Essays Biochem. 2024 Apr 25:EBC20230045. doi: 10.1042/EBC20230045. Online ahead of print.

ABSTRACT

The expression of metabolic proteins is controlled by genetic circuits, matching metabolic demands and changing environmental conditions. Ideally, this regulation brings about a competitive level of metabolic fitness. Understanding how cells can achieve a robust (close-to-optimal) functioning of metabolism by appropriate control of gene expression aids synthetic biology by providing design criteria of synthetic circuits for biotechnological purposes. It also extends our understanding of the designs of genetic circuitry found in nature such as metabolite control of transcription factor activity, promoter architectures and transcription factor dependencies, and operon composition (in bacteria). Here, we review, explain and illustrate an approach that allows for the inference and design of genetic circuitry that steers metabolic networks to achieve a maximal flux per unit invested protein across dynamic conditions. We discuss how this approach and its understanding can be used to rationalize Escherichia coli's strategy to regulate the expression of its ribosomes and infer the design of circuitry controlling gene expression of amino-acid biosynthesis enzymes. The inferred regulation indeed resembles E. coli's circuits, suggesting that these have evolved to maximize amino-acid production fluxes per unit invested protein. We end by an outlook of the use of this approach in metabolic engineering applications.

PMID:38662439 | DOI:10.1042/EBC20230045

Essays Biochem. 2024 Apr 25:EBC20230102. doi: 10.1042/EBC20230102. Online ahead of print.

ABSTRACT

Computational biology is a diverse research field that has gained increasing importance over the last two decades. Broadly, it aims to apply computational approaches to advance our understanding of biological systems. This can take place on multiple levels, for example, by creating computational models of specific biological systems, by developing algorithms that assist in the analysis of experimental data, or by investigating fundamental biological design principles through modelling. The articles in this special issue highlight and review four such distinct applications of computational biology.

PMID:38662436 | DOI:10.1042/EBC20230102

Appl Microbiol Biotechnol. 2024 Apr 25;108(1):310. doi: 10.1007/s00253-024-13111-8.

ABSTRACT

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

PMID:38662130 | DOI:10.1007/s00253-024-13111-8

J Virol. 2024 Apr 25:e0036324. doi: 10.1128/jvi.00363-24. Online ahead of print.

ABSTRACT

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

PMID:38661384 | DOI:10.1128/jvi.00363-24

iScience. 2024 Apr 10;27(5):109478. doi: 10.1016/j.isci.2024.109478. eCollection 2024 May 17.

ABSTRACT

There is increasing interest in applying resilience concepts at different scales of biological organization to address major interdisciplinary challenges from cancer to climate change. It is unclear, however, whether resilience can be a unifying concept consistently applied across the breadth of the biological sciences, or whether there is limited capacity for integration. In this review, we draw on literature from molecular biology to community ecology to ascertain commonalities and shortcomings in how resilience is measured and interpreted. Resilience is studied at all levels of biological organization, although the term is often not used. There is a suite of resilience mechanisms conserved across biological scales, and there are tradeoffs that affect resilience. Resilience is conceptually useful to help diverse researchers think about how biological systems respond to perturbations, but we need a richer lexicon to describe the diversity of perturbations, and we lack widely applicable metrics of resilience.

PMID:38660410 | PMC:PMC11039332 | DOI:10.1016/j.isci.2024.109478

New Phytol. 2024 May;242(4):1404-1407. doi: 10.1111/nph.19759.

NO ABSTRACT

PMID:38659109 | DOI:10.1111/nph.19759

Sci Rep. 2024 Apr 24;14(1):9397. doi: 10.1038/s41598-024-58367-2.

