Chlorella-enriched hydrogels protect against myocardial damage and reactive oxygen species production in anin vitroischemia/reperfusion model using cardiac spheroids.

Microalgae have emerged as promising photosynthetic microorganisms for biofabricating advanced tissue constructs, with improved oxygenation and reduced reactive oxygen species production. However, their use in the engineering of human tissues has been limited due to their intrinsic growth requirements, which are not compatible with human cells. In this study, we first formulated alginate-gelatin (AlgGel) hydrogels with increasing densities ofChlorella vulgaris. Then, we characterised their mechanical properties and pore size. Finally, we evaluated their effects on cardiac spheroid (CS) pathophysiological response under control and ischemia/reperfusion (I/R) conditions. Our results showed that the addition ofChlorelladid not affect AlgGel mechanical properties, while the mean pore size significantly decreased by 35% in the presence of the 107cells mL-1microalgae density. Under normoxic conditions, the addition of 107Chlorellacells mL-1significantly reduced CS viability starting from 14 days in. No changes in pore size nor CS viability were measured for hydrogels containing 105and 106Chlorellacells mL-1. In our I/R model, allChlorella-enriched hydrogels reduced cardiac cell sensitivity to hypoxic conditions with a corresponding reduction in reactive oxygen species (ROS) production, as well as protected against I/R-induced reduction in cell viability. Altogether, our results support a promising use ofChlorella-enriched Alg-Gel hydrogels for cardiovascular tissue engineering. .

Decorin, an exercise-induced secretory protein, is associated with improved prognosis in breast cancer patients but does not mediate anti-tumorigenic tissue crosstalk in mice.

BACKGROUND: Regular exercise can reduce incidence and progression of breast cancer, but the mechanisms for such effects are not fully understood. METHODS: We used a variety of rodent and human experimental model systems to determine whether exercise training can reduce tumor burden in breast cancer and to identify mechanism associated with any exercise training effects on tumor burden. RESULTS: We show that voluntary wheel running slows tumor development in the mammary specific polyomavirus middle T antigen overexpression (MMTV-PyMT) mouse model of breast cancer but only when mice are not housed alone. We identify the proteoglycan decorin as a contraction-induced secretory factor that systemically increases in patients with breast cancer immediately following exercise. Moreover, high expression of decorin in tumors is associated with improved prognosis in patients, while treatment of breast cancer cells in vitro with decorin reduces cell proliferation. Notwithstanding, when we overexpressed decorin in murine muscle or injected recombinant decorin systemically into mouse models of breast cancer, elevated plasma decorin concentrations did not result in higher tumor decorin levels and tumor burden was not improved. CONCLUSION: Exercise training is anti-tumorigenic in a mouse model of luminal breast cancer, but the effect is abrogated by social isolation. The proteoglycan decorin is an exercise-induced secretory protein, and tumor decorin levels are positively associated with improved prognosis in patients. The hypothesis that elevated plasma decorin is a mechanism by which exercise training improves breast cancer progression in humans is not, however, supported by our pre-clinical data since elevated circulating decorin did not increase tumor decorin levels in these models.

Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient.

Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of macroscale shape as well as size and microscale spatial heterogeneity, within cell-laden hydrogels. In this study, a novel diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient and found to respond to the heterogeneity in stiffness through the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics thus finds application mimicking native shape-to-function relationship in the context of tissue engineering and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.

How to present and summarize a scientific journal article.

The success of a journal club hinges on the presentation of articles that are both relevant and scientifically robust. It's insufficient for presenters to merely read through an article and highlight a few points without a clear focus. A strong presentation should thoroughly describe the relevance and validity of the study, offer a critique, suggest how further research might address the issue, and discuss the implications for patient care. Selecting the right article is crucial. It is recommended to begin the presentation with a case scenario to emphasize the article's clinical relevance and to revisit the case at the conclusion of the presentation. The components of the article presentation should include background information, methodology and results, and the authors' discussion. Additionally, the presenter should critique the article's validity, noting any potential biases, evaluating the risks and costs of the proposed intervention, and assessing how well the article supports its hypothesis. The presentation should conclude with a summary statement that includes conclusions, implications, and future directions. Having a structured process for journal club presentations guides presenters and ensures that attendees derive maximum benefit from the educational activity. This organized approach fosters a deeper understanding and encourages critical thinking among participants.

