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Zinc finger proteins (ZNF), a unique yet diverse group of proteins, play pivotal roles in fundamental cellular mechanisms including transcription regulation, chromatin remodeling, protein/RNA homeostasis, and DNA repair. Consequently, the mis regulation of ZNF proteins can result in a variety of human diseases, ranging from neurodevelopmental disorders to several cancers. Considering the promising results of DNA damage repair (DDR) inhibition in the clinic, as a therapeutic strategy for patients with homologous recombination (HR) deficiency, identifying other potential targetable DDR proteins as emerged vulnerabilities in resistant tumor cells is essential, especially when considering the burden of acquired drug resistance. Importantly, there are a growing number of studies identifying new ZNFs and revealing their significance in several DDR pathways, highlighting their great potential as new targets for DDR-inhibition therapy. Although, there are still many uncharacterized ZNF-containing proteins with unknown biological function. In this review, we highlight the major classes and observed biological functions of ZNF proteins in mammalian cells. We briefly introduce well-known and newly discovered ZNFs and describe their molecular roles and contributions to human health and disease, especially cancer. Finally, we discuss the significance of ZNFs in DNA repair mechanisms, their potential in cancer therapy and advances in exploiting ZNF proteins as future therapeutic targets for human disease.
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Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.
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Making a measurement over millions of nanoparticles or exposed crystal facets seldom reports on reactivity of a single nanoparticle or facet, which may depart drastically from ensemble measurements. Within the past 30 years, science has moved toward studying the reactivity of single atoms, molecules, and nanoparticles, one at a time. This shift has been fueled by the realization that everything changes at the nanoscale, especially important industrially relevant properties like those important to electrocatalysis. Studying single nanoscale entities, however, is not trivial and has required the development of new measurement tools. This review explores a tale of the clever use of old and new measurement tools to study electrocatalysis at the single entity level. We explore in detail the complex interrelationship between measurement method, electrocatalytic material, and reaction of interest (e.g., carbon dioxide reduction, oxygen reduction, hydrazine oxidation, etc.). We end with our perspective on the future of single entity electrocatalysis with a key focus on what types of measurements present the greatest opportunity for fundamental discovery.
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The frequency of unprovoked shark bites is increasing worldwide, leading to a growing pressure for mitigation measures to reduce shark-bite risk while maintaining conservation objectives. Personal shark deterrents are a promising and non-lethal strategy that can protect ocean users, but few have been independently and scientifically tested. In Australia, bull (Carcharhinus leucas), tiger (Galeocerdo cuvier), and white sharks (Carcharodon carcharias) are responsible for the highest number of bites and fatalities. We tested the effects of two electric deterrents (Ocean Guardian's Freedom+ Surf and Freedom7) on the behaviour of these three species. The surf product reduced the probability of bites by 54% across all three species. The diving product had a similar effect on tiger shark bites (69% reduction) but did not reduce the frequency of bites from white sharks (1% increase), likely because the electrodes were placed further away from the bait. Electric deterrents also increased the time for bites to occur, and frequency of reactions and passes for all species tested. Our findings reveal that both Freedom+ Surf and Freedom7 electric deterrents affect shark behaviour and can reduce shark-bite risk for water users, but neither product eliminated the risk of shark bites entirely. The increasing number of studies showing the ability of personal electric deterrents to reduce shark-bite risk highlights personal protection as an effective and important part of the toolbox of shark-bite mitigation measures.
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Mordeduras e Picadas , Tubarões , Animais , Tubarões/fisiologia , Mordeduras e Picadas/prevenção & controle , Austrália , Conservação dos Recursos Naturais/métodos , Humanos , EletricidadeRESUMO
The innate immune system eliminates bloodstream pathogens such as Escherichia coli in part through complement protein deposition and subsequent bacterial death (i.e., "serum killing"). Some E. coli strains have developed mechanisms to resist serum killing, though the extent of variation in serum killing among bloodstream infection (BSI) isolates and the clinical impact of this variation is not well understood. To address this issue, we developed a novel assay that uses flow cytometry to perform high throughput serum bactericidal assays (SBAs) with E. coli BSI isolates (n = 183) to define the proportion of surviving bacteria after exposure to serum. We further determined whether E. coli resistance to serum killing is associated with clinical outcomes (e.g., in-hospital attributable mortality, in-hospital total mortality, septic shock) and bacterial genotype in the corresponding patients with E. coli BSI. Our novel flow cytometry-based SBA performed similarly to a traditional SBA, though with significantly decreased hands-on bench work. Among E. coli BSI isolates, the mean proportion that survived exposure to 25% serum was 0.68 (Standard deviation 0.02, range 0.57-0.93). We did not identify associations between E. coli resistance to serum killing and clinical outcomes in our adjusted models. Together, this study describes a novel flow cytometry-based approach to the bacterial SBA that allowed for high-throughput testing of E. coli BSI isolates and identified high variability in resistance to serum killing among a large set of BSI isolates.
