BACKGROUND:To maximize the utilization of cold-stored platelets (CSPs) in preclinical research, the use of small-volume storage bags (mini-bags) or tubes would allow multiple treatment groups to be investigated from a single component. It is necessary to determine the effect of using downscaled storage formats on CSP quality and function. METHODS:Mini-bags were prepared by vacuum sealing platelet storage bags to ¼ the standard size. Apheresis platelets (n = 8) in 40% plasma/60% PAS-E (SSP+) were aseptically aliquoted into mini-bags (30 mL) or polypropylene tubes (15 mL; CSP-tubes), and the remaining volume (~190 mL) was retained in the original bag (control). Platelets were refrigerated (2-6°C), and in vitro quality parameters were assessed over 21 days. RESULTS:Metabolic capacity was altered for CSP-tubes, with a significant decline in pH, mitochondrial membrane depolarization, and rapid glycolysis. CSP-tubes had greater storage-related activation, as evidenced by a higher proportion of annexin-V-positive platelets and a greater number of CD61+/annexin-V+ extracellular vesicles (EVs). Similarly, platelet functional capacity in CSP-tubes was compromised, with reduced clot strength compared to controls. CSPs stored in mini-bags remained comparable to controls, with the exception of a greater proportion of annexin-V-positive platelets and EVs at day 21. Importantly, despite these changes, no functional deficits were observed in mini-bag components. CONCLUSIONS:Storage in tubes markedly impairs the metabolic, structural, and functional quality of CSPs, indicating that this approach is unsuitable. In comparison, mini-bag storage preserves key in vitro parameters, supporting a scalable strategy that maximizes the utilization of CSPs for preclinical evaluation.

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BACKGROUND AND OBJECTIVES:Reduced metabolic activity of cold-stored platelets (CSPs) has led to the assumption that agitation is not required during cold storage. However, guidelines from the Food and Drug Administration state that agitation is 'optional' and there is a degree of international variability described in the literature. The effect of agitation on the quality of CSPs stored in platelet additive solution (PAS) for an extended storage period has not been established. MATERIALS AND METHODS:Matched apheresis platelets (n = 8) in 40% plasma/60% PAS-E (SSP+) were refrigerated (2-6°C) and either non-agitated (control), continuously agitated, or periodically agitated. In vitro quality parameters were assessed at defined intervals over 21 days. RESULTS:Agitation did not alter the metabolic properties of CSPs. The abundance of platelet glycoproteins (GPIIIa, GPIbα, GPVI) decreased, while activation markers (activated GPIIb/IIIa, fibrinogen binding, P-selectin and phosphatidylserine) increased during cold storage; however, no significant differences were observed between groups. The functional capacity of CSPs was the same regardless of agitation, with no differences in aggregation response (collagen-induced and adenosine diphosphate [ADP]-induced), or viscoelastic properties (clotting time and maximum clot strength). Platelets stored with continuous agitation developed visible aggregates; which were observed in 88% of components at Day 14, and 100% of components by Day 21. Aggregates were not observed in non-agitated or periodically agitated components. CONCLUSION:Continuous agitation did not improve the metabolic or functional properties of CSPs and led to the development of aggregates. These findings confirm that non-agitated storage is best practice for CSPs stored in PAS, reinforcing the operational simplicity of cold storage.

BACKGROUND:Multi-drug resistant Gram-negative bacteria (GNB) are major contributors to the antimicrobial resistance (AMR) burden. AMR mechanisms are primarily mediated by proteoforms; therefore, proteomic analyses of GNB offers a significant advantage in understanding the mechanisms of AMR. A large portion of these mechanisms are mediated by membrane proteins; however, they are often difficult to extract due to their hydrophobic nature and complex interactions with other components of the cell membrane. To extract the greatest number of proteoforms, an efficient homogenisation protocol is required to effectively disrupt the rigid cell wall and membrane. METHODS:Using Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, we systematically compared the extraction efficiency of bead-beating with flash frozen and lyophilized cell pellets. RESULTS:We demonstrate that lyophilization improves bead-beating extraction methods by increasing the detection of membrane proteins. We detected numerous unique membrane proteins in each bacterial isolate, including ABC transporters and proteins involved in lipopolysaccharide synthesis, when lyophilizing prior to bead-beating, compared to only flash-freezing. CONCLUSIONS:As membrane proteins play a central role in AMR mechanisms, this improvement in their isolation and identification will aid in understanding the resistance and molecular mechanisms associated with multi-drug resistant GNB.

