An integrated toolkit for human microglia functional genomics.

BACKGROUND: Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9. METHODS: Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . RESULTS: Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. CONCLUSIONS: Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.

Multiple energy X-ray imaging of metal oxide particles inside gingival tissues.

BACKGROUND: Periodontal disease affects over 50% of the global population and is characterized by gingivitis as the initial sign. One dental health issue that may contribute to the development of periodontal disease is foreign body gingivitis (FBG), which can result from exposure to some kinds of foreign metal particles from dental products or food. OBJECTIVE: We design a novel, portable, affordable, multispectral X-ray and fluorescence optical microscopic imaging system dedicated to detecting and differentiating metal oxide particles in dental pathological tissues. A novel denoising algorithm is applied. We verify the feasibility and optimize the performance of the imaging system with numerical simulations. METHODS: The designed imaging system has a focused X-ray tube with tunable energy spectra and thin scintillator coupled with an optical microscope as detector. A simulated soft tissue phantom is embedded with 2-micron thick metal oxide discs as the imaged object. GATE software is used to optimize the systematic parameters such as energy bandwidth and X-ray photon number. We have also applied a novel denoising method, Noise2Sim with a two-layer UNet structure, to improve the simulated image quality. RESULTS: The use of an X-ray source operating with an energy bandwidth of 5 keV, X-ray photon number of 108, and an X-ray detector with a 0.5 micrometer pixel size in a 100 by 100-pixel array allowed for the detection of particles as small as 0.5 micrometer. With the Noise2Sim algorithm, the CNR has improved substantially. A typical example is that the Aluminum (Al) target's CNR is improved from 6.78 to 9.72 for the case of 108 X-ray photons with the Chromium (Cr) source of 5 keV bandwidth. CONCLUSIONS: Different metal oxide particles were differentiated using Contrast-to-Noise ratio (CNR) by utilizing four different X-ray spectra.

Evolving Advance Care Planning in a Health Ecosystem: The Kaiser Permanente Experience.

BACKGROUND: Advance care planning is an integral part of supporting patients through serious illness and end-of-life care. PROBLEM: Several components of advance care planning may be too inflexible to account for patients' changing disease and evolving goals as serious illness progresses. Health systems are starting to implement processes to address these barriers, though implementation has varied. PROPOSED SOLUTION: In 2017, Kaiser Permanente introduced Life Care Planning (LCP), incorporating advance care planning dynamically into concurrent disease management. LCP provides a framework for identifying surrogates, documenting goals, and eliciting patient values across disease progression. LCP provides standardized training to facilitate communication and utilizes a centralized section within the electronic health record for longitudinal documentation of goals. OUTCOMES: More than 6000 physicians, nurses, and social workers have been trained in LCP. Over one million patients have engaged in LCP since its inception, with over 52% of patients age 55+ having a surrogate designated. There is evidence of high treatment concordance with patients' desired wishes (88.9%), with high rates of advance directive completion as well (84.1%).

A Case Report of Rapidly Lethal Acute Respiratory Distress Syndrome Secondary to Coronavirus Disease 2019 Viral Pneumonia.

As of April 2020, the coronavirus 2019 (COVID-19) pandemic has resulted in more than 210,000 deaths globally. The most common cause of death from COVID-19 is acute respiratory failure. We report the case of a 78-year-old female with a history of hypertension, cerebrovascular accident (CVA), type 2 diabetes mellitus, and sarcoidosis, who presented to the emergency department with one day of dyspnea. The patient experienced a rapid decline in respiratory function and was intubated in the intensive care unit (ICU), meeting the Berlin criteria for severe acute respiratory distress syndrome (ARDS). Chest radiography revealed diffuse bilateral coalescent opacities, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA swab test was positive for COVID-19. The patient experienced acute kidney injury with uptrending creatinine levels and remained lethargic and unresponsive throughout her ICU stay, suggestive of potential hypoxic brain injury. In light of the patient's poor clinical status, age, and significant comorbidities, prognosis was conveyed about medical futility and patient's family agreed to terminal extubation and the patient expired peacefully, exactly one week from hospital admission. This case report highlights the speed at which severe ARDS can present and contribute to end-organ dysfunction in COVID-19 patients.

