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The dielectric properties of biological cells can be used to gain information on their physiology and morphology. This paper reports the first measurements of the dielectric spectra of viable and non-viable cells over the full beta-dispersion (interfacial) frequency range. Dielectrophoresis (DEP) single cell in-flow techniques were employed to quantitatively measure the Clausius-Mossotti factor spectrum of individual cells over the 300 kHz–400 MHz range, covering both the MF and UHF DEP cross-over frequencies. Experiments were performed on Chinese hamster ovary (CHO) cells, one set cultured in growth media, the other in nutrient depleted media to induce apoptotic cells. Both cell states were measured using multi-frequency DEP flow cytometry, which provides the equivalent complex dielectric permittivity of individual cells. The measured dielectric spectra facilitate determination of a cell's morphology and the dielectric properties of its intracellular compartments, and are used to develop multi-shell dielectric models of viable and non-viable cells. The developed dielectric models can aid in biosensor design, in interpretation of bulk biomedia measurements where the heterogeneity in cell population can be masked, and in relating measured dielectric responses of cells to stimuli with changes in cellular physiology.

The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the approaches that can be used to produce a preferred glycan profile or a single glycoform is through chemoenzymatic remodeling during the isolation of a mAb. Here we describe protocols that can be utilized to produce preferred glycoforms that include galactosylated, agalactosylated, or sialylated glycoforms following isolation of a mAb. Methods for analysis and assignment of structures of the samples following glycoengineering are also described. Chemoenzymatic modeling of mAb glycans has the potential for scale-up and to be introduced into biomanufacturing of mAbs with higher specific therapeutic activities.

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+ /K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

The dielectric properties of cells are directly related to their morphological and physiological properties and can be used to monitor their status when exposed to stress conditions. In this work, dual-frequency dielectrophoresis (DEP) cytometry was employed to measure changes in the membrane capacitance and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells during the progression of starvation-induced apoptosis. Our dual-frequency DEP cytometer enables simultaneous measurement of multiple dielectric properties of single cells and identification of their state (viable or apoptotic) within a heterogeneous sample. We employed one frequency to determine each cell's viability state and the other frequency to characterize the change in membrane capacitance or cytoplasm conductivity. Cells were starved by incubation in a medium lacking glucose and glutamine and monitored every 12 h over a 64 h period. Our results showed a subpopulation of early apoptotic cells emerged after 40 h in the starvation medium, which rapidly increased during the next 12 h. After 52 h, a complete transition from viable to apoptotic state was observed. Analyzing the subpopulation of viable cells over the first 52 h showed that the membrane capacitance gradually declined from an initial value of 2.0 to 1.2 μF/cm2, and was 0.9 μF/cm2 for apoptotic cells. The cytoplasm conductivity of viable cells initially remained constant and then declined from 0.40 to 0.27 S/m after 40 h, coinciding with onset of apoptotic processes. A dramatic decrease in cytoplasm conductivity from 0.27 to 0.07 S/m was observed after 52 h, corresponding to apoptotic cells. As membrane capacitance is related to membrane morphology and cytoplasm conductivity is related to intracellular ion concentrations, the results indicate that during controlled starvation the cell membrane smooths gradually whereas intracellular ion concentrations are initially maintained near homeostatic levels until a later dramatic decline occurs.

Dielectrophoresis (DEP) cytometry was used to investigate changes in the membrane capacitance, membrane conductance, and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells subjected to thermal stress. CHO cells were exposed to temperatures ranging from 37 °C to 50 °C for 15 min and measured immediately after exposure. Heat-treated cells were measured at frequencies over the $\beta $ -dispersion region to obtain the dielectric parameters of a multishell cell model. A microwave DEP cytometer measuring DEP-induced translation of many individual heat-treated cells at selected frequencies was used to determine the Clausius–Mossotti factor and subsequently cell dielectric parameters. Results reveal that temperature-induced stress is accompanied by decrease in cell membrane capacitance from 1.75 to $0.71~\mu \text{F}$ /cm2, suggesting alteration in morphological features of the cell membrane surface. Results also show an insignificant decline in cytoplasm conductivity for treatment up to 46 °C. A significant decline in cytoplasm conductivity, from an initial value of 0.34–0.22 S/m was observed for 50 °C. The effect of suspending cells in low-conductivity DEP media was also investigated.

