Get Vegucated ! 7 Ways to a Successful Plant-based Diet

Screen Shot 2015-01-09 at 10.21.50 PM

A vegetarian diet is not limiting. Reducing or eliminating animal products means opening up an exciting world of variety in vegetables and grains, beans and pulses, nuts, seeds and fruits that can now take center stage on your plate. Becoming a vegetarian can support your health, meet nutritional needs, and achieve lifestyle goals including: weight loss, reduced blood cholesterol, increase energy levels, boost fertility, improve skin quality, and contribute to a glowing pregnancy.

There are many types of vegetarian diets and the only way to know what really works for you is to try it out! Most people would benefit tremendously from adjusting to a more plant-based diet of whole foods. Being vegetarian is about celebrating the abundant vibrant foods that nature offers us, embracing them for their vitamins and phytochemicals that contribute to our well-being.

 5 Vegetarian Types:

Raw Vegan– a person who eats only plant based foods that haven’t been cooked or heated over 118 degrees.

Vegan– a vegetarian that eats only plant based foods and does not consume any animal products

Lacto vegetarian– a vegetarian that consumes milk or milk products, but not eggs

Lacto-ovo vegetarian– a vegetarian that consumes milk, milk products, eggs,

Flexitarian– a person who consumes a mostly plant based diet with occasional amounts of meat, poultry, fish

7 Steps to a Successful Vegetarian Diet

Make a Commitment- Just say “I commit to eating a plant-based diet to support the well-being of my body and soul”. Making any drastic shifts in our lives requires a lot of support and a huge commitment to stay on track. It helps to set an intention or a goal, for instance “I will decrease my intake of meat to once a week” in an effort to reduce cholesterol” or I will reduce my dairy intake to boost my fertility.”

Self Experiment- Try things out for yourself. If you are experimenting, try all the vegetarian types on for size. See what happens if you consume more greens, or eat more fruit. Only you will know what works. How do you measure what works or doesn’t? Based it on how you feel after eating. Ask yourself are you energized or are you feeling sluggish? Take notes and keep a food diary during your transition process.

Practice Meat-less Mondays- Meatless Mondays is an international initiative from John Hopkins’ Bloomberg School of Public Health that encourages people to cut down their consumption of meats and saturated fat to improve their health and the health of the planet. Consuming plant-based proteins will maintain lean muscle and provide you with adequate energy.

Eat Your Greens- Our ancient ancestors could eat up to 6 pounds of leaves per day! They would forage and gather leaves and eat them while on the go. Can you imagine eating a grocery bag full of greens daily? Few of us even eat the minimum USDA requirement of 3 cups of leafy greens per week. Dark green leafy vegetables are, calorie for calorie, the most concentrated source of nutrition of any food. They are a rich source of minerals (including iron, calcium, potassium, and magnesium) and vitamins, including vitamins K, C, E, and many of the B vitamins. They also provide a variety of phytonutrients including beta-carotene, lutein, and zeaxanthin, which protect our cells from damage. Dark green leaves even contain small amounts of Omega 3 fats. They help to regulate blood sugar and break down fats, and cleanse the blood. Start packing in the greens at every meal, eat more salad and even cooked greens like kale, collards, chard, and mustards.

Cut Dairy- Lots of people avoid dairy because of lactose intolerance, the saturated fat, cholesterol, and the hormones in the milk. Dairy consumption is also linked to type 1 Diabetes. A frequent question most vegetarians are asked is: where do you get your calcium? Milk only contains traces of calcium if cows are grass-fed. Nowadays cows are pent up in factory farms with no space to graze on grass and they are fed grain instead. Milk is a product that is designed for the consumption of a baby calf, so that it grow from about 65 pounds at birth to about 500 lbs within a year’s time. Breast milk is the optimal form of nutrition for a newborn human baby. Once you have been weaned from the breast, you should not be drinking cow’s milk. Calcium loss is prevented through a healthy diet and lifestyle- exercising to build bone density, getting adequate sunlight to absorb vitamin D, eating fruits and veggies, decreasing caffeine intake, decreasing sodium intake. Get your calcium form green leafy veggies.

Eat Ancestral foods- When eating a vegetarian diet, and any health supportive diet for that matter, it is important to incorporate foods that our ancestors ate. Ancient grains like quinoa, kamut, millet, brown rice etc. Greens, fruits, nuts, beans, seeds, high quality oils, these have all been around for a very long time. Explore flavors from your cultural background and connect with healthy foods that your ancestors ate.

Read everything- Labels, literature, recipes, everything. Learn all you can about what’s happening with our food from the issues of factory farming, to genetically modified foods, from food access, to local foodstuffs. Read and make your own informed choices about what to eat and where to source your food from.

Are you ready to adjust your diet? What changes will you make? How vegucated are you?


• Open Access

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple ‘Gala’ were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab-resistant apple ‘Florina’ (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07- and 0.002-fold compared with the natural expression of ‘Florina’, this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab-susceptible ‘Gala’. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site–such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.

Cisgenesis is a promising tool to rapidly deploy disease resistance in apple. By adding only the gene(s) of interest, and therefore the corresponding phenotypic trait(s) from a crossable donor to an existing cultivar, it circumvents most of the problems encountered during traditional resistance breeding (Jacobsen and Schouten, 2008). Addition of a single trait by genetic engineering to an apple plant maintains all the characteristics of the original cultivar (Borejsza-Wysocka et al., 2010).

Decreased susceptibility of the popular cultivar ‘Gala’ to the fungus V. inaequalis, which causes apple scab, is a highly desirable trait, as it would reduce fungicide input during the production of fruits of this cultivar. This, in turn, would be advantageous for the environment, the consumer and the producer (Gessler, 2011). Cisgenic scab-resistant apple lines may cause less concerns compared with a transgenic product, for example allergenicity caused by the product of the inserted gene, as the trait is already present in traditionally bred scab-resistant cultivars (e.g. ‘Florina’, ‘Santana’) and avoids the generation of a completely new cultivar with unpredictable agronomic qualities. So far, the only scab resistance gene to be cloned is HcrVf2 (newly Rvi6), originally derived from Malus floribunda 821 and was shown to incite scab resistance in the susceptible apple ‘Gala’ under the control of the 35S promoter (Belfanti et al., 2004) and under its own native promoter (Szankowski et al., 2009). However, the plants used in these studies still contained the selection marker gene nptII and foreign regulatory sequences. Joshi et al. (2011) also generated transgenic lines with HcrVf1 and HcrVf2 (newly Rvi6) using the vector pMF1 and their own regulatory sequences.

