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Abstract Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

Many drugs have been discontinued during phase II/III breast cancer clinical trials due to lack of clinical efficacy, indicating shortcomings in predictive value of preclinical data. Nutrient availability in the tumour cell microenvironment and the dimensionality of in vitro tumour cells likely impact on drug responsiveness. Global proteomics experiments were conducted to assess the impact of nutrient availability and dimensionality of culture. Protein set enrichment analyses identified “pathways in cancer”, “focal adhesion” and “ECM receptor in interaction” related to cell culture dimensionality in MDA-MB-231 cells. In MCF-7 cells, 4 pathways were influenced by medium composition, and 2 pathways were influenced by cell culture dimensionality (2D vs. 3D). These pathways were also identified using KEGG analyses. Eight drugs were selected for investigation according to the differential expression of their putative or known target proteins. The influence of medium composition on drug effectiveness was explored using the “Melbourne Medium” (MM), developed to have nutritionally physiological levels of metabolites as compared with conventional (hyper-nutritional) cell culture medium (CM). The influence of dimensionality on drug effectiveness was also explored, using an innovative 3D viability assessment combining automated confocal microscopy and image analysis. Dimensionality of culture appeared to have a greater influence on the proteome and drug effects than variation in nutrient levels. The number of differentially expressed proteins in the different media was greater in 2D than 3D. We conclude that the risk of qualifying inactive compounds in preclinical assessment may be mitigated using additional models incorporating physiological media and 3-dimensionality.

Abstract Digital reconstruction has been instrumental in deciphering how in vitro neuron architecture shapes information flow. Emerging approaches reconstruct neural systems as networks with the aim of understanding their organization through graph theory. Computational tools dedicated to this objective build models of nodes and edges based on key cellular features such as somata, axons, and dendrites. Fully automatic implementations of these tools are readily available, but they may also be purpose-built from specialized algorithms in the form of multi-step pipelines. Here we review software tools informing the construction of network models, spanning from noise reduction and segmentation to full network reconstruction. The scope and core specifications of each tool are explicitly defined to assist bench scientists in selecting the most suitable option for their microscopy dataset. Existing tools provide a foundation for complete network reconstruction, however more progress is needed in establishing morphological bases for directed/weighted connectivity and in software validation.

Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.

ABSTRACTAmong the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.IMPORTANCEThe opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

The sexual blood stages of Plasmodium falciparum develop through five morphologically distinct stages culminating in mature crescent-shaped gametocytes that can be transmitted from the mammalian host to the mosquito vector. Here, Li et al. apply different microscopy and tomography approaches to characterize how the microtubule organizing center and cytoplasmic and nuclear microtubules are organized and oriented during these different stages in the absence of genome replication and mitosis.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron–glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

To investigate the effects of microwave (MW) treatment on hays nutritive value, five types of hay (50 g) were treated with MW for 0 (control), 20, 40, 60, and 80 s (0, 440, 880, 1320, and 1760 kJ kg−1 of MW energy, respectively) and analyzed for nutritive value prior to scanning by an electron microscope to observe microstructure changes. The dry matter (DM) % of hays were increased with increasing treatment time (p < 0.001). The improved DM and digestible organic matter in the DM were recorded from MW treated lucerne (60 s), wheat (40 s), and canola (20 s) (p < 0.001), which might have been due to the MW ruptured cell wall. The MW energy required for maximal DM digestibility improvement was linearly related to control hay crude protein content (r2 = 0.79; p < 0.001). Overall, the study showed MW treatment can increase some hays’ digestibility. In addition, the crude protein content from control hay may be a proxy to indicate DM digestibility improvement.

Abstract Background Perfluoroalkyl and polyfluoroalkyl substances (PFAS) exposure has been associated with metabolic diseases, however, the underlying molecular pathogenesis remains to be understood. Integrated PFAS and lipidomic analysis has the potential to identify alterations in lipid metabolism pathways for exposome research. Methods A targeted LC-MS/MS method was developed for the quantification of 14 PFAS from human plasma samples (n = 96). Concurrently, high coverage lipidomics was conducted for the quantification of 665 lipid species in the same plasma samples. Linear regression models were implemented to study the association of PFAS with plasma lipidome. Results Women had lower levels of PFAS compared to men and Asian-Indians had lower levels of PFAS compared to both Chinese and Malay subjects. PFAS were positively associated with a number of lipid species from lysophospholipid, ceramide and triacylglycerol lipid classes. Phosphatidylinositol, acylcarnitine and sphingosine-1-phosphate were negatively associated with PFAS. Association studies revealed both shared and distinct relationship of PFAS with plasma lipids. Conclusions We demonstrate that the circulating levels of PFAS vary with age, ethnicity and sex within a multi-ethnic Asian population with potential implications in future biomonitoring and mitigation. Our comprehensive lipidomics methodology and association studies enabled us to characterize the relationship of circulating PFAS and lipidomic profiles. These results will help in better understanding of the molecular basis of PFAS exposure on human health outcomes.