ABSTRACT

While philanthropic support for science has increased in the past decade, there is limited quantitative knowledge about the patterns that characterize it and the mechanisms that drive its distribution. Here, we map philanthropic funding to universities and research institutions based on IRS tax forms from 685,397 non-profit organizations. We identify nearly one million grants supporting institutions involved in science and higher education, finding that in volume and scope, philanthropy is a significant source of funds, reaching an amount that rivals some of the key federal agencies like the NSF and NIH. Our analysis also reveals that philanthropic funders tend to focus locally, indicating that criteria beyond research excellence play an important role in funding decisions, and that funding relationships are stable, i.e. once a grant-giving relationship begins, it tends to continue in time. Finally, we show that the bipartite funder-recipient network displays a highly overrepresented motif indicating that funders who share one recipient also share other recipients and we show that this motif contains predictive power for future funding relationships. We discuss the policy implications of our findings on inequality in science, scientific progress, and the role of quantitative approaches to philanthropy.

PMID:38658598 | DOI:10.1038/s41598-024-58367-2

Nat Commun. 2024 Apr 24;15(1):3444. doi: 10.1038/s41467-024-47593-x.

ABSTRACT

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

PMID:38658549 | DOI:10.1038/s41467-024-47593-x

Nat Commun. 2024 Apr 24;15(1):3307. doi: 10.1038/s41467-024-47308-2.

ABSTRACT

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

PMID:38658525 | DOI:10.1038/s41467-024-47308-2

Lancet Rheumatol. 2024 May;6(5):e279-e290. doi: 10.1016/S2665-9913(24)00059-6.

ABSTRACT

BACKGROUND: Childhood Sjögren's disease is a rare, underdiagnosed, and poorly-understood condition. By integrating machine learning models on a paediatric cohort in the USA, we aimed to develop a novel system (the Florida Scoring System) for stratifying symptomatic paediatric patients with suspected Sjögren's disease.

METHODS: This cross-sectional study was done in symptomatic patients who visited the Department of Pediatric Rheumatology at the University of Florida, FL, USA. Eligible patients were younger than 18 years or had symptom onset before 18 years of age. Patients with confirmed diagnosis of another autoimmune condition or infection with a clear aetiological microorganism were excluded. Eligible patients underwent comprehensive examinations to rule out or diagnose childhood Sjögren's disease. We used latent class analysis with clinical and laboratory variables to detect heterogeneous patient classes. Machine learning models, including random forest, gradient-boosted decision tree, partial least square discriminatory analysis, least absolute shrinkage and selection operator-penalised ordinal regression, artificial neural network, and super learner were used to predict patient classes and rank the importance of variables. Causal graph learning selected key features to build the final Florida Scoring System. The predictors for all models were the clinical and laboratory variables and the outcome was the definition of patient classes.

FINDINGS: Between Jan 16, 2018, and April 28, 2022, we screened 448 patients for inclusion. After excluding 205 patients due to symptom onset later than 18 years of age, we recruited 243 patients into our cohort. 26 patients were excluded because of confirmed diagnosis of a disorder other than Sjögren's disease, and 217 patients were included in the final analysis. Median age at diagnosis was 15 years (IQR 11-17). 155 (72%) of 216 patients were female and 61 (28%) were male, 167 (79%) of 212 were White, and 20 (9%) of 213 were Hispanic, Latino, or Spanish. The latent class analysis identified three distinct patient classes: class I (dryness dominant with positive tests, n=27), class II (high symptoms with negative tests, n=98), and class III (low symptoms with negative tests, n=92). Machine learning models accurately predicted patient class and ranked variable importance consistently. The causal graphical model discovered key features for constructing the Florida Scoring System.

INTERPRETATION: The Florida Scoring System is a paediatrician-friendly tool that can be used to assist classification and long-term monitoring of suspected childhood Sjögren's disease. The resulting stratification has important implications for clinical management, trial design, and pathobiological research. We found a highly symptomatic patient group with negative serology and diagnostic profiles, which warrants clinical attention. We further revealed that salivary gland ultrasonography can be a non-invasive alternative to minor salivary gland biopsy in children. The Florida Scoring System requires validation in larger prospective paediatric cohorts.