U.S. Healthcare Insurance Market Concentration from 2001 to 2016: Increased Growth in Direct Written Premiums and Overall Decreased Market Consolidation.

With the establishment of state-based health insurance marketplaces, how U.S. health insurers are responding to market pressures and influencing premiums have represented important questions. We made novel use of the Standard and Poor's (S&P) Financial, a Wall Street financial dataset platform, to analyze trends in market capitalization and total direct written premiums (DWPs) from 2001 to 2016 of the top 5, 10, and 25 health insurance companies. Our results indicate that the market concentration of publicly traded companies has remained relatively stable over the past decade. The top 5, 10, and 25 health insurance companies were 43.5%, 57.5%, and 78.6% of the total market share in 2001 and 39.4%, 52.9%, and 72.8% in 2016, respectively. DWPs have grown nearly four-fold from $177 billion to $631 billion at a compounded annual rate of 8.8%, consistent with overall healthcare sector growth. Aggregating state-specific data, the overall U.S. health insurance market has become slightly less consolidated over recent years, as measured using the population-weighted Herfindahl-Hirschman index, a measure for market concentration, falling from 3,817 to 2,174 during this time period. As health insurance costs place a growing burden on American families, additional efforts are needed to study the impact on choice, quality, access, cost, and value to patients and providers from evolving health insurance markets.

Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes.

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide-RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates.

Ribonucleoprotein (RNP) granules are membraneless liquid condensates that dynamically form,dissolve, and mature into a gel-like state in response to a changing cellular environment. RNP condensation islargely governed by promiscuous attractive inter-chain interactions mediated by low-complexity domains(LCDs). Using an archetypal disordered RNP, fused in sarcoma (FUS), here we study how molecular crowdingimpacts the RNP liquid condensation. We observe that the liquid⁻liquid coexistence boundary of FUS islowered by polymer crowders, consistent with an excluded volume model. With increasing bulk crowderconcentration, the RNP partition increases and the diffusion rate decreases in the condensed phase.Furthermore, we show that RNP condensates undergo substantial hardening wherein protein-dense dropletstransition from viscous fluid to viscoelastic gel-like states in a crowder concentration-dependent manner.Utilizing two distinct LCDs that broadly represent commonly occurring sequence motifs driving RNP phasetransitions, we reveal that the impact of crowding is largely independent of LCD charge and sequence patterns.These results are consistent with a thermodynamic model of crowder-mediated depletion interaction, whichsuggests that inter-RNP attraction is enhanced by molecular crowding. The depletion force is likely to play akey role in tuning the physical properties of RNP condensates within the crowded cellular space.

Does the incidence of sore throat postoperatively increase with the use of a traditional intubation blade or the GlideScope?

STUDY OBJECTIVE: The GlideScope video-guided laryngoscope is an alternative standard of care for rescue laryngoscopies when direct laryngoscopy is unsuccessful. During postoperative checks by an anesthesiologist, it was noticed that patients who reported sore throat often required GlideScope laryngoscopy. Consequently, it is difficult to determine whether postoperative sore throats are caused by irritation inflicted by multiple laryngoscopic attempts or the actual utilization of the GlideScope itself. The goal of this study was to determine whether the use of the GlideScope leads to a greater or lesser incidence of sore throat when compared with traditional laryngoscope blades used for intubation. DESIGN: Eligible patients scheduled for elective inpatient surgeries requiring endotracheal tube intubation were enrolled into this single-blinded prospective cohort study. χ(2) Test, Fisher exact test, and t tests were used to compare differences across the primary end point and other demographic categories. SETTING: Operating rooms and postanesthesia recovery unit, Albany Medical Center, Albany, NY. PATIENTS: There were a total of 151 patients with American Society of Anesthesiologists grades 1 to 3 included in the study. INTERVENTIONS: Eighty-one patients were randomized to a control group that received traditional laryngoscopy via Macintosh/Miller blades and 70 patients received video-guided intubation via the GlideScope. MEASUREMENTS: The incidence of postoperative sore throat was recorded via a yes/no questionnaire within 24 hours after extubation. Secondary parameters such as provider type, sex, and perceived difficulty were also recorded. MAIN RESULTS: There was no significant difference in the proportion of patients reporting sore throat by type of blade used (Mac/Miller 36.3% vs GlideScope 32.4%, P = .619). For secondary outcomes, women were significantly more likely to report sore throat as compared with men (men 24.3% vs women 43.2%, P = .015), and the provider type was significantly associated with the occurrence of postoperative sore throat (attendings 26.8% vs certified registered nurse anesthetists 52.3% vs third-year clinical anesthesia residents 30%, P = .012). CONCLUSIONS: Use of the GlideScope videolaryngoscopy was not significantly associated with increased occurrence of postoperative sore throat when compared with traditional intubation techniques. Our results may enable more trainees to acquire intubation skills with the GlideScope during an initial intubation attempt in patients with American Society of Anesthesiologist grades 1 to 3, with optimization of patient satisfaction in respect to postoperative sore throats. In addition, a provider's choice of intubation technique based on either Macintosh/Miller blades or the GlideScope does not significantly impact a patient's risk of postoperative sore throat.

