Soy hydrolysate mimic autocrine growth factors effect of conditioned media to promote single CHO-K1 cell proliferation.

The increasingly competitive biopharmaceutical industry requires companies to focus on rapid and low-cost cell line development. Single-cell cloning (SCC) is a critical and high-value process for cell line development, and typically problematic because single cell proliferates slowly when cultivated at low cell densities. Conditioned media (CM) provide autocrine growth factors to facilitate single cell proliferation, thus improve SCC efficiency. However, conditioned media (CM) are not a feasible solution for industrial cell line development due to variation and cross-contamination concerns. Here, we have found an improvement in the SCC efficiency similar to CM when soy hydrolysate was supplemented in SCC media. Therefore, we concluded that hydrolysate can mimic the autocrine growth factor(s) effect to improve cloning efficiency observed in CM.

Public knowledge of how to use an automatic external defibrillator in out-of-hospital cardiac arrest in Hong Kong.

INTRODUCTION: The survival rate of out-of-hospital cardiac arrest in Hong Kong is low. A long delay between collapse and defibrillation is a contributing factor. Public access to defibrillation may shorten this delay. It is unknown, however, whether Hong Kong's public is willing or able to use an automatic external defibrillator. This study aimed to evaluate public knowledge of how to use an automatic external defibrillator in out-of-hospital cardiac arrest. METHODS: A face-to-face semi-structured questionnaire survey of the public was conducted in six locations with a high pedestrian flow in Hong Kong. RESULTS: In this study, 401 members of the public were interviewed. Most had no training in first aid (65.8%) or in use of an automatic external defibrillator (85.3%). Nearly all (96.5%) would call for help for a victim of out-of-hospital cardiac arrest but only 18.0% would use an automatic external defibrillator. Public knowledge of automatic external defibrillator use was low: 77.6% did not know the location of an automatic external defibrillator in the vicinity of their home or workplace. People who had ever been trained in both first aid and use of an automatic external defibrillator were more likely to respond to and help a victim of cardiac arrest, and to use an automatic external defibrillator. CONCLUSION: Public knowledge of automatic external defibrillator use is low in Hong Kong. A combination of training in first aid and in the use of an automatic external defibrillator is better than either one alone.

Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool.

OBJECTIVE: To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples. RESULTS: A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)-HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R (2) = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC-HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R (2) = 0.992). This method was further validated by a 1500 l production process. CONCLUSION: SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.

Use of triple correlations for the sign determinations of expansion coefficients of symmetric approximations to the diffraction volumes of regular viruses.

An X-ray free electron laser is a new source of x-rays some 10 × 10(9) times brighter than any previous X-ray source, giving rise to the possibility of structure determination of individual biological particles without crystallization. Some of the earliest samples used in the X-ray free electron laser are viruses because they are about the largest of reproducible bioparticles. We show how common virus near-symmetries can be exploited to find a first approximation to their structures to give a starting point for a perturbation approach to determine their structures.

Fiber diffraction without fibers.

Postprocessing of diffraction patterns of completely randomly oriented helical particles, as measured, for example, in so-called "diffract-and-destroy" experiments with an x-ray free electron laser can yield "fiber diffraction" patterns expected of fibrous bundles of the particles. This will allow "single-axis alignment" to be performed computationally, thus obviating the need to do this by experimental means such as forming fibers and laser or flow alignment. The structure of such particles may then be found by either iterative phasing methods or standard methods of fiber diffraction.

Intra-operative correction of acidosis, coagulopathy and hypothermia in combat casualties with severe haemorrhagic shock.

We assessed acidosis, coagulopathy and hypothermia, before and after surgery in 51 combat troops operated on for severe blast injury. Patients were transfused a median (IQR [range]) of 27 (17-38 [5-84]) units of red cell concentrate, 27 (16-38 [4-83]) units of plasma, 2.0 (0.5-3.5 [0-13.0]) units of cryoprecipitate and 4 (2-6 [0-17]) pools of platelets. The pH, base excess, prothrombin time and temperature increased: from 7.19 (7.10-7.29 [6.50-7.49]) to 7.45 (7.40-7.51 [7.15-7.62]); from -9.0 (-13.5 to -4.5 [-28 to -2]) mmol.l⁻¹ to 4.5 (1.0-8.0 [-7 to +11]) mmol.l⁻¹; from 18 (15-21 [9-24]) s to 14 (11-18 [9-21]) s; and from 36.1 (35.1-37.1 [33.0-38.1]) °C to 37.4 (37.0-37.9 [36.0-38.0]) °C, respectively. Contemporary intra-operative resuscitation strategies can normalise the physiological derangements caused by haemorrhagic shock.

The UK military experience of thoracic injury in the wars in Iraq and Afghanistan.