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Infecções por Escherichia coli , Escherichia coli , Citometria de Fluxo , Humanos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/imunologia , Bacteriemia/microbiologia , Bacteriemia/imunologia , Bacteriemia/mortalidade , Atividade Bactericida do Sangue , Masculino , FemininoRESUMO
A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + ß barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.
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Corantes , Oxirredução , Peroxidases , Multimerização Proteica , Streptomyces lividans , Streptomyces lividans/enzimologia , Peroxidases/química , Peroxidases/metabolismo , Corantes/química , Corantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Especificidade por Substrato , Microscopia Crioeletrônica , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismoRESUMO
The intestinal microbiota has a pervasive influence on mammalian innate immunity fortifying defenses to infection in tissues throughout the host. How intestinal microbes control innate defenses in systemic tissues is, however, poorly defined. In our opinion, there are three core challenges that need addressing to advance our understanding of how the intestinal microbiota controls innate immunity systemically: first, deciphering how signals from intestinal microbes are transmitted to distal tissues; second, unraveling how intestinal microbes prime systemic innate immunity without inducing widespread immunopathology; and third, identifying which intestinal microbes control systemic immunity. Here, we propose answers to these problems which provide a framework for understanding how microbes in the intestine can regulate innate immunity systemically.
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Microbioma Gastrointestinal , Microbiota , Animais , Humanos , Imunidade Inata , Intestinos , Mucosa Intestinal , MamíferosRESUMO
Friction taper stitch welding (FTSW) is a novel technique that uses multiple inserts to conceal surface crack in a given substrate. The inserts are rotated and forced to fill the crack as plasticized material, and forge with the substrate in solid-state. The process is well suited for alloys such as duplex stainless steel, which suffers degradation of properties during fusion welding. A detailed experimental and theoretical investigation is presented here on FTSW of a duplex stainless steel (DSS). The experimental results show the presence of a ferrite-rich phase along the interface. The results computed by the numerical process model reveal a direct influence of thermal cycle in the amount of ferrite along the joint interface. The welded joint shows near homogeneous structure and properties similar to those of the substrate.
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Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from -150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.
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Elétrons , Geobacter , Hidroquinonas/metabolismo , Geobacter/metabolismo , Proteínas de Bactérias/química , Transporte de Elétrons , Oxirredução , Citocromos c/metabolismo , Quinonas/metabolismoRESUMO
Introduction: In this study, we demonstrate the feasibility of yeast surface display (YSD) and nextgeneration sequencing (NGS) in combination with artificial intelligence and machine learning methods (AI/ML) for the identification of de novo humanized single domain antibodies (sdAbs) with favorable early developability profiles. Methods: The display library was derived from a novel approach, in which VHH-based CDR3 regions obtained from a llama (Lama glama), immunized against NKp46, were grafted onto a humanized VHH backbone library that was diversified in CDR1 and CDR2. Following NGS analysis of sequence pools from two rounds of fluorescence-activated cell sorting we focused on four sequence clusters based on NGS frequency and enrichment analysis as well as in silico developability assessment. For each cluster, long short-term memory (LSTM) based deep generative models were trained and used for the in silico sampling of new sequences. Sequences were subjected to sequence- and structure-based in silico developability assessment to select a set of less than 10 sequences per cluster for production. Results: As demonstrated by binding kinetics and early developability assessment, this procedure represents a general strategy for the rapid and efficient design of potent and automatically humanized sdAb hits from screening selections with favorable early developability profiles.
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In this study, we generated a novel library approach for high throughput de novo identification of humanized single-domain antibodies following camelid immunization. To achieve this, VHH-derived complementarity-determining regions-3 (CDR3s) obtained from an immunized llama (Lama glama) were grafted onto humanized VHH backbones comprising moderately sequence-diversified CDR1 and CDR2 regions similar to natural immunized and naïve antibody repertoires. Importantly, these CDRs were tailored toward favorable in silico developability properties, by considering human-likeness as well as excluding potential sequence liabilities and predicted immunogenic motifs. Target-specific humanized single-domain antibodies (sdAbs) were readily obtained by yeast surface display. We demonstrate that, by exploiting this approach, high affinity sdAbs with an optimized in silico developability profile can be generated. These sdAbs display favorable biophysical, biochemical, and functional attributes and do not require any further sequence optimization. This approach is generally applicable to any antigen upon camelid immunization and has the potential to significantly accelerate candidate selection and reduce risks and attrition rates in sdAb development.
