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A thorough characterisation of the genetics, physiology and metabolism of Escherichia coli has led to the availability of a large number of strains and vectors suitable for recombinant protein expression. Despite the relative ease in using E. coli for achieving amplified expression of many recombinant proteins, for some proteins this can be a frustrating and time-consuming process leading to very low expression or no expression at all. This is especially true for membrane proteins, which introduce additional challenges. A number of factors can be considered and optimised for achieving required levels of amplified expression of recombinant proteins in E. coli that are broadly classified as host strain, expression vector and growth conditions. In this paper we summarise these factors and consolidate the common challenges encountered and approaches to overcome them, focusing in particular on cases where there is low amplified expression or no expression at all of the desired recombinant protein, due to various reasons.
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Clonagem Molecular/métodos , Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: Seasonal/perennial allergic conjunctivitis is the most common allergic conjunctivitis, usually with acute manifestations when a person is exposed to allergens and with typical signs and symptoms including itching, redness, and tearing. The clinical signs and symptoms of allergic conjunctivitis are mediated by the release of histamine by mast cells. Histamine antagonists (also called antihistamines) inhibit the action of histamine by blocking histamine H1 receptors, antagonising the vasoconstrictor, and to a lesser extent, the vasodilator effects of histamine. Mast cell stabilisers inhibit degranulation and consequently the release of histamine by interrupting the normal chain of intracellular signals. Topical treatments include eye drops with antihistamines, mast cell stabilisers, non-steroidal anti-inflammatory drugs, combinations of the previous treatments, and corticosteroids. Standard treatment is based on topical antihistamines alone or topical mast cell stabilisers alone or a combination of treatments. There is clinical uncertainty about the relative efficacy and safety of topical treatment. OBJECTIVES: The objective of this review was to assess the effects of topical antihistamines and mast cell stabilisers, alone or in combination, for use in treating seasonal and perennial allergic conjunctivitis. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2014, Issue 7), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2014), EMBASE (January 1980 to July 2014), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 17 July 2014. We also searched the reference lists of review articles and relevant trial reports for details of further relevant publications. SELECTION CRITERIA: We included randomised controlled trials (RCTs) comparing topical antihistamine and mast cell stabilisers, alone or in combination, with placebo, no treatment or to any other antihistamine or mast cell stabiliser, or both, that examined people with seasonal or perennial allergic conjunctivitis, or both. The primary outcome was any participant-reported evaluation (by questionnaire) of severity of four main ocular symptoms: itching, irritation, watering eye (tearing), and photophobia (dislike of light), both separately and, if possible, by an overall symptom score. We considered any follow-up time between one week and one year. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed risk of bias. Disagreements were resolved by discussion among review authors and the involvement of a third review author. We followed standard methodological approaches used by Cochrane. MAIN RESULTS: We identified 30 trials with a total of 4344 participants randomised, with 17 different drugs or treatment comparisons. The following antihistamines and mast cell stabilisers were evaluated in at least one RCT: nedocromil sodium or sodium cromoglycate, olopatadine, ketotifen, azelastine, emedastine, levocabastine (or levocabastine), mequitazine, bepotastine besilate, combination of antazoline and tetryzoline, combination of levocabastine and pemirolast potassium. The most common comparison was azelastine versus placebo (nine studies).We observed a large variability in reporting outcomes. The quality of the studies and reporting was variable, but overall the risk of bias was low. Trials evaluated only short-term effects, with a range of treatment of one to eight weeks. Meta-analysis was only possible in one comparison (olopatadine versus ketotifen). There was some evidence to support that topical antihistamines and mast cell stabilisers reduce symptoms and signs of seasonal allergic conjunctivitis when compared with placebo. There were no reported serious adverse events related to the use of topical antihistamine and mast cell stabilisers treatment. AUTHORS' CONCLUSIONS: It seems that all reported topical antihistamines and mast cell stabilisers reduce symptoms and signs of seasonal allergic conjunctivitis when compared with placebo in the short term. However, there is no long-term data on their efficacy. Direct comparisons of different antihistamines and mast cell stabilisers need to be interpreted with caution. Overall, topical antihistamines and mast cell stabilisers appear to be safe and well tolerated. We observed a large variability in outcomes reported. Poor quality of reporting challenged the synthesis of evidence.
