A case report of cardiopulmonary exercise testing and incentive spirometry-guided breathing pattern alteration for a severely tachypnoeic kyphoscoliotic patient undergoing major thoracic surgery for suspected lung malignancy.

A case report of a severely dyspnoeic kyphoscoliotic patient intended for an elective major thoracic surgery for suspected lung malignancy. With baseline near maximal breathing frequency and shallow breaths and poor lung mechanics, the first encountered anaesthetist considered this patient too high risk for lobectomy. This case illustrated the application of cardiopulmonary exercise testing to provide an objective assessment of the patient's functional capacity, ventilatory efficiency and delineation of modifiable respiratory components that guide the formulation of individualized prehabilitation programme. It also depicted the perioperative role of an off-label use of incentive spirometry in providing visual feedbacks and led to subsequent assessment breathing pattern alternation. Patient underwent the lung resection uneventfully and returned to normal lifestyle on postoperative day 4.

Drosophila Nhe2 overexpression induces autophagic cell death.

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Cardiovascular manifestations of Erdheim-Chester disease: A narrative review with two cases.

Erdheim-Chester disease (ECD) is a rare 'L' (Langerhans) group histiocytic neoplasm that affects a multitude of organ systems, causing osteosclerotic bone lesions, periaortic encasement ('coated' aorta), retroperitoneal fibrosis involving kidneys and ureters ('hairy kidney'), and infiltration of the central nervous system. Cardiovascular involvement can occur in up to 70% of patients and is usually found during computed tomography/magnetic resonance imaging evaluation. When present, cardiovascular symptoms can have wide variability in presentation from asymptomatic to pericarditis, fatal cardiac tamponade, myocardial infarction, conduction abnormalities, heart failure, renal artery stenosis, and claudication. Cardiac involvement found on imaging includes right atrial pseudotumor, right atrioventricular groove infiltration, and pericardial effusions. ECD can involve the large- and medium-sized arteries, often seen as periarterial thickening (commonly coating the aorta) with stenosis/occlusion. Although more cardiovascular ECD cases have begun to be published in the literature, more data are needed on the outcomes of these patients, as well as how cardiovascular manifestations respond to treatment of ECD.

Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis.

Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the inhibitor SHIN1, and by AICAR supplementation. Mechanistically, the metabolically driven differentiation is independent of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine 5'-monophosphate-activated protein kinase (AMPK) and is instead mediated by protein kinase C. Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate deficiency-induced anemia.

Regulation of mast cells by overlapping but distinct protein interactions of Siglec-6 and Siglec-8.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 and Siglec-8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. METHODS: Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec-6 and Siglec-8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non-IgE-mediated MC activation in Siglec-6- or Siglec-8-expressing transgenic mice. RESULTS: Siglec-6 and Siglec-8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec-6 and Siglec-8 interacted with a large cluster of proteins involved in IgE and non-IgE-mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL-4 and IL-33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec-6 and Siglec-8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec-6. Indeed, Siglec-6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti-Siglec-6 antibody significantly inhibited SCF-mediated MC activation more in comparison to targeting Siglec-8. CONCLUSION: These data demonstrate a central role for Siglec-6 and Siglec-8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec-6 has a role distinct from that of Siglec-8 in regulating MC function and represents a distinct potential therapeutic target in mast cell-driven diseases.

Guideline-based and restricted fluid resuscitation strategy in sepsis patients with heart failure: A systematic review and meta-analysis.

