A Retrospective Cohort Study Comparing the Functional and General Health Outcomes of Staged vs Simultaneous Bilateral Primary Total Knee.

BACKGROUND: Bilateral primary total knee replacements (TKRs) can be performed simultaneously (under the same anaesthetic) or staged (on separate occasions). Patients who undergo simultaneous biltateral TKRs only have one anaesthetic, operation and hospital stay. This reduces overall time in pain, rehabilitation time and total days in hospital with cost-saving implications. Despite the benefits, simultaneous bilateral TKRs are not routinely performed due to fear of worse outcomes. AIM: This paper assesses the long-term functional and general health outcomes between the two approaches. METHODS: We undertook a retrospective cohort study. Patients who underwent a bilateral TKR in our centre from 2012-2014 were included. The validated Oxford Knee and EuroQol scores were used to assess function and general health outcomes. These were assessed at baseline and 1-year along with satisfaction scores. RESULTS: 41 simultaneous bilateral TKRs and 43 staged bilateral TKRs met the inclusion criteria. Patients were age and sex matched. The Oxford Knee Score increase at one year was equivalent in both groups (20.1 vs. 18.1, p=0.170), suggesting no difference in long-term function. The EuroQol Score increase at one year was equivalent in both groups (0.41 vs. 0.48, p=0.350), suggesting no difference in long-term general health. Satisfaction was comparable between the two groups (88.8 vs. 86.2, p=0.52). CONCLUSIONS: There is no difference in functional and general health outcomes between staged and simultaneous bilateral knee replacements. In patients with bilateral knee arthritis, simultaneous bilateral TKRs should be considered.

Very-large-scale production of antibodies in plants: The biologization of manufacturing.

Gene technology has facilitated the biologization of manufacturing, i.e. the use and production of complex biological molecules and systems at an industrial scale. Monoclonal antibodies (mAbs) are currently the major class of biopharmaceutical products, but they are typically used to treat specific diseases which individually have comparably low incidences. The therapeutic potential of mAbs could also be used for more prevalent diseases, but this would require a massive increase in production capacity that could not be met by traditional fermenter systems. Here we outline the potential of plants to be used for the very-large-scale (VLS) production of biopharmaceutical proteins such as mAbs. We discuss the potential market sizes and their corresponding production capacities. We then consider available process technologies and scale-down models and how these can be used to develop VLS processes. Finally, we discuss which adaptations will likely be required for VLS production, lessons learned from existing cell culture-based processes and the food industry, and practical requirements for the implementation of a VLS process.

Extraction and downstream processing of plant-derived recombinant proteins.

Plants offer the tantalizing prospect of low-cost automated manufacturing processes for biopharmaceutical proteins, but several challenges must be addressed before such goals are realized and the most significant hurdles are found during downstream processing (DSP). In contrast to the standardized microbial and mammalian cell platforms embraced by the biopharmaceutical industry, there are many different plant-based expression systems vying for attention, and those with the greatest potential to provide inexpensive biopharmaceuticals are also the ones with the most significant drawbacks in terms of DSP. This is because the most scalable plant systems are based on the expression of intracellular proteins in whole plants. The plant tissue must therefore be disrupted to extract the product, challenging the initial DSP steps with an unusually high load of both particulate and soluble contaminants. DSP platform technologies can accelerate and simplify process development, including centrifugation, filtration, flocculation, and integrated methods that combine solid-liquid separation, purification and concentration, such as aqueous two-phase separation systems. Protein tags can also facilitate these DSP steps, but they are difficult to transfer to a commercial environment and more generic, flexible and scalable strategies to separate target and host cell proteins are preferable, such as membrane technologies and heat/pH precipitation. In this context, clarified plant extracts behave similarly to the feed stream from microbes or mammalian cells and the corresponding purification methods can be applied, as long as they are adapted for plant-specific soluble contaminants such as the superabundant protein RuBisCO. Plant-derived pharmaceutical proteins cannot yet compete directly with established platforms but they are beginning to penetrate niche markets that allow the beneficial properties of plants to be exploited, such as the ability to produce 'biobetters' with tailored glycans, the ability to scale up production rapidly for emergency responses and the ability to produce commodity recombinant proteins on an agricultural scale.

