Structure, regulation, and functional diversity of microvilli on the apical domain of epithelial cells.

Microvilli are actin-based structures found on the apical aspect of many epithelial cells. In this review, we discuss different types of microvilli, as well as comparisons with actin-based sensory stereocilia and filopodia. Much is known about the actin-bundling proteins of these structures; we summarize recent studies that focus on the components of the microvillar membrane. We pay special attention to mechanisms of membrane microfilament attachment by the ezrin/radixin/moesin family and regulation of this protein family. We also discuss the NHERF family of scaffolding proteins that are found in microvilli and their role in microvilli regulation. Microvilli on cultured cells are not static structures, and their dynamics and those of their components are discussed. Finally, we mention diseases related to microvilli and outline questions that our current knowledge will allow the field to address in the near future.

Ezrin, radixin, and moesin are dispensable for macrophage migration and cellular cortex mechanics.

The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.

Crb3 is required to organize the apical domain of multiciliated cells.

Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane.

The ERM-1 membrane-binding domain directs erm-1 mRNA localization to the plasma membrane in the C. elegans embryo.

mRNA localization and transport are integral in regulating gene expression. In Caenorhabditis elegans embryos, the maternally inherited mRNA erm-1 (Ezrin/Radixin/Moesin) becomes concentrated in anterior blastomeres. erm-1 mRNA localizes within those blastomeres to the plasma membrane where the essential ERM-1 protein, a membrane-actin linker, is also found. We demonstrate that the localization of erm-1 mRNA to the plasma membrane is translation dependent and requires its encoded N-terminal, membrane-binding (FERM) domain. By perturbing translation through multiple methods, we found that erm-1 mRNA localization at the plasma membrane persisted only if the nascent peptide remained in complex with the translating mRNA. Indeed, re-coding the erm-1 mRNA coding sequence while preserving the encoded amino acid sequence did not disrupt erm-1 mRNA localization, corroborating that the information directing mRNA localization resides within its membrane-binding protein domain. A single-molecule inexpensive fluorescence in situ hybridization screen of 17 genes encoding similar membrane-binding domains identified three plasma membrane-localized mRNAs in the early embryo. Ten additional transcripts showed potential membrane localization later in development. These findings point to a translation-dependent pathway for localization of mRNAs encoding membrane-associated proteins.

Impaired breakdown of Herwig's epithelial root sheath disturbs tooth root development.

BACKGROUND: Wnt/ß-catenin signaling plays a variety of roles in both the dental epithelium and mesenchyme at most stages of tooth development. In this study, we verified the roles of Hertwig's epithelial root sheath (HERS) breakdown in tooth root development. This breakdown results in formation of epithelial cell rests of Malassez (ERM). RESULTS: Following induction of ß-catenin stabilization in the epithelium of developing tooth at the moment of HERS breakdown, HERS failed to break down for ERM formation. HERS with stabilized ß-catenin was altered into a multicellular layer enveloping elongated root dentin with higher expression of junctional proteins such as Zo-1 and E-cadherin. Importantly, this impairment of HERS breakdown led to arrest of further root elongation. In addition, the portion of root dentin enveloped by the undissociated HERS remained in a hypomineralized state. The odontoblasts showed ectopically higher expression of pyrophosphate regulators including Ank and Npp1, whereas Tnap expression was unchanged. CONCLUSIONS: Our data suggest that Wnt/ß-catenin signaling is decreased in HERS for ERM formation during root development. Furthermore, ERM formation is important for further elongation and dentin mineralization of the tooth roots. These findings may provide new insight to understand the contribution of ERM to root formation.

Phosphorylated ERM regulates meiotic maturation in mouse oocytes.

The cytoskeleton of mammal oocytes provides structural support to the plasma membrane and contributes to critical cellular dynamic processes such as nuclear positioning, germinal vesicle breakdown, spindle orientation, chromosome segregation, polar body extrusion, and transmembrane signaling pathways. The ERM family (ezrin, radixin and moesin) well known as membrane-cytoskeletal crosslinkers play a crucial role in organizing plasma membrane domains through their capacity to interact with transmembrane proteins and the underlying cytoskeleton. Recent works mainly focused on the structural analysis of the ERM family members and their binding partners, together with multiple functions in cell mitosis, have significantly advanced our understanding of the importance of membrane-cytoskeletal interactions. In the present study, we documented that p-ERM was expressed and localized at cortical and nucleus during mouse oocyte meiosis. p-ERM and microfilaments were colocalized from GV to MII during mouse oocyte maturation. After being treated with cytochalasin B (CB), the F-actin was disassembled. Meanwhile, p-ERM exhibited a diffuse cytoplasmic distribution and no special staining was detected in either the oocyte membrane or condensed chromosomes. p-ERM depletion by trim-away caused the meiotic procedure arrest with a significantly lower polar body extrusion rate. Collectively, these data demonstrate that the subcellular distribution of p-ERM is correlated with microfilaments. Meanwhile, the p-ERM contributes to the first polar extrusion but does not regulate the microfilament assembly.

