Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam-sterilizable dielectricspectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell-culture processes. Technological challenges still exist, however. For some cell lines, the technique’s accuracy in predicting the VCV from probepermittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency-scanning data from the dielectric-spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single-frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed-batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency-scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape-characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health.
The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.
High-throughput screening of a small-molecule library identified a 5-triazolo-2-arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3). Such inhibitors are of interest due to PFKFB3's control of the important glycolytic pathway used by cancer cells to generate ATP. A series of analogues was synthesized to study structure-activity relationships key to enzyme inhibition. Changes to the triazolo or pyridazinone rings were not favoured, but limited-size substitutions on the aryl ring provided modest increases in potency against the enzyme. Selected analogues and literature-described inhibitors were evaluated for their ability to suppress the glycolytic pathway, as detected by a decrease in lactate production, but none of these compounds demonstrated such suppression at non-cytotoxic concentrations.
Sleeve gastrectomy has increased in popularity over the last five years and it is likely to supersede gastric banding. Nevertheless, it is unclear whether vitamin B12 supplementation is required after surgery. The aim of this short report is to identify any vitamin B12 deficiency and highlight the necessity of post laparoscopic sleeve gastrectomy vitamin B12 monitoring.
Tracheal intubation in the Intensive Care Unit (ICU) can be challenging as patients often have anatomic and physiologic characteristics that make intubation particularly difficult. Video laryngoscopy (VL) has been shown to improve first attempt success compared to direct laryngoscopy (DL) in many clinical settings and may be an option for ICU intubations.
The first laparoscopic cholecystectomy was performed in 1985 and since then the growth of technology and innovation in laparoscopic surgery has been phenomenal. Today, operations can be performed through a single incision in the umbilicus or through a natural orifice such as the vagina, leaving no visible scars. Major operations such as colonic resections are now routinely performed using a laparoscopic approach. The benefits of laparoscopic surgery include reduced postoperative pain, improved cosmetic results and patient satisfaction, and reduced hospital stays (Perrin & Fletcher 2004).
Sampling of particle-phase organic carbon (OC) from diesel engines is complicated by adsorption and evaporation of semivolatile organic carbon (SVOC), defined as positive and negative artifacts, respectively. In order to explore these artifacts, an integrated organic gas and particle sampler (IOGAPS) was applied, in which an XAD-coated multichannel annular denuder was placed upstream to remove the gas-phase SVOC and two downstream sorbent-impregnated filters (SIFs) were employed to capture the evaporated SVOC. Positive artifacts can be reduced by using a denuder but particle loss also occurs. This paper investigates the IOGAPS with respect to particle loss, denuder efficiency, and particle-phase OC artifacts by comparing OC, elemental carbon (EC), SVOC, and selected organic species, as well as particle size distributions. Compared to the filterpack methods typically used, the IOGAPS approach results in estimation of both positive and negative artifacts, especially the negative artifact. The positive and negative artifacts were 190 microg/m3 and 67 microg/m3, representing 122% and 43% of the total particle OC measured by the IOGAPS, respectively. However particle loss and denuder break-through were also found to exist. Monitoring particle mass loss by particle number or EC concentration yielded similar results ranging from 10% to 24% depending upon flow rate. Using the measurements of selected particle-phase organic species to infer particle loss resulted in larger estimates, on the order of 32%. The denuder collection efficiencyfor SVOCs at 74 L/min was found to be less than 100%, with an average of 84%. In addition to these uncertainties the IOGAPS method requires a considerable amount of extra effort to apply. These disadvantages must be weighed against the benefits of being able to estimate positive artifacts and correct, with some uncertainty, for the negative artifacts when selecting a method for sampling diesel emissions.
This work shows development of a scale up correlation using computational fluid dynamic (CFD) simulations for floating solids drawdown operation in stirred tanks. Discrete phase modeling (DPM) simulations were used in conjunction with the lab scale experimental measurements to develop a semi-empirical correlation for the prediction of rate of drawdown of floating solid particles. The rate was correlated to average liquid velocity at the free liquid surface. Since, this correlation is based on a fundamental hydrodynamic parameter, velocity, rather than an operating parameters such as the impeller speed, it can be used for a variety of impeller types and tank geometries. The correlation was developed based on the data obtained from the 2L tank using four different tank designs and was validated against the data obtained from the 10L scale tank. The correlation was further extended to the pilot and the commercial scale tanks ranging from 40L to 4000L scale based solely on the CFD model.