Prevalence and consequences of delayed diagnosis of anterior cruciate ligament ruptures.

PURPOSE: Rupture of the anterior cruciate ligament (ACL) is a common injury, often presenting with a typical injury pattern. Historically, the literature indicates that the accuracy of diagnosis of ACL ruptures is poor at the initial medical consultation. The aims of this study were to determine: the mechanism of injury; changes in accuracy of diagnosis of ACL ruptures at initial presentation over the last decade; and the effect of subsequent delay in diagnosis and definitive treatment. METHODS: A prospective cohort of one hundred and thirty-two consecutive patients who underwent ACL reconstruction between 2005 and 2009 were analysed. The median age of the patients was 18 years (12-57). Sixteen patients were excluded due to chronic ACL injury. RESULTS: One hundred and sixteen patients (117 ACL ruptures) were included in the analysis. A typical injury pattern was documented in 87 (74.4 %) of cases. The most common sporting activities associated with an ACL injury were football (35.3 %), skiing (21.6 %) and rugby (10.3 %). The majority of patients (67.5 %) sought medical attention within 1 week from time of injury. The correct diagnosis of an ACL rupture was made in 33 cases (28.2 %) at the initial medical consultation. The diagnosis was made following medical consultation in 13 (11.1 %) of cases with the use of magnetic resonance imaging and 6 (5.1 %) cases at arthroscopy. The median time to diagnosis was 6 weeks (0-192), and the median time to ACL reconstruction was 24 weeks (1-240). A delay in diagnosis of >6 months was associated with a medial meniscal tear rate of 72.2 % compared to 23.1 % if the diagnosis was made within 4 months of the injury (p < 0.05). CONCLUSIONS: Despite a 'typical' mechanism of injury leading to ACL rupture, the rate of initial diagnosis in the UK still remains poor. This often leads to an unnecessary delay in the diagnosis and subsequent treatment and increases the risk of secondary injury to the knee. A delay in diagnosis of >6 months was associated with an increased medial meniscal tear rate. Patients who present with a 'typical' injury pattern should therefore be referred for further assessment by a knee specialist within 6 weeks. LEVEL OF EVIDENCE: IV.

Can plant biotechnology help break the HIV-malaria link?

The population of sub-Saharan Africa is at risk from multiple, poverty-related endemic diseases. HIV and malaria are the most prevalent, but they disproportionately affect different groups of people, i.e. HIV predominantly affects sexually-active adults whereas malaria has a greater impact on children and pregnant women. Nevertheless, there is a significant geographical and epidemiological overlap which results in bidirectional and synergistic interactions with important consequences for public health. The immunosuppressive effects of HIV increase the risk of infection when individuals are exposed to malaria parasites and also the severity of malaria symptoms. Similarly, acute malaria can induce a temporary increase in the HIV viral load. HIV is associated with a wide range of opportunistic infections that can be misdiagnosed as malaria, resulting in the wasteful misuse of antimalarial drugs and a failure to address the genuine cause of the disease. There is also a cumulative risk of toxicity when antiretroviral and antimalarial drugs are given to the same patients. Synergistic approaches involving the control of malaria as a strategy to fight HIV/AIDS and vice versa are therefore needed in co-endemic areas. Plant biotechnology has emerged as a promising approach to tackle poverty-related diseases because plant-derived drugs and vaccines can be produced inexpensively in developing countries and may be distributed using agricultural infrastructure without the need for a cold chain. Here we explore some of the potential contributions of plant biotechnology and its integration into broader multidisciplinary public health programs to combat the two diseases in developing countries.

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.

Randomized dose-controlled study of topiramate as first-line therapy in epilepsy.