The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1-3 Gal that may be immunogenic. We have looked at the effects of a commonly used media supplements of manganese, galactose and uridine (MGU) on Mab production from CHO and NS0 cells in enhancing galactosylation and sialylation as well as the generation of these non-human glycan epitopes. In the absence of the MGU supplement, the humanized IgG1 antibody (Hu1D10) produced from NS0 cells showed a low level of mono- and di-sialylated structures (SI:0.09) of which 75 % of sialic acid was Neu5Gc. The chimeric human-llama Mab (EG2-hFc) produced from CHO cells showed an equally low level of sialylation (SI: 0.12) but the Neu5Gc content of sialic acid was negligible (

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90). It was obtained from cell culture with and without a fucosylation inhibitor, and treated with EndoS which cleaves between the two core N-acetyl glucosamine (GlcNAc) residues. It is the first time that this combined approach with a unique mass spectrometer was used to measure 146 Da differences as part of a large intact dimeric antibody. Results showed that in the dimer, both heavy chains were fucosylated on the core GlcNAc of the Fc Asn site equivalent to Asn297. In the presence of the fucosylation inhibitor, fucosylation was lost on both subunits. Following reduction, monomers were analyzed and the masses obtained corroborated the dimer results. Dimeric EG2-hFc Mab treated with PNGase F, to deglycosylate the protein, was also measured by MS for mass comparison. In spite of the success of fucosylation level measurements, the experimental masses of deglycosylated dimers and GlcNAc-Fuc bearing dimers did not correspond to masses of our sequence of reference (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90; ; ), which prompted experiments to determine the protein backbone sequence. Digest mixtures from trypsin, GluC, as well as trypsin + GluC proteolysis were analyzed by matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS. A few variations were found relative to the reference sequence, which are discussed in detail herein. These measurements allowed us to build a new "experimental" sequence for the EG2-hFc samples investigated in this work, although there are still ambiguities to be resolved in this new sequence. MALDI-MS/MS also confirmed the fucosylation pattern in the Fc tryptic peptide EEQYNSTYR.

Summary Background Catheter-based renal denervation has significantly reduced blood pressure in previous studies. Following a positive pilot trial, the SPYRAL HTN-OFF MED (SPYRAL Pivotal) trial was designed to assess the efficacy of renal denervation in the absence of antihypertensive medications. Methods In this international, prospective, single-blinded, sham-controlled trial, done at 44 study sites in Australia, Austria, Canada, Germany, Greece, Ireland, Japan, the UK, and the USA, hypertensive patients with office systolic blood pressure of 150 mm Hg to less than 180 mm Hg were randomly assigned 1:1 to either a renal denervation or sham procedure. The primary efficacy endpoint was baseline-adjusted change in 24-h systolic blood pressure and the secondary efficacy endpoint was baseline-adjusted change in office systolic blood pressure from baseline to 3 months after the procedure. We used a Bayesian design with an informative prior, so the primary analysis combines evidence from the pilot and Pivotal trials. The primary efficacy and safety analyses were done in the intention-to-treat population. This trial is registered at ClinicalTrials.gov , NCT02439749 . Findings From June 25, 2015, to Oct 15, 2019, 331 patients were randomly assigned to either renal denervation (n=166) or a sham procedure (n=165). The primary and secondary efficacy endpoints were met, with posterior probability of superiority more than 0·999 for both. The treatment difference between the two groups for 24-h systolic blood pressure was −3·9 mm Hg (Bayesian 95% credible interval −6·2 to −1·6) and for office systolic blood pressure the difference was −6·5 mm Hg (−9·6 to −3·5). No major device-related or procedural-related safety events occurred up to 3 months. Interpretation SPYRAL Pivotal showed the superiority of catheter-based renal denervation compared with a sham procedure to safely lower blood pressure in the absence of antihypertensive medications. Funding Medtronic.

Background Over 28 000 individuals were infected with Ebola virus during the West Africa (2013–2016) epidemic, yet there has been criticism of the lack of robust clinical descriptions of Ebola virus disease (EVD) illness from that outbreak. Objectives To perform a meta-analysis of published data from the epidemic to describe the clinical presentation, evolution of disease, and predictors of mortality in individuals with EVD. To assess the quality and utility of published data for clinical and public health decision-making. Data sources Primary articles available in PubMed and published between January 2014 and May 2017. Eligibility Studies that sequentially enrolled individuals hospitalized for EVD and that reported acute clinical outcomes. Methods We performed meta-analyses using random-effect models and assessed heterogeneity using the I2 method. We assessed data representativeness by comparing meta-analysis estimates with WHO aggregate data. We examined data utility by examining the availability and compatibility of data sets. Results In all, 3653 articles were screened and 34 articles were included, representing 16 independent cohorts of patients (18 overlapping cohorts) and at least 6168 individuals. The pooled estimate for case fatality rate was 51% (95% CI 46%–56%). However, pooling of estimates for clinical presentation, progression, and predictors of mortality in individuals with EVD were hampered by significant heterogeneity, and inadequate data on clinical progression. Our assessment of data quality found that heterogeneity was largely unexplained, and data availability and compatibility were poor. Conclusions We have quantified a missed opportunity to generate reliable estimates of the clinical manifestations of EVD during the West Africa epidemic. Clinical data standards and data capture platforms are urgently needed.