Heparanase mediates a novel mechanism in lapatinib-resistant brain metastatic breast cancer.

Heparanase (HPSE) is the dominant mammalian endoglycosidase and important tumorigenic, angiogenic, and pro-metastatic molecule. Highest levels of HPSE activity have been consistently detected in cells possessing highest propensities to colonize the brain, emphasizing the therapeutic potential for targeting HPSE in brain metastatic breast cancer (BMBC). Lapatinib (Tykerb) is a small-molecule and dual inhibitor of human epidermal growth factor receptor1 and 2 (EGFR and HER2, respectively) which are both high-risk predictors of BMBC. It was approved by the US Food and Drug Administration for treatment of patients with advanced or metastatic breast cancer. However, its role is limited in BMBC whose response rates to lapatinib are significantly lower than those for extracranial metastasis. Because HPSE can affect EGFR phosphorylation, we examined Roneparstat, a non-anticoagulant heparin with potent anti-HPSE activity, to inhibit EGFR signaling pathways and BMBC onset using lapatinib-resistant clones generated from HER2-transfected, EGFR-expressing MDA-MB-231BR cells. Cell growth, EGFR pathways, and HPSE targets were assessed among selected clones in the absence or presence of Roneparstat and/or lapatinib. Roneparstat overcame lapatinib resistance by inhibiting pathways associated with EGFR tyrosine residues that are not targeted by lapatinib. Roneparstat inhibited the growth and BMBC abilities of lapatinib-resistant clones. A molecular mechanism was identified by which HPSE mediates an alternative survival pathway in lapatinib-resistant clones and is modulated by Roneparstat. These results demonstrate that the inhibition of HPSE-mediated signaling plays important roles in lapatinib resistance, and provide mechanistic insights to validate the use of Roneparstat for novel BMBC therapeutic strategies.

Increased red blood cell transfusions are associated with worsening outcomes in pediatric heart transplant patients.

BACKGROUND: Red blood cell (RBC) transfusions are associated with increased morbidity. Children receiving heart transplants constitute a unique group of patients due to their risk factors. Although previous studies in nontransplant patients have focused primarily on the effects of postoperative blood transfusions, a significant exposure to blood occurs during the intraoperative period, and a larger percentage of heart transplant patients require intraoperative blood transfusions when compared with general cardiac surgery patients. We investigated the relationship between clinical outcomes and the amount of blood transfused both during and after heart transplantation. We hypothesized that larger amounts of RBC transfusions are associated with worsening clinical outcomes in pediatric heart transplant patients. METHODS: A database comprising 108 pediatric patients undergoing heart transplantation from 2004 to 2010 was queried. Preoperative and postoperative clinical risk factors, including the amount of blood transfused intraoperatively and 48 hours postoperatively, were analyzed. The outcome measures were length of hospital stay, duration of tracheal intubation, inotrope score, and major adverse events. Bivariate and multivariate analyses were performed to control for simultaneous risk factors and determine outcomes in which the amount of blood transfused was an independent risk factor. RESULTS: Ninety-four patients with complete datasets were included in the final analysis. Eighty-eight percent received RBC transfusions, with a median transfusion amount of 38.7 mL/kg. A multivariate analysis correcting for 8 covariate risk factors, including the Index for Mortality Prediction After Cardiac Transplantation, age, weight, United Network for Organ Sharing status, warm and cold ischemia time, repeat sternotomy, and pretransplant hematocrit, showed RBC transfusions were independently associated with increased length of intensive care unit stay (means ratio = 1.34; 95% confidence interval, 1.03-1.76; P = 0.03), and increased inotrope score in the first postoperative 24 hour (mean ratio = 1.26; 95% confidence interval, 1.04-1.52; P = 0.04). Patients suffering major adverse events received significantly larger median amounts of blood RBC transfusions (P = 0.002). Transfusions >60 mL/kg were also associated with increased risk of major adverse events (accuracy 76%) including postoperative sepsis, extracorporeal membrane oxygenation, open chest, dialysis, and graft failure. CONCLUSION: The majority of pediatric patients undergoing orthotropic heart transplantation receive RBC transfusions, with the largest amount transfused in the operating room. Escalating amounts of RBC transfusions are independently associated with increased length of intensive care unit stay, inotrope scores, and major adverse events. Since heart allografts are a limited resource, improvement in the blood transfusion and conservation practices can enhance clinical outcomes in pediatric heart transplant patients.