Evaluating the Long-Term Neurologic Sequelae Among Trauma Patients who Received Flexion-Extension Radiographs.

Introduction: This study evaluated the presence of neurologic sequelae among trauma patients after flexion-extension (F/E) radiographs. Methods: Authors of the study conducted a retrospective review of patients (age ≥ 14 years) with a Glasgow Coma Score of 15 who sustained a blunt traumatic injury and received F/E radiographs. Radiographic scans were defined as positive, negative, inconclusive, or incomplete. The neurologic status of each patient was assessed before and after the F/E radiographs, and at discharge and follow-up. Results: Of the 501 patients included in the analysis, 84.6% (n = 424) had negative F/E radiographs, and 3.2% (n = 16) had positive F/E radiographs. Ten percent (n = 51) of patients had incomplete F/E radiographs, and 2.0% (n = 10) were inconclusive due to the inability to rule out a ligamentous injury. Three patients (0.6%) had MRI-confirmed ligamentous injuries, all of which had initial incomplete F/E radiographs due to pain. No patient had a documented neurological deficit before or after the F/E exam. Three patients with an initial negative F/E radiograph returned to the clinic with symptoms of neurologic sequelae. Two of these patients had symptom resolution with no further issues at future follow-up appointments. The third patient was found to have chronic neurologic symptoms after further evaluation. Conclusions: The inclusion of F/E exams in cervical spine clearance protocols did not demonstrate any new long-term iatrogenic neurologic injuries. Consideration should be given to performing MRIs on patients with incomplete F/E radiographs that cannot rule out a ligamentous injury.

Type XII collagen is elevated in serum from patients with solid tumors: a non-invasive biomarker of activated fibroblasts.

Understanding the tumor microenvironment (TME) and extracellular matrix (ECM) is crucial in cancer research due to their impact on tumor progression. Collagens, major ECM components, regulate cell signaling and behavior. Of the 28 reported collagens, type XII collagen is known to be vital for ECM organization. Over-produced by cancer-associated fibroblasts (CAFs), its upregulation correlates with poor survival in various cancers. This study aimed to develop an ELISA for quantifying circulating type XII collagen as a cancer biomarker. A specific ELISA targeting the C-terminal of type XII collagen was developed and used to analyze serum samples from cancer patients (n = 203) and healthy controls (n = 33). Additionally, type XII collagen expression was assessed in CAFs and normal fibroblasts (NFs) from different tissues, both under TGF-ß stimulated and non-stimulated conditions. The nordicPRO-C12 ELISA demonstrated robustness and specificity for type XII collagen. PRO-C12 levels were significantly elevated in patients with various cancers compared to healthy controls and effectively distinguished between cancer patients and controls. Findings were validated using gene expression data. Furthermore, Western blot analysis revealed increased type XII collagen expression in both CAFs and NFs upon TGF-ß1 stimulation, suggesting a potential role of TGF-ß1 in modulating the expression of type XII collagen in cancerous and normal tissue microenvironments. This study unveils a promising avenue for harnessing PRO-C12 as a non-invasive serum biomarker, enabling the quantification of type XII collagen fragments in cancer patients. Further investigations are warranted to explore the potential of PRO-C12 across different cancer types and disease stages, shedding light on its multifaceted role in cancer development.

Why do patients with cancer die?

Cancer is a major cause of global mortality, both in affluent countries and increasingly in developing nations. Many patients with cancer experience reduced life expectancy and have metastatic disease at the time of death. However, the more precise causes of mortality and patient deterioration before death remain poorly understood. This scarcity of information, particularly the lack of mechanistic insights, presents a challenge for the development of novel treatment strategies to improve the quality of, and potentially extend, life for patients with late-stage cancer. In addition, earlier deployment of existing strategies to prolong quality of life is highly desirable. In this Roadmap, we review the proximal causes of mortality in patients with cancer and discuss current knowledge about the interconnections between mechanisms that contribute to mortality, before finally proposing new and improved avenues for data collection, research and the development of treatment strategies that may improve quality of life for patients.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

miRNAs in pancreatic cancer progression and metastasis.