INTRODUCTION: Thoracic injury during warfare is associated with a high incidence of morbidity and mortality. This study examines the pattern and mortality of thoracic wounding in the counter-insurgency conflicts of Iraq and Afghanistan, and outlines the operative and decision making skills required by the modern military surgeon in the deployed hospital setting to manage these injuries. METHODS: The UK Joint Theatre Trauma Registry was searched between 2003 and 2011 to identify all patients who sustained battle-related thoracic injuries admitted to a UK Field Hospital (Role 3). All UK soldiers, coalition forces and local civilians were included. RESULTS: During the study period 7856 patients were admitted because of trauma, 826 (10.5%) of whom had thoracic injury. Thoracic injury-related mortality was 118/826 (14.3%). There were no differences in gender, age, coalition status and mechanism of injury between survivors and non-survivors. Survivors had a significantly higher GCS, Revised Trauma Score and systolic blood pressure on admission to a Role 3 facility. Multivariable regression analysis identified admission systolic blood pressure less than 90, severe head or abdominal injury and cardiac arrest as independent predictors of mortality. CONCLUSIONS: Blast is the main mechanism of thoracic wounding in the recent conflicts in Iraq and Afghanistan. Thoracic trauma in association with severe head or abdominal injuries are predictors of mortality, rather than thoracic injury alone. Deploying surgeons require training in thoracic surgery in order to be able to manage patients appropriately at Role 3.

Nontherapeutic laparotomy in combat casualties.

BACKGROUND: The selective nonoperative management of ballistic abdominal injury remains contentious, particularly in the military setting. The exigencies of military practice have traditionally favored a more liberal approach to abdominal exploration. The driver for selective nonoperative management is the avoidance of morbidity incurred by nontherapeutic intervention. However, the incidence and complications of nontherapeutic laparotomy (NTL) in the military setting are not known. METHODS: All UK military patients undergoing a laparotomy following battlefield trauma were identified from the UK Joint Theatre Trauma Registry. Procedures were classed as therapeutic laparotomy (TL) or NTL. Demographics, admission physiology, injury pattern, and mortality were compared, and complications in the NTL group were determined by Joint Theatre Trauma Registry and case record review. RESULTS: Between March 2003 and March 2011, 130 (7.2%) of 1,813 combat wounded UK service personnel underwent a laparotomy. A total of 103 (79.2%) were considered TL, and 27 (20.8%) were NTL. There was no difference in demographic distribution or mechanism of injury. Patients undergoing TL were more likely to be hypotensive (systolic blood pressure, <90 mm Hg; p = 0.015) and have a reduced consciousness level (Glasgow Coma Scale [GCS] score ≤ 8; p = 0.006). There was a greater abdominal injury burden in the TL group (p < 0.001). There was no difference in severe extra-abdominal injury (Abbreviated Injury Scale [AIS] score ≥ 3), overall Injury Severity Score (ISS) and New ISS (NISS) scores, or mortality. Of the 27 patients who underwent NTL, 7 (25.9%) developed complications. CONCLUSION: During the past decade, trauma laparotomy has become a relatively uncommon procedure. The NTL rate is also relatively low. This finding could be explained by the fact that selective nonoperative management is used more widely in the military setting than previously thought or that very few military injuries are amenable to nonoperative management. NTL is associated with a significant risk of complications and should therefore be minimized but not at the expense of missing a life-threatening intra-abdominal injury. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Reconstructing an icosahedral virus from single-particle diffraction experiments.

The first experimental data from single-particle scattering experiments from free electron lasers (FELs) are now becoming available. The first such experiments are being performed on relatively large objects such as viruses, which produce relatively low-resolution, low-noise diffraction patterns in so-called "diffract-and-destroy" experiments. We describe a very simple test on the angular correlations of measured diffraction data to determine if the scattering is from an icosahedral particle. If this is confirmed, the efficient algorithm proposed can then combine diffraction data from multiple shots of particles in random unknown orientations to generate a full 3D image of the icosahedral particle. We demonstrate this with a simulation for the satellite tobacco necrosis virus (STNV), the atomic coordinates of whose asymmetric unit is given in Protein Data Bank entry 2BUK.

New light on disordered ensembles: ab initio structure determination of one particle from scattering fluctuations of many copies.

We report on the first experimental ab initio reconstruction of an image of a single particle from fluctuations in the scattering from an ensemble of copies, randomly oriented about an axis. The method is applicable to identical particles frozen in space or time (as by snapshot diffraction from an x-ray free electron laser). These fluctuations enhance information obtainable from an experiment such as conventional small angle x-ray scattering.

Beyond the crystallization paradigm: structure determination from diffraction patterns from ensembles of randomly oriented particles.

We amplify on the principles of the method we have recently proposed for recovering an oversampled diffraction pattern of a single particle from measured diffraction patterns from multiple particles in orientations related by rotation about an axis parallel to the incident radiation. We propose an alternative method of phasing a reference resolution ring by means of a non-negativity constraint on the diffraction intensities, point out the need for caution about enantiomeric ambiguities in the reconstruction of a diffraction pattern from its angular correlations, and show that converged correlations may be deduced by appropriate averaging of even very noisy data.

A lack of contact of sperm with accessory sex gland secretions deregulates DNA methylation and imprinted gene expression in rodent embryos.

