoic acid-mediated anterior-posterior patterning, mimicking the developmental processes found in the post-heart tube stage primitive heart. Moreover, we provide proof-of-concept demonstration of the value of this system for disease modeling by exploring the effects of ondansetron, a drug administered to pregnant women and associated with congenital heart defects. These findings constitute a significant technical advance for synthetic heart development and provide a powerful tool for cardiac disease modeling.

PMID:38086920 | DOI:10.1038/s41467-023-43999-1

Sci Rep. 2023 Dec 12;13(1):22089. doi: 10.1038/s41598-023-49017-0.

ABSTRACT

Microorganisms are ubiquitously distributed in nature and usually appear as biofilms attached to a variety of surfaces. Here, we report the development of a thick biofilm in the drain pipe of several standard laboratory ice machines, and we describe and characterise, through culture-dependent and -independent techniques, the composition of this oligotrophic microbial community. By using culturomics, 25 different microbial strains were isolated and taxonomically identified. The 16S rRNA high-throughput sequencing analysis revealed that Bacteroidota and Proteobacteria were the most abundant bacterial phyla in the sample, followed by Acidobacteriota and Planctomycetota, while ITS high-throughput sequencing uncovered the fungal community was clearly dominated by the presence of a yet-unidentified genus from the Didymellaceae family. Alpha and beta diversity comparisons of the ice machine microbial community against that of other similar cold oligotrophic and/or artificial environments revealed a low similarity between samples, highlighting the ice machine could be considered a cold and oligotrophic niche with a unique selective pressure for colonisation of particular microorganisms. The recovery and analysis of high-quality metagenome-assembled genomes (MAGs) yielded a strikingly high rate of new species. The functional profiling of the metagenome sequences uncovered the presence of proteins involved in extracellular polymeric substance (EPS) and fimbriae biosynthesis and also allowed us to detect the key proteins involved in the cold adaptation mechanisms and oligotrophic metabolic pathways. The metabolic functions in the recovered MAGs confirmed that all MAGs have the genes involved in psychrophilic protein biosynthesis. In addition, the highest number of genes for EPS biosynthesis was presented in MAGs associated with the genus Sphingomonas, which was also recovered by culture-based method. Further, the MAGs with the highest potential gene number for oligotrophic protein production were closely affiliated with the genera Chryseoglobus and Mycobacterium. Our results reveal the surprising potential of a cold oligotrophic microecosystem within a machine as a source of new microbial taxa and provide the scientific community with clues about which microorganisms are able to colonise this ecological niche and what physiological mechanisms they develop. These results pave the way to understand how and why certain microorganisms can colonise similar anthropogenic environments.

PMID:38086912 | DOI:10.1038/s41598-023-49017-0

Nat Commun. 2023 Dec 12;14(1):8224. doi: 10.1038/s41467-023-43969-7.

ABSTRACT

Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

PMID:38086853 | DOI:10.1038/s41467-023-43969-7

Nat Commun. 2023 Dec 13;14(1):8260. doi: 10.1038/s41467-023-43917-5.

ABSTRACT

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet's ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

PMID:38086839 | DOI:10.1038/s41467-023-43917-5

Sci Rep. 2023 Dec 12;13(1):22020. doi: 10.1038/s41598-023-49179-x.

ABSTRACT

Chromosome conformation capture (3C) sequencing approaches, like Hi-C or micro-C, allow for an unbiased view of chromatin interactions. Most analysis methods rely on so-called interaction matrices, which are derived from counting read pairs in bins of fixed size. Here, we propose the Voronoi diagram, as implemented in Voronoi for chromosome conformation capture data visualization (v3c-viz) to visualize 3C data. The Voronoi diagram corresponds to an adaptive-binning strategy that adapts to the local densities of points. In this way, visualization of data obtained by moderate sequencing depth pinpoint many, if not most, interesting features such as high frequency contacts. The favorable visualization properties of the Voronoi diagram indicate that the Voronoi diagram as density estimator can be used to identify high frequency contacts at a resolution approaching the typical size of enhancers and promoters. v3c-viz is available at https://github.com/imbbLab/v3c-viz .

PMID:38086827 | DOI:10.1038/s41598-023-49179-x

Small. 2023 Dec 12:e2310206. doi: 10.1002/smll.202310206. Online ahead of print.

