Co-Culture of Gut Bacteria and Metabolite Extraction Using Fast Vacuum Filtration and Centrifugation.

This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell-cell interactions, cross-feeding, and the metabolic interactions between different bacteria, fungi, and microbial consortia which enable the mimicking of complex co-habitant conditions. This protocol highlights a robust reproducible physiologically relevant culture and extraction protocol for the co-culture of gut bacterium. The novel extraction steps are conducted without using quenching and cell disruption through bead-cell methods, freeze-thaw cycles, and sonication, which tend to affect the physical and biochemical properties of intracellular metabolites and secretome. The extraction procedure of inoculated bacterial co-cultures and monocultures use fast vacuum filtration and centrifugation. The extraction methodology is fast, effective, and robust, requiring 4 h to complete.

Microplastic-induced hepatic adverse effects evaluated in advanced quadruple cell human primary models following three weeks of repeated exposure.

Nano and microplastics are defined as particles smaller than 100 nm and 5 mm respectively. The widespread production and use of plastics in everyday life has resulted in significant accumulation of plastic debris in the environment. Over the last two decades there are increased concerns regarding the potential entry and accumulation of plastics in the human body with ingestion being one of the most important routes of exposure. However, the magnitude and nature of potential toxic effects of plastic exposure to human health is not yet fully understood. The liver is the body's principal detoxification organ and critically to this study recognized as the main accumulation site for particulates. In this study as the first of its kind the health impacts of long term low repeated polystyrene microplastics (1 and 5 µm) exposure was investigated in a functionally active 3D liver microtissue model, composed of primary human hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells. The highlight from the data includes microplastic-induced dose (3.125-25 µg/ml) and time dependent (up to 504 h) increase in cell death and inflammation manifested by enhanced release of IL6, IL8 and TNF-α. The exposure to repeated dosing of the plastics also resulted in notable pathology manifested as aberrant tissue architecture, such as dilated bile canaliculi and large lipid droplets inside the hepatic cells. This toxicity matched extremely well to the accumulation of the materials with the cells of microtissue predominately in the organ macrophages. This study highlights the real issue and danger of microplastic exposure with potential for long-term accumulation and adverse effects of non-biodegradable plastics within the liver.

Endobronchial Ultrasound Diagnosis of a Malignant Superior Vena Cava Tumor Thrombus Extending Into the Right Atrium: An Unusual Cause of Recurrent Syncope.

The superior vena cava (SVC) is mainly responsible for the return of blood flow from the head, upper limbs, and neck into the right atrium. The large vein can be subject to extrinsic tumor compression and invasive intraluminal tumors-metastatic and mediastinal tumors that can lead to complete or partial occlusion. SVC occlusion can also result from chronic inflammation or scarring of the vessel iatrogenically by pacemaker wires or venous access ports used for chemotherapy, long-term antibiotics, or hemodialysis. Patients with SVC occlusion present with a constellation of clinical abnormalities that make up SVC syndrome. SVC syndrome includes varying degrees of facial fullness, neck and upper extremity swelling, dyspnea, and classically dilated collateral veins in the upper chest. Very rarely do patients present with syncope, hoarseness, dysphagia, or acute encephalopathy. The diagnosis of SVC syndrome is best established on imaging such as CT Chest with contrast; however, on rare occasions, it can be discovered by endobronchial ultrasound. We present an unusual presentation of SVC syndrome- primarily presenting as frequent syncopal episodes- diagnosed via endobronchial ultrasound.

An Unusual Case of Hypoxia: A Case of Right-to-Left Interatrial Shunting in a Patient With a Patent Foramen Ovale and Normal Pulmonary Pressure.

A patent foramen ovale (PFO) is an embryological remnant. Hypoxia in the setting of a PFO is generally attributed to pulmonary hypertension resulting in an increase in right atrial pressure and mixing of venous blood from the right atrium with blood in the left atrium resulting in a right-to-left interatrial shunt (RLIAS), thus deoxygenating it. We present a case of a 64-year-old male with a past medical history of coronary artery disease (CAD) who presented with two weeks of dyspnea on exertion and intermittent chest pressure. He was found to be hypoxic at 87% (normal >95%) with largely normal workup except for left anterior descending (LAD) stenosis, which was stented, and a PFO that was found on transesophageal echocardiogram with normal pulmonary artery pressure (PAP). This case of hypoxia in the setting of a PFO without pulmonary hypertension puts into question the pathophysiology of hypoxia in a PFO and RLIAS.

Incidental Diagnosis of Pulmonary Embolism in Asymptomatic Patient Using Endobronchial Ultrasound (EBUS) During Mediastinal Lymphadenopathy Assessment.