Made available in DSpace on 2020-12-12T01:32:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification. Science and Technology Institute Federal University of São Paulo (UNIFESP) Viral Immunology Laboratory Butantan Institute Medical School São Paulo State University (UNESP) National Institute for Biotechnology Research and Training (NIBRT) Medical School São Paulo State University (UNESP)

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

Humans infected with H7N9 avian influenza viruses can result in severe pneumonia and acute respiratory syndrome with an approximately 40% mortality rate, and there is an urgent need to develop an effective vaccine to reduce its pandemic potential. In this study, we used a novel PELC/CpG adjuvant for recombinant H7HA (rH7HA) subunit vaccine development. After immunizing BALB/c mice intramuscularly, rH7HA proteins formulated in this adjuvant instead of an alum adjuvant elicited higher IgG, hemagglutination-inhibition, and virus neutralizing antibodies in sera; induced higher numbers of H7HA-specific IFN-γ-secreting T cells and antibody secreting cells in spleen; and provided improved protection against live virus challenges. Our results indicate that rH7HA proteins formulated in PELC/CpG adjuvant can induce potent anti-H7N9 immunity that may provide useful information for H7N9 subunit vaccine development.

Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HEK293, glycosylated/de-glycosylated) on cytostasis, cell death (MTT, and Guava-ViaCount® flow-cytometry) and apoptotic signalling (Western blot of Cdk2, histone H3, procaspase-3, FADD, cleaved PARP, and caspase-3) was examined. Hydrophilic Interaction Liquid Chromatography determined the structure of N-linked glycans present in HEK293-expressed hIFNγ (hIFNγ-HEK). PEO1 was more sensitive to hIFNγ than SKOV3, but responses were dose-dependent and expression platform/glycosylation status-independent, whereas SKOV3 responded to mammalian-expressed hIFNγ in a dose-independent manner, only. Complex-type oligosaccharides dominated the N-glycosylation pattern of hIFNγ-HEK with some terminal sialylation and core fucosylation. Cleaved PARP and cleaved caspase-3 were not detected in either cell line, but FADD was expressed in SKOV3 with levels increased following treatment. In conclusion, hIFNγ did not induce apoptosis in either cell line. Mammalian- expressed hIFNγ increased cell death in the drug-resistant SKOV3. The presence of FADD in SKOV3, which may inhibit apoptosis through activation of NF-κB, could serve as a novel therapeutic target.

BACKGROUND: While cardiac rehabilitation (CR) is recommended for all patients after an acute cardiac event, limitations exist in reach. OBJECTIVE: The purpose of the current study was to develop and pilot a flexible online CR program based on self-management principles "Help Yourself Online." METHODS: The program was designed as an alternative to group-based CR as well as to complement traditional CR. The program was based on existing self-management resources developed previously by the Heart Research Centre. Twenty-one patients admitted to Cabrini Health for an acute cardiac event were recruited to test the program. The program was evaluated using qualitative and quantitative methods. RESULTS: Quantitative results demonstrated that patients believed the program would assist them in their self-management. Qualitative evaluation, using focus group and interview methods with 15 patients, showed that patients perceived the online CR approach to be a useful instrument for self-management. CONCLUSIONS: Broader implications of the data include the acceptability of the intervention, timing of intervention delivery, and patients' desire for additional online community support.

IntroductionRates of chronic pain in military personnel are disproportionately high. Chronic pain is often associated with mental health and substance use disorders as comorbid conditions, making treatment of chronic pain complex. Mindfulness-based interventions (MBIs) are a promising behavioral approach to managing chronic pain and psychosocial sequelae. The unique nature of the military context may require adaptations to original MBIs for successful delivery in active-duty military populations. This study adapted the mindfulness-based stress reduction (MBSR) program to create a mindfulness training program that was relevant to active-duty Army personnel experiencing chronic pain. This article delineates the adaptation process employed to modify the MBSR program to the military context and discusses the resulting training program.Materials and MethodsThe adaptation process consisted of three iterative stages: 1) Drafting the preliminary intervention protocol with recommendations from stakeholders, including military healthcare providers; 2) Refining the preliminary protocol after pretesting the sessions with research team members and a military Veteran advisory committee; and 3) Delivering the preliminary protocol to one cohort of active-duty Soldiers with chronic pain, collecting feedback, and further refining the intervention protocol.ResultsMilitary-related adaptations to MBSR addressed three areas: military culture, language and terminology, and practical and logistical factors relevant to implementation in the military setting. This adaptation process resulted in a live, online program with six, weekly, sessions. Feedback from a military Veteran advisory committee resulted in modifications, including increasing military-relevant examples; preliminary testing with the target population resulted in additional modifications, including shortening the sessions to 75 min and structuring discussions more efficiently.ConclusionsThe adaptation process was successful in generating an engaging mindfulness training program that was highly relevant to the military context. Obtaining input from stakeholders, such as military healthcare providers and active-duty soldiers, and iterative feedback and modification, were key to the process. Moreover, the program was designed to maintain the integrity and core elements of MBIs while adapting to military culture. A future randomized controlled trial design will be used to evaluate the effectiveness of the intervention in improving chronic pain in military personnel. This program is responsive to the military’s call for nonpharmacologic treatments for chronic pain that are easily accessible. If effective, the mindfulness program has the potential for widespread dissemination to complement standard care for Service Members experiencing chronic pain.