Asthma is a chronic inflammatory respiratory disease well-known to demonstrate sexual dimorphism in incidence and severity, although the mechanisms causing these differences remain incompletely understood. RPS4X and RPS4Y1 are X and Y-chromosome-linked genes coding ribosomal subunits previously associated with inflammation, airway remodelling and asthma medication efficacy. Particularly, RPS4Y1 has been under-investigated within the context of disease, with little examination of molecular mechanisms and pathways regulated by this gene. The ribosome, a vital cellular machinery, facilitates the translation of mRNA into peptides and then proteins. Imbalance or dysfunction in ribosomal components may lead to malfunctioning proteins. Using CRISPR-Cas9 knockout cellular models for RPS4Y1 and RPS4X, we characterised the function of RPS4Y1 in the context of the asthma-relevant processes, inflammation and fibrosis. No viable RPS4X knockouts could be generated. We highlight novel molecular mechanisms such as specific translation of IL6 and tenascin-C mRNA by RPS4Y1 containing ribosomes. Furthermore, an RPS4Y1-centric gene signature correlates with clinical lung function measurements, specifically in adult male asthma patients. These findings inform the current understanding of sex differences in asthma, as females do not produce the RPS4Y1 protein. Therefore, the pathologically relevant functions of RPS4Y1 may contribute to the complex sexually dimorphic pattern of asthma susceptibility and progression.

Trophoblast organoids can provide crucial insights into mechanisms of placentation, however their potential is limited by highly variable extracellular matrices unable to reflect in vivo tissues. Here, we present a bioprinted placental organoid model, generated using the first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol (PEG) matrix. Bioprinted or Matrigel-embedded organoids differentiate spontaneously from cytotrophoblasts into two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs). Bioprinted organoids are driven towards EVT differentiation and show close similarity with early human placenta or primary trophoblast organoids. Inflammation inhibits proliferation and STBs within bioprinted organoids, which aspirin or metformin (0.5 mM) cannot rescue. We reverse the inside-out architecture of ACH-3P organoids by suspension culture with STBs forming on the outer layer of organoids, reflecting placental tissue. Our bioprinted methodology is applicable to trophoblast stem cells. We present a high-throughput, automated, and tuneable trophoblast organoid model that reproducibly mimics the placental microenvironment in health and disease.

There is no safe level of exposure to air pollution, including particulate matter smaller than 2.5 µm (PM2.5), to human health. Whilst it is well known that exposure to heavily polluted air is associated with several liver disorders, it is unclear how long-term exposure to low-level traffic-derived PM2.5 affects liver health. BALB/c mice (5 weeks, male) were exposed to traffic-derived PM2.5 (10 µg/mouse/day, intranasally) daily for 4, 8 and 12 weeks. Markers of inflammation and fibrosis were measured at each time point. Changes in liver proteome and lipid profiles were measured using proteomics and lipidomics at 12 weeks. Low-dose PM2.5 exposure increased macrophage infiltration, pro-inflammatory cytokine production, and increased collagen deposition at 12 weeks. Despite liver lipid metabolism being increased, the abundance of triglycerides, precursor diacylglycerols, and ceramide was also significantly increased by PM2.5 exposure, whereas glycogen content was reduced. Proteomics analysis revealed 64 proteins to be significantly changed in PM2.5-exposed mice, and KEGG pathway enrichment analysis indicated their involvement in lipid metabolism, alcohol-related liver disease, neutrophil extracellular trap formation, and transcriptional dysregulation related to cancer. In conclusion, prolonged exposure to low-dose traffic-derived PM2.5 promotes pathological changes in the liver, suggestive of an increased risk of metabolic dysfunction-associated fatty liver disease. Future studies can enable the identification of the signalling pathways underlying low-dose PM2.5-induced lipid accumulation in the liver.

Metabolomics analyses enable the examination and identification of endogenous biochemical reaction products, revealing information on the metabolic pathways and processes active within a living cell or organism. Determination of metabolic shifts can provide important information on a treatment or disease. Unlike other omics fields that typically have analytes of the same chemical class with common building blocks, those that fall under the nomenclature of metabolites encompass a wide array of different compounds with very diverse physiochemical properties. Development of a comprehensive metabolomic pipeline therefore can be a troublesome and complicated process for the analyst. Often single liquid chromatography-mass spectrometry methods on unfractionated samples are carried out in order to be time-efficient, however this could potentially produce data with a low number of identifiable metabolites. In the present studies, we developed a comprehensive polar metabolomics pipeline for cell-based metabolomics. SH-SY5Y neuroblastoma cells were selected as the sample matrix for method development since they are one of the most widely used cell lines for human neurotoxicity studies. This was accomplished by investigating and optimising different mass spectrometry source and chromatographic conditions to enhance the signal of polar metabolites. Optimised hydrophilic interaction liquid chromatography (HILIC) based metabolomic methods at different pH values were examined in positive, negative, and polarity switching modes to determine which combination yielded the highest number of confidently identified metabolites. Additionally, the use of sequentially running two methods was also compared to determine the degree of overlap and whether there is merit in running two separate methods on one sample. It was determined that solvent switching between two optimised methods, acidic chromatographic conditions in positive mode and basic chromatographic conditions in negative mode, yielded the highest number of unique identifiable metabolites. This could be run in a single analytical batch due to the large pH range of the column. A quick switch method in-between each method allowed both conditioning the column and preparation of the MS source conditions for the sequential method.