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Antialérgicos/administração & dosagem , Conjuntivite Alérgica/tratamento farmacológico , Antagonistas dos Receptores Histamínicos/administração & dosagem , Mastócitos/efeitos dos fármacos , Histamina/metabolismo , Humanos , Mastócitos/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Estações do AnoRESUMO
Lymphoid tissue inducer cells (LTi) play an important role in the development of lymphoid tissue in embryos. Adult CD4(+)CD3(-) LTi-like cells present a similar phenotype and gene expression to their embryonic counterpart and have important roles in CD4(+) T-cell memory and lymphoid tissue recovery following viral infection. However, adult LTi-like cells are heterogeneous populations and the factors that regulate their survival and accumulation within secondary lymphoid organs remain unclear, in particular whether the T-zone stroma is involved. Here we report the identification and characterization of a distinct subset of podoplanin(+) murine splenic stromal cells that support adult LTi-like cell survival. We have identified and isolated CD45(-)podoplanin(+) stromal cell populations which have a similar but distinct phenotype to T-zone reticular cells in LN. CD45(-)podoplanin(+) fibroblast-like cells mediate LTi-like cell survival in vitro; surprisingly this was not dependent upon IL-7 as revealed through blocking Ab experiments and studies using LTi-like cells unable to respond to gamma chain cytokines. Our findings show that adult LTi-like cells require extrinsic signals from podoplanin(+) splenic stromal cells to survive and suggest that IL-7 is not necessary to mediate their survival in the adult spleen.
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Interleucina-7/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Estromais/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofluorescência , Interleucina-7/genética , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Tecido Linfoide/citologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Baço/citologia , Células Estromais/citologia , Linfócitos T Auxiliares-Indutores/citologia , Fatores de TempoRESUMO
Brittle cornea syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the collagen type I/collagen type V ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of collagen type I, offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus in which the cornea thins progressively. This article has an associated First Person interview with the first author of the paper.
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Proteínas de Ligação a DNA , Anormalidades da Pele , Animais , Córnea , Proteínas de Ligação a DNA/genética , Anormalidades do Olho , Humanos , Instabilidade Articular/congênito , Camundongos , Mutação/genética , Anormalidades da Pele/genética , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
PURPOSE: To audit and analyse the accuracy of current biometric formulae on refractive outcomes following cataract surgery in patients with axial length less than 22 mm. METHODS: A total of 84 eyes from 84 patients with axial length <22 mm were identified from consecutive patients undergoing cataract surgery retrospectively at a single university hospital. All subjects had biometry using the IOLMaster (Carl Zeiss Meditec, Inc, Dublin, CA, USA) and a Sensar AR40 intraocular lens implant (Abbott Medical Optics, CA, USA). One eye from each patient was randomly selected for inclusion. Prediction errors were calculated by comparing expected refraction from optimized formulas (SRK/T, Hoffer Q, Haigis and Holladay 1) to postoperative refraction. A national survey of ophthalmologists was conducted to ascertain biometric formula preference for small eyes. RESULTS: The mean axial length was 21.00 ± 0.55 mm. Mean error was greatest for Hoffer Q at -0.57 dioptres. There was no significant difference in mean absolute error between formulae. SRK/T achieved the highest percentage of outcomes within 0.5 dioptres (45.2%) and 1 dioptre (76.2%) of target. Shallower anterior chamber depth was associated with higher mean absolute error for SRK/T (p = 0.028), Hoffer Q (p = 0.003) and Haigis (p = 0.016) but not Holladay (p = 0.111). CONCLUSION: SRK/T had the highest proportion of patients achieving refractive results close to predicted outcomes. However, there was a significant association between a shallower anterior chamber depth and higher mean absolute error for all formulae except Holladay 1. This suggests that anterior chamber depth with axial length should be considered when counselling patients about refractive outcome.