OBJECTIVES: To examine whether a fluid resuscitation strategy based on guidelines (at least 30 mL/kg IV crystalloids) vs. a restrictive approach with <30 mL/kg within three hours affects in-hospital mortality in patients with sepsis and a history of heart failure (HF). DATA SOURCES: On 03/07/2023, we searched Embase, PubMed, and Scopus for peer-reviewed papers and abstracts using the PRISMA guidelines. STUDY SELECTION: The language was limited to English. Studies published since 2016 included if they had sepsis patients with a history of HF, or a subgroup of patients with HF, and in-hospital mortality data on these patients that did or did not meet the 30 mL/kg by 3 h (30 × 3) goal. Duplicate studies, studies that focused on a broader period than 3 h from the diagnosis of sepsis or without mortality breakdown for HF patients or with unrelated title/abstract, or without an IRB approval were excluded. DATA EXTRACTION: In-hospital mortality data was taken from the final studies for HF patients with sepsis who did or did not meet the 30 × 3 goal. DATA SYNTHESIS: The meta-analysis was performed using the Review Manager 5.4 program with ORs as the effect measure. The ProMeta program version 3.0 was used to evaluate the publication bias. Egger's linear regression and Berg and Mazumdar's rank correlation was used to evaluate the publication bias. The result was visually represented by a funnel plot. To estimate the proportion of variance attributable to heterogeneity, the I2 statistic was calculated. RESULTS: The search yielded 26,069 records, which were narrowed down to 4 studies. Compared to those who met the 30 × 3 goal, the <30 × 3 group had a significantly higher risk of in-hospital mortality (OR = 1.81, 95% CI = 1.13-2.89, P = 0.01). CONCLUSIONS: Restrictive fluid resuscitation increased the risk of in-hospital mortality in HF patients with sepsis. More rigorous research is required to determine the optimal fluid resuscitation strategy for this population.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity.

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

A roadmap to high-resolution standard microcoil MAS NMR spectroscopy for metabolomics.

Current microcoil probe technology has emerged as a significant advancement in NMR applications to biofluids research. It has continued to excel as a hyphenated tool with other prominent microdevices, opening many new possibilities in multiple omics fields. However, this does not hold for biological samples such as intact tissue or organisms, due to the considerable challenges of incorporating the microcoil in a magic-angle spinning (MAS) probe without relinquishing the high-resolution spectral data. Not until 2012 did a microcoil MAS probe show promise in profiling the metabolome in a submilligram tissue biopsy with spectral resolution on par with conventional high-resolution MAS (HR-MAS) NMR. This result subsequently triggered a great interest in the possibility of NMR analysis with microgram tissues and striving toward the probe development of "high-resolution" capable microcoil MAS NMR spectroscopy. This review gives an overview of the issues and challenges in the probe development and summarizes the advancements toward metabolomics.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

CRISPR screening in cancer stem cells.

Cancer stem cells (CSCs) are a subpopulation of tumor cells with self-renewal ability. Increasing evidence points to the critical roles of CSCs in tumorigenesis, metastasis, therapy resistance, and cancer relapse. As such, the elimination of CSCs improves cancer treatment outcomes. However, challenges remain due to limited understanding of the molecular mechanisms governing self-renewal and survival of CSCs. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screening has been increasingly used to identify genetic determinants in cancers. In this primer, we discuss the progress made and emerging opportunities of coupling advanced CRISPR screening systems with CSC models to reveal the understudied vulnerabilities of CSCs.

Optochemical Control of mTOR Signaling and mTOR-Dependent Autophagy.

As an important regulator of cell metabolism, proliferation, and survival, mTOR (mammalian target of rapamycin) signaling provides both a potential target for cancer treatment and a research tool for investigation of cell metabolism. One inhibitor for both mTORC1 and mTORC2 pathways, OSI-027, exhibited robust anticancer efficacy but induced side effects. Herein, we designed a photoactivatable OSI-027 prodrug, which allowed the release of OSI-027 after light irradiation to inhibit the mTOR signaling pathway, triggering autophagy and leading to cell death. This photoactivatable prodrug can provide novel strategies for mTOR-targeting cancer therapy and act as a new tool for investigating mTOR signaling and its related biological processes.

A Combinatorial CRISPR-Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment.

Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell's metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Metabolic NMR mapping with microgram tissue biopsy.