Prolonged exposure to PM2.5 is recognised as a significant risk factor for the development of chronic pulmonary diseases, and was first recognised in countries with high levels of air pollution (e.g. as occurs in Asia). However, it is important to note that there is no safe level of PM2.5. As such, the detrimental effects on the lungs caused by low levels of PM2.5, as found in Europe and North America, are often overlooked. The objective of this study was to determine how a low level of PM2.5 exposure affects lung integrity. Male mice (Balb/c, 6 weeks of age) were subjected to daily exposure to 5 μg/ml PM2.5 (nasal installation) with or without the supplement of vitamin C (VC) in drinking water (1.5 g/L), while those in the sham group received saline exposure. The addition of VC effectively prevented the elevation of total reactive oxygen species (ROS), mitochondrial ROS, and inflammation induced by PM2.5 exposure. Furthermore, in-vitro studies showed that PM2.5 reduced the cell viability of human epithelial cells (BEAS-2B cells), while increasing their susceptibility to oxidative stress-induced cell injury and inflammatory responses. The pre-treatment with VC significantly mitigated the adverse effects of PM2.5 exposure on cell viability, inflammatory response, mitochondrial ROS levels, and mitochondrial loss in BEAS-2B cells. These findings suggest that daily exposure to a low level of PM2.5 possesses adverse effects on lung health through inflammatory response and oxidative stress-induced mitochondrial loss. VC supplement can effectively protect the lungs from the adverse effects of low-level PM2.5 exposure.

Postmortem interval (PMI) estimation is a critical component of forensic investigations as it can help establish a timeline of events and assist in reducing a pool of missing persons or potential suspects. Despite its importance, the accuracy of PMI estimation remains challenging due to the complexity of decomposition and limitations of current methods, particularly in the later postmortem period. Proteomics has shown potential as a more objective method, with current studies taking an untargeted approach to identify informative peptide targets. Previous studies in our laboratory analysed human muscle tissue and identified 12 peptide ratios, consisting of 19 individual peptides, that were correlated with decomposition time. This study aimed to optimise the detection of these 19 peptide targets using a targeted liquid chromatography triple quadrupole mass spectrometry (LC-QQQ) method suited for casework applications and instrument platforms commonly found in forensic laboratories. Of the 19 peptide targets, 17 were optimised to be reproducible. Additionally, a longitudinal analysis in authentic human samples was conducted over a 14-day period postmortem to monitor detection throughout the decomposition process. All but one of the targets produced interpretable results, and data analysis was performed to generate peptide ratios. After log2 transformation, two of the previously proposed peptide ratios showed linear trends. Overall, this study has shown that a targeted LC-QQQ method is a viable system for the detection of PMI peptide targets from human postmortem muscle tissue and has demonstrated the potential for this type of analysis to be validated and applied in routine forensic casework.

Advances in methodologies and technologies over the past decade have led to an unprecedented depth of analysis of a cell's biomolecules, with entire genomes able to be sequenced in hours and up to 10,000 transcripts or ORF products (proteins) able to be quantified from a single cell. Methods for analysing individual omes are now optimised, reliable and robust but are often performed in isolation with other biomolecules considered contaminants. However, there is a growing body of systems biology studies that aim to study multiple omes from the same sample. This review details the current state of the "multi-omics" field, trying to define what the field is, the methodologies employed and the challenges facing researchers in this field. It also critically evaluates whether these approaches are "fit-for-purpose" and how the field needs to evolve to enhance our understanding of how biomolecules from distinct omes interact with one another to alter cellular phenotype in response to change.

