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Phenylpropanoid sucrose esters (PSEs) are plant-derived metabolites that exist widely in medicinal plants and possess important bioactivities. Their precise synthesis is challenging due to the distinct and diverse substitution patterns at the sugar framework, and it is scarcely reported. Orthogonal protection/deprotection strategies for disaccharides are more complex and less developed than those for monosaccharides. We disclose a precise synthesis of PSEs starting from 2,1′:4,6-di-O-diisopropylidene sucrose 7via an orthogonal protection/deprotection and selective cinnamoylation strategy. We demonstrate the strategy for the synthesis of several PSEs cinnamoylated at the O-3 and O-4′ positions of diisopropylidene sucrose 7. The strategy is enabled by a carefully selected and synergistic set of protecting groups and deprotecting agents under the optimized conditions. It potentially gives access to the ∼150 reported PSEs and opens the door for the custom synthesis of unnatural PSEs for industrial applications. The reported work also presents a viable strategy for the general orthogonal protection/deprotection of disaccharides for the precise synthesis of other classes of phenylpropanoid esters and related compounds. Nanyang Technological University Published version We thank the Interdisciplinary Graduate School and the School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore for financial support (CoE, Startup Grant).

Introduction: Feruloyl Sucrose Esters (FSEs) are a class of Phenylpropanoid Sucrose Esters (PSEs) widely distributed in plants. They were investigated as potential selective Alpha Glucosidase Inhibitors (AGIs) to eliminate the side effects associated with the current commercial AGIs. The latter effectively lowers blood glucose levels in diabetic patients but causes severe gastrointestinal side effects. Methods: Systematic structure-activity relationship (SAR) studies using in silico, in vitro and in vivo experiments were used to accomplish this aim. FSEs were evaluated for their in vitro inhibition of starch and oligosaccharide digesting enzymes α-glucosidase and α amylase followed by in silico docking studies to identify the binding modes. A lead candidate, FSE 12 was investigated in an STZ mouse model. Results: All active FSEs showed desired higher % inhibition of α-glucosidase and desired lower inhibition of α -amylase in comparison to AGI gold standard acarbose. This suggests a greater selectivity of the FSEs towards α -glucosidase than α -amylase, which is proposed to eliminate the gastrointestinal side effects. From the in vitro studies, the position and number of the feruloyl substituents on the sucrose core, the aromatic ‘OH’ group, and the diisopropylidene bridges were key determinants of the % inhibition of α - glucosidase and α -amylase. In particular, the diisopropylidene bridges are critical for achieving inhibition selectivity. Molecular docking studies of the FSEs corroborates the in vitro results. The molecular docking studies further reveal that the presence of free aromatic ‘OH’ groups and the substitution at position 3 on the sucrose core are critical for the inhibition of both the enzymes. From the in vitro and molecular docking studies, FSE 12 was selected as a lead candidate for validation in vivo. The oral co-administration of FSE 12 with starch abrogated the increase in post-prandial glucose and significantly reduced blood glucose excursion in STZ-treated mice compared to control (starch only) mice. Conclusion: Our studies reveal the potential of FSEs as selective AGIs for the treatment of diabetes, with a hypothetical reduction of side effects associated with commercial AGIs. Ministry of Education (MOE) Nanyang Technological University Submitted/Accepted version This work has been funded by the Nanyang Techno logical University, College of Engineering Start Up Grant. In addition, this work was also supported by the Singapore Ministry of Education, Tier 1 grant (2019- T1-001-059) (YA).