FUNDING: National Institute of Dental and Craniofacial Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Heart, Lung, and Blood Institute, and Sjögren's Foundation.

PMID:38658114 | DOI:10.1016/S2665-9913(24)00059-6

Int J Biol Macromol. 2024 Apr 22:131763. doi: 10.1016/j.ijbiomac.2024.131763. Online ahead of print.

ABSTRACT

Hsp16.3 plays a vital role in the slow growth of Mycobacterium tuberculosis via its chaperone function. Many secretory proteins, including Hsp16.3 undergo acetylation in vivo. Seven lysine (K) residues (K64, K78, K85, K114, K119, K132 and K136) in Hsp16.3 are acetylated inside pathogen. However, how lysine acetylation affects its structure, chaperone function and pathogen's growth is still elusive. We examined these aspects by executing in vitro chemical acetylation (acetic anhydride modification) and by utilizing a lysine acetylation mimic mutant (K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q). Far- and near-UV CD measurements revealed that the chemically acetylated proteins(s) and acetylation mimic mutant has altered secondary and tertiary structure than unacetylated/wild-type protein. The chemical modification and acetylation mimic mutation also disrupted the oligomeric assembly, increased surface hydrophobicity and reduced stability of Hsp16.3, as revealed by GF-HPLC, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding and urea denaturation experiments, respectively. These structural changes collectively led to an enhancement in chaperone function (aggregation and thermal inactivation prevention ability) of Hsp16.3. Moreover, when the H37Rv strain expressed the acetylation mimic mutant protein, its growth was slower in comparison to the strain expressing the wild-type/unacetylated Hsp16.3. Altogether, these findings indicated that lysine acetylation improves the chaperone function of Hsp16.3 which may influence pathogen's growth in host environment.

PMID:38657928 | DOI:10.1016/j.ijbiomac.2024.131763

Cell Chem Biol. 2024 Apr 12:S2451-9456(24)00127-2. doi: 10.1016/j.chembiol.2024.03.011. Online ahead of print.

ABSTRACT

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands joined by a linker, enabling them to simultaneously bind with an E3 ligase and a protein of interest (POI) and trigger proteasomal degradation of the POI. Limitations of PROTAC include lack of potent E3 ligands, poor cell selectivity, and low permeability. AS1411 is an antitumor aptamer specifically recognizing a membrane-nucleus shuttling nucleolin (NCL). Here, we repurpose AS1411 as a ligand for an E3 ligase mouse double minute 2 homolog (MDM2) via anchoring the NCL-MDM2 complex. Then, we construct an AS1411-NCL-MDM2-based PROTAC (ANM-PROTAC) by conjugating AS1411 with large-molecular-weight ligands for "undruggable" oncogenic STAT3, c-Myc, p53-R175H, and AR-V7. We show that the ANM-PROTAC efficiently penetrates tumor cells, recruits MDM2 and degrades the POIs. The ANM-PROTAC achieves tumor-selective distribution and exhibits excellent antitumor activity with no systemic toxicity. This is a PROTAC with built-in tumor-targeting and cell-penetrating capacities.

PMID:38657608 | DOI:10.1016/j.chembiol.2024.03.011

Comput Biol Med. 2024 Apr 22;175:108507. doi: 10.1016/j.compbiomed.2024.108507. Online ahead of print.

ABSTRACT

BACKGROUND: Post COVID-19 Condition (PCC), characterized by lingering symptoms post-acute COVID-19, poses clinical challenges, highlighting the need to understand its underlying molecular mechanisms. This meta-analysis aims to shed light on the transcriptomic landscapes and sex-specific molecular dynamics intrinsic to PCC.

METHODS: A systematic review identified three studies suitable for comprehensive meta-analysis, encompassing 135 samples (57 PCC subjects and 78 recovered subjects). We performed meta-analysis on differential gene expression, a gene set enrichment analysis of Reactome pathways, and weighted gene co-expression network analysis (WGCNA). We performed a drug and disease enrichment analysis and also assessed sex-specific differences in expression patterns.