OBJECTIVES: To evaluate the efficacy and tolerability of topiramate as monotherapy, using a dose-controlled study design. MATERIALS AND METHODS: We conducted a multinational, randomized, double-blind trial in adults and children (> or =6 years old) with epilepsy that was not being treated when randomized to 400 or 50 mg/day topiramate as target maintenance dosages. In addition to > or =2 lifetime unprovoked seizures, patients had to have one or two partial-onset seizures or generalized-onset tonic-clonic seizures in the 3-month retrospective baseline. The primary efficacy end point was time to first seizure; a secondary efficacy measure was the seizure-free rate at 6 months and 1 year. Double-blind treatment continued until 6 months after the last patient was randomized. RESULTS: Kaplan-Meier survival analyses for time to first seizure (intent-to-treat, n = 470) favored 400 mg/day over 50 mg/day (P = 0.0002) as a target maintenance dosage. The first evaluation point with a significant difference (P = 0.046) favoring the higher dose was at day 14 when patients were receiving 100 or 25 mg/day. The probability of being seizure-free at 6 months was 83% in patients randomized to 400 mg/day and 71% in those randomized to 50 mg/day (P = 0.005). Seizure-free rates at 12 months were 76% and 59%, respectively (P = 0.001). Differences favoring the higher dose were significant in patients with partial-onset seizures (P = 0.009) and in those with generalized-onset tonic-clonic seizures (P = 0.005). The most common dose-related adverse events were paresthesia, weight loss, and decreased appetite. Discontinuations due to cognitive-related adverse events were 2% in the 50-mg group and 7% in the 400-mg group. Overall, 7% and 19%, respectively, discontinued with adverse events during the median treatment duration of 9 months. CONCLUSION: Topiramate is effective as monotherapy in adults and children. Because a therapeutic effect emerges during titration, clinicians should adjust dosages in step-wise fashion with intermediate stopping points, e.g., 100 mg/day, to evaluate patient response and achieve the optimal maintenance dosage.

Superficial radial nerve damage due to Kirschner wiring of the radius.

For displaced fractures of the radius, the use of Kirschner wires (K Wires) is accepted practice either alone or to supplement external fixation. Complications related to K wires include infection, migration and damage to tendons and nerves. We set out to investigate to incidence of superficial radial nerve damage due to radial styloid K wires. Injury to the sensory branch of the superficial radial nerve was seen in eight (20%) out of the 40 patients. K wiring is a popular technique to help maintain anatomic reduction of distal radial fractures. It has the advantage of being a semi-closed procedure, which is simple to perform. However, the morbidity of nerve damage is often underestimated and can be avoided.

Echocardiographic assessment of the right ventricular stress-velocity relationship under normal and chronic overload conditions.

UNLABELLED: The effects of chronic volume or pressure overload on the velocity of right ventricular ejection have not been previously well defined. We hypothesized that, as formerly shown for the left ventricle, there would be a direct relationship between the velocity of ejection and an estimate of systolic wall stress. METHODS: Echocardiograms of asymptomatic patients, not on cardiac medications, with either an isolated secundum atrial septal defect > or = 5 mm in diameter or isolated pulmonic stenosis with a peak instantaneous pressure gradient > or = 20 mmHg, were reviewed. Forty-one patients with an atrial septal defect and 34 with pulmonary stenosis met criteria, and were compared to age-matched normal controls. Total subjects were 127 with ages ranging from 1 day to 54 years. Right ventricular monoplane ejection fraction, ejection time corrected for heart rate (ETc), mean normalized systolic ejection rate (MNSERc) and meridianal peak-systolic wall stress (WSps) were measured. RESULTS: Compared to controls, ejection fractions were not significantly different, but WSps averaged 81% and 110% higher, ETc 8% and 9% longer, and MNSERc 5% and 9% slower in the atrial septal defect and pulmonary stenosis groups, respectively. Among all subjects WSps had a significant linear correlation with ETc (r = 0.61, P < 0.01), MNSERc (r =-0.46, P < 0.01), and ejection fraction (r =-0.19, P < 0.05). CONCLUSIONS: Increases in WSps cause an incremental slowing of MNSERc in the right ventricle, with a relationship that is linear over a wide range of normal and abnormal loading conditions.

Native and artificial reticuloplasmins co-accumulate in distinct domains of the endoplasmic reticulum and in post-endoplasmic reticulum compartments.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Transgenic rice plants expressing the ferredoxin-like protein (AP1) from sweet pepper show enhanced resistance to Xanthomonas oryzae pv. oryzae.