Increased oxidative DNA damage is observed in sperm devoid of contact with accessory sex gland (ASG) secretion. After fertilization, these sperm may produce abnormal embryos. In this study, we investigated the possibility that the pattern of DNA methylation and imprinted gene expression in these embryos may be perturbed. Epididymal sperm, uterine sperm, and embryonic day 13 (E13) embryos were collected from hamster and mouse. The extent of global DNA methylation was determined with an antibody against methylcytosine using an embryo DNA dot. The sperm and embryo Gtl2 promoter and H19 differential methylated region (DMR) were subject to bisulfite sequencing. Expression of their reciprocally activated genes Dlk1 and Igf2 was quantified by real-time PCR. Genome-wide DNA hypo-methylation in both hamster and mouse embryos sired by males without ASG was observed. The imprinting pattern of fetal mouse Gtl2 promoter and fetal hamster H19 DMR were also disrupted while the expression of Dlk1 and Igf2 was dysregulated in the hamster embryo. This study suggests that a lack of contact of sperm with the ASG secretion disrupts genome-wide DNA methylation and also affects the DNA methylation pattern of imprinted genes in embryos.

Absence of paternal accessory sex gland secretions disturbs epigenetic reprogramming and expression of Igf2 and Dlk1 in golden hamster embryos.

Accessory sex gland (ASG) secretion is known to exert an effect on sperm that is heritable in hamster embryos. We hypothesized that ASG secretion changes the sperm epigenome, which in turn is propagated in sired embryos. To test our hypothesis, we produced male hamsters that were devoid of either all ASG (TX) or only the ventral lobe of the prostate gland (VPX). A sham-operated control group (SH) was also established. These males were mated with normal females; uterine sperm, fertilized oocytes, and pre-implantation embryos were harvested from the females after mating. Epididymal sperm were collected at the end of experiments. Immunofluorescent staining was performed on these harvested specimens using antibodies against 5-methylcytosine, Dnmt1, Dnmt3a, Dnmt3b, protamine 1, protamine 2, and aectyl-H4K5. Expression of Igf2 and Dlk1 were analyzed by real-time RT PCR and in situ hybridization. We demonstrated that the DNA methylation pattern changed dynamically in SH, TX, and VPX fertilized oocytes. In VPX and TX embryos, DNA demethylation was slower and remethylation was delayed when compared with SH embryos. In addition, Dnmt3b expression was also abnormal. When sperm from VPX and TX males were exposed to whole ASG secretion in vivo, the resulting embryos all methylated normally. Immunofluorescent staining revealed that there was no difference in protamine packaging of uterine sperm from VPX and TX males. The staining also showed a lower level of acetyl-H4K5 expression in the male pronuclei of TX produced embryos. Furthermore, the VPX and TX embryos also expressed higher levels Igf2, and Dlk1. We concluded that interactions between ASG and sperm affected: (1) histone acetylation in male pronuclei; (2) DNA methylation in fertilized oocytes; and (3) Igf2 and Dlk1 expression embryos.

Impact of legislative changes on patterns of antipsychotic prescribing and self-poisoning in Scotland: 2000-06.

Recently, national guidelines have advocated greater use of atypical rather than typical antipsychotics in the treatment of schizophrenia. In addition, there have been safety concerns regarding the potential cardiotoxicity of certain antipsychotics taken in overdose. This has led regulatory authorities in the United Kingdom to restrict the use of thioridazine. The overall impact of these legislative changes on patterns of antipsychotic prescribing has received comparatively little attention. Therefore, we sought to examine the effects on community prescribing practices, and to determine whether this was accompanied by changes in patterns of antipsychotic poisoning. Between 2000-03, there was a rapid decline in the use of typical antipsychotics, whereas the use of atypical antipsychotics increased. The prevalence of atypical and typical antipsychotic prescribing has been approximately equal between 2003-06. During the same study period, hospital admissions due to typical antipsychotic poisoning also declined, however, the effects lagged behind changes in prescribing practice by 2-3 years. These data indicate that legislative changes that restrict the use of thioridazine and other typical antipsychotics are associated with a measurable reduction in the number of hospital admissions due to overdose with these agents.

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells.

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells.

By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48h after a 30min cocaine exposure compared to control cells, and that this was followed by cell death at 72h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain.

Oxidative stress in the aging murine olfactory bulb: redox proteomics and cellular localization.

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine.

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.

Redox proteomics identification of oxidatively modified brain proteins in inherited Alzheimer's disease: an initial assessment.

OBJECTIVE: To identify oxidatively modified proteins in brains of persons with inherited Alzheimer's disease. METHODS: Redox proteomics was used to identify oxidatively modified brain proteins in persons with mutations in the genes for presenilin-1 (PS-1). RESULTS: An initial redox proteomics assessment of oxidatively modified proteins from brains of individuals with PS-1 mutations was performed. These PS1 mutations, Q222H and M233T, are completely penetrant causing early-onset familial AD as previously reported in these Australian families. We show that oxidative modifications of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), gamma-enolase, actin, and dimethylarginine dimethylaminohydrolase 1 (DMDMAH-1) are present in the brain of familial AD subjects. CONCLUSIONS: These initial results suggest that oxidatively modified proteins are important common features in both familial and sporadic AD.