Cisgenic lines of apple ‘Gala’ carrying the Rvi6 scab resistance gene have been reported recently (Vanblaere et al., 2011). The endogenous apple scab resistance gene Rvi6 with its native regulatory sequences was inserted into the genome of the susceptible apple ‘Gala’. Cisgenic plant lines were generated through the Agrobacterium-mediated transformation method developed by Schaart et al. (2004), which consists of a two-step process: transgenic lines containing the Rvi6 gene and an excisable cassette carrying the marker fusion gene codA/nptII and the RecLBD gene were first generated. Then, a chemical activation of the recombination led to excision of the cassette resulting in a truly cisgenic genotype. During this process, Vanblaere et al. (2011) reported the regeneration of ten transgenic lines, eight of them amplifying–in addition to the target gene Rvi6 and plant selection gene codA/nptII–sequences corresponding to the bacterial selection gene nptIII present on the backbone of the pMF1-vector. Two transgenic lines without backbone (T11.1 and T12.1) and one (T7.1) with backbone were selected for detailed analysis, together with the cisgenic lines (C7.1.49, C11.1.53, C12.1.49) derived from the aforementioned transgenic lines. Following recombination, no amplification of the nptIII gene was obtained from the cisgenic lines, suggesting that the corresponding vector backbone sequences were excised during the recombination step resulting in cisgenic line C7.1.49. Copy number assessment by Southern blot hybridization showed a single integration of the marker gene codA/nptII used for selection in each of the three transgenic selected mother lines (Vanblaere et al., 2011).

Cisgenesis in apple as reported here has been achieved by an Agrobacterium-mediated transformation technique that randomly inserts the cisgene of interest into the genome of the target cultivar by T-DNA transfer to the host cell. Due to the process of the integration, genomic rearrangements and the interruption of important functional genes or pathways can occur, which could alter the phenotype, quality and traits of the plant (Rosati et al., 2008). The random integration of the T-DNA into different loci in the genome can also be responsible for variation in the transcriptional activity of the inserted gene, thereby altering its phenotypic expression (Day et al., 2000). Identification of genomic sequences flanking the integration site allows detailed investigation of the transition sequences. Moreover, the availability of the ‘Golden Delicious’ genome (Velasco et al., 2010) coupled with EST and gene prediction information may allow estimation of where the integration took place in the genome.

The legal regulation of GM plants in Europe (e.g. (EC) 1829/2003) requires molecular characterization, including the amount of vector and/or donor nucleic acid remaining in the modified organism, sequence data for the flanking regions, level of expression of the specific protein resulting from the genetic modification and information on the organization of the inserted genetic material at the insertion site and should specifically aim to identify whether the genetic modification(s) raise(s) any issues regarding the potential for producing new toxins or allergens (EFSA 2011).

In this study, we report the examination of the insertion site of the T-DNA (based on the ‘Golden Delicious’ genome, which is a parent of ‘Gala’) and the T-DNA/plant genomic DNA junction of these lines. Southern blot hybridization with a probe designed for the cisgene (Rvi6) was used to determine copy number insertions of the T-DNA in the mother transgenic lines and in the derived cisgenic lines. A quantitative real-time (qRT)-PCR analysis was performed to assess the gene expression in the trans- and cisgenic plants compared with ‘Florina’ (a traditionally bred cultivar carrying the Rvi6 gene). Moreover, scab resistance was evaluated visually as well as by qPCR. The generated data serves as further characterization of the cisgenic lines, as requested for biosafety assessment based on European GMO regulations.

Complete backbone integration in six of 10 transgenic ‘Gala’ lines

Backbone integration was investigated in the lines by PCR. Amplification of one or more of the four amplicons distributed along the pMF1 backbone sequence used for transformation indicated genomic integration of the corresponding sequences. Six of the eight transgenic lines (T7.1, T7.2, T7.3, T7.4, T8.1 and T11.2; Figure 1 and Table 2), which contain backbone sequences as mentioned by Vanblaere et al. (2011), were found to amplify all four amplicons. This indicates that most of the sequences of the pMF1 vector backbone were integrated in these lines either as a single sequence or fragmented in several parts (truncated copies). The other two transgenic lines (T8.2, T8.3) produced only two of the four amplicons, indicating only partial integration of the vector backbone sequences (Table 2). Transgenic lines T11.1 and T12.1 did not amplify any of the amplicons on the backbone, leading to the assumption that only the T-DNA was inserted. In the putative cisgenic lines–‘C'7.1.17, ‘C'7.1.63, ‘C'8.2.7, ‘C'8.3.4, ‘C'8.3.6–at least one of the four PCR products indicating the presence of the vector backbone were amplified following the recombination step, indicating that these lines are not truly cisgenic (and therefore marked as ‘C’), as they contain foreign genes. Cisgenic lines C7.1.3, C7.1.49, C11.1.53 and C12.1.49 were found to be free from backbone integration as no backbone amplicon was detected (data not shown).

Figure 1.

Schematic representation of the linearized pMF1 with the apple scab resistance gene Rvi6. Black arrows indicate the approximate position of the primers used in this work. Primer sequences are shown in Table 1.

Determination of T-DNA border junctions and the genomic point of T-DNA integration in lines T7.1, T11.1 and T12.1

The product of the inverse PCR (iPCR) of line T7.1 was sequenced, and a 379 bp sequence flanking the insertion site was obtained. This sequence matched with the genome of ‘Golden Delicious’ chromosome 4 (contig MDC017356.126) and indicated integration into an exon of the putative transcript MDP0000250224, containing a cyclin-like F-box domain. Primers T7.1_a and T7.1_c (Table 1) were designed for this sequence and amplified a 200 bp amplicon in ‘Gala’.