The diagnosis of pulmonary embolism (PE) needs clinical manifestations and radiological findings. CT angiography (CTA) of pulmonary vessels is the gold standard of diagnosis of PE. However, endobronchial ultrasound (EBUS) can be a reliable and accurate alternative method of diagnosis in patients who are not candidates for CTA. Invasiveness and high cost are still the major limitations for EBUS, however, they should be considered in the appropriate population in future practice. We present a case of a 62-year-old asymptomatic male diagnosed with PE during EBUS for mediastinal lymph node assessment and biopsy.

Transverse Sinus Thrombosis Secondary to Internal Jugular Vein Abnormalities.

Cerebral sinus venous thrombosis (CSVT) is a rare disorder that presents with highly variable neurological manifestations ranging from headache, confusion, seizures, coma to stroke-like symptoms. It predominantly affects young adults and children, with female predilection. We are presenting a case of 59-year-old female with left-sided transverse and sigmoid sinus thrombosis most likely secondary to dehydration on top of chronically diminutive left transverse sinus and internal jugular vein.

Treatment of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown cause, occurring in adults and limited to the lungs. In the past, treatment was aimed at minimizing inflammation and slowing the progression of inflammation to fibrosis. However, the underlying lesion in IPF may be more fibrotic than inflammatory, explaining why few patients respond to anti-inflammatory therapies and the prognosis remains poor. In this review of literature, we will be focusing on main lines of treatment including current medications, supportive care, lung transplantation evaluation, and potential future strategies of treatment.

A Rare Cause of Exudative Pleural Effusion in a Female.

Yellow nail syndrome is an extremely rare syndrome that presents with a clinical triad of thickened yellow nails, lymphedema, and recurring pulmonary manifestations (pleural effusion, chronic cough, or bronchiectasis), usually in the population above the age of 50 years. We describe a case of yellow nail syndrome in a 48-year-old lady who presented with the typical classical triad of this syndrome.

Protamine Sulfate Neutralization Profile of Various Dosages of Bovine, Ovine and Porcine UFHs and Their Depolymerized Derivatives in Non-Human Primates.

INTRODUCTION: Currently used unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cow (Bovine) and sheep (Ovine). Protamine sulphate (PS) is an effective inhibitor of heparin and is used clinically to neutralize both LMWH and UFH. In this study, we determined the PS neutralization profile of these agents in non-human primate model using anti-Xa and anti-IIa methods. MATERIAL AND METHODS: UFHs obtained from bovine, ovine and porcine mucosal tissues and their respective depolymerized LMWHs were administered at both, gravimetric (0.5 mg/kg) and potency adjusted (100 U/kg) dosages regimen intravenously to individual groups of primates in cross over studies. PS was administered at a fixed dosage and the relative neutralization of these anticoagulants was measured utilizing amidolytic anti-Xa and anti-IIa methods. RESULTS: These studies have demonstrated that, the equi-gravimetric dosages of BMH, PMH and OMH have comparable PS neutralization profiles. At potency adjusted dosages, all UFHs were completely neutralized by PS. Although comparable, the LMWHs were not fully neutralized by PS in both the anti-Xa and anti-IIa assays. PS was more efficient in neutralizing the anti-IIa effects of LMWHs. CONCLUSION: Heparins of diverse origins showed comparable neutralization profiles by PS in the amidolytic anti-Xa and anti-IIa assays.

July Effect on Mortality and Complications in Patients With ARDS in US Teaching Hospitals.

BACKGROUND: Each July, teaching hospitals in the United States experience an influx of new resident and fellow physicians. It has been theorized that this occurrence may be associated with increased patient mortality, complication rates, and health care resource use, a phenomenon known as the "July effect." OBJECTIVE: To assess the existence of a July effect in clinical outcomes of patients with acute respiratory distress syndrome (ARDS) receiving mechanical ventilation in the intensive care unit in US teaching hospitals. METHODS: The National Inpatient Sample database was queried for all adult patients with ARDS who received mechanical ventilation from 2012 to 2014. Using a multivariate difference-in-differences (DID) model, differences in mortality, ventilator-associated pneumonia, iatrogenic pneumothorax, central catheter-associated bloodstream infection, and Clostridium difficile infection were compared between teaching and nonteaching hospitals during April-May and July-August. RESULTS: There were 70 535 and 43 175 hospitalizations meeting study criteria in teaching and nonteaching hospitals, respectively. Multivariate analyses revealed no differential effect on the rates of all-cause inpatient mortality (DID, 0.66; 95% CI, -0.42 to 1.75), C difficile infection (DID, 0.29; 95% CI, -0.19 to 0.78), central catheter-associated bloodstream infection (DID, 0.14; 95% CI, -0.04 to 0.33), iatrogenic pneumothorax (DID, 0.00; 95% CI, -0.25 to 0.24), ventilator-associated pneumonia (DID, 0.22; 95% CI, -0.05 to 0.49), and any complication (DID, 0.60; 95% CI, -0.01 to 1.20) for July-August versus April-May in teaching hospitals compared with nonteaching hospitals. CONCLUSION: This study did not show a differential July effect on mortality outcomes and complication rates in ARDS patients receiving mechanical ventilation in teaching hospitals compared with nonteaching hospitals.