Many inorganic nanoparticles have been prepared and their behaviors in living systems are investigated. Yet, common electrolytes such as NaCl have been left out of this campaign. The underlying assumption is that electrolyte nanoparticles would quickly dissolve in water and behave similarly as their constituent salts. Herein, we challenge this preconception. Our study shows that NaCl nanoparticles (SCNPs) but not salts are highly toxic to cancer cells. This is because SCNPs enter cells through endocytosis, bypassing cell regulations on ion transport. When dissolved inside cancer cells, SCNPs cause a surge of osmolarity and rapid cell lysis. Interestingly, normal cells are much more resistant to the treatment due to their relatively low sodium levels. Unlike conventional chemotherapeutics, SCNPs cause immunogenic cell death or ICD. In vivo studies show that SCNPs not only kill cancer cells but also boost an anticancer immunity. Our discovery opens up a new perspective on nanoparticle-based therapeutics.

To investigate the feasibility of conducting a rehabilitation program for patients following surgery for abdomino-pelvic cancer. A non-randomised controlled before-and-after study. Patients who had undergone surgery for stage I–III abdomino-pelvic cancer (colorectal, gynaecological or prostate cancer) were recruited. The rehabilitation group (n = 84) received an 8-week, bi-weekly education and exercise program conducted by a physiotherapist, exercise physiologist, health psychologist and dietician, supplemented by exercise diaries and telephone coaching sessions. The comparator group (n = 104) completed postal questionnaires only. Feasibility measures, functional exercise capacity, muscle strength, physical activity levels, pelvic floor symptoms, anxiety and depression, health-related quality of life (HRQoL) and self-efficacy were measured at baseline (time 1), immediately post-intervention (time 2) and at 6 months post-baseline (time 3) and compared within- and between-groups. The consent rate to the rehabilitation program was 24%. Eighty-one percent of the rehabilitation group attended 85–100% of 16 scheduled sessions. Overall satisfaction with the program was 96%. Functional exercise capacity, handgrip strength in males, bowel symptoms, physical activity levels, depression and HRQoL were significantly improved in the rehabilitation group (p

INTRODUCTION The scene-size-up is a crucial first step in the response to a mass casualty incident (MCI). Unmanned aerial vehicles (UAV) may potentially enhance the scene-size-up with real-time visual feedback during chaotic, evolving or inaccessible events. We performed this study to test the feasibility of paramedics using UAV video from a simulated MCI to identify scene hazards, initiate patient triage, and designate key operational locations. METHODS We simulated an MCI, including 15 patients plus 4 hazards, on a college campus. A UAV surveyed the scene, capturing video of all patients, hazards, surrounding buildings and streets. We invited attendees of a provincial paramedic meeting to participate. Participants received a lecture on Sort-Assess-Lifesaving Interventions-Treatment/Transport (SALT) Triage and MCI scene management principles. Next, they watched the UAV video footage. We directed participants to sort patients according to SALT Triage Step One, identify injuries, and to localize the patients within the campus. Additionally, we asked them to select a start point for SALT Triage Step Two, identify and locate hazards, and designate locations for an Incident Command Post, Treatment Area, Transport Area and Access/Egress routes. The primary outcome was the number of correctly allocated triage scores. RESULTS Ninety-six individuals participated. Mean age was 35 years (SD 11); 46% (44) were female and 49% (47) were Primary Care Paramedics. Most participants (79; 82%) correctly sorted at least 12 of 15 patients. Increased age was associated with decreased triage accuracy [-0.04(-0.07, -0.01); p = 0.031]. Fifty-two (54%) correctly localized 12 or more patients to a 27 × 20m grid area. Advanced paramedic certification, and local residency were associated with improved patient localization [2.47(0.23,4.72); p = 0.031], [3.36(1.10,5.61); p = 0.004]. The majority of participants (70; 81%) chose an acceptable location to start SALT Triage Step Two and 75 (78%) identified at least 3 of 4 hazards. Approximately half (53; 56%) of participants appropriately designated 4 or more of 5 key operational areas. CONCLUSION This study demonstrates the ability of UAV technology to remotely facilitate the scene size-up in an MCI. Additional research is required to further investigate optimal strategies to deploy UAVs in this context.

In mammalian cells cytoplasm ion concentrations and hence cytoplasm conductivity is an important indicator of their physiological state. Changes in the cytoplasm conductivity has been associated with physiological changes such as progression of cancer and apoptosis. In this work, a model that predicts the effects of physiological changes in ion transport on the cytoplasm conductivity of Chinese hamster ovary (CHO) cells is demonstrated. We determined CHO-specific model parameters, Na+/K+ ATPase pumps and ion channels densities, using a flux assay approach. The obtained sodium (PNa), potassium (PK) and chloride (PCl) permeability and Na+/K+ ATPase pump density were estimated to be 5.6 × 10−8 cm/s, 5.6 × 10−8 cm/s, 3.2 × 10−7 cm/s and 2.56 × 10−11 mol/cm2, respectively. The model was tested by comparing the model predictions with the experimentally determined temporal changes in the cytoplasm conductivity of Na+/K+ ATPase pump inhibited CHO cells. Cells’ Na+/K+ ATPase pumps were inhibited using 5 mM Ouabain and the temporal behavior of their cytoplasm conductivity was measured using dielectrophoresis cytometry. The measured results are in close agreement with the model-calculated values. This model will provide insight on the effects of processes such as apoptosis or external media ion concentration on the cytoplasm conductivity of mammalian cells.