Table 1. Primer sequences
Primer Sequence (5′–3′)

Similarly, a 343 bp sequence flanking the insertion site was obtained for line T11.1, and BLAST analysis of this sequence against the ‘Golden Delicious’ genome revealed a match to a sequence on chromosome 12 (contig MDC021013.168). In this line, the T-DNA is inserted in a putative ORF (MD00G455290) to which the gene ontology term ‘microtubule cytoskeleton organization’ has being assigned. The 343 bp sequence obtained from untransformed ‘Gala’ using primers T11.1_b/T11.1_d revealed that two different alleles were amplified. For line T12.1, two different sequences of 188 bp and 924 bp flanking the insertion site were obtained by iPCR and matched Golden Delicious genomic sequences on chromosomes 11 (MDC004866.187) and 14 (MDC018357.590). A third sequence of 193 bp was found in line 12.1 flanking the recombination site 1 (RS1) of the transformation vector and corresponded to a Tnos, a terminator sequence present on the pMF1 vector. Primers T12.1_a/T12.1_d developed on both regions flanking the insertion site failed to amplify an amplicon from ‘Gala’ DNA, thus indicating that the two corresponding sequences are noncontiguous in both ‘Golden Delicious’ and ‘Gala’–the insertion site of the T-DNA in plant line T12.1 could therefore not be determined.

PCR was then attempted on the transgenic and cisgenic lines, using the HcrVf2term2 primer in combination with the primers developed on the corresponding sequence flanking the left border (T7.1_c, T11.1_d and T12.1_d, respectively). In both transgenic T7.1 and cisgenic C7.1.49, an amplicon of approximately 1.2 kbp was amplified. An amplicon of about 500 bp was obtained from line C11.1.53, while no amplification was observed in line T11.1 and in untransformed ‘Gala’.

Sequencing of the PCR product amplified in line T7.1 with primers pmf_IPCR_9/T7.1_a showed that the right border and the PacI restriction site used for cloning were not integrated. The integrated T-DNA starts at the promoter of the Rvi6 gene, runs over the marker fusion gene cassette, LB, the whole pMF1 backbone, RB, Rvi6 gene, Tnos terminator sequence and is interrupted at approximately 1/5 of the end of the RecLBD gene. The partial integration of RecLBD is demonstrated by successful amplification using primers Rec1/T7.1_c but not with primer pair Rec2/T7.1_c (Figure 1, Figure 2 and Table 1). In the cisgenic line C7.1.49, following recombination the left border (LB) is also missing. Sequencing of the PCR products amplified in line T11.1 and C11.1.53 with primers pmf_IPCR_9/T11.1_b showed no integration of the right border and the PacI restriction site as well as the first five nucleotides of the Rvi6 (Figure 1 and Table 1). The left border is also absent, and the recombination site (RS) next to the left border is truncated. A minimal integration of foreign DNA (72 bp), resulting from the vector cloning site and the recombination site, was found in cisgenic plant line C11.1.53 (Figure 3).

Figure 2.

Schematic overview of the regeneration of two different genotypes from the same transgenic plant line T7.1, in which a whole copy of the T-DNA was integrated. The recombination between different RS is called type 1 (C7.1.49, C7.1.3 g), and the recombination between two copy of the same RS is called type 2 (‘C'7.1.63, ‘C'7.1.17a).

Figure 3.

Schematic overview of the regeneration a cisgenic plant line, C11.1.53, from transgenic line T11.1.

Copy number of Rvi6 estimated by Southern hybridization

Lines T7.1 (~16 000 bp) and C11.1.53 (~9000 bp) had one additional band for Rvi6 compared with ‘Gala’ (Figure 4). Lines T11.1 (~16000 bp and ~9000 bp), T12.1 (~10500 bp and ~3900 bp) and C12.1.49 (~3900 bp and ~2700 bp) possessed two additional bands for Rvi6. For the cisgenic line C7.1.49, there was no specific band for Rvi6 visible.

Figure 4.

Copy number analysis by Southern blotting with a probe derived from Rvi6. Genomic DNA of transgenic lines T7.1, T11.1 and T12.1 and cisgenic lines C7.1.49, C11.1.53 and C12.1.49 digested by XhoI. pMF1 with Rvi6 was added as positive control and ‘Gala’ as a control to distinguish between Rvi6 and its homologues. A DIG-labelled marker VII was added to estimate the band sizes. Arrows indicate additional bands in comparison with ‘Gala’.

Expression level of Rvi6 in the cisgenic lines compared with ‘Florina’

The expression level of Rvi6 in all trans- and cisgenic lines, as measured by quantitative real-time PCR, was below the expression level of the scab-resistant apple ‘Florina’. Rvi6 expression level in lines T7.1 and C7.1.49 was 14-fold lower compared with the control, while in lines T11.1 and C11.1.53, Rvi6 is 300 and 450 times less expressed, respectively. Rvi6 showed sixfold lower expression in line T12.1, whereas in C12.1.49, it was 200 times less expressed than in ‘Florina’. Very low Rvi6 expression could be observed for ‘Gala’, corresponding to 0.000749 times the expression in ‘Florina’ and indicating a nonspecific amplification as ‘Gala’ lacks Rvi6. To display the different expression levels on the same graph, a log-scale transformation was used (Figure 5).

Figure 5.

Relative expression analysis of Rvi6 compared with ‘Florina’. ‘Florina’ contains the Rvi6 gene and is a conventionally bred apple scab-resistant cultivar. The y-axis is on a logarithmic scale, and the ratio of Rvi6 is expressed in the trans- and cisgenic plant lines in comparison with ‘Florina’.

Apple scab resistance evaluation

Susceptible control ‘Gala’ showed abundant sporulation without chlorosis (class 4 symptoms) 21 days after artificial inoculation, whereas the resistant control ‘Florina’ showed no symptoms at all (class 0 symptoms). All three investigated cisgenic lines showed a strong foliar deformation, chlorosis and in some cases also sporulating lesions (class 2-3b symptoms). Results of visual scoring are shown in Table 3.