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Biometria/métodos , Hiperopia/fisiopatologia , Implante de Lente Intraocular , Facoemulsificação , Idoso , Idoso de 80 Anos ou mais , Comprimento Axial do Olho/patologia , Feminino , Humanos , Lentes Intraoculares , Masculino , Óptica e Fotônica , Refração Ocular/fisiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Testes Visuais , Acuidade Visual/fisiologiaRESUMO
PURPOSE: To report a case of Candida albicans endophthalmitis with no identifiable predisposing risk factors. CASE REPORT: A 57-year-old male presented with a 3-day history of worsening floaters and reduced visual acuity. Fundoscopy and optical coherence tomography showed presence of fluffy white preretinal and intraretinal infiltrates. With no past medical history or evidence of immunosuppression but having travelled abroad and suffered from diarrhoea, fungal aetiology was thought to be unlikely and as a result, treatment was commenced for toxoplasma. Despite treatment, his vision did not improve. Initial investigations including inflammatory markers, serology for toxoplasmosis, blood culture, chest radiograph and aqueous sampling could not identify a source of infection. However, polymerase chain reaction results from vitreous sampling revealed C. albicans. As a result, the patient was treated with intravenous voriconazole and intravitreal amphotericin B. As initial clinical improvement was limited, a vitrectomy was performed with further intravitreal amphotericin B. Clinical improvement was rapid following vitrectomy. After repeated Gram staining and culture of infected toenails, Gram-positive yeast cells were isolated. CONCLUSION: Although C. albicans is a frequent cause of endogenous endophthalmitis, patients often have one or more predisposing systemic condition assisting the diagnosis. The present case illustrates that (1) even in the absence of any predisposing risk factors, C. albicans should be considered as a possible differential diagnosis in recalcitrant uveitis, and (2) endogenous candida endophthalmitis can be a result of fungal infections from distant sites such as the toenails.
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OBJECTIVES: This study sought to determine whether procedural factors during percutaneous coronary intervention (PCI) are associated with the occurrence of ischemic stroke or transient ischemic attack (PCI-stroke). BACKGROUND: Stroke is a devastating complication of PCI. Demographic predictors are nonmodifiable. Whether PCI-stroke is associated with procedural factors, which may be modifiable, is unknown. METHODS: We performed a single-center retrospective study of 21,497 PCI hospitalizations between 1994 and 2008. We compared procedural factors from patients who suffered an ischemic stroke or transient ischemic attack related to PCI (n=79) and a control group (n=158), and matched them 2:1 based on a predicted probability of stroke developed from a logistic regression model. RESULTS: PCI-stroke procedures involved the use of more catheters (median: 3 [quarter (Q) 1, Q3: 3, 4] vs. 3 [Q1, Q3: 2, 3], p<0.001), greater contrast volumes (250 ml vs. 218 ml, p=0.006), and larger guide caliber (median: 7-F [Q1, Q3: 6, 8] vs. 6-F [Q1, Q3: 6, 8], p<0.001). The number of lesions attempted (1.7±0.8 vs. 1.5±0.8, p=0.14) and stents placed (1.4±1.2 vs. 1.2±1.1, p=0.35) were similar between groups, but PCI-stroke patients were more likely to have undergone rotational atherectomy (10% vs. 3%, p=0.029). Overall procedural success was lower in the PCI-stroke group compared with controls (71% vs. 85%, p=0.017). Evaluation of the entire PCI population revealed no difference in the rate of PCI-stroke between radial and femoral approaches (0.4% vs. 0.4%, p=0.78). CONCLUSIONS: Ischemic stroke related to PCI is associated with potentially modifiable technical parameters. Careful procedural planning is warranted, particularly in patients at increased risk.