Entomopathogenic nematodes (EPNs) of the families
Heterorhabditidae
and
Steinernematidae
are efficient biological control agents against important insect pests. In vitro liquid culture production ...technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate,
Steinernema jeffreyense,
in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria,
Xenorhabdus khoisanae
. After the successful in vitro production of
S. jeffreyense
-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml
−1
after 14 days, in comparison to 75% and 99,875 IJs ml
−1
respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml
−1
), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.
Summary
Entomopathogenic nematodes (EPN) are successful biological control agents of a variety of soilborne insect pests. They have the potential to be mass-produced, using in vitro liquid culture ...technology, and can be formulated and sold as a biopesticide. To commercialise an EPN-based biopesticide successfully, the method of liquid mass production requires in-depth optimisation to reduce the cost of production and to increase yields, to make it affordable to the farming community. This study attempted to optimise the liquid culture protocol for the South African isolates, Steinernema jeffreyense and S. yirgalemense, by investigating the impact of cheaper medium ingredients on the recovery and yield of the liquid culture process. Studies were conducted by investigating alternative protein, lipid and nitrogen/yeast sources, compared to the more expensive laboratory-grade ingredients currently used. The results showed that egg yolk has no impact on the yield in the case of S. jeffreyense. However, for S. yirgalemense, egg yolk was shown to be a superior protein source to soy and insect-based protein in terms of nematode yield. Moreover, neither canola oil nor olive oil showed a significant difference in the yield of S. yirgalemense, with yeast extract being found to be the optimal nitrogen/yeast source. When comparing the yields with those in other liquid culture research on S. yirgalemense, yields have been successfully increased by 300%, with the cost of the nematode nutrient medium having decreased by 77%. Thus, it is imperative that, prior to a scale up to large bioreactors, the nutrient medium should be optimised to reduce the cost of production.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Entomopathogenic nematodes (EPNs) are a safe insect biological control agent. Key to the implementation of EPNs as a biopesticide is their mass production in shake flasks and bioreactors. For ...commercial application, in vitro liquid culture is the predominant choice, due to the cost and scale of production, and to the ease of downstreaming. The in vitro liquid culture of EPNs begins in Erlenmeyer shake flasks to provide aeration and agitation. The initial liquid culture phase is followed by upscaling to 5-20-L desktop bioreactors and, thereafter, to 80-120-1000-L industrial-scale bioreactors. The ingredients of the liquid culture media, on which symbiotic bacteria and nematode develop, is of great importance for mass-culturing. The diet usually consists of essential nutrients that best replicate the constituency of the natural insect host, such as protein, carbohydrates and lipids. In addition, aeration and agitation are maintained, without causing shear damage due to the rotating impeller blades. Such factors, however, requires different parameters, depending on the EPN species involved, and, moreover, optimisation is required to obtain high yields and quality of infective juveniles. The objective of the current review is to assess the conditions required for optimal EPN production in liquid culture, and how the conditions might be optimised for South African EPN species.
Developing repeatable protocols for the in vitro liquid mass production of entomopathogenic nematodes (EPNs) is a difficult task and depends on the nematode species being cultured. Of critical ...importance is the establishment of a monoxenic population of nematodes as a stock culture for optimisation and experimental purposes. Establishing a new stock inoculum culture flask of pure infective juveniles (IJs) is challenging, particularly for the Heterorhabditis species, due to their affinity for developing into an amphimictic second generation that does not copulate in liquid culture flasks. Developing mass production protocols for multiple EPNs is advisable because different pest insects are susceptible to different species of EPN. This study attempted to mass-produce a South African isolate of Heterorhabditis zealandica and its symbiotic bacteria, Photorhabdus thracensis, using in vitro liquid culture technology methods previously developed for Steinernema species. The results indicate that the pre-culture protocols developed for Steinernema species are applicable to a H. zealandica isolate. Moreover, the results, in terms of the protein source optimisation experiments, confirm that different EPN species have different culture conditions and nutrient requirements, with H. zealandica seeming to prefer soy-based protein instead of egg yolk, having higher recovery and producing more hermaphrodites, using soy protein. This study illustrates the importance of developing dependable and infallible preculture methods, prior to the flask mass production process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To validate our previously identified candidate metabolites, and to assess the ability of these metabolites to predict hypoxic-ischemic encephalopathy (HIE) both individually and combined with ...clinical data.
Term neonates with signs of perinatal asphyxia, with and without HIE, and matched controls were recruited prospectively at birth from 2 large maternity units. Umbilical cord blood was collected for later batch metabolomic analysis by mass spectroscopy along with clinical details. The optimum selection of clinical and metabolites features with the ability to predict the development of HIE was determined using logistic regression modelling and machine learning techniques. Outcome of HIE was determined by clinical Sarnat grading and confirmed by electroencephalogram grade at 24 hours.