This study explores the potential of profiling a microgram-scale soft tissue biopsy by NMR spectroscopy. The important elements of high resolution and high sensitivity for the spectral data are achieved through a unique probe, HR-µMAS, which allowed comprehensive profiling to be performed on microgram tissue for the first time under MAS conditions. Thorough spatially resolved metabolic maps were acquired across a coronal brain slice of rat C6 gliomas, which rendered the delineation of the tumor lesion. The results present a unique ex vivo NMR possibility to analyze tissue pathology that cannot be fully explored by the conventional approach, HR-MAS and in vivo MRS. Aside from the capability of analyzing a small localized region to track its specific metabolism, it could also offer the possibility to carry out longitudinal investigations on live animals due to the feasibility of minimally invasive tissue excision.

A monoclonal antibody to Siglec-8 suppresses non-allergic airway inflammation and inhibits IgE-independent mast cell activation.

In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.

Single-Cell RNA-Seq Reveals that CD9 Is a Negative Marker of Glucose-Responsive Pancreatic ß-like Cells Derived from Human Pluripotent Stem Cells.

To date, it remains unclear if there are specific cell-surface markers for purifying glucose-responsive pancreatic ß-like cells derived from human pluripotent stem cells (hPSCs). In searching for this, we generated an efficient protocol for differentiating ß-like cells from human embryonic stem cells. We performed single-cell RNA sequencing and found that CD9 is a negative cell-surface marker of ß-like cells, as most INS+ cells are CD9-. We purified ß-like cells for spontaneous formation of islet-like organoids against CD9, and found significantly more NKX6.1+MAFA+C-PEPTIDE+ ß-like cells in the CD9- than in the CD9+ population. CD9- cells also demonstrate better glucose responsiveness than CD9+ cells. In humans, we observe more CD9+C-PEPTIDE+ ß cells in the fetal than in the adult cadaveric islets and more Ki67+ proliferating cells among CD9+ fetal ß cells. Taken together, our experiments show that CD9 is a cell-surface marker for negative enrichment of glucose-responsive ß-like cells differentiated from hPSCs.

Cutaneous angiosarcoma of the head and neck resembling rosacea: A case report.

Angiosarcoma is a malignant endothelial cell tumor that involves a variety of anatomic sites with the skin being the most common. Cutaneous angiosarcoma is a diagnostic challenge as it can be confused with lesions such as rosacea, hemangiomas and hematomas. Since the tumor has a propensity for early metastasis and extensive intradermal spread, early diagnostic intervention via punch biopsy may prevent delays in diagnosis and improve tumor resectability and prognosis. We present a case of cutaneous angiosarcoma on the nose and cheeks of a 75-year-old male that resembled rosacea.

A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

Emerging drugs for the treatment of hidradenitis suppurativa.

INTRODUCTION: Hidradenitis suppurativa (HS) is a severe, chronic inflammatory disorder that causes recurrent occlusion of hair follicles in the intertriginous regions of the skin. Mild to moderate HS has been successfully treated with oral antibiotics, topical therapy, and lifestyle modifications. However, moderate to severe HS is known to be refractory to conventional treatments. Wide excision surgery is a treatment option for severe HS, but often leads to functional impairments. Additionally, recurrence is common. The proper management of moderate to severe HS continues to be a challenge to practitioners. AREAS COVERED: A comprehensive literature search was conducted to identify published HS treatments using PubMed databases, in addition, ongoing studies were sought in clinicaltrials.gov. Search terms included 'hidradenitis suppurativa,' 'treatment,' and 'management.' EXPERT OPINION: Although adalimumab is currently the only biologic approved by the United States Food and Drug Administration for treatment of HS, there are many studies underway involving the development of drugs with a variety of immunological targets. Those potential HS therapies in either Phase II or Phase III trials show much promise. Since HS is a complicated disease that involves both pathological and environmental factors, treating HS continues to involve a multidisciplinary approach and monotherapy tends to not be efficacious.