Genetic diversity of macrolides resistant Staphylococcus aureus clinical isolates and the potential synergistic effect of vitamins, C and K3.

BACKGROUND: Macrolide antibiotics have been extensively used for the treatment of Staphylococcus aureus infections. However, the emergence of macrolide-resistant strains of S. aureus has become a major concern for public health. The molecular mechanisms underlying macrolide resistance in S. aureus are complex and diverse, involving both target site modification and efflux pump systems. In this study, we aim to overcome the molecular diversity of macrolide resistance mechanisms in S. aureus by identifying common molecular targets that could be exploited for the development of novel therapeutics. METHODS: About 300 Staphylococcus aureus different isolates were recovered and purified from 921 clinical specimen including urine (88), blood (156), sputum (264), nasal swabs (168), pus (181) and bone (39) collected from different departments in Tanta University Hospital. Macrolide resistant isolates were detected and tested for Multi Drug Resistant (MDR). Gel electrophoresis was performed after the D test and PCR reaction for erm(A), (B), (C), msr(A), and mph(C) genes. Finally, we tried different combinations of Erythromycin or Azithromycin antibiotics with either vitamin K3 or vitamin C. RESULTS: Macrolide resistance S. aureus isolates exhibited 7 major resistance patterns according to number of resistance markers and each pattern included sub patterns or subgroups. The PCR amplified products of different erm genes; analysis recorded different phenotypes of the Staphylococcus aureus isolates according to their different genotypes. In addition, our new tested combinations of Erythromycin and vitamin C, Erythromycin, and vitamin K3, Azithromycin and vitamin C and Azithromycin and vitamin K3 showed significant antibacterial effect when using every antibiotic alone. Our findings provide new insights into the molecular mechanisms of macrolide resistance in S. aureus and offer potential strategies for the development of novel protocols to overcome this emerging public health threat.

The impact of Vitreo-Macular interface abnormalities on the response to Anti-VEGF therapy for centre involving diabetic macular oedema.

BACKGROUND: The influence of Vitreomacular Interface Abnormalities (VMIA) such as Epiretinal Membrane (ERM) and/or vitreomacular traction (VMT) on the response of patients with Centre Involving Diabetic Macular Edema (CIDME) to standard of care Anti-VEGF medications is under-researched. The aims of this study were: 1) To determine the incidence of VMIA at baseline and 12 months amongst treatment naive patients commencing anti-VEGF treatment 2) To compare the response to Anti-VEGF medications at 3 monthly intervals for 12 months in a large cohort of patients with and without VMIA on their baseline OCT scan. Response was determined in terms of: number of injections, central macular thickness and visual acuity. METHODS: A retrospective case notes review of treatment naïve patients with newly diagnosed CIDME. Included patients had been commenced on intravitreal Anti-VEGF injections (ranibizumab or aflibercept) at a single centre. Inclusion criteria were: treatment naïve DME patients with a CMT of 400µ or more receiving anti-VEGF treatment with at least 12 months follow up and in whom macular OCT scans and visual acuity (VA) measurements were available within two weeks of baseline, 3, 6, 9 and 12 months. Exclusion criteria included: previous intravitreal therapy, previous vitrectomy, cataract surgery during the follow-up period, concurrent eye conditions affecting vision or CMT. RESULTS: 119 eyes met the inclusion criteria and underwent analysis. Groups were comparable in their baseline demographics. Baseline CMT measurements were comparable at baseline (417µ and 430µ in the No-VMIA and VMIA groups respectively) and improved to approximately 300µ in both groups. From 6 months CMT continued to improve in the no-VMIA while progressively deteriorating in the VMIA group. Change in CMT was statistically different at 12 months between the 2 groups (108µ and 79µ, p= 0.04). There was a mean of 7 injections after 12 months. CONCLUSION: Our study has shown a 46% incidence of VMIA amongst patients newly diagnosed with centre involving DME undergoing treatment with anti-VEGF injections. We have also demonstrated a significant difference in CMT and VA response to anti-VEGF treatment in patients with and without VMIA. Initial response was similar between the 2 groups up until 6 months. From 6 to 12 months significant differences in treatment response emerged. Differences in clinical response between patients with and without VMIA may help guide further prospective controlled studies and optimise treatment strategies.