Background: While there are intrinsic differences in the pros and cons between plain radiographs and computer tomography, the role of pelvic radiographs in polytrauma management is diminishing as computer tomography scans are becoming more accessible. Previous studies found varying results in the inter-observer agreement in pelvic radiograph interpretations. Objectives: To evaluate inter-observer agreement of classifying pelvic fractures in major trauma patients by emergency physicians, and the inter-method agreement between plain radiographs and computer tomography scans. Methods: Three hundred sixty-nine patients with pelvic trauma were recruited from the trauma registries of four designated trauma centres in Hong Kong, each having one set of anteroposterior pelvic radiographs and pelvic computer tomography scans. Pelvic radiographs were classified by two emergency physicians using Young–Burgess classification, and pelvic computer tomography scans classified by an experienced radiologist. Disagreed pelvic radiographs were evaluated by a senior emergency physician to make a final decision before comparing with computer tomography scans. Cohen’s kappa was used to measure the inter-observer and the inter-method agreements, in the groups ‘mechanism of injury’, ‘stable versus unstable fractures’ and ‘complete classification’. Results: Inter-observer agreements of plain radiograph classification for ‘mechanism of injury’, ‘stable versus unstable fractures’ and ‘complete classification’ were moderate to substantial (κ = 0.72, 0.60 and 0.55, respectively). Inter-method agreement for the three groups between plain radiographs and computer tomography were fair to moderate (κ = 0.42, 0.59 and 0.38, respectively). Conclusion: The inter-method agreement between plain pelvic radiographs and computer tomography scans was fair in classifying pelvic fractures, and moderate in detection of unstable pelvic fractures. If the patient is haemodynamically unstable or when computer tomography is unavailable, it is reasonable to obtain plain radiographs to screen for unstable pelvic fractures to expedite early intervention. A review in the education approach and material of Young–Burgess classification may improve inter-observer agreement.

Students with autism spectrum disorder (ASD) studying in mainstream classrooms have diverse adjustment difficulties in learning, social interaction, and emotion regulation. It is crucial to identify the areas these students find most challenging so that teachers can provide training and support accordingly. We therefore developed, examined, and provided norms for the Learning, Social and Emotion Adaptation Questionnaire-Short Form (LSEAQ-S), a teacher report instrument measuring 53 essential adaptive behaviors for mainstream primary school students in Hong Kong. Teachers completed the LSEAQ-S for three samples of 2,298, 2,690, and 3,305 students with ASD from 204 schools and a sample of 1,869 students without ASD from 112 schools. Our study showed that an 11-factor structure best describes the LSEAQ-S, which has high internal consistency and good convergent validity examined with the Social Responsiveness Scale-Second Edition (SRS-2). Normative data of the LSEAQ-S stratified by gender and grade (grades 1 to 3; grades 4 to 6) are presented. Gender and grade differences were found, with girls with ASD lagging behind their same-gender peers in related skills more than boys with ASD did, across both grade levels and especially in senior grades. The LSEAQ-S, together with its normative data, can reveal students' difficulties and needs, inform intervention priorities, and help monitor training progress. LAY SUMMARY: This study introduces the Learning, Social and Emotion Adaptation Questionnaire-Short Form (LSEAQ-S), a teacher report instrument developed in Hong Kong measuring school adaptation of students with autism spectrum disorder (ASD) in mainstream primary schools. The measure helps education personnel identify behaviors in which a student falls behind his/her peers and facilitate training and support targeting those behaviors. Autism Res 2021, 14: 959-972. © 2020 The Authors. Autism Research published by International Society for Autism Research and Wiley Periodicals LLC.

Background: Body packing is a frequently used method for drug trafficking. Local information about the clinical and radiological features of body packing is lacking. Objectives: To evaluate the radiological features of body packers presenting to a hospital near to the Hong Kong International Airport and to compare the radiological features of solid form versus liquid cocaine. Methods: This was a retrospective cohort study. Medical notes of 269 suspected body packers, presenting to the Accident and Emergency Department of North Lantau Hospital under the detention by the law enforcement personnel from 1st January 2015 to 28th February 2017, were reviewed. The radiological features of body packing were retrospectively evaluated. Results: Sixty-nine cases were confirmed body packers radiographically. Majority of them (81%, 56/69) were cocaine packers. Powder form cocaine (67%, 49/69) was the most popular drug packed, followed by liquid cocaine (15%, 10/69). There was a trend of increasing incidence of liquid cocaine packers. The classical ‘double condom’, ‘tic tac’ and ‘halo’ signs were present in 94%, 72.5% and 42% of cases with radiologically confirmed body packing respectively. The ‘rosette’ sign was only identified in 1 case. Three new radiological signs, the ‘bag of eggs’, ‘lucent triangle’ and ‘black crescent’ sign, were suggested to aid identification of drug packets. The classical ‘tic tac’ sign was absent in all liquid cocaine packing cases (pConclusion: Failure in detecting drug body packing may result in medicolegal consequences. Emergency physicians need to be aware of subtle radiological signs of liquid cocaine packets in the plain abdominal radiography.

Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic “CaaX” motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.