Fifteen of 27 candidate metabolites showed significant alteration in infants with perinatal asphyxia or HIE when compared with matched controls. Metabolomic data predicted the development of HIE with an area under the curve of 0.67 (95% CI, 0.62-0.71). Lactic acid and alanine were the primary metabolite predictors for the development of HIE, and when combined with clinical data, gave an area under the curve of 0.96 (95% CI, 0.92-0.95).
By combining clinical and metabolic data, accurate identification of infants who will develop HIE is possible shortly after birth, allowing early initiation of therapeutic hypothermia.
Possession of an apolipoprotein E (APOE)epsilon4 allele has been shown to be associated with a poor outcome after closed head injury and spontaneous intracerebral hemorrhage but not after ischemic ...stroke. This study assessed the influence of the APOE genotype on outcome in patients admitted to a neurosurgical unit with spontaneous subarachnoid hemorrhage.
A total of 100 patients with spontaneous subarachnoid hemorrhage were studied. Four patients were excluded because the diagnosis of subarachnoid hemorrhage was not confirmed. The incidence of rehemorrhage and delayed ischemia and the outcome at 6 months were determined using the Glasgow Outcome Scale. APOE genotypes were determined by polymerase chain reaction and restriction enzyme digestion.
Allele frequencies in this patient group were 0.04 for epsilon2, 0.86 for epsilon3, and 0.1 for epsilon4. Of 96 patients, 72 had an aneurysmal hemorrhage and 1 had a hemorrhage from an arteriovenous malformation. In 14 patients, the results of angiography were negative, and in 9, no angiogram was performed. Of the 96 patients, 20 had one or more epsilon4 allele. Outcome at 6 months was no worse in patients with one or more epsilon4 allele than in those with no epsilon4 allele (odds ratio, 0.98; 95% confidence interval, 0.35-2.74). None of the 12 patients who experienced delayed ischemic deterioration had an epsilon4 allele. Of the 20 patients with an epsilon4 allele, 3 had a rehemorrhage, as compared with 6 of 76 patients without an epsilon4 allele.
There was underrepresentation of the epsilon4 allele in this group when compared with previously studied cases of subarachnoid hemorrhage with a fatal outcome and with the general population. This suggests that patients with the epsilon4 allele who have a subarachnoid hemorrhage are less likely to be admitted to a neurosurgical unit. This study does not support an association between possession of an epsilon4 allele and poor outcome in patients admitted to a neurosurgical unit with spontaneous subarachnoid hemorrhage, although the wide confidence interval does not preclude a clinically relevant association between APOE genotype and outcome. The findings indicate that an association between genotype and the development of delayed ischemic complications after subarachnoid hemorrhage may be possible.
Background Loss-of-function mutations in the skin barrier protein filaggrin ( FLG ) are a major risk for atopic dermatitis (AD). The pathogenic sequence of disturbances in skin barrier function ...before or during the early development of AD is not fully understood. A more detailed understanding of these events is needed to develop a clearer picture of disease pathogenesis. A robust, noninvasive test to identify babies at high risk of AD would be important in planning early intervention and/or prevention studies. Objectives To ascertain whether a noninvasive measurement of skin barrier function at day 2 after birth and at 2 months predicts the development of AD at 1 year. Furthermore, to determine whether increases in transepidermal water loss (TEWL) predate the development of clinical AD. Methods A total of 1903 infants were enrolled in the Cork Babies After Scope: Evaluating the Longitudinal Impact Using Neurological and Nutritional Endpoints Birth Cohort study from July 2009 to October 2011. Measurements of TEWL were made at birth (day 2) and at 2 and 6 months. The presence of AD was ascertained at 6 and 12 months, and disease severity was assessed by using the SCORing Atopic Dermatitis clinical tool at 6 months and by using both the SCORing Atopic Dermatitis clinical tool and Nottingham Severity Score at 12 months. A total of 1300 infants were genotyped for FLG mutations. Results At 6 months, 18.7% of the children had AD, and at 12 months, 15.53%. In a logistic regression model, day 2 upper quartile TEWL measurement was significantly predictive of AD at 12 months (area under the receiver operating characteristic curve, 0.81; P < .05). Lowest quartile day 2 TEWL was protective against AD at 12 months. An upper quartile 2 month TEWL was also strongly predictive of AD at 12 months (area under the receiver operating characteristic curve, 0.84; P < .05). At both ages, this effect was independent of parental atopy, FLG status, or report of an itchy flexural rash at 2 months. Associations were increased when parental atopy status or child FLG mutation status was added into the linear regression model. Conclusions Impairment of skin barrier function at birth and at 2 months precedes clinical AD. In addition to providing important mechanistic insights into disease pathogenesis, these findings have implications for the optimal timing of interventions for the prevention of AD.