Pathogen leaf colonization by quantitative PCR

Pathogen/host DNA ratio was calculated following qPCR of the DNA extracted from whole leaves. The CCcorr values corresponding to a colonization coefficient expressed as the ratio between pathogen and host DNA evinced from the infected leaves of the investigated cisgenic lines are shown in Figure 6. ‘Florina’, the resistant control, showed an average CCcorr value of 0.007 (N = 2), while the susceptible control ‘Gala’ reached 0.133 (± 0.079, N = 7) 21 days after inoculation. All cisgenic lines had lower values: C7.1.49 showed a CCcorr value of 0.022 (± 0.020, N = 8), C11.1.53 a value of 0.037 (± 0.029, N = 7) and C12.1.49 a CCcorr value of 0.028 (± 0.013, N = 4).

Figure 6.

Colonization coefficient (ratio between pathogen and host DNA per leaf) calculated for the three investigated cisgenic lines C7.1.49, C11.1.53 and C12.1.49 measured by qPCR after Gusberti et al. (2012) 21 days following inoculation with V. inaequalis. ‘Florina’ is a scab-resistant Rvi6-cultivar, while ‘Gala’ is the scab-susceptible genotype used for generation of cisgenic lines.

The European Commission has stated in its latest report that cisgenic crops are becoming more and more accepted in Europe (Gaskell et al., 2010). In the European Union, cisgenics underlie the GMO regulations. We report here molecular investigations on three cisgenic lines of the apple ‘Gala’. The creation of these lines has been described recently (Vanblaere et al., 2011).

Cisgenic line C7.1.49

In this cisgenic line, a single insertion of the T-DNA occurred in chromosome 4 of ‘Gala’, putatively interrupting a predicted exon of a cyclin-like F-box domain. The single insertion was already supported by Vanblaere et al. (2011) using a nptII-specific probe and was confirmed by hybridization with the Rvi6 probe of a 17 kbp band in the mother line T7.1. No extra band is observed in C7.1.49. However, following recombination and subsequent loss of 7.3 kbp of the excisable cassette, the resulting band in the derived cisgenic line C7.1.49 would overlay the 10 kbp band observed in ‘Gala’. PCR primers designed for the flanking sequences amplified a 200 bp band in ‘Gala’, confirming the proximity of the two sequences separated by T-DNA insertion in transgenic and cisgenic lines T7.1 and C7.1.49, respectively. PCR using HcrVf2term and T7.1_c primers suggests that spontaneous recombination already took place before induction by dexamethasone treatment, as both lines amplify a fragment of the size corresponding to the expected one following removal of the excisable cassette (data not shown), while the amplicon expected prior excision could not be amplified probably due to its size of 8.4 kbp. Line C7.1.49 lacks both right and left border sequences. The line still contains the AscI restriction site used for cloning, the RS site and part of the RecLBD gene and its Tnos terminator. This leads to the presence of approximately 1 kbp of nonfunctional DNA sequences belonging to the vector. Although foreign DNA is present, C7.1.49 still falls under the definition of ‘cisgenic’ from Schouten et al. (2006). Therefore, we conclude that T7.1 has two copies of Rvi6, but the final product–the cisgenic line C7.1.49–contains only one copy of Rvi6 (Figure 2). The expression level of Rvi6 in T7.1 and C7.1.49 was sevenfold lower than in ‘Florina’. No difference in expression level was observed between T7.1 and C7.1.49, despite the extra copy of Rvi6 in T7.1. Joshi et al. (2011) also found no variation in the expression level between one or two copies of the gene, concluding that there is no correlation between the copy number and gene expression level, whereas the material they analysed consisted of independently transformed lines with integration in different genomic regions. Despite the low expression level of Rvi6, line C.7.1.49 was found to exhibit a defence reaction varying between class 2 and 3b of Chevalier's classes and the strong foliar deformation that seldom resulted in abundant sporulation (Table 3). Therefore, we conclude that cisgenic line C7.1.49 shows a level of resistance comparable with other Vf genotypes (Tartarini et al., 2000), even if it does not attain the complete resistance found in ‘Florina’.

In the transgenic mother line T7.1, it was deduced that due to the read-through, the complete vector pMF1 containing Rvi6, plus a second partial fragment of about 800 bp of the T-DNA, was integrated leading to the presence of three recombination sites in line T7.1 (Figure 2). An extra recombination site can result in the regeneration of three different plant genotypes after recombination, one for each possible pair of recombination sites. We did not detect any line that still had the RecLBD gene and codA-nptII after activation of the recombinase due to the use of the 5-fluorocytosine in selective regeneration media following dexamethasone treatment (Vanblaere et al., 2011). It was possible to detect lines corresponding to the other two possible genotypes (Figure 2, following recombination type 1 and 2). Type-2 recombination removed the second copy of Rvi6 and the whole backbone, leaving only one copy of the Rvi6 gene, one recombination site and the Tnos terminator sequence with part of the recombination gene (Figure 2). The presence of both types has been proved through PCR in lines C7.1.49 (Type 2), C7.1.3 g (Type 2), ‘C'7.1.17a (Type 1) and ‘C'7.1.63 (Type 1) (Vanblaere et al., 2011). Type-2 recombination regenerates a cisgenic plant, whereas type 1 regenerates a plant that remains transgenic, as it still contains the whole backbone vector DNA with its transgenes.