Klebsiella pneumoniae is a Gram-negative bacterium and a major cause of nosocomial infections such as urinary tract infections (UTIs), pneumonia and meningitis. Although these infections are commonly treated with the β-lactam group of antibiotics and combinations of it, (multi-)drug resistance in K. pneumoniae has steadily increased in the last few decades. Resistance to the β-lactam class of antibiotics is primarily mediated through the activation of narrow and extended-spectrum β-lactamase enzymes and changes within the bacterial cell envelope. Antimicrobial Resistance (AMR) has become a large global public health problem and economic burden. Carbapenem-resistant and extended-spectrum β-lactamase-producing K. pneumoniae have been classified as critical pathogens for which improved diagnostics and treatments are urgently needed. In order to develop new diagnostics and treatments, the resistance profiles of K. pneumoniae on a molecular level needs to be understood. Despite the identification of several genes, transcripts, proteoforms and their associated roles in resistance mechanisms, global resistance phenotypes are steadily evolving and consequently lack comprehensive understanding. This review discusses the role of genomics, transcriptomics, and proteomics in understanding resistance mechanisms and the associated molecular characteristics using carbapenem-resistant and extended-spectrum β-lactamase-producing K. pneumoniae as a model pathogen to further our current understanding of AMR. Furthermore, we also highlight the need for a holistic approach (i.e., combining 'omics technologies and data) to provide an in-depth understanding of the global resistance phenotype of K. pneumoniae in AMR.

ABSTRACT Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions, exemplified by variable colony morphotypes. In Burkholderia pseudomallei , discrete genomic alterations and modulation of gene expression facilitate adaptation. Adapted variants of species within the Burkholderia cepacia complex (Bcc) often lose the pC3 virulence megaplasmid, impacting their colony morphology and their production of virulence factors. In this study, we characterize variants arising in Burkholderia ambifaria clinical isolates using proteomics and phenotypic tests and show that some of them have retained the pC3, indicating a distinct phase variation mechanism at play in this Bcc species. Interestingly, variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) still share similarities in phenotypes controlled by the Cep quorum-sensing (QS) system. We further investigated the role of QS in B. ambifaria HSJ1 phase variation and confirmed that the Cep QS system is important for the emergence of variants. Given that DNA methylation is a key epigenetic factor regulating virulence factors in Burkholderia cenocepacia , we hypothesized that adenosine DNA methylation also governs phase variation in B. ambifaria HSJ1. By deleting the genes encoding putative adenosine DNA methyltransferases, we discovered that an orphan type II DNA methyltransferase inhibits the emergence of phase variants. This study is the first to demonstrate that quorum sensing and adenosine DNA methylation are two antagonistic systems independently controlling phase variation in B. ambifaria . IMPORTANCE Some Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, and people suffering from cystic fibrosis, infection from the Burkholderia cepacia complex (Bcc) can lead to “ cepacia syndrome.” In northern Australia and southeast Asia, melioidosis caused by Burkholderia pseudomallei is prevalent among native population, particularly among people with diabetes, chronic lung or kidney disease or alcoholism. Burkholderia ’s phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing its survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group and is the first to report that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying mechanisms of CMV could lead to the development of targeted therapies against these highly antibiotic-tolerant bacteria.

The bacterial surfaceome, representing the full complement of surface-expressed proteins, is integral to interactions between bacteria, their hosts, and the environment, influencing processes such as adhesion, invasion, immune evasion, and biofilm formation. These proteins, due to their accessibility and vital roles in bacterial physiology, are prime targets for therapeutic intervention. This protocol describes a detailed methodology for mapping bacterial surfaceomes, including enzymatic surface shaving, cell surface biotinylation, solid-phase extraction, and liquid chromatography-tandem mass spectrometry.

Endothelial dysfunction is a hallmark feature of cardiovascular disease (CVD), yet the underlying mechanisms are still poorly understood. This has impeded the development of effective therapies, particularly for peripheral artery disease. FK506-binding protein like (FKBPL) and its therapeutic peptide mimetic, AD-01, are crucial negative regulators of angiogenesis, however their roles in CVD are unknown. In this study, we aimed to elucidate the FKBPL-mediated mechanisms involved in regulating endothelial dysfunction induced by hypoxia or inflammation, and to determine whether AD-01 can effectively restore endothelial function under these conditions. Hindlimb ischemia was induced in mice by ligating the proximal and distal ends of the right femoral artery, and, after three days, the gastrocnemius muscle was collected for immunofluorescence staining, and RNA extraction. A 3D in vitro microfluidics model was developed to determine the endothelial cell migration and impact of FKBPL following treatments with: (i) 24 µM FKBPL targeted siRNA, (ii) 1 mM hypoxia inducible factor (HIF-1)α activator (DMOG), (iii) 50