The identification and characterization of breast cancer CTCs competent for brain metastasis.

Brain metastatic breast cancer (BMBC) is uniformly fatal and increasing in frequency. Despite its devastating outcome, mechanisms causing BMBC remain largely unknown. The mechanisms that implicate circulating tumor cells (CTCs) in metastatic disease, notably in BMBC, remain elusive. We characterize CTCs isolated from peripheral blood mononuclear cells of patients with breast cancer and also develop CTC lines from three of these patients. In epithelial cell adhesion molecule (EpCAM)-negative CTCs, we identified a potential signature of brain metastasis comprising "brain metastasis selected markers (BMSMs)" HER2+ / EGFR+ / HPSE+ / Notch1+. These CTCs, which are not captured by the CellSearch platform because of their EpCAM negativity, were analyzed for cell invasiveness and metastatic competency in vivo. CTC lines expressing the BMSM signature were highly invasive and capable of generating brain and lung metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency, and quantitation were detected in EpCAM- CTCs overexpressing the BMSM signature. The presence of proteins of the BMSM CTC signature was also detected in the metastatic lesions of animals. Collectively, we provide evidence of isolation, characterization, and long-term culture of human breast cancer CTCs, leading to the description of a BMSM protein signature that is suggestive of CTC metastatic competency to the brain.

Heparanase-induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells.

Heparanase is the only mammalian endoglycosidase which has been widely implicated in cancer because of its capability to degrade heparan sulfate chains of heparan sulfate proteoglycans (HSPG). Specifically, the cell surface HSPG syndecan-1 and -4 (SDC1 and SDC4) are modulators of growth factor action, and SDC4 is implicated in cell adhesion as a key member of focal adhesion complexes. We hypothesized that extracellular heparanase modulates brain metastatic breast cancer (BMBC) cell invasiveness by affecting cytoskeletal dynamics, SDC4 carboxy-terminal-associated proteins, and downstream targets. We used two independently derived human BMBC cell systems (MB-231BR and MB-231BR3), which possess distinct cellular morphologies and properties. Highly aggressive spindle-shaped 231BR3 cells changed to a round cell morphology associated with expression of the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1). We showed that GEF-H1 is a new component of the SDC4 signaling complex in BMBC cells. Treatment with heparanase resulted in regulation of the SDC4/protein kinase C α axis while maintaining a constitutive GEF-H1 level. Third, GEF-H1 knockdown followed by cell exposure to heparanase caused a significant regulation of activities of Rac1 and RhoA, which are GEF-H1 targets and fundamental effectors in cell plasticity control. Fourth, L-heparanase augmented expression of ß1 integrin in BMBC cells and of vascular cell adhesion molecule 1 (VCAM1; the major ß1 integrin receptor) in human brain microvascular endothelial cells. Finally, using a newly developed blood-brain barrier in vitro model, we show that BMBC cell transmigration was significantly reduced in GEF-H1 knockdown cells. These findings implicate heparanase in mechanisms of cytoskeletal dynamics and in the cross-talk between tumor cells and vascular brain endothelium. They are of relevance because they elucidate molecular events in the initial steps leading to BMBC onset and capturing distinct roles of latent and active heparanase in the brain microenvironment.