Cisgenic line C11.1.53

In cisgenic line C11.1.53, as well as in transgenic line T11.1, no amplification of vector backbone sequences was observed (Table 2), and it was therefore assumed that the inserted DNA corresponded to the expected one from the right border to the left border. A single insertion was found for line C11.1.53 as corroborated by Southern blot analysis, while results obtained for line T11.1 indicated two copies of Rvi6 (Figure 4). However, the lower band (~9 kbp) corresponds to the band in C11.1.53 and is ~7300 bp smaller than the higher band (~16 kbp) in T11.1. The difference in size between both bands corresponds to the size of the excisable cassette that is deleted during recombination. The lower band in T11.1 most likely results from spontaneous recombination, leading to a chimera composed of cisgenic and transgenic cells. Genomic sequences flanking the right and left border matched the same contig of ‘Golden Delicious’, which is located on linkage group 12. Primers developed for these sequences amplified two 343 bp amplicons in ‘Gala’ corresponding to two alleles. Spontaneous recombination could not be shown to occur by PCR amplification of the fragment encompassing the excisable cassette (primers HcrVf2term2 and T11.1_d). In fact, only line C11.1.53 amplified a fragment of about 0.5 kbp, while no amplification was observed in line T11.1. Very low expression levels were found in T11.1 and C11.1.53 compared with the natural control plant ‘Florina’. A 100- and 500-fold lower expression level of the Rvi6 gene was observed in the transgenic, respectively, cisgenic lines. Despite the low expression of the Rvi6 gene, a sufficient level of resistance, comparable with the one of the cisgenic lines C7.1.49, could be achieved (Table 3 and Figure 6). Possible explanations for the low expression levels are the position on the chromosome where the T-DNA insert is integrated, position-dependent spreading of methylation or condensed chromatin structure from surrounding genomic DNA, or a genomic scanning mechanism that identifies and methylates ‘invading’ DNA (Kooter et al., 1999). However, the aforementioned possible explanations for low expression levels do not explain why there is a significant difference in expression level following recombination between the lines T12.1 and C12.1.49 but not between T7.1 and T7.1.49 or between T11.1 and C11.1.53. Further research will be necessary to address this problem.

Table 2. Results of the PCR amplification of four amplicons scattered on the pMF1 vector's backbone. The position of the amplicons can be evinced from Figure 1
Primer pairs amplifying the vector's backbone
 IPCR_codA_1/pmf_bb2nptIII for/revpmf_bb3/4pmf_bb1/pmf_IPCR9
Table 3. Distribution of V. inaequalis inoculated apple leaves into the different scab symptom classes after Chevalier et al. (1991). The three youngest leaves per shoot have been scored by three independent researchers at day 21 after inoculation (three observations/leaf/shoot). Gala is the susceptible genotype used for transformation, while Florina is a conventionally bred scab-resistant cultivar carrying the Rvi6 gene
Apple genotypeNumber of observationsScab Symptom classes after Chevalier et al. (1991)
Cisgenic 11.1.53785%58%28%4%5%
Cisgenic 12.1.49546%56%28%11%0%
Cisgenic 7.1.499012%63%23%1%0%

Cisgenic line C12.1.49

Southern blot analysis of the line T12.1 with a probe derived from nptII (Vanblaere et al., 2011) showed a single integration, whereas Southern blot analysis with an Rvi6 specific probe revealed two bands. In T12.1, we found a band (~9500 bp) that corresponds to the band (~2500 bp) found in C12.1.49 after a normal recombination, followed by the excision of the cassette of 7300 bp. The second band present in T12.1 and C12.1.49 has the same size, ~3800 bp and is assumed to be part of a second copy of the Rvi6 gene (Figure 4). In this second integration, a truncated T-DNA was integrated only partially without the presence of the excisable cassette because Southern blot analysis with an nptII probe showed only one copy integration of the gene. We assume that two independent integrations took place on possibly different chromosomes/linkage groups based on the localization in the ‘Golden Delicious’ genome. In line T12.1, no backbone sequence amplification was observed. However, the amplification of the flanking regions led to different sequences, located by comparing with the ‘Golden Delicious’ genome, on two different chromosomes (11 and 14). No amplification of the insertion site was achieved, and it is therefore impossible to elucidate either the structure of the integrated DNA(s) or its position in the genome. Gene expression level analysis in transgenic line T12.1 had only a twofold lower expression level compared with the expression level of ‘Florina’, but in C12.1.49, there was a 100-fold lower level of gene expression compared with ‘Florina’ corresponding to a 50-fold lower level of expression than in T12.1 (Figure 5). However, this low expression was sufficient to enhance resistance to apple scab, as shown by the results of visual scoring and qPCR (Figure 6 and Table 3).

Assessment of the ability of pMF1:Rvi6 vector to generate cisgenic lines

This system, used to produce cisgenic plants in apple, was successful in generating cisgenic plant lines. Backbone integration tests indicated a very frequent (80%) read-through over the left border (Table 2). The phenomenon of ‘leaky borders’ has been reported in the past (Kohli et al., 2010; Joshi et al., 2011), but no explanation was found as to why the T-DNA transfer stopped at other sequences than the left border (LB) region. Optimization of the procedure through reduction in the backbone transfer by resolving the ‘leaky border’ problem could be achieved through the use of more effective borders (Kuraya et al., 2004; Rommens et al., 2004). The recombination system used to excise the cassette containing the nptII-codA and RecLBD recombinase was found to be not completely repressed in the absence of dexamethasone. This is supported by the observation of spontaneous recombination events in lines T7.1 and T11.1 leading to chimeras of transgenic and cisgenic genotypes, while for line T12.1, chimerity could not be tested. However, in this case, the spontaneous recombination did not hamper the generation of transgenic and cisgenic lines, due to the use of the positive and negative selection system (Vanblaere et al., 2011). Recombination also led to a reduction in the expression level of the cisgene in two of three lines (C11.1.53 and C12.1.49), whereas for the latter, no data about T-DNA integration extent were obtained. The introgression of Rvi6 led to increased resistance to apple scab, even though no resistance level similar to ‘Florina’ was observed. On one hand, the observed resistance reaction of the classes 2-3b is also found in Vf genotypes with other genetic backgrounds and may be sufficient to incite resistance under field conditions (Tartarini et al., 2000). On the other hand, however, in all cisgenic lines, the expression of Rvi6 was much lower than in the resistant control. This overall low expression level in the three trans- and cisgenic lines could be linked with the native promoter length of 242 bp used. Szankowski et al. (2009) proved that a promoter length of 779 bp has a 100-fold increased expression level compared with a promoter length of 115 bp. The latter promoter induced similar expression to the one observed in ‘Florina’, while it could not induce complete scab resistance. Also, the use of the native terminator could be a limiting factor in overall gene expression level. Szankowski et al. (2009) used a Tnos terminator, which could have a stronger influence on gene transcription termination than the native terminator. Also, the length of the native terminator might influence a more efficient gene transcription termination. Joshi et al. (2011) used the native terminator with a length of more than 2 kbp, whereas our terminator length was a mere 220 bp. However, despite the low expression, partial resistance was observed in our lines: the results of qPCR quantification of the colonization coefficient (CCcorr, Figure 6) indicated that the results obtained 21 dpi for ‘Gala’ and ‘Florina’ were similar to the ones reported by Gusberti et al. (2012). The cisgenic lines C7.1.49, C11.1.53 and C12.1.49 showed CCcorr values similar to those of ‘Milwa’, a moderately resistant cultivar (Gusberti et al., 2012). The present work represents the complete molecular characterization of two of three cisgenic lines reported by Vanblaere et al. (2011). However, such molecular characterization, as requested by the (EC) Regulation No. 1829/2003, may become unnecessary if cisgenic crops are made exempt from GMO regulations as suggested by Schouten et al. (2006).

