By M. Zapotek. Polytechnic University of Puerto Rico. 2019.
With the removal of two non-essential viral genes order arcoxia without a prescription, the maximum insert size is still less than 1 kbp cheap 60 mg arcoxia with mastercard. Each image is at 300 000 × magni- ﬁcation buy arcoxia without prescription, and is reproduced from the following web site: http://www arcoxia 90mg without a prescription. This led to the speculation that geminiviruses could serve as relatively stable transient gene expression vectors – which is particularly important since they infect many crop plants (Stanley, 1993). Geminiviruses, however, tend to undergo high levels of gene rearrangement and deletion during their infective cycle, which is highly undesirable for a cloning vector. This allows for the insertion of multiple foreign genes into the chloroplast that can be expressed from the same polycistronic transcript. The chloroplast is highly polyploid – each chloroplast may contain multiple copies of the genome – and photosynthetic cells may contain many thousands of chloroplasts. Consequently, the expression of genes inserted into the chloroplast can greatly exceed that from a gene in the nucleus. Thus, the chances of spreading a foreign gene inserted into the chloroplast genome during crosses are remote. Successfully transformed chloroplasts initially grow as undifferentiated green calluses, which will, in the presence of appro- priate growth hormones, produce shoots. Both antibiotics are therefore used to ensure that spontaneous spectinomycin-resistance mutants have not arisen. The three leaf fragments on the right-hand side of the plate have become bleached in the presence of both antibi- otics because they are spontaneous spectinomycin resistance. This image was kindly provided by Professor Ralf Bock (Universitat¨ Munster,¨ Germany), and is reproduced from Bock (2001) homologous recombination at precise locations. The foreign gene that is being introduced into the chloroplast must be ﬂanked by sequences homologous to the chloroplast genome itself (Staub and Maliga, 1992). To obtain chloroplast transformants at a reasonable frequency, the foreign gene must be ﬂanked by greater than 400 bp of homologous sequence at both its 5 -and3-ends. Chloroplast genes are themselves transcribed by chloroplast-speciﬁc promoters and use chloroplast-speciﬁc termination signals. Therefore, foreign genes to be expressed in the chloroplast must be placed between suitable promoter and terminator sequences. The chloroplast-speciﬁc antibiotic resistance marker aadA,con- ferring resistance to aminoglycoside type antibiotics such as spectinomycin, is commonly used (Goldschmidt-Clermont, 1991). The kanamycin-resistance gene may also be used, but alternative selectable markers to antibiotic-resistance genes are desirable as there is a risk of transferring antibiotic resistance from the plant to bacteria either in the soil or in the guts of animals that eat the plant. This gene produces an enzyme that converts betaine aldehyde, a toxic compound, into glycine betaine, a non-toxic derivative, and is an effective selectable marker (Daniell, Muthukumar and Lee, 2001). Alternatively, selectable markers may be selectively removed after the construction of the transgenic plant (Iamtham and Day, 2000). For example, the marker can be ﬂanked by loxP sequences and eliminated from the genome using Cre-mediated recombination (Corneille et al. Chloroplast transformation in this way results the transformation of only one, or a few, genome copies within a single plant cell. This produces genetically unstable cells containing a mixture of transformed and wild-type chloroplast genomes. Strong selective pressure (high antibiotic levels) must be applied for two to four cycles of plant regeneration to ensure that cells contain uniform recombinant chloroplasts (Figure 11. This is, however, a time consuming process and imposes limits on the speed at which transformants may be generated. A range of proteins have been produced as a result of chloroplast transfor- mation. These include proteins resulting in insect resistance herbicide resistance and drought tolerance. For example, human somatotropin (a growth hormone that is used to treat pituitary dwarﬁsm) has been expressed in tobacco chloroplasts (Staub et al. The protein accumulates at high levels and is both correctly folded and possesses appropriate disulphide bonds. An extremely attractive extension of this work would be to produce therapeutic proteins in edible plants. Even if therapeutic proteins can be produced in this way, mechanisms by which they can be ingested and maintained in an active form still need to be established. It should also be possible to express recombinant proteins in potato and tomato chloroplasts. For example, tomato plants have been produced expressing the aadA marker gene, where the recombinant protein accumulates in both fruits and leaves (Ruf et al. One unique aspect of chloroplast engineering is the possibility of using operons to express multiple transgenes. Plant cells can contain between 50 and 100 chloroplasts, and each of these contains 10–20 nucleoids. Transformation is likely to result in the alteration of just one of these genomes. A non-homogenous genome content in a chloroplast appears to be unstable, so multiple rounds of regeneration and selection are