Many patients who experience bulimia nervosa (BN) and binge eating disorder (BED) find it hard to access evidence-based treatments. Rates of failure to enter outpatient services following initial assessment are high, as are dropout rates from specialist outpatient eating disorders services.
The United Kingdoms Department of Health has identified reducing delays in patient discharge as a key aim for Health Service development. Laparoscopic cholecystectomy and laparoscopic inguinal hernia repair may be safely performed on a short stay basis, but day case rates remain low, with delays in discharge identified as a major contributing factor. Nurse-led discharge has been widely advocated to speed patient discharge across varied specialities, but objective evidence to support its use is lacking. This study aimed to assess the effectiveness of nurse-led discharge following laparoscopic surgery.
There is increasing interest in understanding the mechanisms underlying the interactions that occur between Mycobacterium tuberculosis and host innate immune cells. These cells express pattern recognition receptors (PRRs) which recognise mycobacterial pathogen-associated molecular patterns (PAMPs) and which can influence the host immune response to the infection. Although many of the PRRs appear to be redundant in the control of M. tuberculosis infection in vivo, recent discoveries have revealed a key, nonredundant, role of the Syk/CARD9 signalling pathway in antimycobacterial immunity. Here we review these discoveries, as well as recent data investigating the role of the Syk/CARD9-coupled PRRs that have been implicated in mycobacterial recognition, including Dectin-1 and Mincle.
C-type lectins are a diverse family of proteins which recognize a wide range of ligands. This review focuses on the Dectin-2 family of C-type lectins that includes Dectin-2, BDCA-2, DCIR, DCAR, Clecsf8 and Mincle whose genes are clustered in the telomeric region of the NK-gene cluster on mouse chromosome 6 and human chromosome 12. These type II receptors are expressed on myeloid and non-myeloid cells and contain a single extracellular carbohydrate recognition domain and have diverse functions in both immunity and homeostasis. DCIR is the only member of the family which contains a cytoplasmic signalling motif and has been shown to act as an inhibitory receptor, while BDCA-2, Dectin-2, DCAR and Mincle all associate with FcRgamma chain to induce cellular activation, including phagocytosis and cytokine production. Dectin-2 and Mincle have been shown to act as pattern recognition receptors for fungi, while DCIR acts as an attachment factor for HIV. In addition to pathogen recognition, DCIR has been shown to be pivotal in preventing autoimmune disease by controlling dendritic cell proliferation, whereas Mincle recognizes a nuclear protein released by necrotic cells. Here we review each of these receptors in detail describing their expression, ligand recognition, signalling and known physiological functions.
Solid amorphous dispersions are frequently used to improve the solubility and, thus, the bioavailability of poorly soluble active pharmaceutical ingredients (APIs). Spray-drying, a well-characterized pharmaceutical unit operation, is ideally suited to producing solid amorphous dispersions due to its rapid drying kinetics. This paper describes a novel flowchart methodology based on fundamental engineering models and state-of-the-art process characterization techniques that ensure that spray-drying process development and scale-up are efficient and require minimal time and API. This methodology offers substantive advantages over traditional process-development methods, which are often empirical and require large quantities of API and long development times. This approach is also in alignment with the current guidance on Pharmaceutical Development Q8(R1). The methodology is used from early formulation-screening activities (involving milligrams of API) through process development and scale-up for early clinical supplies (involving kilograms of API) to commercial manufacturing (involving metric tons of API). It has been used to progress numerous spray-dried dispersion formulations, increasing bioavailability of formulations at preclinical through commercial scales.
CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcR? chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.
Huisgen [3+2] dipolar cycloaddition of 6?-azido-6?-deoxy-?-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing ?-galactosyl ceramide (?-GalCer) analogues in high yield. These ?-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-? and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, ?-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by ?-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.