Experimental procedures

Apple lines

Transgenic apple lines T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3, T11.1, T11.2 and T12.1 were used (Vanblaere et al., 2011). Cisgenic apple lines were regenerated following recombination of the transgenic lines, and, for naming, the prefix T (transgenic) was replaced by C (cisgenic), and the number of each regenerated cisgenic line was added to the name (e.g. C7.1.49 is the 49th regenerated cisgenic line derived from the transgenic mother line T7.1). Cisgenic lines C7.1.49, C7.1.3, C7.1.17, C7.1.63, C11.1.53 and C12.1.49 were previously reported by Vanblaere et al. (2011).

Backbone integration

Genomic plant DNA of trans- and cisgenic ‘Gala’ lines was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. To detect and quantify backbone integrations, several primers were designed to amplify different parts of the backbone of vector pMF1 containing the Rvi6 gene used for transformation (Vanblaere et al., 2011). The primer sequences are listed in Table 1, and their relative position on the linearized vector is shown in Figure 1. Sequences of primers pmf_bb3 and pmf_bb4 have been published previously (Vanblaere et al., 2011). For PCR amplification, the Qiagen multiplex kit (Qiagen, Hilden, Germany) was used following the manufacturer's protocol, with each primer pair applied singularly. To evaluate the possibility of a read-through over the whole backbone (and therefore a second copy of the T-DNA), a primer pair was developed to span the plasmid sequence before and after the right border (RB).

Integration site

For each of the three transgenic lines T7.1, T11.1 and T12.1, genotype inverse PCR (iPCR) was used to isolate genomic regions of ‘Gala’ flanking the RB, as well as the LB of the T-DNA insert in separate reactions, combining for each reaction the suitable restriction endonuclease and a corresponding pair of primers. The method of Triglia et al. (1988) was used with following modifications: 1 μg of DNA was digested in a total volume of 20 μL. For the self-ligation reaction, 100 ng (2 μL) of the digestion mixture was mixed with 10 μL 10X ligation buffer, 10 μL ATP (10 mm), and 4 μL T4 DNA ligase (3U) (New England Biolabs Inc., Beverly, MA) in a total volume of 100 μL and incubated overnight at 16 °C. The ligation reaction was deactivated by heating at 65 °C for 10 min. Five μL was taken from the ligation mixture and added directly to the PCR reaction. PCR reactions consisted of 1X Qiagen Multiplex PCR Master Mix, 0.5X Q-solution and 0.2 μm of each primer in a total volume of 50 μL. PCR reactions were performed applying the manufacturer's protocol conditions (95 °C for 15 min, followed by 35 cycles at 94 °C for 30 sec, 55 °C for 90 sec, 72 °C for 3 min, and a final extension at 72 °C for 10 min). The amplicons were analysed by agarose gel electrophoresis (1%), and a nested PCR was performed in a second PCR reaction. The reaction mixture and conditions were the same as for the first PCR reaction, with 5 μL of the PCR product used as the template. The amplicons were analysed by agarose gel electrophoresis (1%); the bands were cut out of the gel and sequenced (primer sequences are listed in Table 1) using the BigDye terminator kit 3.1 (Applied Biosystems, Foster City, CA). The obtained sequences, after trimming of the T-DNA sequences, were subjected to BLAST analysis against the whole genome sequence of ‘Golden Delicious’ (Velasco et al., 2010) in order to estimate the site of insertion in the Malus genome. Primers (T7.1_a, T7.1_c, T11.1_b, T11.1_d, T12.1_a, T12.1_d) were designed on the genomic DNA regions and used to amplify the corresponding region in untransformed ‘Gala’ to identify single nucleotide polymorphisms (SNPs) between the two alleles of ‘Gala’. In a second step, the primers were used for amplification on DNA of the transgenic ‘Gala’ lines T7.1, T11.1 and T12.1. As the inserted T-DNA (11 kbp) was very large for a normal PCR amplification, only amplicons from the allele in which the integration did not occur should be obtained from these lines. The developed primers were also used separately in combination with primers on the T-DNA (HcrVf2termrev2, pmf_IPCR_9; Table 1) to amplify the transition region between T-DNA and genomic DNA at the right and left borders, respectively. The amplicons were sequenced to verify the extent of the T-DNA integration. Finally, the developed primers were used to find out whether spontaneous recombination events had occurred, amplifying the DNA of the transgenic mother lines T7.1, T11.1 and T12.1. Spontaneous recombination is assumed to occur if the obtained amplicon is smaller in size than the expected T-DNA insert (11 kbp) and is shorter by the length of the excisable cassette (about 7.3 kbp shorter, Vanblaere et al., 2011). In the corresponding cisgenic line, the 11 kbp band should be absent.