A Retrospective Review of Outcomes in Intensive Care Unit Patients Infected With SARS-Cov2 in Correlation to Admission Acute Physiologic Assessment and Chronic Health Evaluation II Scores.

Introduction Coronavirus disease 2019 (COVID-19) has emerged as a global pandemic that has placed an unprecedented burden on intensive care services worldwide. Identification of a reliable risk-stratification tool for COVID-19 patients is necessary for appropriate resource allocation, selection of clinical management pathways, and guidance of goals of care conversations with families and caregivers in the critical care setting. The Acute Physiologic Assessment and Chronic Health Evaluation (APACHE) II scoring system is one of several predictive models used to classify illness severity and estimate mortality risk on admission to the intensive care unit (ICU). Our retrospective study sought to evaluate the prognostic ability of the APACHE II score in COVID-19 patients according to endpoints of mortality and length of stay (LOS) as well as unfavorable clinical outcomes, including development of acute renal failure (ARF) requiring renal replacement therapy (RRT) and acute venous thromboembolic events (VTE). Methods This multicenter retrospective cohort study evaluated a randomized sample of 3,102 patients with confirmed COVID-19 disease admitted to the ICU from January 2020 to May 2020. A total of 395 patients with complete data points for appropriate APACHE II score calculation, absence of the preexisting comorbidities end-stage renal disease, and history of VTE were included. Linear and logistic regression models were employed to evaluate primary outcomes of mortality and LOS as well as secondary outcomes of VTE and ARF requiring continuous renal replacement therapy (CRRT) or hemodialysis (HD). Key results Among the 395 patients enrolled, total percent mortality and mean LOS were 37.0% and 12.92 days, respectively. Primary outcome analysis revealed a statistically significant increase in odds of mortality as well as in mean LOS with every additional point increase in APACHE II score from a baseline of zero. Specifically, for every point increase in the APACHE II score, odds of mortality increased by 12% (p value < 0.001), and average LOS increased by 0.2 days (p value < 0.001). In our secondary outcome analysis, 14.43% and 62.2% of the total sample population developed ARF requiring RRT and VTE, respectively. For every additional point increase in APACHE II score from a baseline of zero, odds of requiring CRRT or HD increased by 10% on average (95% CI (1.06, 1.15); p value < 0.001). Similarly, for every additional point increase in the APACHE II score from a baseline of zero, there was a corresponding increase in odds of VTE by 19% (95% CI (1.14, 1.24); p value < 0.001). Conclusions The APACHE II score is an effective predictive model of in-hospital mortality and unfavorable clinical outcomes, including prolonged LOS, ARF requiring CRRT or HD, and development of VTE. As therapeutic interventions for COVID-19 evolve, application of this risk-stratification tool may guide clinical management decisions in the critical care setting.

Studies on Tissue Factor Pathway Inhibitor Antigen Release by Bovine, Ovine and Porcine Heparins Following Intravenous Administration to Non-Human Primates.

Unfractionated heparin (UFH) is a sulfated glycosaminoglycan that consists of repeating disaccharides, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits the extrinsic pathway of coagulation by inactivating factor Xa and the factor VIIa/TF complex. Most heparins used clinically are derived from porcine intestinal mucosa however, heparins can also be derived from tissues of bovine and ovine origin. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low molecular weight heparins (LMWHs). Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. Bovine mucosal heparins (BMH) are currently being developed for re-introduction to the US market for both medical and surgical indications. Compared to porcine mucosal heparin (PMH), BMH exhibits a somewhat weaker anti-coagulant activity. In this study, we determined the TFPI antigen level following administration of various dosages of UFHs from different origins. These studies demonstrated that IV administration of equigravemetric dosages of PMH and ovine mucosal heparin (OMH) to non-human primates resulted in comparable TFPI antigen release from endothelial cells. In addition, the levels of TFPI were significantly higher than TFPI antigen levels observed after BMH administration. Potency adjusted dosing resulted in comparable TFPI release profiles for all 3 heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations warrant further clinical validation in specific indications.

HSP70iQ435A-Encoding DNA Repigments Vitiligo Lesions in Sinclair Swine.

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells in vitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.