ABSTRACT

Point-of-care testing (POCT) is experiencing a groundbreaking transformation with microfluidic chips, which offer precise fluid control and manipulation at the microscale. Nevertheless, chip design or operation for existing platforms is rather cumbersome, with some even heavily depending on external drivers or devices, impeding their broader utilization. This study develops a unique programmable gravity self-driven microfluidic chip (PGSMC) capable of simultaneous multi-reagent sequential release, multi-target analysis, and multi-chip operation. All necessary reagents are introduced in a single step, and the process is initiated simply by flipping the PGSMC vertically, eliminating the need for additional steps or devices. Additionally, it demonstrates successful immunoassays in less than 60 min for antinuclear antibodies testing, compared to more than 120 min by traditional methods. Assessment using 25 clinically diagnosed cases showcases remarkable sensitivity (96%), specificity (100%), and accuracy (99%). These outcomes underscored its potential as a promising platform for POCT with high accuracy, speed, and reliability, highlighting its capability for automated fluid control.

PMID:38085133 | DOI:10.1002/smll.202310206

mBio. 2023 Dec 12:e0279023. doi: 10.1128/mbio.02790-23. Online ahead of print.

ABSTRACT

The travelers' gut microbiome is potentially assaulted by acute and chronic perturbations (e.g., diarrhea, antibiotic use, and different environments). Prior studies of the impact of travel and travelers' diarrhea (TD) on the microbiome have not directly compared antibiotic regimens, and studies of different antibiotic regimens have not considered travelers' microbiomes. This gap is important to be addressed as the use of antibiotics to treat or prevent TD-even in moderate to severe cases or in regions with high infectious disease burden-is controversial based on the concerns for unintended consequences to the gut microbiome and antimicrobial resistance (AMR) emergence. Our study addresses this by evaluating the impact of defined antibiotic regimens (single-dose treatment or daily prophylaxis) on the gut microbiome and resistomes of deployed servicemembers, using samples collected during clinical trials. Our findings indicate that the antibiotic treatment regimens that were studied generally do not lead to adverse effects on the gut microbiome and resistome and identify the relative risks associated with prophylaxis. These results can be used to inform therapeutic guidelines for the prevention and treatment of TD and make progress toward using microbiome information in personalized medical care.

PMID:38085102 | DOI:10.1128/mbio.02790-23

Plant Cell. 2023 Dec 12:koad308. doi: 10.1093/plcell/koad308. Online ahead of print.

ABSTRACT

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NAC (NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON) transcription factor SOMBRERO. Surprisingly, breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.

PMID:38085063 | DOI:10.1093/plcell/koad308

ACS Synth Biol. 2023 Dec 12. doi: 10.1021/acssynbio.3c00363. Online ahead of print.

ABSTRACT

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

PMID:38084917 | DOI:10.1021/acssynbio.3c00363

J Biomol Struct Dyn. 2023 Dec 12:1-13. doi: 10.1080/07391102.2023.2292294. Online ahead of print.

ABSTRACT

This research is part of the research umbrella regarding the use of watery rose apple leaf [Eugenia aqueum (Burm. F) Alston] in the health sector. Infectious diseases of worms, are still a health problem with a high burden based on the incidence and mortality rates in Southeast Asian countries. Therefore, we tried to create a 3D structure of vital receptors of worms including Nicotinic Cholinergic Receptor (NCR), Acetylcholinesterase Enzymes (AE), and Phosphorylase Enzymes (PE) using homology modeling method through the SWISS-MODEL webserver, then conducted a molecular dynamics simulations using active metabolite of watery rose apple leaf i.e. 2',4'‑dihydroxy‑6'‑methoxy‑3',5'‑dimethylchalcone which was then compared with Piperazine Citrate and Pyrantel Pamoate which are anthelmintic drugs on the market. Based on the results of the study, the structure of the three receptors with high resolution was successfully obtained which was characterized by a good Ramachandran value, which was above 90%. Then the ligand structure was successfully modeled in a three-dimensional model and optimized geometrically using GaussView 5.0.8 and Gaussian09 software. In further, Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) analysis of molecular dynamics simulations ligand against the three receptors, compound 2',4'‑dihydroxy‑6'‑methoxy‑3',5'‑dimethylchalcone showed better stability than Piperazine Citrate and Pyrantel Pamoate at 50 ns simulation, although from the calculation of binding free energy Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) is not better than the two comparisons. It can be concluded that the compound 2',4'‑dihydroxy‑6'‑methoxy‑3',5'‑dimethylchalcone from watery rose apple leaf showed potential as anthelmintic candidates.Communicated by Ramaswamy H. Sarma.