Southern blot hybridization

To confirm the number of integration events obtained by hybridization of the Southern blots using the nptII probe (Vanblaere et al., 2011), a second hybridization using a PCR probe on Rvi6 was performed (primers RT1for and RT2rev (Vanblaere et al., 2011)). Transgenic and cisgenic lines were analysed by Southern blotting as described by Vanblaere et al. (2011). ‘Gala’ was added to distinguish between the Rvi6 homologues and the actual Rvi6 gene. pMF1 with Rvi6 was added as a positive control. Genomic DNA was digested by XhoI, run on an agarose gel, blotted onto a nylon membrane and hybridized using digoxygenin-labelled probes of Rvi6.

Quantitative Real-Time RT–PCR analysis

Quantitative real-time RT–PCR on cDNA was used to assess the relative level of expression of the Rvi6 gene in transgenic and cisgenic lines T7.1, C7.1.49, T11.1, C11.1.53, T12.1 and C12.1.49. As negative and positive control plants, ‘Gala’ and ‘Florina’ were used, respectively. ‘Gala’ does not contain the Rvi6 gene, while ‘Florina’ is a classically bred Vf-cultivar resistant to apple scab carrying the Rvi6 gene. The Rvi6 mRNA expression measured for ‘Florina’ was used as reference for normalizing the expression measured for the other lines. Total RNA was isolated using PureLink™ Plant RNA Reagent (Invitrogen, Paisley, Scotland) and converted into cDNA using the M-MLV reverse transcriptase, RNase H Minus Kit with oligo(dT)18 primers (Promega, Madison, WI, USA) according to the protocol described by Vanblaere et al. (2011). The cDNA was afterwards diluted 1:9. The PCR reaction mix consisted of 6 μL of cDNA, 2X SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 60 nm of each primer in a total volume of 20 μL. All reactions were performed according to the procedure outlined in the manufacturer's instructions (95 °C for 20 sec, followed by 40 cycles at 95 °C for 3 sec, 62 °C for 30 sec.) with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Ct values were obtained using specific primers for Rvi6 (formerly HcrVf2, primers HcrVf2-2273For and HcrVf2-2331Rev; Table 1) and for the reference gene ubiquitin-conjugating enzyme (MDP0000223660; primers UBCforward and UBCreverse, Pagliarani et al., 2013). All reactions were performed in biological and technical triplicates. The average of the three technical replicates was taken over each sample, while the biological replicates were analysed independently from one another. The mathematical model of Pfaffl (2001) was used to calculate the relative expression ratio. The results, including standard deviation, are presented in Figure 2 as an average of the three biological repeats.

Inoculation Tests for Apple Scab Resistance

Scab resistance was tested by inoculating grafted greenhouse plants of each cisgenic line with V. inaequalis. As susceptible controls, grafted plants of ‘Gala’ from an untransformed in vitro culture were used. As resistant controls, grafted plants of ‘Florina’ (not from in vitro culture) were used. At least three plants with one shoot per line were inoculated for each plant line used. Active growing shoots with more than three leaves were inoculated by spraying with a hand-operated mist blower until a fine water film was visible and the inoculated shoots were kept in a plastic tent to maintain the water film for 48 h in the dark at 15 °C. V. inaequalis conidia suspension was obtained from leaves with sporulating scab lesions of susceptible seedlings of the Agroscope ACW breeding programme representing a mixture of genotypes, but not containing a Vf-virulent genotype. Conidia concentration was adjusted to 1.5 × 105 using tap water. After 48 h, the plants were moved into a greenhouse cabin with day/night temperature of 20°/15°, 8-h night period, 70% rH. Symptoms were classified by three independent observers after 21 days in the greenhouse according to Chevalier et al. (1991) with class 0 no visible symptoms, class 2 yellow chlorotic flecks, whose centre could be slightly necrotic, class 3a ‘necrotic lesions and some chlorotic lesions with occasional very slight sporulation’, class 3b ‘clearly sporulating chlorotic and necrotic lesions’ and class 4 abundant sporulation and no necroses. Symptoms were evaluated on the three youngest leaves per shoot (most susceptible). Estimation of fungal colonization was assessed using the qPCR method developed by Gusberti et al. (2012). DNA was extracted from infected leaves (second youngest leaf at the time of inoculation, harvested at the time of visual screening), and the DNA was subjected to qPCR applying two different TaqMan probes targeting host DNA (Elongation factor 1) and pathogen (ATP binding cassette 2). The ratio between pathogen and host DNA (colonization coefficient, CCcorr) was calculated.

The authors wish to acknowledge the financial support by the Swiss National Science Foundation NRP59 and COST Action 864. Contributions of Michele Gusberti, Roberta Paris, Gabriela Ziltener, Ines Hiller, Jan Schaart and Henk Schouten are gratefully acknowledged.


Zimbabwean ivory poachers have killed more than 80 elephants by poisoning water holes with cyanide, endangering one of the world's biggest herds.

Saviour Kasukuwere, environment minister, said on Wednesday the elephants had died in the last few weeks in the Hwange national park, the country's largest, while security forces were preoccupied with a July 31 general election.

"We are declaring war on the poachers," he told Reuters news agency.

"We are responding with all our might because our wildlife, including the elephants they are killing, are part of the natural resources and wealth that we want to benefit the people of Zimbabwe."

Police and rangers had recovered 19 tusks, cyanide and wire snares after a sweep through villages close to the park, which lies just south of Victoria Falls.

Zimbabwe is home to some of Africa's largest herds, with half of its estimated 80,000 elephants thought to be in Hwange.

Kasukuwere, who was appointed to the environment ministry a week ago, said he would push for stiff penalties for convicted poachers, who routinely get less than the nine-year jail term imposed for cattle rustling.

Zimbabwe is working to revive its tourism industry, including its wildlife sector, which has suffered years of decline blamed by some on long-serving President Robert Mugabe's economic policies.