Background: Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory skin disorder known to result in significant psychological symptoms and impaired quality of life. However, most of these studies are limited to western countries, with limited data from other sociocultural regions. Objective: To understand the psychosocial burden of HS in the Asian context, by exploring the correlation between objective disease measures with psychosocial health and work productivity. Methods: A prospective single-center questionnaire study was conducted. A total of 45 patients with HS completed a questionnaire and examination by a dermatologist. Results: Higher objective disease severity scores (Hurley, physician global assessment, International Hidradenitis Suppurative Severity, and modified Sartorius) correlated with poorer quality of life (Dermatology Life Quality Index and Patient Global Assessment), increased anxiety and depression (hospital anxiety and depression scale) and at-work productivity loss. There was no significant correlation between objective disease severity and other domains Work productivity and activity impairment score or self-esteem (Rosenburg Self-Esteem Scale). Limitations: The main limitation of our study is the small sample size, assessment at a single time point, and lack of control cohort. Conclusion: Our findings demonstrate the impact of HS on a patient’s psychosocial well-being and emphasize the importance of treating patients from a holistic standpoint.

A 53-year-old lady underwent a left total knee arthroplasty. She developed a dermatitis over the left knee on postoperative day 5, which worsened over the next 2 weeks and spread to the trunk despite regular topical corticosteroids and oral antihistamines. Physical examination revealed an erythematous plaque over the left knee and urticated plaques over the neck. She was given a course of oral and topical corticosteroids with resolution. Subsequent patch testing showed a showing ++ reactions to gold and nickel, and + reactions to copper, palladium, rhodium, titanium, vanadium, zinc, and hydroquinone. Orthopedic implants contain metal alloys, which may include nickel, cobalt. Hypersensitivity to implants allergy may arise from the metal alloy or bone cement. Metal hypersensitivity reactions (MHR) can manifest as a local or systemic contact dermatitis weeks to months following exposure. The role of MHR in contributing to prosthesis failure is conflicting. In patients with no history of metal allergy, pre-implant patch testing is not routinely recommended as a positive patch test does not consistently predict in vivo metal-induced complication. MHR may be managed conservatively with good outcomes. However, in patients with MHR and implant failure, or in a preoperative patient with a proven and clinically relevant hypersensitivity, replacement of the implant, or implant with a titanium or oxidized zirconium alloy should be considered.

Do lymphatic vessels support cancer cells? Or are they vessels that help suppress cancer development? It is known that the lymphatic system is a vehicle for tumor metastasis and that the lymphangiogenic regulator VEGF-C supports the tumor. One such role of VEGF-C is the suppression of the immune response to cancer. The lymphatic system has also been correlated with an increase in interstitial fluid pressure of the tumor microenvironment. On the other hand, lymphatic vessels facilitate immune surveillance to mount an immune response against tumors with the support of VEGF-C. Furthermore, the activation of lymphatic fluid drainage may prove to filter and decrease tumor interstitial fluid pressure. In this review, we provide an overview of the dynamic between lymphatics, cancer, and tumor fluid pressure to suggest that lymphatic vessels may be used as an antitumor therapy due to their capabilities of immune surveillance and fluid pressure drainage. The application of this potential may help to prevent tumor proliferation or increase the efficacy of drugs that target cancer.

Objective To explore the phenomenon of topical corticosteroid (TCS) phobia and comprehensively understand the factors driving TCS concerns, in particular pertaining to steroid addiction and withdrawal.Design Prospective qualitative study using 1:1 in-depth semistructured interviews and analysed using grounded theory.Participants Patients with a prior experience of TCS use for a dermatological condition recruited from a tertiary academic dermatology clinic, or through word of mouth and online social media platforms.Results 26 participants encompassing those with positive, neutral and negative opinions towards TCS were interviewed. 13 reported having topical steroid addiction or withdrawal. The drivers of TCS concerns could be categorised into seven themes: attitudes towards TCS (comprising beliefs and knowledge about TCS), availability of alternatives, treatment inconvenience, personality, patient’s ongoing evaluation of clinical response to TCS, doctor–patient relationship and healthcare-seeking behaviour. Of mention, patients placed high value and trust on their own experiences with TCS, such as their perceived experienced side effects. The doctor who failed to acknowledge the patient’s opinions and instead emphasised the safety of TCS was often viewed as dismissive, resulting in a deteriorating patient–doctor relationship.Conclusion Provision of knowledge and education is important but may be ineffective if the basis for TCS concern regarding safety is reasonable, or when the patient has a firmly established belief supporting his/her concern. In such instances, failure to acknowledge and respect the patient’s decision to avoid TCS could worsen the doctor–patient relationship.

Oral lichen planus (LP) is a common manifestation in patients with LP; however, isolated lip LP is rare and may mimic other conditions such as lichenoid drug eruptions, actinic cheilitis, and early carcinoma in situ in the absence of typical skin lesions. Actinic lichen planus (ALP) is a variant of LP occurring on light-exposed areas in patients with dark skin. We report the case of a Chinese female with isolated ALP of the lower lip, mimicking herpes simplex infection at presentation. The presence of prognathism, involvement of the lower lip, and flares associated with sunlight reinforces the role of sun exposure in the development of this condition.

RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONOSFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches. [ABSTRACT FROM AUTHOR]