required to produce a homogenous population of transgenic chloroplasts (often termed homoplasmic)within the plant cell. Therefore, multiple transgene expression relies on either the crossing of plants containing single transgenes, or the concurrent transfer of transgenes (Daniell and Dhingra, 2002). In the chloroplast, most genes are transcribed as polycistronic messages, therefore multiple foreign genes may be expressed within the same transgene. We will discuss the use of the Bacillus thuringiensis cry system below, but this approach results in very high levels of insecticidal protein accumulation – over 45% total soluble protein – with potent effects on insect pests. The lack of transfer of either the gene or its lethal protein product into pollen eliminates the danger of harming non-target and beneﬁcial insects. Chloroplast engineering therefore appears to be a safe and environmentally friendly alternative to nuclear gene transfer for the plant biotechnology industry (Maliga, 2002). Selective breeding programmes have been used to generate varieties yield- ing better nutritional qualities, higher yields, or improvements that can aid cultivation and harvesting of the crop. Genetic engineering does, however, provide the opportunity to alter the properties of a plant in a directed fashion. Some examples of commercially released genetically altered plants are listed in Table 11. This is particularly relevant to the transportation of tomatoes, where any damage can make the fruit unsellable. One of these, encoding the enzyme polygalacturonase, is involved in the slow break-down of the polygalacturonic acid component of cell walls in the fruit pericarp. However, the longer the enzyme is able to act on the cell walls, the softer and more over-ripe fruit will become. Therefore, if the effects of the enzyme can be delayed then the fruit will ripen more slowly and, as a result, tomatoes can be left on the plant for longer to accumulate greater ﬂavour. Tomatoes have been engineered so that they express less of the polygalacturonase enzyme. This was achieved through the insertion of the antisense sequence to a 5 -region of the polygalacturonase gene into the tomato genome. Expression of the antisense sequence was driven from the cauliﬂower mosaic virus 35S promoter, and the construct was inserted into tomato cells using Agrobacterium (Smith et al. The resulting transgenic tomatoes expressed reduced levels (6 per cent) of the polygalacturonase gene in comparison to their wild-type counterparts, and the fruit could be stored for prolonged periods before beginning to spoil. Bacillus thuringiensis is a Gram-positive spore-forming bacterium that synthe- sizes a large cytoplasmic crystal containing insecticidal toxins. Different strains of the bacterium produce toxins that are effective against different insect species. The crystal protein is highly insoluble so it is relatively safe to humans, higher animals and most insects. Once it has been solubilized in the insect gut, the protoxin is cleaved by a gut protease to produce an active toxin, termed δ-endotoxin, of about 60 kDa. It binds to the midgut epithelial cells, creating pores in the cell membranes and leading to equilibration of ions. As a result, the gut is rapidly immobilized, the epithelial cells lyse, the larva stops feeding, and the gut pH is lowered by equilibration with the blood pH. The structure of the Bacillus thuringiensis δ-endotoxin (Li, Carroll and Ellar, 1991) enables the bacterial spores to germinate, and the bacterium can then invade the host, causing a lethal septicaemia. Several crops have been engineered to contain a copy of the Bacillus thuringiensis cry1Ac gene, encoding the protoxin (Table 11. In addition, the gene has been expressed at very high levels in the chloroplasts of tomato plants, resulting plants that are resistant to a range of insect pests (McBride et al.
Patients with cervical aortic arches often present with a pulsating neck mass due to the aortic arch purchase generic arcoxia. Patients may have symptoms of tracheoesophageal compression due to an associated vascular ring cheap arcoxia 60mg online, including stridor and recurrent respiratory infections (16 cheap arcoxia 120 mg visa,29) arcoxia 60mg line. They also may develop symptoms of arch obstruction due to kinking or coarctation of the arch. Chest x-ray may demonstrate mediastinal widening, an absence of the aortic knob in the usual location, and an increase in the width of the retropharyngeal space if the cervical arch extends well into the neck (29,132). Barium esophagram may demonstrate a retroesophageal indentation due to the aortic arch coursing posterior to the esophagus. Surgical intervention which is indicated only in symptomatic patients may include division of a vascular ring, resection of a stenotic or aneurysmal area of the arch, or reconstruction of the aortic arch (130). Pulmonary Artery Sling In a left pulmonary artery sling, the left pulmonary artery arises from the right pulmonary artery instead of the pulmonary trunk. It courses toward the left lung between the esophagus and the trachea and compresses the esophagus at its anterior aspect (Fig. The trachea is caught in a vice, with the main pulmonary artery abutting its anterior surface, and the left pulmonary artery abutting its right and posterior surface. The major bronchi may