KEY FINDINGS: A clear difference was observed in the transcriptomic profiles of PCC subjects, with 530 differentially expressed genes (DEGs) identified. Enrichment analysis revealed that the altered pathways were predominantly implicated in cell cycle processes, immune dysregulation and histone modifications. Antioxidant compounds such as hesperitin were predominantly linked to the hub genes of the DEGs. Sex-specific analyses highlighted disparities in DEGs and altered pathways in male and female PCC patients, revealing a difference in the expression of ribosomal proteins. PCC in men was mostly linked to neuro-cardiovascular disorders, while women exhibited more diverse disorders, with a high index of respiratory conditions.

CONCLUSION: Our study reveals the intricate molecular processes underlying PCC, highlighting that the differences in molecular dynamics between males and females could be key to understanding and effectively managing the varied symptomatology of this condition.

PMID:38657468 | DOI:10.1016/j.compbiomed.2024.108507

Annu Rev Plant Biol. 2024 Apr 24. doi: 10.1146/annurev-arplant-070623-111458. Online ahead of print.

ABSTRACT

Development is a chain reaction in which one event leads to another until the completion of a life cycle. Phase transitions are milestone events in the cycle of life. LEAFY COTYLEDON1 (LEC1), ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 proteins, collectively known as LAFL, are master transcription factors (TFs) regulating seed and other developmental processes. Since the initial characterization of the LAFL genes (44, 71, 98-100), more than three decades of active research has generated tremendous amounts of knowledge about these TFs, whose roles in seed development and germination have been comprehensively reviewed (63, 43, 65, 81). Recent advances in cell biology and genetic and genomic tools have allowed the characterization of the LAFL regulatory networks in previously challenging tissues at a higher throughput and resolution in reference species and crops. In this review, we provide a holistic perspective by integrating advances at the epigenetic, transcriptional, posttranscriptional, and protein levels to exemplify the spatiotemporal regulation of the LAFL networks in Arabidopsis seed development and phase transitions, and we briefly discuss the evolution of these TF networks.

PMID:38657282 | DOI:10.1146/annurev-arplant-070623-111458

J Clin Oncol. 2024 Apr 24:JCO2301983. doi: 10.1200/JCO.23.01983. Online ahead of print.

ABSTRACT

PURPOSE: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC.

MATERIALS AND METHODS: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs.

RESULTS: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers.

CONCLUSION: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

PMID:38657187 | DOI:10.1200/JCO.23.01983

ACS Synth Biol. 2024 Apr 24. doi: 10.1021/acssynbio.3c00542. Online ahead of print.

ABSTRACT

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. This study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

PMID:38657170 | DOI:10.1021/acssynbio.3c00542

PLoS Comput Biol. 2024 Apr 24;20(4):e1011800. doi: 10.1371/journal.pcbi.1011800. Online ahead of print.

ABSTRACT

Biochemical signaling pathways in living cells are often highly organized into spatially segregated volumes, membranes, scaffolds, subcellular compartments, and organelles comprising small numbers of interacting molecules. At this level of granularity stochastic behavior dominates, well-mixed continuum approximations based on concentrations break down and a particle-based approach is more accurate and more efficient. We describe and validate a new version of the open-source MCell simulation program (MCell4), which supports generalized 3D Monte Carlo modeling of diffusion and chemical reaction of discrete molecules and macromolecular complexes in solution, on surfaces representing membranes, and combinations thereof. The main improvements in MCell4 compared to the previous versions, MCell3 and MCell3-R, include a Python interface and native BioNetGen reaction language (BNGL) support. MCell4's Python interface opens up completely new possibilities for interfacing with external simulators to allow creation of sophisticated event-driven multiscale/multiphysics simulations. The native BNGL support, implemented through a new open-source library libBNG (also introduced in this paper), provides the capability to run a given BNGL model spatially resolved in MCell4 and, with appropriate simplifying assumptions, also in the BioNetGen simulation environment, greatly accelerating and simplifying model validation and comparison.