Background Transcutaneous exposure to food allergens can lead to food sensitization (FS)/food allergy (FA). We measured skin barrier function in early infancy and related it to the later development ...of FS/FA at age 2 years. Objective We sought to examine the relationship between early life skin barrier function and FA in infancy. Methods Infants in the Babies After Scope: Evaluating the Longitudinal Impact Using Neurological and Nutritional Endpoints (BASELINE) birth cohort had transepidermal water loss (TEWL) measured in the early newborn period and at 2 and 6 months of age. At age 2 years, infants had FS/FA screening with skin prick tests and oral food challenges. Results One thousand nine hundred three infants were enrolled. One thousand three hundred fifty-five were retained to age 2 years, and 1260 underwent FS screening. FS was present in 6.27% (79/1260; 95% CI, 4.93% to 7.61%), and FA prevalence was 4.45% (56/1258; 95% CI, 3.38% to 5.74%). Egg was the most prevalent allergen (2.94%), followed by peanut (1.75%) and cow's milk (0.74%). Day 2 upper-quartile TEWL (>9 gwater /m2 /h) was a significant predictor of FA at age 2 years (odds ratio OR, 4.1; 95% CI, 1.5-4.8). Seventy-five percent of children with FA at 2 years of age had day 2 TEWL in the upper quartile. Even in those without atopic dermatitis (AD), infants with upper-quartile day 2 TEWL were 3.5 times more likely to have FA at 2 years than infants in the lowest quartile (95% CI, 1.3-11.1; P = .04). Conclusion Neonatal skin barrier dysfunction predicts FA at 2 years of age, supporting the concept of transcutaneous allergen sensitization, even in infants who do not have AD. TEWL could be used for stratifying infants in the first few days of life before development of AD or FA for targeted intervention studies to potentially alter the atopic march.
Three thousand seven hundred and eighty-three patients with non-malignant hypertension attending the Glasgow Blood Pressure Clinic between 1968 and 1983 were followed prospectively for an average of ...6.5 years. Left ventricular hypertrophy (LVH) was present at the outset in 34.5% of the men, and 12.8% had ST-T changes. The corresponding figures for women were 21.5% and 8.8%. The prevalence of LVH increased with the severity of hypertension and was higher for a given blood pressure level in men than in women. All-cause age-adjusted mortality, expressed as deaths per 1000 patient-years, was 27.6 for men with normal electrocardiographs, 43.2 for men with LVH only (P less than 0.001) and 56.9 for men with LVH and ST-T changes (P less than 0.001). Similar trends were seen in women. The excess risk associated with LVH, with or without ST-T changes, could not be explained by age, increased blood pressure at referral to the clinic, or smoking habit, when these factors were considered either separately or in combination (regression analysis). Thus, our study demonstrates that LVH, with or without ST-T changes is an independent risk factor for mortality in hypertensive patients.
Adjusted-dose warfarin is highly efficacious for prevention of ischaemic stroke in patients with atrial fibrillation (AF). However, this treatment carries a risk of bleeding and the need for frequent ...medical monitoring. We sought an alternative that would be safer and easier to administer to patients with AF who are at high-risk of thromboembolism.
1044 patients with AF and with at least one thromboembolic risk factor (congestive heart failure or left ventricular fractional shortening ≤25%, previous thromboembolism, systolic blood pressure of more than 160 mm Hg at study enrolment, or being a woman aged over 75 years) were randomly assigned either a combination of low-intensity, fixed-dose warfarin (international normalised ratio INR 1·2–1·5 for initial dose adjustment) and aspirin (325 mg/day) or adjusted-dose warfarin (INR 2·0–3·0). Drugs were given open-labelled.
The mean INR during follow-up of patients taking combination therapy (n=521) was 1·3, compared with 2·4 for those taking adjusted-dose warfarin (n=523). During follow-up, 54% of INRs in patients taking combination therapy were 1·2–1·5 and 34% were less than 1·2. The trial was stopped after a mean follow-up of 1·1 years when the rate of ischaemic stroke and systemic embolism (primary events) in patients given combination therapy (7·9% per year) was significantly higher than in those given adjusted-dose warfarin (1·9% per year) at an interim analysis (p<0·0001), an absolute reduction of 6·0% per year (95% CI 3·4, 8·6) by adjusted-dose warfarin. The annual rates of disabling stroke (5·6%
vs 1·7%, p=0·0007) and of primary event or vascular death (11·8%
vs 6·4%, p=0·002), were also higher with combination therapy. The rates of major bleeding were similar in both treatment groups.
Low-intensity, fixed-dose warfarin plus aspirin in this regimen is insufficient for stroke prevention in patients with non-valvular AF at high-risk for thromboembolism; adjusted-dose warfarin (target INR 2·0–3·0) importantly reduces stroke for high-risk patients.
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