Lipid cochleates are gaining increasing interest as drug-carriers. However, their preparation relies on conventional batch processes that are complex, time consuming and lack batch-to-batch reproducibility; presenting a bottleneck for clinical translation. We report an efficient continuous preparation process for artemisinin-loaded cochleates (ART-cochleates) using inexpensive off-the-shelf flow focusing device. By carefully controlling the flow focusing parameters, we showed along with the mechanism that, ART-cochleates of uniform and tuneable size (~180 nm in width and ~1030 nm in length) were obtained with low dispersity (0.18 in width and 0.27 in length), narrow size distribution and high reproducibility compared to the batch process. The device achieved high throughput of 11.5 g/day with ART encapsulation of 64.24 ± 2.5% and loading of 83.37 ± 3.68 mg ART/g of cochleates. Art-cochleates were non-toxic and showed sustained in-vitro release of ART with effective transepithelial permeability across intestinal Caco-2 monolayer (~60% and ~25% transport for pure ART and ART-cochleates, respectively) resulting in better in-vitro bioavailability. The off-the-shelf device is envisioned to be highly promising platform for continuous and high-throughput manufacturing of drug-loaded cochleates in a controlled and reproducible manner. It has potential to enable clinical translation of drug-loaded cochleates with predicable drug release, absorption and bioavailability. Nanyang Technological University We thank Nanyang Technological University, Singapore for financial help (RG 13/18).

Cinnamoyl sucrose esters (CSEs) were evaluated as AGIs and their enzyme inhibition activity and potency were compared with gold standard acarbose. The inhibition activity of the CSEs against &alpha, glucosidase and &alpha, amylase depended on their structure including the number of the cinnamoyl moieties, their position, and the presence or absence of the acetyl moieties. The inhibitory values of the CSEs 2&ndash, 9 generally increases in the order of mono-cinnamoyl moieties &lt, di-cinnamoyl &le, tri-cinnamoyl &lt, tetra-cinnamoyl. This trend was supported from both in vitro and in silico results. Both tetra-cinnamoyl CSEs 5 and 9 showed the highest &alpha, glucosidase inhibitory activities of 77 &plusmn, 5%, 74 &plusmn, 9%, respectively, against acarbose at 27 &plusmn, 4%, and highest &alpha, amylase inhibitory activities of 98 &plusmn, 2%, 99 &plusmn, 1%, respectively, against acarbose at 93 &plusmn, 2%. CSEs 3, 4, 6, 7, 8 showed desired higher inhibition of &alpha, glucosidase than &alpha, amylase suggesting potential for further development as AGIs with reduced side effects. Molecular docking studies on CSEs 5 and 9 attributed the high inhibition of these compounds to multiple &pi, &pi, interactions and favorable projection of the cinnamoyl moieties (especially O-3 cinnamoyl) in the enzyme pockets. This work proposes CSEs as new AGIs with potentially reduced side effects.

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3',5'-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3',5'-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.

Massive upper gastrointestinal bleeding is an uncommon presentation of Burkitt’s lymphoma in a patient with HIV/AIDS, and is seldom reported in the literature. A 39-year-old man who has sex with men presented with abdominal pain and massive haematemesis and a rapid drop in haemoglobin level to 4.8 g/dL. Upper gastrointestinal endoscopy showed a large blood clot in the stomach, and an emergency laparotomy was performed because of unstable haemodynamics. This showed active bleeding from multiple tumours in the fundus and body of the stomach. The patient underwent gastrectomy and gastric biopsy confirmed Burkitt’s lymphoma. Further tests showed lymphoma involvement in bone marrow and central nervous system. The patient tested positive for HIV, and had a CD4 count of 212 cells/mm3 and viral load of 18,000 copies/mL at diagnosis. He was commenced on a chemotherapy regimen of CODOX-M/IVAC, and highly active antiretroviral therapy consisting of indinavir, stavudine and lamivudine. The major side effect was peripheral neuropathy. Infective complications during chemotherapy were controlled by broad-spectrum antibiotics and anti-fungal agents. Complete remission of the lymphoma was achieved after the chemotherapy and remission was maintained for more than 14 years.