We employed dielectrophoresis (DEP) to investigate changes in the membrane capacitance and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells induced by thermal stress. CHO cells were exposed to temperatures ranging from 42 °C to 50°C for 15 minutes. The DEP response of heat-treated cells were measured at frequencies over the $\beta$ -dispersion region using a microwave DEP cytometer. The results reveal that temperature induced stress is accompanied by decrease in the cell membrane capacitance, and decline in the ionic content of cytoplasm.

Nutrient depletion in fed-batch cultures and at the end of batch cultures is among the main causes of stress on cells and a trigger of apoptosis. In this study, we investigated changes in the cytoplasm conductivity of Chinese hamster ovary (CHO) cells under controlled starvation. Employing a single-cell dielectrophoresis (DEP) cytometer, we measured the DEP response of CHO cells incubated in a medium without glucose and glutamine over a 48-h period. Using the measured data in conjunction with numerical simulations, we determined the cytoplasm conductivity of viable and apoptotic cell subpopulations. The results show that a small subpopulation of apoptotic cells emerges after 24 to 36 h of starvation and increases rapidly over a short period of time,12 h. The apoptotic cells have a dramatically lower cytoplasm conductivity, ∼0.05 S/m, than viable cells, ∼0.45 S/m. Viability of starvation cultures was measured by fluorescent cytometry, DEP cytometry, and trypan blue exclusion assays. DEP, Annexin V, caspase-8, and 7-AAD assays show a similar decline in viability after 36 h of starvation and indicate a very low viability after 48 h. Trypan blue exclusion assay fails to detect early-stage viability decline and estimates a much higher viability after 48 h.

Rationale In the expression of recombinant proteins, an important parameter to control or influence is their level of sialylation. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) methods tend to either underestimate (positive mode) or overestimate (negative mode) the content of sialylated vs. neutral glycans in glycoproteins. Esterification methods have been developed for free sialylated glycans and sialylated Asn-glycans, allowing these acidic groups to ionize with the same efficiency as neutral sugars. Methods Here we describe a method which modifies glycopeptides by esterification. This simple procedure is applied to glycopeptides isolated from tryptic digests of monoclonal antibodies (mAbs), some highly sialylated. To better understand the effect of esterification on the peptide backbone, synthetic EEQYNSTYR was esterified and studied by tandem mass spectrometry (MS/MS). Acetamidation of EEQYNSTYR was also studied as some mAb samples had been overalkylated prior to tryptic digestion. Results As a general trend, ethyl-esterification or lactonization is observed for each sialic acid on glycoforms of EEQYNSTYR (the N-glycosylated tryptic peptide of IgG Fc), depending on the branching position of the sialic acid (α2,3 or α2,6). Esterification also affects the carboxyl groups in the peptide, including the C-terminal COOH. Conclusions For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies. Copyright © 2015 John Wiley & Sons, Ltd.

The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol continues with gel electrophoresis. Bands containing the heavy chain are cut and isolated from the gel followed by tryptic digestion to obtain peptides and glycopeptides. The enrichment of glycopeptides by C18 chromatography is described followed by characterization using positive and negative modes MALDI-MS and MS/MS. An exoglycosidase, beta-galactosidase, is used to verify anomericity of linkages in the glycan portion of glycopeptides. In the last step, glycans are detached from glycopeptides using PNGase F labelled with phehylhydrazine and characterized by MALDI-MS/MS. This workflow is reported for the first time for this particular antibody and presents a valuable approach for the analysis of N-glycans on most antibodies and glycoproteins.

Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric human-llama monoclonal antibody secreted by CHO cells. The cells were inoculated into media containing varying concentrations of glucose. Glucose depletion occurred in cultures with an initial glucose 5.5 mM and seeded at low density (2.5 x 10(5) cells/mL) or at high cell inoculum (2.5 x 10(6) cells/mL) at higher glucose concentration (up to 25 mM). Glucose-depleted cultures produced non-glycosylated Mabs (up to 51%), lower galactosylation index (GI