Small non-coding RNA or microRNA (miRNA) are critical regulators of eukaryotic cells. Dysregulation of miRNA expression and function has been linked to a variety of diseases including cancer. They play a complex role in cancers, having both tumour suppressor and promoter properties. In addition, a single miRNA can be involved in regulating several mRNAs or many miRNAs can regulate a single mRNA, therefore assessing these roles is essential to a better understanding in cancer initiation and development. Pancreatic cancer is a leading cause of cancer death worldwide, in part due to the lack of diagnostic tools and limited treatment options. The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), is characterised by major genetic mutations that drive cancer initiation and progression. The regulation or interaction of miRNAs with these cancer driving mutations suggests a strong link between the two. Understanding this link between miRNA and PDAC progression may give rise to novel treatments or diagnostic tools. This review summarises the role of miRNAs in PDAC, the downstream signalling pathways that they play a role in, how these are being used and studied as therapeutic targets as well as prognostic/diagnostic tools to improve the clinical outcome of PDAC.

How people learn: insights for medical faculty.

To increase medical students' and residents' understanding and retention, faculty need to teach from a knowledge standpoint and understanding of how individuals learn. We know from cognitive information processing that learners remember only a small portion of what they read or hear but remember up to 90% of information when strong active learning modalities are included. Faculty also need to be aware of different learning styles-kinesthetic, visual, and auditory-and ensure that they are including methods that can reach all learners. The cognitive and information processing theories of learning provide insights to educators related to building on prior knowledge from learning and limiting the number of points taught so learners can process and retain the information. Strategies such as a flipped classroom model and question clicker technology can assist in reaching learning goals. Fundamental conditions for learning include awareness, interest, motivation, relevance, engagement, reinforcement, and support.

The collagen landscape in cancer: profiling collagens in tumors and in circulation reveals novel markers of cancer-associated fibroblast subtypes.

Cancer-associated fibroblasts (CAFs) deposit and remodel collagens in the tumor stroma, impacting cancer progression and efficacy of interventions. CAFs are the focus of new therapeutics with the aim of normalizing the tumor microenvironment. To do this, a better understanding of CAF heterogeneity and collagen composition in cancer is needed. In this study, we sought to profile the expression of collagens at multiple levels with the goal of identifying cancer biomarkers. We investigated the collagen expression pattern in various cell types and CAF subtypes in a publicly available single-cell RNA sequencing (RNA-seq) dataset of pancreatic ductal adenocarcinoma. Next, we investigated the collagen expression profile in tumor samples across cancer types from The Cancer Genome Atlas (TCGA) database and evaluated if specific patterns of collagen expression were associated with prognosis. Finally, we profiled circulating collagen peptides using a panel of immunoassays to measure collagen fragments in the serum of cancer patients. We found that pancreatic stellate cells and fibroblasts were the primary producers of collagens in the pancreas. COL1A1, COL3A1, COL5A1, COL6A1 were expressed in all CAF subtypes, whereas COL8A1, COL10A1, COL11A1, COL12A1 were specific to myofibroblast CAFs (myCAF) and COL14A1 specific to inflammatory CAFs (iCAF). In TCGA database, myCAF collagens COL10A1 and COL11A1 were elevated across solid tumor types, and multiple associations between high expression and worse survival were found. Finally, circulating collagen biomarkers were elevated in the serum of patients with cancer relative to healthy controls with COL11A1 (myCAF) having the best diagnostic accuracy of the markers measured. In conclusion, CAFs express a noncanonical collagen profile with specific collagen subtypes associated with iCAFs and myCAFs in PDAC. These collagens are deregulated at the cellular, tumor, and systemic levels across different solid tumors and associate with survival. These findings could lead to new discoveries such as novel biomarkers and therapeutic targets. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The role of peroxidasin in solid cancer progression.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in cancer.

Intra-abdominal packing does not increase infection risk or mandate longer presumptive antibiotic therapy.