bifurcate normally (type 1), or there may be a bridging bronchus extending from the left bronchus to supply the right lung (type 2) (134). In this setting, the right mainstem bronchus may supply only the right upper lobe, with the remainder of the lung supplied by a bridging bronchus, or there may be complete absence of the right bronchial tree, with the entire lung supplied by a bridging bronchus. In type 2, tracheal stenosis is common, and there are abnormal cartilaginous rings that surround the trachea. Tracheal bifurcation is lower in type 2 than type 1, occurring at T6 rather than T4–5 (135), though a recent study has called this into question (136). The left pulmonary artery arises from the right pulmonary artery and courses posterior to the trachea and anterior to the esophagus (not depicted). Developmentally, the proximal pulmonary arteries arise from the sixth aortic arches. The distal pulmonary arteries arise from two postbranchial arteries that develop from the capillary plexus that surrounds the lung buds. If the postbranchial components fail to connect to the sixth aortic arches, they will connect to a nearby artery. Left pulmonary artery slings are rare and thought to develop because the left postbranchial component of the pulmonary artery failed to connect to the left sixth aortic arch, and instead connected to the right sixth aortic arch via the right postbranchial component of the pulmonary artery. Thus, the left pulmonary artery courses from the right pulmonary artery, between the trachea and esophagus to the left side (139). Right pulmonary artery slings are extremely rare, and thought only to occur in the presence of left bronchial isomerism (140). In one study, of 59 patients presenting with symptomatic great arterial anomalies over a 20-year period, 10% had anomalous left pulmonary artery origins (7). Of known cases, a male predominance has been reported, with a male to female ratio of 3:2 (138). Associated Congenital Heart Disease Left pulmonary artery slings have been reported in association with ventricular septal defects, atrial septal defects, persistent left superior vena cava, persistent arterial duct, and tetralogy of Fallot (141,142). There is an increased incidence of imperforate anus in patients with type 2 pulmonary slings, with 14% of patients with a type 2 pulmonary sling affected in one study (135). Esophageal atresia and other gastrointestinal disorders have also been reported (141,143,144,145). Clinical Manifestations The major clinical concern of left pulmonary artery slings is the respiratory symptoms related to the tracheal compression by the pulmonary artery, and abnormal development of the bronchial tree. Type 2 left pulmonary artery slings are especially problematic as they are associated with hypoplasia of the right lung and worse tracheobronchial stenosis (135). Symptoms include stridor, pneumonia, respiratory distress, and respiratory failure. A large review of patients with a pulmonary vascular sling found that 90% presented in infancy (138). Symptoms include dyspnea, wheeze, stridor, cyanosis, apnea, and respiratory failure (138,146). Some patients are asymptomatic initially, with the diagnosis made incidentally (143,146), or they present in adolescence or adulthood, with patients reported to present with wheeze, hemoptysis, dyspnea, cough, or choking sensation (146,147,148). There may also be hyperinflation of the lung secondary to obstructive emphysema, usually affecting the right lung, but it may be bilateral or left sided (138). On barium esophagram, pulmonary slings cause anterior indentation, due to the pulmonary artery coursing between the esophagus and trachea, unlike vascular rings, which are associated with posterior and lateral indentation (138). However, barium esophagram has a low sensitivity, missing more than 20% of cases in one series (138). While bronchoscopy can identify the presence of tracheal rings, it is unable to evaluate the distal bronchi due to the stenosis. Also, bronchoscopy is invasive and not without risk as it can cause edema and worsen any respiratory distress already present (136). Important anatomic features include the location and degree of tracheal–bronchial stenosis, and whether the stenosis is focal or diffuse. However, it often requires sedation, which may not be advisable in a patient experiencing respiratory symptoms (136). Computational fluid dynamics analysis has been proposed to evaluate the effect of the tracheobronchial stenosis on the airway, though the clinical utility remains to be seen (149). Echocardiograms can diagnose the pulmonary sling, but are unable to assess the bronchial anatomy. They are an important part of the workup, however, to assess for any associated intracardiac disease (139). Management and Outcome Pulmonary artery slings carry significant morbidity and mortality. In one review, 7 of 27 patients died, 4 preoperatively and 3 after tracheoplasty (136). Surgical management includes anastomosis of the left pulmonary artery to the pulmonary trunk and tracheoplasty (136). One approach to tracheal stenosis is slide tracheoplasty, where the trachea is transected at the level of the stenosis, a vertical P. In a recent study of 18 patients with tracheal stenosis, 8 due to pulmonary sling, 1 died postoperatively, 2 patients required reoperation for recurrent tracheal stenosis, 