Platelets are transfused to patients to prevent and stop bleeding. Conventionally, platelets are stored at room temperature (RT; 20–24 °C), however, this limits their shelf life to 5–7 days, due to an increased risk of bacterial proliferation at RT. In recent years, cold storage (2–6 °C) of platelets has regained interest, largely due to the potential to extend the shelf life up to 21 days. The historical use of cold-stored platelets and decades of foundational research has made their (re)implementation possible, with cold-stored platelets already being transfused in several countries, including the United States and Norway. However, as efforts continue to expand implementation, it is becoming increasingly evident that variations in processing methods from collection to transfusion, including the collection platform, storage solution and additional component modifications, may alter platelet characteristics during cold storage. This variability is largely overlooked and there is a need to recognize how these differences may affect clinical outcomes post-transfusion. This review outlines the assumptions that have been made regarding cold-stored platelets and discusses areas that require further consideration in an effort to inform future research and best practice.

Signet Ring Cell Carcinoma (SRCC) is a rare and highly malignant form of adenocarcinoma with increasing incidence and poor prognosis due to late diagnosis and limited treatment options. We employed Deep Visual Proteomics (DVP), which combines AI-directed cell segmentation and classification with laser microdissection and ultra-high sensitivity mass spectrometry, for cell-type-specific proteomic analysis of SRCC across the bladder, prostate, seminal vesicle, and a lymph node of a single patient. DVP identified significant alterations in DNA damage response (DDR) proteins, particularly within the ATR and mismatch repair (MMR) pathways, indicating replication stress as a crucial factor in SRCC mutagenicity. Additionally, we observed substantial enrichment of immune-related proteins, reflecting high levels of cytotoxic T lymphocyte infiltration and elevated PD-1 expression. These findings suggest that pembrolizumab immunotherapy may be more effective than conventional chemotherapy for this patient. Our results provide novel insights into the proteomic landscape of SRCC, identify potential targets, and open up for personalized therapeutic strategies in managing SRCC.

Corals at very early life stages face high mortality bottlenecks, limiting natural stock-recruitment and the success of restoration and aquaculture strategies reliant on sexual reproduction. Nutrient availability and quality, particularly lipids, are critical for larval survival, dispersal, and settlement, yet the specific lipids influencing early-stage fitness remain unclear. Since measuring lipid depletion in non-surviving larvae is impractical, we developed a targeted supplementation approach to identify nutrients that enhance larval fitness. Supplementing Acropora spathulata larvae with nanoparticle-encapsulated triacylglycerols and sterols significantly improved swimming distance and speed, indicating enhanced dispersal potential. Supplementation with sterols, which are essential for cell structure and metamorphosis, also increased settlement rates. Furthermore, juveniles developed from larvae fed with sterols and omega-3-rich fish oils demonstrated superior performance under controlled and thermal stress conditions during the first six months, while those supplemented with Calanus oil also enhanced survival under elevated temperatures. Lipid profiling revealed key sphingolipids, glycerophospholipids, and fatty acids likely responsible for the improved fitness of fed larvae. Our approach highlights the critical role of lipid composition and availability during coral early life stages and demonstrates the potential of targeted nutritional supplementation to address nutrient deficiencies, enhance coral resilience, and support restoration and aquaculture efforts.

BACKGROUND:Cold storage (2-6°C) preserves the aggregatory and clot formation properties of platelets beyond the standard 7-day shelf life. Comparatively little is known about the effect of cold storage on clot resolution functions, which are necessary to ensure normal hemostatic balance is maintained. STUDY DESIGN AND METHODS:Double-dose apheresis platelets (n = 8) were collected using the Trima apheresis platform (40% plasma/60% PAS-E). Platelets were sampled on Day 1 post-collection and allocated to room temperature (RT; 20-24°C) or cold storage (2-6°C). In vitro testing was performed over a 21-day period. RESULTS:The coagulation profile of cold-stored platelets was better maintained throughout storage compared to RT platelets. Cold-stored platelets showed increased thrombin generation potential (faster lag time, higher peak, and increased ETP) compared to RT platelets throughout storage. By viscoelastic testing, the IN-test and EX-test clotting times (CT) were maintained over cold storage for 21 days, while the clot strength (MCF) decreased by approximately 15%. The lysis time (LT) was unchanged during cold storage. Macroscopically, less clot retraction and a higher percentage of internal lysis were observed in cold-stored platelets; however, susceptibility to external lysis was not affected. CONCLUSION:Hemostasis requires a delicate balance between clot formation and resolution, and we have shown that the key functional properties of cold platelets stored in PAS-E are well-regulated during extended storage for 21 days.

Background Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. Results The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. Conclusions These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.