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Anticorpos de Domínio Único , Humanos , Imunização , Biblioteca Gênica , Antígenos , Regiões Determinantes de Complementaridade/químicaRESUMO
The intestine is the primary colonisation site for carbapenem-resistant Enterobacteriaceae (CRE) and serves as a reservoir of CRE that cause invasive infections (e.g. bloodstream infections). Broad-spectrum antibiotics disrupt colonisation resistance mediated by the gut microbiota, promoting the expansion of CRE within the intestine. Here, we show that antibiotic-induced reduction of gut microbial populations leads to an enrichment of nutrients and depletion of inhibitory metabolites, which enhances CRE growth. Antibiotics decrease the abundance of gut commensals (including Bifidobacteriaceae and Bacteroidales) in ex vivo cultures of human faecal microbiota; this is accompanied by depletion of microbial metabolites and enrichment of nutrients. We measure the nutrient utilisation abilities, nutrient preferences, and metabolite inhibition susceptibilities of several CRE strains. We find that CRE can use the nutrients (enriched after antibiotic treatment) as carbon and nitrogen sources for growth. These nutrients also increase in faeces from antibiotic-treated mice and decrease following intestinal colonisation with carbapenem-resistant Escherichia coli. Furthermore, certain microbial metabolites (depleted upon antibiotic treatment) inhibit CRE growth. Our results show that killing gut commensals with antibiotics facilitates CRE colonisation by enriching nutrients and depleting inhibitory microbial metabolites.
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Actinobacteria , Enterobacteriáceas Resistentes a Carbapenêmicos , Neoplasias Intestinais , Humanos , Animais , Camundongos , Antibacterianos/farmacologia , Bacteroidetes , Escherichia coli , NutrientesRESUMO
The intestinal microbiota regulates immunity across organ systems. Which symbionts control systemic immunity, the mechanisms they use, and how they avoid widespread inflammatory damage are unclear. We uncover host tolerance and resistance mechanisms that allow Firmicutes from the human microbiota to control systemic immunity without inducing immunopathology. Intestinal processing releases Firmicute glycoconjugates that disseminate, resulting in release of cytokine IL-34 that stimulates macrophages and enhances defenses against pneumonia, sepsis, and meningitis. Despite systemic penetration of Firmicutes, immune homeostasis is maintained through feedback control whereby IL-34-mediated mTORC1 activation in macrophages clears polymeric glycoconjugates from peripheral tissues. Smaller glycoconjugates evading this clearance mechanism are tolerated through sequestration by albumin, which acts as an inflammatory buffer constraining their immunological impact. Without these resistance and tolerance mechanisms, Firmicutes drive catastrophic organ damage and cachexia via IL-1ß. This reveals how Firmicutes are safely assimilated into systemic immunity to protect against infection without threatening host viability.
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Firmicutes , Microbiota , Humanos , Simbiose , Tolerância Imunológica , Citocinas , Interleucinas , Imunidade InataRESUMO
[This corrects the article DOI: 10.1039/D2SC06553C.].
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Deep sequencing has revealed that the 16S rRNA gene composition of the human microbiome can vary between populations. However, when existing data are insufficient to address the desired study questions due to limited sample sizes, Dirichlet mixture modeling (DMM) can simulate 16S rRNA gene predictions from experimental microbiome data. We examined the extent to which simulated 16S rRNA gene microbiome data can accurately reflect the diversity within that identified from experimental data and calculate the power. Even when experimental and simulated datasets differed by less than 10%, simulation by DMM consistently overestimates power, except when using only highly discriminating taxa. Admixtures of DMM with experimental data performed poorly compared to pure simulation and did not show the same correlation with experimental data p-value and power values. While multiple replications of random sampling remain the favored method of determining the power, when the estimated sample size required to achieve a certain power exceeds the sample number, then simulated samples based on DMM can be used. We introduce an R-Package, MPrESS, to assist in power calculation and sample size estimation for a 16S rRNA gene microbiome dataset to detect a difference between populations. MPrESS can be downloaded from GitHub.