PMID:38656994 | DOI:10.1371/journal.pcbi.1011800

J Am Soc Nephrol. 2024 Apr 24. doi: 10.1681/ASN.0000000000000353. Online ahead of print.

ABSTRACT

BACKGROUND: The transcription factor Grainyhead-like 2 (GRHL2) plays a crucial role in maintaining the epithelial barrier properties of the renal collecting duct and is essential for osmoregulation. We noticed a reduction in GRHL2 expression in cysts derived from the collecting ducts in kidneys affected by Autosomal Dominant Polycystic Kidney Disease (ADPKD). However, the specific role of GRHL2 in cystic kidney disease remains unknown.

METHODS: The functional role of the transcription factor Grhl2 in the context of cystic kidney disease was examined through analysis of its expression pattern in patient samples with ADPKD and generating a transgenic cystic kidney disease (TCKD) mouse model by overexpressing the human proto-oncogene c-MYC in kidney collecting ducts. Next, TCKD mice bred with collecting duct-specific Grhl2 knockout mice (Grhl2KO). The resulting TCKD-Grhl2KO mice and their littermates were examined by various types of histological and biochemical assays and gene profiling analysis via RNA-seq.

RESULTS: A comprehensive examination of kidney samples from patients with ADPKD revealed GRHL2 downregulation in collecting duct-derived cyst epithelia. Comparative analysis of TCKD and TCKD-Grhl2KO mice exhibited that the collecting duct-specific deletion of Grhl2 resulted in markedly aggravated cyst growth, worsened kidney dysfunction, and shortened life span. Furthermore, transcriptomic analyses indicated sequential downregulation of kidney epithelial cyst development regulators (Frem2, Muc1, Cdkn2c, Pkd2, and Tsc1) during cyst progression in kidneys of TCKD-Grhl2KO mice which included presumed direct Grhl2 target genes.

CONCLUSIONS: These results suggest GRHL2 as a potential progression modifier, especially for cysts originating from collecting ducts.

PMID:38656794 | DOI:10.1681/ASN.0000000000000353

Anal Chem. 2024 Apr 24. doi: 10.1021/acs.analchem.4c00651. Online ahead of print.

ABSTRACT

Immunoassays serve as powerful diagnostic tools for early disease screening, process monitoring, and precision treatment. However, the current methods are limited by high costs, prolonged processing times (>2 h), and operational complexities that hinder their widespread application in point-of-care testing. Here, we propose a novel centrifugo-pneumatic reciprocating flowing coupled with spatial confinement strategy, termed PRCM, for ultrafast multiplexed immunoassay of pathogens on a centrifugal microfluidic platform. Each chip consists of four replicated units; each unit allows simultaneous detection of three targets, thereby facilitating high-throughput parallel analysis of multiple targets. The PRCM platform enables sequential execution of critical steps such as solution mixing, reaction, and drainage by coordinating inherent parameters, including motor rotation speed, rotation direction, and acceleration/deceleration. By integrating centrifugal-mediated pneumatic reciprocating flow with spatial confinement strategies, we significantly reduce the duration of immune binding from 30 to 5 min, enabling completion of the entire testing process within 20 min. As proof of concept, we conducted a simultaneous comparative test on- and off-the-microfluidics using 12 negative and positive clinical samples. The outcomes yielded 100% accuracy in detecting the presence or absence of the SARS-CoV-2 virus, thus highlighting the potential of our PRCM system for multiplexed point-of-care immunoassays.

PMID:38656793 | DOI:10.1021/acs.analchem.4c00651

Biol Open. 2024 Apr 15;13(4):bio060415. doi: 10.1242/bio.060415. Epub 2024 Apr 24.

ABSTRACT

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

PMID:38656788 | DOI:10.1242/bio.060415