PMID:38084758 | DOI:10.1080/07391102.2023.2292294

J Agric Food Chem. 2023 Dec 12. doi: 10.1021/acs.jafc.3c06868. Online ahead of print.

ABSTRACT

Because food flavor is perceived through a combination of odor and taste, an analytical method that covers both dimensions would be very beneficial for mapping the consistent product quality over the entirety of a manufacturing process. Such a method, so-called "unified flavor quantitation", has been successfully applied to several different food products in recent years. The simultaneous detection of aroma and taste compounds by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) enables the analysis and quantification of an enormously large number of compounds in a single run. To evaluate the limits of this method, chocolate, a high-fat, complex matrix, was selected. In 38 distinct commercial chocolate samples, 20 flavor-active acids, aldehydes, and sugars were analyzed after a simple, rapid extraction step followed by derivatization with 3-nitrophenylhydrazine using a single UHPLC-MS/MS method. The results obtained highlight the great potential of the "unified flavor quantitation" approach and demonstrate the possibility of high-throughput quantitation of key aroma- and taste-active molecules in a single assay.

PMID:38084639 | DOI:10.1021/acs.jafc.3c06868

Annu Int Conf IEEE Eng Med Biol Soc. 2023 Jul;2023:1-4. doi: 10.1109/EMBC40787.2023.10340045.

ABSTRACT

An accurate prediction of breast cancer is essential to help physicians make appropriate treatment recommendations to reduce the chance of excessive treatment, avoiding unnecessary anxiety for patients. Cancer prognosis is highly related to patients' genomic features, which are high-dimensional in nature. In this study, we utilize a systems biology feature selector for dimension reduction to select 20 prognostic biomarkers that are considered closely related to breast cancer prognosis from the high dimensional RNA Sequencing (RNA-Seq) data. Furthermore, we establish a graph neural network (GNN) and a multi-layer perception (MLP) graph-level readout method to better extract the underlying gene interactions from the corresponding gene interaction network (GIN). With the help of GINs, the model performs the best among all baseline models, especially in the area under the precision-recall curve (AUPRC) by as large as 23%. The results demonstrate that our approach using GNNs can successfully extract high-dimensional and complicated interactions within genomic data.

PMID:38083007 | DOI:10.1109/EMBC40787.2023.10340045

Genome Biol. 2023 Dec 11;24(1):286. doi: 10.1186/s13059-023-03127-0.

ABSTRACT

Long-read RNA sequencing has emerged as a powerful tool for transcript discovery, even in well-annotated organisms. However, assessing the accuracy of different methods in identifying annotated and novel transcripts remains a challenge. Here, we present SQANTI-SIM, a versatile tool that wraps around popular long-read simulators to allow precise management of transcript novelty based on the structural categories defined by SQANTI3. By selectively excluding specific transcripts from the reference dataset, SQANTI-SIM effectively emulates scenarios involving unannotated transcripts. Furthermore, the tool provides customizable features and supports the simulation of additional types of data, representing the first multi-omics simulation tool for the lrRNA-seq field.

PMID:38082294 | DOI:10.1186/s13059-023-03127-0

Nat Genet. 2023 Dec 11. doi: 10.1038/s41588-023-01589-3. Online ahead of print.

ABSTRACT

Most fresh bananas belong to the Cavendish and Gros Michel subgroups. Here, we report chromosome-scale genome assemblies of Cavendish (1.48 Gb) and Gros Michel (1.33 Gb), defining three subgenomes, Ban, Dh and Ze, with Musa acuminata ssp. banksii, malaccensis and zebrina as their major ancestral contributors, respectively. The insertion of repeat sequences in the Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 RGA2 (resistance gene analog 2) promoter was identified in most diploid and triploid bananas. We found that the receptor-like protein (RLP) locus, including Foc race 1-resistant genes, is absent in the Gros Michel Ze subgenome. We identified two NAP (NAC-like, activated by apetala3/pistillata) transcription factor homologs specifically and highly expressed in fruit that directly bind to the promoters of many fruit ripening genes and may be key regulators of fruit ripening. Our genome data should facilitate the breeding and super-domestication of bananas.