Librarians' Electronic Resource Reviews Network (LERRN): a free citation database for resource reviews.

Electronic resource reviews written by librarians are a valuable way to identify potential content platforms and stay current on new resources. Resource-focused articles can also assist with learning about useful features, training others, and marketing to potential user groups. However, articles evaluating or highlighting innovative uses of resources may be published in disparate journals or online platforms and are not collocated. Small or solo-staffed libraries may not subscribe to library and information sciences databases or journals that contain reviews of electronic resources. And many of these reviews or other useful articles are open access. With this in mind, the main aim of the LERRN citation database was to create a freely available citation database that brings together electronic resource reviews and other content that can assist librarians in appraising and using electronic resources.

Intracellular and extracellular moesins differentially regulate Src activity and ß-catenin translocation to the nucleus in breast cancer cells.

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and ß-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as ß-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, ß-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and ß-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and ß-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and ß-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during Caenorhabditiselegans development.

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.

Plasmid-mediated drug resistance in mycobacteria: the tip of the iceberg?

Macrolides, such as clarithromycin, are crucial in the treatment of nontuberculous mycobacteria (NTM). NTM are notoriously innately drug resistant, which has made the dependence on macrolides for their treatment even more important. Not surprisingly, resistance to macrolides has been documented in some NTM, including Mycobacterium avium and Mycobacterium abscessus, which are the two NTM species most often identified in clinical isolates. Resistance is mediated by point mutations in the 23S ribosomal RNA or by methylation of the rRNA by a methylase (encoded by an erm gene). Chromosomally encoded erm genes have been identified in many of the macrolide-resistant isolates, but not in Mycobacterium chelonae. Now, Brown-Elliott et al. (J Clin Microbiol 61:e00428-23, 2023, https://doi.org/10.1128/JCM.00428-23) describe the identification of a new erm variant, erm(55), which was found either on the chromosome or on a plasmid in highly macrolide-resistant clinical isolates of M. chelonae. The chromosomal erm(55) gene appears to be associated with mobile elements; one gene is within a putative transposon and the second is in a large (37 kb) insertion/deletion. The plasmid carrying erm(55) also encodes type IV and type VII secretion systems, which are often linked on large mycobacterial plasmids and are hypothesized to mediate plasmid transfer. While the conjugative transfer of the erm(55)-containing plasmid between NTM has yet to be demonstrated, the inferences are clear, as evidenced by the dissemination of plasmid-mediated drug resistance in other medically important bacteria. Here, we discuss the findings of Brown-Elliott et al., and the potential ramifications on treatment of NTM infections.

Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55).

Macrolides are a mainstay of therapy for infections due to nontuberculous mycobacteria (NTM). Among rapidly growing mycobacteria (RGM), inducible macrolide resistance is associated with four chromosomal 23S rRNA methylase (erm) genes. Beginning in 2018, we detected high-level inducible clarithromycin resistance (MICs of ≥16µg/mL) in clinical isolates of Mycobacterium chelonae, an RGM species not previously known to contain erm genes. Using whole-genome sequencing, we identified a novel plasmid-mediated erm gene. This gene, designated erm(55)P, exhibits <65% amino acid identity to previously described RGM erm genes. Two additional chromosomal erm(55) alleles, with sequence identities of 81% to 86% to erm(55)P, were also identified and designated erm(55)C and erm(55)T. The erm(55)T is part of a transposon. The erm(55)P allele variant is located on a putative 137-kb conjugative plasmid, pMchErm55. Evaluation of 133 consecutive isolates from 2020 to 2022 revealed 5 (3.8%) with erm(55). The erm(55)P gene was also identified in public data sets of two emerging pathogenic pigmented RGM species: Mycobacterium iranicum and Mycobacterium obuense, dating back to 2008. In both species, the gene appeared to be present on plasmids homologous to pMchErm55. Plasmid-mediated macrolide resistance, not described previously for any NTM species, appears to have spread to multiple RGM species. This has important implications for antimicrobial susceptibility guidelines and treatment of RGM infections. Further spread could present serious consequences for treatment of other macrolide-susceptible RGM. Additional studies are needed to determine the transmissibility of pMchErm55 and the distribution of erm(55) among other RGM species.

The antimicrobial activity of cethromycin against Staphylococcus aureus and compared with erythromycin and telithromycin.

