Sialylated Glycan Bindings from SARS-CoV-2 Spike Protein to Blood and Endothelial Cells Govern the Severe Morbidities of COVID-19.

Consistent with well-established biochemical properties of coronaviruses, sialylated glycan attachments between SARS-CoV-2 spike protein (SP) and host cells are key to the virus's pathology. SARS-CoV-2 SP attaches to and aggregates red blood cells (RBCs), as shown in many pre-clinical and clinical studies, causing pulmonary and extrapulmonary microthrombi and hypoxia in severe COVID-19 patients. SARS-CoV-2 SP attachments to the heavily sialylated surfaces of platelets (which, like RBCs, have no ACE2) and endothelial cells (having minimal ACE2) compound this vascular damage. Notably, experimentally induced RBC aggregation in vivo causes the same key morbidities as for severe COVID-19, including microvascular occlusion, blood clots, hypoxia and myocarditis. Key risk factors for COVID-19 morbidity, including older age, diabetes and obesity, are all characterized by markedly increased propensity to RBC clumping. For mammalian species, the degree of clinical susceptibility to COVID-19 correlates to RBC aggregability with p = 0.033. Notably, of the five human betacoronaviruses, the two common cold strains express an enzyme that releases glycan attachments, while the deadly SARS, SARS-CoV-2 and MERS do not, although viral loads for COVID-19 and the two common cold infections are similar. These biochemical insights also explain the previously puzzling clinical efficacy of certain generics against COVID-19 and may support the development of future therapeutic strategies for COVID-19 and long COVID patients.

Consecutive nursing shifts and the risk of hypoglycemia in critically ill patients who are receiving intravenous insulin: a multicenter study.

STUDY OBJECTIVES: Intensive care unit nurses commonly work multiple consecutive 12-hour shifts that leave little time for sleep between work shifts. Working multiple consecutive shifts could compromise vigilance and patient care, especially with respect to managing high-risk medications such as insulin infusions. We hypothesized that as the number of consecutive shifts worked by nurses increases, the rate of hypoglycemia in patients who are receiving an insulin infusion would also increase. METHODS: We identified patients who had hypoglycemia (glucose ≤ 3.5 mmol/L, 63 mg/dL) between December 2008 and December 2009 in 3 intensive care units in Vancouver, British Columbia, Canada. For each hypoglycemic event, we counted the number of shifts worked on consecutive days during the previous 72 hours by the bedside nurse who was caring for the patient at the time of hypoglycemia (case shift). For each case shift, we identified up to 3 control shifts (24, 48, and 72 hours before the hypoglycemic event in the same patient when there were no hypoglycemic events) and counted the number of consecutive shifts worked by those nurses in the previous 72 hours. This analysis allowed us to control for patient-associated confounders. Conditional logistic regression was used to determine the association between number of consecutive shifts worked and occurrence of hypoglycemic events. RESULTS: A total of 282 hypoglycemic events were identified in 259 patients. For 191 events, we were able to identify 1 or more control shifts. Compared with nurses who had not worked a shift in the preceding day, the odds ratio of a hypoglycemic event was 1.68 (95% confidence interval: 1.12-2.52), 2.16 (95% confidence interval:1.25-3.73), and 2.54 (95% confidence interval: 1.28-5.06) for nurses who were working their second, third, or fourth consecutive shift, respectively. CONCLUSIONS: Working multiple consecutive nursing shifts is associated with increased risk of hypoglycemic events in patients in an intensive care unit.

Chromatographic assay to probe the binding energy and mechanisms of homologous proteins to surface-bound ligands.

Probing the affinity of a ligand for homologous protein targets currently relies on laborious assays that need special equipment and high amounts of isolated, highly pure proteins. Herein we present the use of pISep, an integrated buffer system and modeling package, as an analytical method to rapidly and accurately probe the binding strength and mechanisms of homologous proteins to surface-bound ligands. To demonstrate our method, we utilized the four subclasses of human immunoglobulin G (IgG) as model homologous protein targets and the IgG-binding peptide HWRGWV as model ligand. Following IgG adsorption on a HWRGWV-Toyopearl adsorbent, the pISep buffer system was used to run uncoupled dual elution gradients of pH (from pH 8.5 to 2.5) and either isocratic or time dependent salt concentration. Both the sequence and partial overlap of elution times (IgG4 > IgG3 ≥ IgG1 > IgG2) was found to match closely the values of binding strength (KD) determined with both in silico docking simulations and isothermal titration calorimetry experiments. pISep gradients performed at different values of ionic strengths provided a means to compare the contribution of hydrophobic vs. electrostatic interactions to the IgG-peptide affinity. The shifts in retention times indicated that, among the various components of the binding energy, the hydrophobic interaction dominates in the binding of IgG2 and IgG4, whereas the binding of IgG1 and IgG3 features a balance of electrostatic and hydrophobic modes. These findings were also confirmed by the in silico analysis of the complexes formed by HWRGWV and the Fc fragment of the IgG subclasses. Collectively, these results indicate that the retention times on pISep elution gradients - in particular peak max, overlap, and shift under different conditions - directly correlate to the strength and nature of protein-ligand interactions. This work demonstrates the effectiveness of the pISep toolbox for probing the differential binding of homologous proteins to a reference ligand and informing the optimization of platform processes for the purification and fractionation of biotherapeutics.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 2: Computer controlled pH gradients in the presence of urea provide improved separation of proteins: Stability influenced anion and cation exchange chromatography.