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Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone, although its clinical impact on patients with bloodstream infection (BSI) is incompletely understood. This study aims to further define the risk factors, clinical outcomes, and bacterial genetics associated with ST131 BSI. A prospectively enrolled cohort study of adult inpatients with E. coli BSI was conducted from 2002 to 2015. Whole-genome sequencing was performed with the E. coli isolates. Of the 227 patients with E. coli BSI in this study, 88 (39%) were infected with ST131. Patients with E. coli ST131 BSI and those with non-ST131 BSI did not differ with respect to in-hospital mortality (17/82 [20%] versus 26/145 [18%]; P = 0.73). However, in patients with BSI from a urinary tract source, ST131 was associated with a numerically higher in-hospital mortality than patients with non-ST131 BSI (8/42 [19%] versus 4/63 [6%]; P = 0.06) and increased mortality in an adjusted analysis (odds ratio of 5.85; 95% confidence interval of 1.44 to 29.49; P = 0.02). Genomic analyses showed that ST131 isolates primarily had an H4:O25 serotype, had a higher number of prophages, and were associated with 11 flexible genomic islands as well as virulence genes involved in adhesion (papA, kpsM, yfcV, and iha), iron acquisition (iucC and iutA), and toxin production (usp and sat). In patients with E. coli BSI from a urinary tract source, ST131 was associated with increased mortality in an adjusted analysis and contained a distinct repertoire of genes influencing pathogenesis. These genes could contribute to the higher mortality observed in patients with ST131 BSI.
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Infecções por Escherichia coli , Sepse , Infecções Urinárias , Sistema Urinário , Adulto , Humanos , Escherichia coli/genética , Estudos de Coortes , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Antibacterianos , beta-Lactamases/genéticaRESUMO
BACKGROUND: Our work aims to investigate the mechanical properties of the human posterior rectus sheath in terms of its ultimate tensile stress, stiffness, thickness and anisotropy. It also aims to assess the collagen fibre organisation of the posterior rectus sheath using Second-Harmonic Generation microscopy. METHODS: For mechanical analysis, twenty-five fresh-frozen samples of posterior rectus sheath were taken from six different cadaveric donors. They underwent uniaxial tensile stress testing until rupture either in the transverse (n = 15) or longitudinal (n = 10) plane. The thickness of each sample was also recorded using digital callipers. On a separate occasion, ten posterior rectus sheath samples and three anterior rectus sheath samples underwent microscopy and photography to assess collagen fibre organisation. FINDINGS: samples had a mean ultimate tensile stress of 7.7 MPa (SD 4.9) in the transverse plane and 1.2 MPa (SD 0.8) in the longitudinal plane (P < 0.01). The same samples had a mean Youngs modulus of 11.1 MPa (SD 5.0) in the transverse plane and 1.7 MPa (SD 1.3) in the longitudinal plane (P < 0.01). The mean thickness of the posterior rectus sheath was 0.51 mm (SD 0.13). Transversely aligned collagen fibres could be identified within the posterior sheath tissue using Second-Harmonic Generation microscopy. INTERPRETATION: The posterior rectus sheath displays mechanical and structural anisotropy with greater tensile stress and stiffness in the transverse plane compared to the longitudinal plane. The mean thickness of this layer is around 0.51 mm - consistent with other studies. The tissue is constructed of transversely aligned collagen fibres that are visible using Second-Harmonic Generation microscopy.
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Parede Abdominal , Humanos , Resistência à Tração , Anisotropia , Módulo de Elasticidade , Colágeno , Estresse MecânicoRESUMO
The vast majority of pelvic and intra-abdominal surgery is undertaken through at least one incision, through either the linea alba or the rectus sheath. These connective tissue layers are formed from the aponeuroses of the rectus muscles (anterior and posterior rectus sheath) and are vital for the structural integrity of the abdominal wall. Poor healing of these connective tissues after surgery can lead to significant morbidity for patients, who can develop unsightly and painful incisional hernias. Fibroblasts within the rectus sheath are responsible for laying down and remodeling collagen during the healing process after surgery. Despite their importance for this healing process, such cells have not been studied in vitro. In order to carry out such work, researchers must first be able to isolate these cells from human tissue and culture them successfully so they may be used for experimentation. This article provides an extensive and detailed protocol for the isolation, culture, cryopreservation, and thawing of human rectus sheath fibroblasts (RSFs). In our hands, this protocol develops confluent cultures of primary fibroblasts within 2 weeks, and sufficient cultures ready for freezing and storage after a further 2 to 4 weeks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs Alternate Protocol: Collagenase digestion of human rectus sheath and isolation of RSFs, digestion in flask Support Protocol: Cryopreservation and thawing of human RSFs.