BACKGROUND: This study aims to explore the antibacterial activity of cethromycin against Staphylococcus aureus (S. aureus), and its relationship with multilocus sequence typing (MLST), erythromycin ribosomal methylase (erm) genes and macrolide-lincosamide-streptogramin B (MLSB) phenotypes of S. aureus. RESULTS: The minimum inhibitory concentrations (MICs) of cethromycin against 245 S. aureus clinical isolates ranged from 0.03125 to ≥ 8 mg/L, with the resistance of 38.8% in 121 methicillin-resistant S. aureus (MRSA). This study also found that cethromycin had strong antibacterial activity against S. aureus, with the MIC ≤ 0.5 mg/L in 55.4% of MRSA and 60.5% of methicillin-sensitive S. aureus (MSSA), respectively. The main MLSTs of 121 MRSA were ST239 and ST59, and the resistance of ST239 isolates to cethromycin was higher than that in ST59 isolates (P = 0.034). The top five MLSTs of 124 MSSA were ST7, ST59, ST398, ST88 and ST120, but there was no difference in the resistance of MSSA to cethromycin between these STs. The resistance of ermA isolates to cethromycin was higher than that of ermB or ermC isolates in MRSA (P = 0.016 and 0.041, respectively), but the resistance of ermB or ermC isolates to cethromycin was higher than that of ermA isolates in MSSA (P = 0.019 and 0.026, respectively). The resistance of constitutive MLSB (cMLSB) phenotype isolates to cethromycin was higher than that of inducible MLSB (iMLSB) phenotype isolates in MRSA (P < 0.001) or MSSA (P = 0.036). The ermA, ermB and ermC genes was mainly found in ST239, ST59 and ST1 isolates in MRSA, respectively. Among the MSSA, the ermC gene was more detected in ST7, ST88 and ST120 isolates, but more ermB genes were detected in ST59 and ST398 isolates. The cMLSB phenotype was more common in ST239 and ST59 isolates of MRSA, and was more frequently detected in ST59, ST398, and ST120 isolates of MSSA. CONCLUSION: Cethromycin had strong antibacterial activity against S. aureus. The resistance of MRSA to cethromycin may had some clonal aggregation in ST239. The resistance of S. aureus carrying various erm genes or MLSB phenotypes to cethromycin was different.

Ericoid mycorrhizal fungi mediate the response of ombrotrophic peatlands to fertilization: a modeling study.

Ericaceous shrubs adapt to the nutrient-poor conditions in ombrotrophic peatlands by forming symbiotic associations with ericoid mycorrhizal (ERM) fungi. Increased nutrient availability may diminish the role of ERM pathways in shrub nutrient uptake, consequently altering the biogeochemical cycling within bogs. To explore the significance of ERM fungi in ombrotrophic peatlands, we developed the model MWMmic (a peat cohort-based biogeochemical model) into MWMmic-NP by explicitly incorporating plant-soil nitrogen (N) and phosphorus (P) cycling and ERM fungi processes. The new model was applied to simulate the biogeochemical cycles in the Mer Bleue (MB) bog in Ontario, Canada, and their responses to fertilization. MWMmic_NP reproduced the carbon(C)-N-P cycles and vegetation dynamics observed in the MB bog, and their responses to fertilization. Our simulations showed that fertilization increased shrub biomass by reducing the C allocation to ERM fungi, subsequently suppressing the growth of underlying Sphagnum mosses, and decreasing the peatland C sequestration. Our species removal simulation further demonstrated that ERM fungi were key to maintaining the shrub-moss coexistence and C sink function of bogs. Our results suggest that ERM fungi play a significant role in the biogeochemical cycles in ombrotrophic peatlands and should be considered in future modeling efforts.

Surgical Management of Diabetic Macular Edema.

PURPOSE OF REVIEW: Diabetic macular edema (DME) is the accumulation of fluid in the extracellular space within the macula and is a major cause of visual impairment among patients with diabetes. First-line treatment for DME includes pharmacotherapy with intravitreal anti-vascular endothelial growth factor medications and intravitreal corticosteroids. Alternative therapeutic strategies include laser photocoagulation for non-center involving DME, and surgical options such as pars plana vitrectomy (PPV) with or without internal limiting membrane (ILM) peel in cases with vitreoretinal interface anomalies or DME refractory to pharmacotherapy, and the Port Delivery System (PDS) for sustained release of anti-vascular endothelial growth factor (VEGF) medication. Our aim is to review the existing literature on surgical management of DME including imaging changes in chronic DME and the clinical relevance of surgical intervention. RECENT FINDINGS: Imaging changes associated with DME and a worse prognosis include disorganization of the retinal layer, disruption of both the external limiting membrane (ELM) and ellipsoid zone, and vitreomacular interface abnormalities. Studies involving pars plana vitrectomy with and without ILM peel show anatomic improvement but may not always be associated with significant change in visual outcomes. Early studies lacked detailed imaging of the retinal layers and PPV was likely performed as a last resort. In addition, the novel PDS is surgically implanted into the pars plana and works as a drug reservoir with controlled release of drug. However, it has been recalled in patients with wet age-related macular degeneration due to issues with dislodgement. Surgical interventions for DME include pars plana vitrectomy with and without ILM peel and new surgical therapies for DME such as the PDS and subretinal gene therapy have the potential to reduce the risk of DME progression.

Antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii isolated from patients with mastadenitis.

To investigate the antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii (C. kroppenstedtii) isolated from patients with mastadenitis. Ninety C. kroppenstedtii clinical isolates were obtained from clinical specimens in 2018-2019. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed by the broth microdilution method. The resistance genes were detected using PCR and DNA sequencing. The results of antimicrobial susceptibility testing indicated that the resistance rates of C. kroppenstedtii to erythromycin and clindamycin, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole were 88.9%, 88.9%, 67.8%, 62.2%, and 46.6%, respectively. None of the C. kroppenstedtii isolates was resistant to rifampicin, linezolid, vancomycin, or gentamicin. The gene of erm(X) was detected in all clindamycin and erythromycin-resistant strains. The gene of sul(1) and tet(W) were detected among all trimethoprim sulfamethoxazole-resistant strains and tetracycline-resistant strains, respectively. Furthermore, 1 or 2 amino acid mutations (mainly single mutation) were observed in the gyrA gene among ciprofloxacin-resistant strains.

Commutability evaluation of candidate reference materials and ERM-DA470k/IFCC for immunoglobulin M using two international approaches.

BACKGROUND: This study aimed to assess the commutability of frozen pooled human serum (PHS), high concentration of Immunoglobulin M (IgM) pure diluted materials (HPDM), commercialized pure materials (CPM), and dilutions of ERM-DA470k/IFCC in IgM detection using the CLSI and IFCC approaches, to support standardization or harmonization of IgM measurement. METHODS: Twenty-four serum samples, relevant reference materials (PHS, HPDM, CPM), and different ERM-DA470k/IFCC dilutions were analyzed in triplicate using six routine methods. The commutability of the relevant reference materials was carried out following CLSI EP30-A and IFCC bias analysis. RESULTS: According to the CLSI approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 13, 15, 13, and 8 of 15 assay combinations, respectively. Using the IFCC approach, low, medium, and high concentrations of PHS, HPDM, and CPM were commutable on 10, 11, 9, 15, and 10 of 15 assay combinations, respectively. The ERM-DA470k/IFCC dilutions with D-PBS and RPMI-1640 Medium were commutable on 13 of 15 assay combinations according to CLSI and were commutable on all 15 assay combinations using IFCC approach. CONCLUSIONS: High concentration of PHS were commutable on all six detection systems using the CLSI approach. Low and medium concentration of PHS showed unsatisfied commutability. HPDM, not CPM have good commutability, has the potential to become reference materials. ERM-DA470k/IFCC diluted with different medium showed different commutability.

Exploring Yersinia ruckeri (O1 Biotype 2) infection in three early life-stages of rainbow trout.

Enteric redmouth disease (ERM) caused by the enterobacterium Yersinia ruckeri poses a significant threat to salmonid aquaculture globally. Despite decades of experimental infection studies, key knowledge gaps remain regarding the onset of disease susceptibility and mechanisms of immunity during early developmental stages, undermining disease management efforts in all susceptible life-stages. In this study, a series of immersion challenges were conducted, challenging and re-challenging rainbow trout Oncorhynchus mykiss (Walbaum) at 7, 14 and 51 d post-hatch (dph; mean weights = 0.085, 0.1 and 2.0 g respectively) to high concentrations (1.72 × 107-1.1 × 108 CFU) of Y. ruckeri at 15°C. This study indicates the hitherto unknown initial point of susceptibility to infection as the time of first ingestion of exogenous food (14 dph), and shows that individuals surviving primary challenge at 14 dph are significantly more likely to survive re-challenge at 51 dph compared with naive individuals (hazard ratio = 1.446, p = 0.032). Other key findings include large variation in mortality between different development-stages, from 21.1% at 14 dph to 81.2% at 51 dph, and novel age-dependent symptoms not reported previously. Results from this study enhance our understanding of ERM in juvenile rainbow trout and inform the development of improved aquatic animal health management strategies, thereby contributing to the productivity and sustainability of salmonid aquaculture into the future.

Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance.

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.