This paper details the use of a method of creating controlled pH gradients (pISep) to improve the separation of protein isoforms on ion exchange (IEX) stationary phases in the presence of various isocratic levels of urea. The pISep technology enables the development of computer controlled pH gradients on both cationic (CEX) and anionic (AEX) IEX stationary phases over the very wide pH range from 2 to 12. In pISep, titration curves generated by proportional mixing of the acidic and basic pISep working buffers alone, or in the presence of non-buffering solutes such as the neutral salt NaCl (0-1M), polar organics such as urea (0-8M) or acetonitrile (0-80 Vol%), can be fitted with high fidelity using high order polynomials which, in turn allows construction of a mathematical manifold %A (% acidic pISep buffer) vs. pH vs. [non-buffering solute], permitting precise computer control of pH and the non-buffering solute concentration allowing formation of dual uncoupled liquid chromatographic (LC) gradients of arbitrary shape (Hirsh and Tsonev, 2012 [1]). The separation of protein isoforms examined in this paper by use of such pH gradients in the presence of urea demonstrates the fractionation power of a true single step two dimensional liquid chromatography which we denote as Stability-Influenced Ion Exchange Chromatography (SIIEX). We present evidence that SIIEX is capable of increasing the resolution of protein isoforms difficult to separate by ordinary pH gradient IEX, and potentially simplifying the development of laboratory and production purification strategies involving on-column simultaneous pH and urea unfolding or refolding of targeted proteins. We model some of the physics implied by the dynamics of the observed protein fractionations as a function of both urea concentration and pH assuming that urea-induced native state unfolding competes with native state electrostatic interaction binding to an IEX stationary phase. Implications for in vivo protein-membrane interactions are discussed.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 1 - Large increases in isoform resolution of human transferrin by use of dual simultaneous independent gradients of pH & acetonitrile on a mixed bed (anion exchange plus reversed phase) stationary phase.

We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding.

Multiple, simultaneous, independent gradients for versatile multidimensional liquid chromatography. Part I: Theory.

The general method for constructing coupled dual gradients in liquid chromatography (LC) is to begin by filling a reservoir A with a solution of one mobile phase (MP) component at concentration [c(1)(A)] and a second MP component at concentration [c(2)(A)], followed by filling a reservoir B with a solution containing MP component one at concentration [c(1)(B)] and the second MP component at concentration [c(2)(B)]. In another scenario the reservoirs A and B are filled with solutions of only one MP component at different concentrations [c(1)(A)] and [c(1)(B)] and the two solutions are titrated to a different pH value: pH (A) for the reservoir A and pH (B) for the reservoir B respectively. In either case, mixing of flows from the two reservoirs varies the concentrations of the two MP components (MP solutes) or the concentration of one MP component and pH along a particular compositional curve producing an eluent with two compositionally coupled gradients. This is a kind of a two dimensional LC utilizing dual simultaneous dependent gradients (DSDGs) wherein two parameters affecting the binding free energy of an analyte to a stationary phase (SP) are being altered simultaneously. Such a DSDG suffers from a significant limitation in that the gradient concentration of the two solutes or the concentration of one MP component and the pH cannot be varied independently. The only way to attain an optimal multigradient LC system, that promises a remarkable increase in chromatographic resolution of complex analyte mixtures, is to uncouple the multiple (dual) gradients, making each independent of the other(s). In this paper the theory of uncoupling of n such gradients, n ≥ 2 is developed. It is shown that for n solutes 2(n) reservoirs are required in concert with an LC eluent delivery system capable of freely apportioning the flows among the reservoirs according to equations we develop here. We go on to predict a substantial increase in chromatographic resolution when applying dual simultaneous independent gradients (DSIGs) of salt and pH to fractionate difficult to separate proteins. This prediction is naturally explained by the electrostatic interaction theory of protein binding to an ion exchanger. In subsequent experimental papers it will be shown that the algorithms presented here properly instruct a quad pump HPLC system to produce well controlled independent simultaneous gradients of pH and non-buffering solutes with attendant significant gain in chromatographic resolution of complex mixtures of protein isoforms.

Improving selectivity in multimodal chromatography using controlled pH gradient elution.

Externally generated pH gradients are employed on a multimodal cation exchange chromatographic resin to improve the selectivity for a mixture of model proteins. By combining controlled pH gradients with the unique selectivities arising from the multiple interaction types exhibited by the multimodal resin, the separation of the protein mixture is significantly improved as compared to linear salt gradient operation. Several gradient conditions are explored and a shallow gradient from pH 3.8 to 5.5 is shown to be able to resolve the proteins. This work provides proof of concept for the use of pH gradients in multimodal chromatography and sets the stage for future applications.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.