2 required tracheostomy for tracheomalacia, and 13 were asymptomatic (150). For patients with long segments of stenosis, tracheoplasty including use of a pericardial patch and costal cartilage has been advocated, with some success but with a high rate of complications including infection and patch dehiscence (151). Postoperative pulmonary stenosis is also a known concern, with 74% of patients developing pulmonary artery stenosis, of whom 45% required at least one reintervention (153). Anomalous Origin of the Left or Right Pulmonary Artery from Aorta Anomalous origin of either the right or left pulmonary artery from the ascending aorta is rare malformation, with the former being more common. These lesions should be differentiated from discontinuous pulmonary arteries, where one of the branches is supplied by an arterial duct (154). The lesion has been called hemitruncus, though this is a misnomer because there are two semilunar valves, rather than the common truncal valve necessary to diagnose common arterial trunk (154). There is frequently associated intracardiac disease, most commonly a ventricular septal defect which may exacerbate the problem (154). The anomalous pulmonary artery acts as a very large aortopulmonary collateral, shunting much of the oxygenated blood back to the lungs and creating a volume load to the left heart. It also exposes the pulmonary vascular bed to systemic arterial pressures, resulting in severe pulmonary vascular obstructive disease if uncorrected (154). Patients may present with tachypnea, failure to gain weight appropriately, respiratory distress, and congestive heart failure (154,155). Surgical treatment includes reimplantation of the pulmonary artery to the pulmonary trunk. Outcome after early intervention is reported to be good, though surgical or percutaneous reintervention to address aortic or pulmonary stenosis is not uncommon (154). Brachiocephalic Artery Compression of the Trachea Rarely, the brachiocephalic artery may arise more posteriorly than normal, and course anterior to the trachea, thereby compressing the trachea (Fig. However, some may present in infancy or childhood with symptoms of apnea, chronic cough, respiratory infection, dyspnea, stridor, or wheeze.
Fungal endocarditis is relatively unusual in children although it is one of the most feared forms of endocarditis generic 60 mg arcoxia overnight delivery. Candida species are the most common organisms recovered order arcoxia pills in toronto; Aspergillus species discount arcoxia 60mg with mastercard, Torulopsis glabrata order 60mg arcoxia free shipping, and some other fungi (Histoplasma, Coccidioides, Cryptococcus) also have been reported. Fungal endocarditis is often noted in narcotic addicts or after cardiac surgery, but it also occurs in immunocompromised individuals and in neonates. In neonates, this infection may be a complication of modern intensive care measures, including hyperalimentation fluid infusion, use of broad- spectrum antibiotics for a prolonged time, and extended use of indwelling venous catheters. The mortality rate from fungal endocarditis is high, even with intensive medical and surgical therapy (6,14). Approximately 5% to 10% of patients with endocarditis have negative blood cultures. Many of these individuals demonstrate subsequent proof of endocarditis, either in the operating room or at necropsy. Consultation with the clinical microbiologist is invaluable in looking for unusual and fastidious organisms as molecular methods may be required in addition to standard blood cultures (16). The clinician should carefully evaluate such cases for the possibility of other diseases. Careful clinical and pathologic studies of endocarditis have defined the underlying structural cardiac or great vessel abnormalities that are the most frequent sites of infection. A review of available reports by Steckelberg and Wilson (17) suggests that the incidence of endocarditis in the general population is approximately five cases per 100,000 person-years. In high-risk groups, the incidence is substantially higher (300 to 2,160 cases per 100,000 person-years). The incidence in the moderate risk groups ranges from 50 to 440 cases per 100,000 person-years. Virtually all vegetations occur in areas where there is a pressure gradient with resulting turbulence of blood flow. Turbulent blood flow produced by certain types of congenital or acquired heart disease, such as flow from a high- to low-pressure chamber or across a narrowed orifice (e. Intact cardiac endothelium is a poor stimulator of blood coagulation and is weakly receptive to bacterial attachment, whereas damaged or denuded endothelium is a potent inducer of thrombogenesis. This provides an environment to which bacteria can adhere and eventually form an infected vegetation. Such catheters may traumatize the endocardium or valvular endothelium, exposing the subendothelial collagen. Use of a polyethylene catheter in a rabbit model, for example, yielded important information. Very shortly after the vascular endothelium is injured by the catheter, platelets and fibrin will adhere to the site of injury. This meshwork continues to grow with further accumulation of platelets and fibrin; very few leukocytes are involved. Following the initial deposition of platelets and fibrin, thrombus formation occurs. Certain bacteria such as