Legionella pneumophila is a pathogen, causing severe pneumonia in humans called Legionnaires' disease. AnkC (LegA12) is a poorly characterized 495-residue effector protein conserved in multiple Legionella species. Here, we report the crystal structure of a C-terminally truncated AnkC (2-384) at 3.2 A resolution. The structure shows seven ankyrin repeats (ARs) with unique structural features. AnkC forms a dimer along the outer surface of loops between ARs. The dimer exists both in the crystal form and in solution, as shown by analytical ultracentrifugation. This is the first example of ARs as a dimerization module as opposed to solely a protein interaction domain. In addition, a novel α-helix insert between AR3-AR4 is positioned across the surface opposite the ankyrin groove. Sequence conservation suggests that the ankyrin groove of AnkC is a functional site that interacts with binding targets. This ankyrin domain structure is an important step towards a functional characterization of AnkC.

Legionella pneumophila is a pathogen causing severe pneumonia in humans called Legionnaires’ disease. Lem22 is a previously uncharacterized effector protein conserved in multiple Legionella strains. Here, we report the crystal structure of Lem22 from the Philadelphia strain, also known as lpg2328, at 1.40 Å resolution. The structure shows an up-and-down three-helical bundle with a significant structural similarity to a number of protein-binding domains involved in apoptosis and membrane trafficking. Sequence conservation identifies a putative functional site on the interface of helices 2 and 3. The structure is an important step towards a functional characterization of Lem22.

Objectives To examine the safety and tolerability of an active rehabilitation program for adolescents who are slow to recover from a sport-related concussion, and secondarily to estimate the treatment effect for this intervention. Design Single-site, parallel, open-label, randomized controlled trial comparing treatment as usual (TAU) to TAU plus active rehabilitation. Setting Outpatient concussion clinic. Participants Adolescents (N=19) aged 12 to 18 years with postconcussion symptoms lasting ≥1 month after a sports-related concussion. Interventions TAU consisted of symptom management and return-to-play advice, return-to-school facilitation, and physiatry consultation. The active rehabilitation program involved in-clinic subsymptom threshold aerobic training, coordination exercises, and visualization and imagery techniques with a physiotherapist (mean, 3.4 sessions) as well as a home exercise program, over 6 weeks. Main Outcome Measures A blinded assessor systematically monitored for predetermined adverse events in weekly telephone calls over the 6-week intervention period. The treating physiotherapist also recorded in-clinic symptom exacerbations during aerobic training. The Post-Concussion Symptom Scale was the primary efficacy outcome. Results Nineteen participants were randomized, and none dropped out of the study. Of the 12 adverse events detected (6 in each group), 10 were symptom exacerbations from 1 weekly telephone assessment to the next, and 2 were emergency department visits. Four adverse events were referred to an external safety committee and deemed unrelated to the study procedures. In-clinic symptom exacerbations occurred in 30% (9/30) of aerobic training sessions, but resolved within 24 hours in all instances. In linear mixed modeling, active rehabilitation was associated with a greater reduction on the Post-Concussion Symptom Scale than TAU only. Conclusions The results support the safety, tolerability, and potential efficacy of active rehabilitation for adolescents with persistent postconcussion symptoms.

Structural analysis reveals how a bacterial enzyme catalyses attachment of the protein tag ubiquitin to host proteins, illuminating a process that allows pathogenic bacteria to subvert host-cell function. Structural analysis reveals how a bacterial enzyme catalyses attachment of the protein tag ubiquitin to host proteins, illuminating a process that allows pathogenic bacteria to subvert host-cell function.

Objective To review and compare initial high resolution computed tomography (HRCT) findings in patients with metapneumovirus pneumonia and severe acute respiratory syndrome (SARS-Coronovirus). Materials and methods 4 cases of metapneumovirus pneumonia (mean age of 52.3 years) in an institutional outbreak (Castle Peak Hospital) in 2008 and 38 cases of SARS-coronovirus (mean age of 39.6 years) admitted to Tuen Mun hospital during an epidemic outbreak in 2003 were included. HRCT findings of the lungs for all patients were retrospectively reviewed by two independent radiologists. Results In the metapneumovirus group, common HRCT features were ground glass opacities (100%), consolidation (100%), parenchymal band (100%), bronchiectasis (75%). Crazy paving pattern was absent. They were predominantly subpleural and basal in location and bilateral involvement was observed in 50% of patients. In the SARS group, common HRCT features were ground glass opacities (92.1%), interlobular septal thickening (86.8%), crazy paving pattern (73.7%) and consolidation (68%). Bronchiectasis was not seen. Majority of patient demonstrated segmental or lobar in distribution and bilateral involvement was observed in 44.7% of patients. Pleural effusion and lymphadenopathy were of consistent rare features in both groups. Conclusion Ground glass opacities, interlobular septal thickening and consolidations were consistent HRCT manifestations in both metapneumovirus infection and SARS. The presence of bronchiectasis (0% in SARS) may point towards metapneumovirus while crazy paving pattern is more suggestive of SARS.