BACKGROUND: Damage control laparotomy allows for resuscitation and reversal of coagulopathy with improved mortality. In-tra-abdominal packing is often used to limit hemorrhage. Temporary abdominal closure is associated with increased rates of subse-quent intra-abdominal infection. The effect of increased duration of antibiotics is unknown on these infection rates. We sought to determine the role of antibiotics in damage control surgery. METHODS: A retrospective analysis of all trauma patients requiring damage control laparotomy on admission to an ACS verified level one trauma center from 2011 to 2016 was performed. Demographic and clinical data including ability and time to attain primary fascial closure, as well as complication rates, were recorded. The primary outcome measure was intra-abdominal abscess formation following damage control laparotomy. RESULTS: Two-hundred and thirty-nine patients underwent DCS during the study period. A majority were packed (141/239, 59.0%). No differences existed in demographics or injury severity between groups, and infection rates were similar (30.5% vs. 38.8%, P=0.18). Patients with infection were more likely to have suffered gastric injury (23.3% vs. 6.1%, P=0.003) than those without complication. There was no significant association between gram negative and anaerobic (Odds Radio [OR] 0.96, 95% confidence interval [CI] 0.87-1.05) or antifungal therapy (OR 0.98, 95% CI 0.74-1.31) and infection rate, regardless of duration on multivariate regression CONCLUSION: Our study offers the first review of the effect of antibiotic duration on intra-abdominal complications following DCS. Gastric injury was more commonly identified in patients who developed intra-abdominal infection. Duration of antimicrobial therapy does not affect infection rate in patients who are packed following DCS.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

Contribution of animal models to the mechanistic understanding of Alternative Pathway and Amplification Loop (AP/AL)-driven Complement-mediated Diseases.

This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti-complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease.

Extracellular matrix profiles determine risk and prognosis of the squamous cell carcinoma subtype of non-small cell lung carcinoma.

BACKGROUND: Squamous cell carcinoma (SqCC) is a subtype of non-small cell lung cancer for which patient prognosis remains poor. The extracellular matrix (ECM) is critical in regulating cell behavior; however, its importance in tumor aggressiveness remains to be comprehensively characterized. METHODS: Multi-omics data of SqCC human tumor specimens was combined to characterize ECM features associated with initiation and recurrence. Penalized logistic regression was used to define a matrix risk signature for SqCC tumors and its performance across a panel of tumor types and in SqCC premalignant lesions was evaluated. Consensus clustering was used to define prognostic matreotypes for SqCC tumors. Matreotype-specific tumor biology was defined by integration of bulk RNAseq with scRNAseq data, cell type deconvolution, analysis of ligand-receptor interactions and enriched biological pathways, and through cross comparison of matreotype expression profiles with aging and idiopathic pulmonary fibrosis lung profiles. RESULTS: This analysis revealed subtype-specific ECM signatures associated with tumor initiation that were predictive of premalignant progression. We identified an ECM-enriched tumor subtype associated with the poorest prognosis. In silico analysis indicates that matrix remodeling programs differentially activate intracellular signaling in tumor and stromal cells to reinforce matrix remodeling associated with resistance and progression. The matrix subtype with the poorest prognosis resembles ECM remodeling in idiopathic pulmonary fibrosis and may represent a field of cancerization associated with elevated cancer risk. CONCLUSIONS: Collectively, this analysis defines matrix-driven features of poor prognosis to inform precision medicine prevention and treatment strategies towards improving SqCC patient outcome.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Dynamic dentin: A quantitative microscopic assessment of age and spatial changes to matrix architecture, peritubular dentin, and collagens types I and III.

To investigate age and site-related changes to human dentin collagen, sound human teeth collected from donors aged 13-29 (young) and 50-74 (aged) years (n = 9/group) were cut to shallow and deep sites. Dentin collagen orientation and fibril bundling was investigated using the Picrosirius Red (PSR) stain observed under cross-polarized light microscopy (Pol), and collagen distribution was investigated using Confocal Laser Scanning Microscopy (CLSM). Collagen types III to I distribution in peritubular dentin (PTD) was revealed using Herovici stain and brightfield microscopy. Image analysis software and linear mixed modelling quantified outcomes. In situ dentin collagen was observed using Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM). The PSR-Pol analysis revealed less coherently aligned and more bundled collagen fibrils in aged dentin (P = 0.005). Deep inner dentin collagen in both groups were less coherently aligned with reduced bundling. Regardless of age, CLSM showed collagen distribution remained stable; and more collagen type III was detectable in PTD located in inner dentin (Young: P = 0.006; Aged: P = 0.008). Observations following Xe PFIB-SEM cross-sectioning showed apatite-like deposits surrounding large intratubular collagen fibers, and evidence of smaller intertubular dentin collagen fibrils in situ. In conclusion, aging changes collagen network architecture, but not distribution or content.