staphylococci and streptococci, commonly implicated in endocarditis, are potent stimuli of platelet aggregation. In addition, the lysosomal granules of platelets may release hydrolytic enzymes or other active proteins that may potentiate the process. The exception to this is valvular aortic stenosis, where the site of the vegetation is usually on the ventricular side of the aortic valve. A possible explanation for this finding is that in almost all instances of aortic stenosis there is at least some degree of aortic insufficiency. The ability of various microorganisms to adhere to specific sites determines the location of the infection. Numerous bacterial surface components present in streptococci, staphylococci, and enterococci have been shown in animal models of endocarditis to function as critical adhesions. Bacteria adhering to the vegetation stimulate further deposition of fibrin and platelets on their surface. In endocarditis caused by a-hemolytic streptococci, the large colonies of bacteria become encased in an organizing mass of fibrin. The fibrin barrier has a direct effect on two important factors in the defense against infection: the prevention of the invasion by phagocytic leukocytes and the difficulty in penetration of the vegetation by antimicrobial agents. For reasons that are not fully appreciated, this type of vegetation formation does not frequently occur with some of the more virulent bacteria, such as S. There have been substantial gains within the past several years in the understanding of the pathogenesis of endocarditis largely because of the availability of newer molecular biologic techniques. These techniques have allowed the examination of individual virulence factors of gram-positive cocci and the investigation of important host–cell interactions with microorganisms. Several specific surface structures of staphylococci, streptococci, and enterococci have been identified as markers of virulence (19). Transient bacteremia can be induced by trauma to a mucosal surface during various dental, oral, and surgical procedures; however, spontaneous bacteremia may also occur (17). The bacteremia associated with various tissue manipulations, including dental and surgical procedures, has been carefully studied. Spontaneous bacteremia also has been noted to occur after tooth brushing, chewing hard foods, or other daily life events. Many dental procedures have been associated with bacteremia, particularly procedures known to induce gingival or mucosal bleeding. Transient bacteremia caused by viridans group streptococci and other oral microflora following tooth extraction may reach 80%. In experimental animals, large doses of bacteria are generally needed to induce endocarditis. Following successful medical therapy, the cardiac lesions of endocarditis usually heal, although important residua can remain. Experimental studies in rabbits suggest that the resolution process includes endothelialization of the affected surface; phagocytosis of bacterial debris, sometimes with calcification; and subsequent organization by fibroblasts. Resulting hemodynamic abnormalities depend on the site of infection, the specific damage caused by the active vegetation, and the size and location of the abscess. The immediate consequences of endocarditis, including vegetation formation, hemodynamic alterations, and the clinical syndrome, are only part of an evolving complex disease entity. Distal manifestations of the disease in the past were considered to be the results of embolic phenomena. It is now recognized that additional mechanisms are involved in the pathogenesis of endocarditis that lead to peripheral manifestations. Many important extracardiac findings in endocarditis are related to immunologic mechanisms. Rheumatoid factor is present for 6 weeks or longer in sera of about half of the patients with endocarditis. Rheumatoid factor is more frequently found in patients with endocarditis related to a-hemolytic streptococci or coagulase-negative staphylococci (low-virulence organisms) than in those caused by S. There is also correlation of the duration of infection with the presence of this antiglobulin. Immunologic mechanisms also underlie other clinical manifestations of the disease including the skin, subcutaneous tissues, and eye findings noted below (Clinical Features). This is due to extended exposure to foreign antigen, and these immune complexes disappear after successful antimicrobial therapy. Although deposition of immune complexes in renal parenchyma can occur, their precise role in pathogenesis has not been fully defined. The nephritis seen in patients with endocarditis may manifest itself microscopically as either focal or diffuse glomerulonephritis. In the focal lesion, there is often segmental fibrinoid necrosis of isolated lobules of the glomerular tuft. In the more diffuse form, there is marked cellular proliferation with interstitial round cell infiltrates. Immunofluorescence studies show granular deposits in the glomerular basement membrane and mesangium, usually associated with complement and immunoglobulin G (IgG) deposits, although IgA, IgM, and fibrinogen also have been demonstrated. Urinalysis results may be normal, but hematuria, cylindruria, and pyuria have been reported. Compromise of renal function may occur and appears to be more common in adults than in children.