Dynamic nutrient feeding to control glutamine at low levels in protein-free fed-batch cultures of 293-human embryonic kidney (HEK) cells achieved cell concentrations of 6×10 6 cells/ml. This represented a 4-fold improvement in cell concentration compared to batch cultures. Reduction in glutamine and glucose consumption, as well as lactate and ammonia production, were also observed in these fed-batch cultures. High virus production titers of 3×10 11 pfu/ml were achieved in fed-batch cultures which were 10,000-fold higher than batch cultures. An investigation of the transcriptional regulation of the metabolic changes associated with the batch and the low-glutamine fed-batch cultures using DNA microarray was conducted. This analysis provides better understanding of the transcriptional regulatory mechanism resulting in the observed physiological changes. Transcriptional profiling of cells from the mid-exponential, late exponential and stationary phases of both the batch and fed-batch were undertaken using an 18,000 element human chip. Transcriptional profiles were ontologically classified to provide a global view of the genetic changes. Furthermore, a pathway-oriented analysis focusing on cellular metabolism was conducted to reveal the dynamic regulation of genes related to amino acid metabolism, tRNA synthetases, TCA cycle, electron transport chain and glycolysis.

Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.

Chinese Hamster ovary (CHO) cells are regarded as one of the "work-horses" for complex biotherapeutics production. In these processes, loss in culture viability occurs primarily via apoptosis, a genetically controlled form of cellular suicide. Using our "in-house" developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis of batch and fed-batch CHO cell cultures, the genetic circuitry that regulates and executes apoptosis induction were examined. During periods of high viability, most pro-apoptotic genes were down-regulated but upon loss in viability, several early pro-apoptotic signaling genes were up-regulated. At later stages of viability loss, we detected late pro-apoptotic effector genes such as caspases and DNases being up-regulated. This sequential regulation of apoptotic genes showed that DNA microarrays could be used as a tool to study apoptosis. We found that in batch and fed-batch cultures, apoptosis signaling occurred primarily via death receptor- and mitochondria-mediated signaling pathways rather than endoplasmic reticulum-mediated signaling. These insights provide a greater understanding of the regulatory circuitry of apoptosis during cell culture and allow for subsequent targeting of relevant apoptosis signaling genes to prolong cell culture.

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15K CHO cDNA microarray chip, whereas proteomic analysis was performed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.

Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.

As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-gamma). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-gamma yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-gamma. However, very low glutamine (

Objective To describe the feasibility and safety of a randomised controlled trial (RCT) comparing active rehabilitation (AR) to treatment as usual (TAU) in adolescents with persistent symptoms following a sport-related concussion. Design Partially blinded, pilot RCT. Setting Outpatient Rehabilitation Hospital. Participants 13 adolescents enrolled and completed the study (10 girls, 3 boys). All were between the ages of 12–18 and were symptomatic for greater than 4 weeks. Participants were excluded if they had more than 1 concussion in the last year, any active mental health disorder, or if their concussion occurred more than 1 year from enrolment. Intervention Participants were randomly allocated to TAU (occupational therapy and school recommendations) or AR. AR included aerobic exercise, positive visualisation techniques, and sport-specific exercises. AR sessions were supervised by a physiotherapist, and a blinded assessor monitored all participants weekly. Outcome measures Investigators monitored symptoms using the Post-Concussion Symptom Inventory. Secondary outcome measures included activity level, balance, mood, fatigue, and return to school. Main results 125 individuals were screened over a 1.25 year period. Of the 22% of individuals deemed eligible, 15 declined to participate. Most commonly, individuals were excluded due to being unreachable, uninterested, or having more than one concussion. No drop outs occurred. An external committee reviewed two adverse events in TAU and 1 adverse event in the AR group. All adverse events were unrelated to the intervention and study procedures. Conclusions Based on this small pilot study, exercise appeared safe in this sample of youth with persistent symptoms. Competing interests None.