Purpose The majority of cemented knee replacements worldwide are performed with a tourniquet. The benefits of surgery in TKR without a tourniquet have been well described but questions have been raised about the ability to prepare the bone for cementation, and take up of a tourniquet-less technique has been slow. A laboratory based model was constructed to directly compare the quality of cementing between tourniquet and tourniquet-less knee replacement supplemented by metaphyseal suction and a cement gun. Methods A Sawbone model to represent the metaphyseal proximal tibia was constructed. The model allowed the inflow of simulated blood and the use of metaphyseal suction. The study compared four different techniques; the use of a tourniquet, no tourniquet, no tourniquet with cancellous suction and no tourniquet with cancellous suction and a cement gun. Each subtype of experiment was repeated 5 times. Quality of cementation was assessed using a calibrated engineering planimeter. Results This model has shown that combining the use of metaphyseal suction and a cement gun but without the use of a tourniquet offers significantly better cement penetration (p Conclusions This study offers further in vitro evidence to the argument that tourniquet-less surgery when supplemented by appropriate techniques can achieve a good cement–bone interface, whilst avoiding the additional risk of using a tourniquet to perform the procedure.

Non-human mammalian cells such as CHO have been used predominantly for the production of biopharmaceuticals including monoclonal antibodies (Mabs). Although the glycosylation profile of these products is 'human-like' there is still the possibility of immunogenic epitopes such as α-Gal and Neu5Gc. Human cell lines have now been designed for high productivity of recombinant proteins and ensuring authentic glycosylation patterns. The control of glycosylation on such proteins is important for the efficacy of recombinant biopharmaceuticals as well as the immunogenic properties of viral vaccines such as influenza. We are now starting to understand some of the relationships between the structure of glycans and the function bestowed on the associated protein. This has promoted cell culture technologies for the targeted control of glycosylation to produce pre-determined glycan profiles of secreted products.

In this paper we present the use of a dielectrophoresis (DEP) cytometry technique to study changes in single cells upon application of a particular stimulus. In one case we measure single CHO cells immediately after exposure to pulsed electric fields. Our results show a significant change in the dielectric response of cells after application of sufficiently strong pulses. In another study we investigate the effect of nutrient deficiency on CHO cells. Many individual cells are measured as they undergo starvation due to lack of nutrients in their medium. Our DEP measurement results, after 48 hours of starvation, show a substantial change in the dielectric response that correlates with the viability of the cells. By the examples we present in the paper, we demonstrate the potential of our DEP cytometry technique in applications such as monitoring the effect of drugs or developing optimal protocols for cell electroporation.

We employed dielectrophoresis (DEP) to investigate changes in the dielectric properties of single Chinese hamster ovary (CHO) cells induced by nutrient depletion. CHO cells were concurrently incubated in media with and without glucose and glutamine. After 54 hours, cells were identified as viable and non-viable by trypan-blue exclusion test. The DEP response of single viable and non-viable cells were measured at frequencies over a 100 kHz-300 MHz frequency range using a wide-band DEP cytometer. The results reveal that the induced stress is accompanied by decrease in the cell membrane capacitance, decline in the ion content of cytoplasm, and decrease in the high frequency Clausius-Mossotti factor associated with cytoplasm permittivity.

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3–5 μm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.

The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.

Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By optimizing the timing and dose of 3-MA treatment, the cell-specific productivity was increased 4-fold, resulting in 2-fold increased total MAb production. The positive effect of the 3-MA treatment appeared to be reduced when the amino acid feed concentration was increased 5-fold. Further investigation revealed that by slowly increasing osmolality up to ∼450 mOsm/kg, both the cell-specific productivity and the total MAb almost doubled. This effect was replicated with a DUXB-based CHO cell line expressing a human–llama chimeric antibody. The positive effect of gradually increasing osmolality was then combined with the positive effects of the 3-MA treatment, however their combined effect were not additive. Thus, either increased osmolality or 3-MA treatment were equally effective in increasing MAb-CHO cell fed-batch production on the cell lines tested. Analysis of protein glycosylation showed that both of these fed-batch modifications did not substantially influence the overall glycan profiles of the MAb product.

Background Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused primarily by mutations in the low-density lipoprotein receptor gene. Familial hypercholesterolemia is characterized by exceedingly high levels of low-density lipoprotein cholesterol (LDL-C) and subsequent premature coronary heart disease. Homozygous FH (HoFH) is less prevalent, but more severe, than heterozygous FH. Current treatment options include dietary therapy, lipid-lowering agents (eg, statins), and/or LDL-C apheresis. Purpose Despite the available treatment options, patients with FH rarely attain treatment goals. This review will focus on 2 novel agents, lomitapide and mipomersen, with recently approved US Food and Drug Administration (FDA) labeling for use in patients with HoFH. Conclusions Lomitapide and mipomersen are 2 agents with novel mechanisms of action and the ability to significantly lower LDL-C, apolipoprotein B, and non-high-density lipoprotein cholesterol levels. A black box warning exists for lomitapide and mipomersen regarding the risk for transaminase elevations and hepatic steatosis. Furthermore, these agents are currently restricted for use only in patients with HoFH and have been required by the FDA to participate in a Risk Evaluation and Mitigation Strategy. Clinical implications These new agents offer additional treatment options for clinicians managing patients with HoFH, but it remains uncertain whether lomitapide and mipomersen will gain FDA approval for use in patients with heterozygous FH or in the general population. Cost and concern for the risk for hepatotoxicity will remain limiting factors to these agents being more widely used.

Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

The first attempts at culturing animal cells in vitro made use of biological fluids, such as serum or tissue extracts. It was in the 1950s that a scientific approach was adopted to determine the defined nutrients required for mammalian cell growth. The idea of a chemically defined media was pioneered by Eagle, who determined the minimum ingredients that were essential for the growth of a number of human cell lines. This led to the development of Eagle’s minimal essential media that consisted of 13 amino acids, 8 vitamins and 6 ionic species [1]. This formulation appeared to provide the requirements for the growth of a number of isolated cell lines if supplemented with animal-sourced serum. Higher cell densities were obtainable by increasing the component concentrations of Eagle’s minimal essential media and became established through basal formulations such as Dulbecco’s modification of Eagles medium (DMEM). Clonal cell growth of selected cell lines was obtained by enrichment with an enhanced range of nutritional components, largely through the early work of Ham to produce the well-known Ham’s F-12 medium, Sato had the ingenious idea of combining these two approaches to blend a basal media formulation – DMEM/F-12 – that has become widely used for the growth of multiple cell lines to high density [2]. However, despite the inclusion of up to 70 components in these well-defined basal media formulations, supplementation with dialyzed serum (~10%) is necessary to provide sustained growth of most cell lines.

To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off-line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells between different dielectric sub-populations, rather than assuming a homogeneous dielectric population. This shows that bulk dielectric probes are sensitive to the early apoptotic changes in cells. DEP cytometry offers a novel and unique technology for analyzing and characterizing mammalian cells based on their dielectric properties, and suggests a potential application of the device as a low-cost, label-free, electronic monitor of physiological changes in cells.

Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-β- N -acetylglucosaminidase-treated KIF (KIF+E) to generate single- N -acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development. IMPORTANCE Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.

Endovascular aneurysm repair (EVAR) is now the preferred procedure for abdominal aortic aneurysm repair. As a result of the need for fluoroscopy during EVAR, radiation exposure is a potential hazard. We studied the quantity of radiation delivered during EVAR to identify risks for excessive exposure. Fluoroscopy time, contrast volume used, and procedural details were recorded prospectively during EVARs. Using data collected from similar EVARs, an equation was derived to calculate approximate dose-area product (DAP) from fluoroscopy time. DAP values were then compared between procedures in which a relevant postdeployment procedure (PDP) was necessary intraoperatively with those without. Clinical data on 17 patients were collected. The mean age of patients was 68 (±9) years. Fluoroscopy times and approximate DAP values were found to be significantly higher in the seven patients with a PDP compared with the 10 patients without an intraoperative PDP (31.2 [±9.6] vs 22.7 [±6.0] minutes, P = 0.033 and 537 [±165] vs 390 [±103] Gy-cm2, P = 0.033, respectively). The average amount of contrast volume used was not significantly different between groups. Radiation emitted during EVARs with PDPs was significantly greater relative to those without PDPs. Device design and operators should thus aim to decrease PDPs and to minimize fluoroscopy time.

We present a dielectric model and its parameters for Chinese hamster ovary (CHO) cells based on a double-shell structure which includes the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm. Employing a dielectrophoresis (DEP) based technique and a microfluidic system, the DEP response of many single CHO cells is measured and the spectrum of the Clausius-Mossotti factor is obtained. The dielectric parameters of the model are then extracted by curve-fitting to the measured spectral data. Using this approach over the 0.6-10 MHz frequency range, we report the values for CHO cells' membrane permittivity, membrane thickness, cytoplasm conductivity, nuclear envelope permittivity, and nucleoplasm conductivity. The size of the cell and its nuclei are obtained using optical techniques.

Background IgGs that possess a2,6-sialylated Fc-glycans are thought to be responsible for the anti-inflammatory properties of intravenous immunoglobulins (IVIGs) [1], through a mechanism which has not been elucidated yet. The impact of this sialylation on the classic IgG’s effector functions also remains unclear [2,3]. The understanding of these mechanisms has been impeded by the heterogeneity of the sialylated glycan species together with the relative rarity of a2,6-sialylated IgGs. N-glycan sialylation is a post-translational modification where a sialic acid (SA) residue is added to the terminal galactose residue (Gal) of a growing glycan chain. The SA type (NANA or NGNA), the nature of the linkage between the SA and the Gal (a2,3 or a2,6), or the number of glycan antennae being sialylated, may vary according to the IgG subtype, the host cell in which it is expressed and the cell culture environment. A closer attention must thus be paid to the precise sialylation profiles leading to the reduced ADCC activity and to the gain of anti-inflammatory properties. In humans, 10 to 15% of the circulating IgG1s are sialylated, carrying mostly complex di-antennary glycans with two Gal and one a2,6-linked SA residue (G2FS(6)1, where G stands for galactose, F for fucose and S(6) for a2,6SA). Most of the therapeutic monoclonal antibodies (mabs) are produced in Chinese hamster ovary (CHO) cells, which have a glycosylation machinery close to that of humans, but possess only a2,3-sialyltransferases (ST3) whereas humans have both a2,3and a2,6-sialyltransferases. The Fc domain of mabs produced in CHO typically possesses N-glycans with low galactosylation and very low sialylation (0-2% of a2,3-sialylated glycans). In this study, we show that the a2,6-sialylation of IgG1’s Fc domain can be efficiently achieved by the transient coexpression of the human b1,4-galactosyltransferase 1 (GT) and a2,6-sialyltransferase 1 (ST6) in CHO cells, whereas the expression of one or the other glycosyltransferase alone does not significantly improve sialylation [4]. The process allows for the production of milligrams of human-like sialylated mabs within two weeks. We present a panel of four orthogonal assays for the fine characterization of the mabs’ glycoprofile that are in very good agreement with each other.