We used particle bombardment to cotransform mature seed-derived rice callus (Oryza sativa L., ssp. japonica, cv. Eyi 105) with plasmids containing the linked marker genes gusA and hpt, and the ap1 gene encoding an amphipathic protein previously shown to delay the hypersensitive response induced in non-host plants by the pathogen Pseudomonas syringae pv. syringae (Pss). Thirty-two independent lines of transgenic rice plants were regenerated, and 27 of these lines carried all three transgenes as shown by molecular analysis. A bacterial blight inoculation test was carried out on ten lines. In each case, plants carrying the ap1 gene showed enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo) race 6 at various levels. This suggests the ap1 gene could be a useful candidate for genetic engineering strategies in rice to provide bacterial blight resistance.

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns.

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice.

We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns, including a novel form of transcriptional silencing involving methylation of cytosine residues only at non-conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another.

The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor.

Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion in Caenorhabditis elegans requires only the unc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure. unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently in Xenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.

Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors.

1. Regional distinctions in GABA type A (GABAA) miniature IPSC responses are thought to be determined by postsynaptic receptor composition. The kinetics of receptor activation and deactivation were studied using rapid exchange (100 micros) of GABA at excised patches containing recombinant (alpha1beta1gamma2 or alpha2beta1gamma2) and native (cortical) GABAA receptors. 2. Receptors activated by brief (< 1 ms) pulses of GABA demonstrated a characteristic current response, hereby referred to as the 'receptor system response'. System response properties included agonist concentration-dependent peak amplitudes and concentration-independent maximal rates of activation and deactivation. Receptor subtypes were characterized functionally and phenotyped using the system response characteristics. 3. System responses obtained for alpha1beta1gamma2 receptors exhibited a single phenotype while alpha2beta1gamma2 receptors exhibited either a predominant slow deactivation (type I) or a relatively infrequent faster (type II) phenotype. Receptor system responses of alpha2beta1gamma2 receptors reached peak currents twice as fast as those of alpha1beta1gamma2 receptors (0.5 versus 1.0 ms) but decayed 2 or 6 times more slowly (taulong of approximately 190 and 62 ms for type I and II alpha2beta1gamma2, and approximately 34 ms for alpha1beta1gamma2 receptors). 4. Receptor system responses from cultured fetal mouse cortical neurons could be statistically separated and classified into five major types with little intragroup variability, primarily based on variations in the current deactivation phases. 5. Receptors subjected to pharmacological modulation exhibited alterations in system response properties consistent with known mechanisms of action, such that distinctions between binding and gating modulations were possible. 6. Brief agonist exposure places limits on receptor activation and deactivation response kinetics. Consequently, receptor system responses may be used to characterize and functionally phenotype an excised patch receptor population. Furthermore, since synaptic exposure to transmitter is postulated to be similarly brief, IPSC kinetics may reflect a functional fingerprint of synaptic receptors.

The methylglutamate, SYM 2081, is a potent and highly selective agonist at kainate receptors.

The methylglutamate analog (2S,4R)-4-methylglutamate (SYM 2081) has been shown to potently displace high affinity [3H]kainate binding to cortical tissue and to recombinant kainate receptors, and to evoke rapidly desensitizing responses in electrophysiological recordings. We have used two electrode voltage clamp recordings to compare the potency and efficacy of SYM 2081 with other alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor agonists at homomeric kainate and AMPA receptors expressed in Xenopus oocytes. In the presence of concanavalin A to reduce agonist induced desensitization at kainate receptors, SYM 2081 was a potent agonist at homomeric kainate receptors composed of the GluR5 and GluR6 subunit, with an EC50 of 0.12 +/- 0.02 and 0.23 +/- 0.01 microM, respectively. SYM 2081 was highly selective for kainate receptors, the EC50 for activation of AMPA receptors composed of the GluR1 and GluR3 subunits was 132 +/- 44 and 453 +/- 57 microM, respectively. Other methylglutamate analogs were tested for kainate receptor agonist activity. Methylglutamate compounds with the methyl group at the 2 or 3 position of glutamate were inactive indicating that positioning of the methyl group at the 4 position was essential for agonist activity. Of the four stereoisomers of 4-methylglutamate, SYM 2081 (2S,4R) was the most potent agonist. The (2R,4R) isomer was estimated to be 20-fold and the (2S,4S)-isomer approximately 1000-fold less potent than SYM 2081. These results indicate that SYM 2081 is a potent and selective agonist at kainate receptors, and thus will be a useful ligand for evaluating the role of kainate receptors in central nervous system function and disease.