PMID:38082204 | DOI:10.1038/s41588-023-01589-3

Genes Immun. 2023 Dec 11. doi: 10.1038/s41435-023-00224-9. Online ahead of print.

ABSTRACT

Immune evasion is a hallmark of cancer, yet the underlying mechanisms are often unknown in many patients. Using single-cell transcriptomics analysis, we previously identified the co-stimulator CD58 as part of a cancer cell-intrinsic immune checkpoint resistance signature in patient melanoma tissue. We subsequently validated CD58 loss as a driver of immune evasion using a patient-derived co-culture model of cancer and cytotoxic tumor-infiltrating lymphocytes in a pooled single-cell perturbation experiment, where we additionally observed concurrent upregulation of PD-L1 protein expression in melanoma cells with CD58 loss. In our most recent study, we uncovered the mechanisms of immune evasion mediated by CD58 loss, including impaired T cell activation and infiltration within tumors, as well as inhibitory signaling by PD-L1 via a shared regulator, CMTM6. Thus, cancer cell-intrinsic reduction of CD58 represents a multi-faceted determinant of immune evasion. Furthermore, its reciprocal interaction with PD-L1 via CMTM6 provides critical insights into how co-inhibitory and co-stimulatory immune cues are regulated.

PMID:38082156 | DOI:10.1038/s41435-023-00224-9

Sci Rep. 2023 Dec 11;13(1):21906. doi: 10.1038/s41598-023-49225-8.

ABSTRACT

Gastric cancer (GC) is a leading cause of mortality for many people. Cancer's initiating factors are poorly understood. miR-21 has a crucial function in several malignancies, particularly GC. Furthermore, it has been shown that miR-21 is critical for the emergence and advancement of GC. This work intends to identify new genes which expression is associated with the activity of mir-21 in GC and to investigate the effect of downregulation of mir-21 on these genes and gastric tumorigenesis. We utilized the gene expression profiles of GCs from an Array database (GSE13911) from the Gene Expression Omnibus (GEO) dataset to find differentially expressed genes (DEGs) between control and gastric cancer groups. Using weighted gene correlation network analysis (WGCNA) in R, the Gene co-expression network was reconstructed. The microRNA-mRNA network was then reconstructed using the miRWalk database, and by investigating the microRNA-mRNA network, the genes that have an association with mir-21 were found. To implement the functional investigation, MKN and AGS cell lines were transfected with anti-miR-21 next. Subsequently, MTT proliferation was utilized to assess the cell's vitality. qRT-PCR was then used to evaluate the anticipated levels of gene expression in both GC cell lines. This study discovered and predicted CCL28, NR3C2, and SNYPO2 as the targets of miR-21 (GC), which are downregulated through gastric tumorigenesis, showing great potential as therapeutic and diagnostic targets. The suppression of miR-21 in gastric GC cells led to the inhibition of cell proliferation and decreased expression of CCL28, NR3C2, and SNYPO2 genes. This study established that miR-21, via downregulating these genes, contributes significantly to the development of GC. In addition, systems biology techniques identified CCL28, NR3C2, and SNYPO2 genes as possible GC surveillance and therapy components.

PMID:38081950 | DOI:10.1038/s41598-023-49225-8

Nat Commun. 2023 Dec 11;14(1):8198. doi: 10.1038/s41467-023-43624-1.

ABSTRACT

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus, catalyze hydrolysis of β-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-like bond of the four membered β-lactam ring of nitrocefin, an antibiotic β-lactam surrogate. Examination of the catalytic activities of several PSMα3 variants allowed mapping of the active sites on the amyloid fibrils' surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine arrays. Molecular dynamics simulations further illuminate the structural features of β-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed β-lactams by PSMα3 fibrils, and illustrating antibiotic degradation in actual S. aureus biofilms and live bacteria environments. Overall, this study unveils functional amyloids as catalytic agents inducing degradation of β-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms.

PMID:38081813 | DOI:10.1038/s41467-023-43624-1

Metab Eng. 2023 Dec 9:S1096-7176(23)00176-3. doi: 10.1016/j.ymben.2023.12.003. Online ahead of print.

ABSTRACT

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

PMID:38081506 | DOI:10.1016/j.ymben.2023.12.003