PurposeTo introduce femtosecond laser wound design combined with riboflavin/ultraviolet light-A (UVA) collagen cross-linking at the wound for penetrating (PKP) and anterior lamellar keratoplasty (ALK). Primary outcomes were intraocular pressure (IOP in mmHg) at burst point for the PKP group, and tensile strength (kPa) until dehiscence for the ALK group.MethodsHuman corneoscleral rims (N=20) were mounted on artificial anterior chambers. PKP specimens underwent FUR, femtosecond laser-cut without cross-linking, or conventional corneal transplantation. PKP maximum burst IOP with progressive suture removal was assessed by a digital manometer, in triplicate and by three observers. ALK involved whole human globes (N=10) divided into three groups using a 200-micron, 8mm diameter donor lenticule, with or without cross-linking. Cross-linked specimens were exposed to UVA light (3mW/cm2 irradiance, 3.4J, 370nm wavelength) for 30min with 0.1% riboflavin (20% Dextran) applied every 2-min. ALK tensile strength was determined using a digital tensiometer.ResultsIn PKP, burst IOP was 31.32mmHg greater for corneas that underwent the UVA-riboflavin treatment than for those that did not (p

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q P with increasing cell densities up to a threshold of >1 × 108 cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q P any further. A positive linear correlation (r 2 = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 106 to 3 × 108 cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.

Developmental pathways have been shown to be important in the initiation and progression of cancer in various tissues. We showed that the transcription factor SOX9 is expressed in the epithelia of the mouse embryonic prostate and is required for proper prostate development. We have performed an in vivo investigation into the role of SOX9 in prostate cancer in mouse and human. Studies on Pten and Nkx3.1 mutant mice show that cells with an increased level of SOX9 appear within the epithelia at the early stages of prostate neoplasia and this high expression correlates with all stages of neoplastic progression. Using genetically modified mice we show that overexpression of SOX9 in prostate epithelia leads to an increase in cell proliferation without inducing hyperplasia. In mice that were heterozygous for the conditional mutant allele of Pten, overexpression of SOX9 gave rise to an earlier induction of high-grade prostate intraepithelial neoplasia. Consistent with this role, loss of Sox9 in prostate epithelia led to a decrease in proliferating cells in normal and in homozygous Pten mutant mice with prostate neoplasia. Analysis of a cohort of 880 human prostate cancer samples showed that SOX9 expression is associated with increasing Gleason grades and higher Ki67 staining. These studies identify SOX9 as part of a developmental pathway that is reactivated in prostate neoplasia where it is involved in regulating proliferation and suggests it can contribute to carcinogenesis in specific genetic contexts.

Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Production of single glycoforms remains a challenge despite their value as therapeutics. The authors report a method of sequential enzymatic-based changes to antibodies while immobilized on an affinity column. Various antibodies (monoclonal and polyclonal) are isolated on Protein A or G columns and their glycans modified by sequential addition of enzymes for a desired transformation. Galactosylated antibodies (>90% yield) are produced by a one stage reaction process with sialidase to remove any sialic acid residues and addition of galactose with galactosyltransferase and UDP-Gal. Sialylated antibodies (>90%) are produced by a 2 stage conversion involving α(2,3) sialidase and galactosyltransferase followed by treatment with α(2,6) sialyltransferase in the presence of CMP-NANA. By this method, >90% of a disialylated human-llama antibody (EG2-hFc) and equimolar quantities of monosialylated and disialylated forms of human antibodies (αIL8-hFc and human polyclonal) are produced. Such high levels of sialylation are very difficult to obtain by typical cell culture methods. This method of transformation while the antibody is held on a solid-phase column is superior to previous methods because it allows a series of enzymatic steps without the need for intermediate purification. This is an efficient and rapid method to generate therapeutic antibodies with predefined glycosylation profiles. This should also assist in investigating the structure-function relationship of antibody glycans to find the desired glycosylation profile for high functional activity. With further optimization the method can be used to modify antibodies in large-scale manufacturing.