Source: Reuters


risk management in healthcare

A recently released report from the FBI has found an increase in the number of active shooter incidents in the U.S. since 2000. Many hospitals and schools conduct active shooter training. However, 40 percent of active shooting incidents happen in a business location, suggesting that other businesses should also establish an active shooter plan and provide training. Below are a few elements that we believe are essential in active shooter preparedness and response.
1. Risk Assessment
Assessing risks of an active shooter can predict the likelihood of an incident as well as help to establish evacuation routes and other procedures.

2. Access Control
Identifying potential threats before they enter the building could decrease the likelihood of a shooting incident.
3. Surveillance
If there is an active shooter in the building, seconds matter and early detection is essential. If a possible threat is identified on the premises remote surveillance may allow security personnel to locate and respond more quickly.
4. Mass Notification/Communication
Notifying personnel in the building may help in the safe evacuation or lockdown of a facility. This should also include communication within security teams, local law enforcement, and other emergency services.
5. Trained Personnel
Security should have active shooter training, however, they are not the only entity that should be trained. Administrators, personnel, and other frequent visitors (i.e. students) should be trained on what to do in the incident of an active shooter.
6. Delegated Authority Figures
Even though you may have gone through training, in an unpredictable and intense situation like an active shooter there is likely to be chaos. A delegated line of authority or leaders can help contain chaos in many emergency situations.

The above are just a few useful elements in active shooter response. However, it is important to consult security industry specialists to assist in risk assessment, plan creation, and routine training.

Photo by U.S. Naval War College


Basic Calculations for Navigation

in Navigation

Before a cross-country flight, a pilot should make common calculations for time, speed, and distance, and the amount of fuel required.

Converting Minutes to Equivalent Hours

Frequently, it is necessary to convert minutes into equivalent hours when solving speed, time, and distance problems. To convert minutes to hours, divide by 60 (60 minutes = 1 hour). Thus, 30 minutes is 30/60 = 0.5 hour. To convert hours to minutes, multiply by 60. Thus, 0.75 hour equals 0.75 x 60 = 45 minutes.

Time T = D/GS

To find the time (T) in flight, divide the distance (D) by the GS. The time to fly 210 NM at a GS of 140 knots is 210 ÷ 140, or 1.5 hours. (The 0.5 hour multiplied by 60 minutes equals 30 minutes.) Answer: 1:30.

Distance D = GS X T

To find the distance flown in a given time, multiply GS by time. The distance flown in 1 hour 45 minutes at a GS of 120 knots is 120 x 1.75, or 210 NM.

To find the GS, divide the distance flown by the time required. If an aircraft flies 270 NM in 3 hours, the GS is 270 ÷ 3 = 90 knots.

Converting Knots to Miles Per Hour

Another conversion is that of changing knots to miles per hour (mph). The aviation industry is using knots more frequently than mph, but it might be well to discuss the conversion for those that use mph when working with speed problems. The NWS reports both surface winds and winds aloft in knots. However, airspeed indicators in some aircraft are calibrated in mph (although many are now calibrated in both miles per hour and knots). Pilots, therefore, should learn to convert wind speeds that are reported in knots to mph.

A knot is 1 nautical mile per hour (NMPH). Because there are 6,076.1 feet in 1 NM and 5,280 feet in 1 SM, the conversion factor is 1.15. To convert knots to miles per hour, multiply speed in knots by 1.15. For example: a wind speed of 20 knots is equivalent to 23 mph.

Most flight computers or electronic calculators have a means of making this conversion. Another quick method of conversion is to use the scales of NM and SM at the bottom of aeronautical charts.

Fuel Consumption

Aircraft fuel consumption is computed in gallons per hour. Consequently, to determine the fuel required for a given flight, the time required for the flight must be known. Time in flight multiplied by rate of consumption gives the quantity of fuel required. For example, a flight of 400 NM at a GS of 100 knots requires 4 hours. If an aircraft consumes 5 gallons an hour, the total consumption is 4 x 5, or 20 gallons.

The rate of fuel consumption depends on many factors: condition of the engine, propeller/rotor pitch, propeller/rotor revolutions per minute (rpm), richness of the mixture, and particularly the percentage of horsepower used for flight at cruising speed. The pilot should know the approximate consumption rate from cruise performance charts, or from experience. In addition to the amount of fuel required for the flight, there should be sufficient fuel for reserve.

Flight Computers

Up to this point, only mathematical formulas have been used to determine such items as time, distance, speed, and fuel consumption. In reality, most pilots use a mechanical or electronic flight computer. These devices can compute numerous problems associated with flight planning and navigation. The mechanical or electronic computer has an instruction book that probably includes sample problems so the pilot can become familiar with its functions and operation. [Figure 15-18]

Another aid in flight planning is a plotter, which is a protractor and ruler. The pilot can use this when determining true course and measuring distance. Most plotters have a ruler which measures in both NM and SM and has a scale for a sectional chart on one side and a world aeronautical chart on the other. [Figure 15-18]

Figure 15-18. A plotter (A), the computational and wind side of a mechanical flight computer (B), and an electronic flight computer (C).

51+CmORESYL._SX373_BO1,204,203,200_Learn more about flight navigation with the Aviator’s Guide to Navigation. One of the best books on the market to refine your navigation skills. The Fourth Edition of this comprehensive guide explains the full range of air navigation innovations, covering all the new technologies and tools that have emerged in the past ten years.

Comments on this entry are closed.

Previous post:

Next post:


Find Now

Law Enforcement, Policing and Investigation Degrees

Law enforcement is a major part of the criminal justice system. This area basically comprises of law enforcement agencies that enforce, regulate law, and capture criminals. The police department and border patrol is an example of a few of the main law enforcement agencies. These agencies can be found working at local, county, state, and federal level. Every society has a law enforcement sector that is working towards minimizing criminal activity. The field offers a wide range of career opportunities to individuals who want to serve their fellow citizens and help make the society a safer place to live in.

Law Enforcement Degrees

In the past few years, the demand for law enforcement degrees has increased. Many students are opting for careers in law enforcement. Institutes in all states that specialize in criminal justice education are now offering a wide range of law enforcement degree programs. This field can be studied at graduate, undergraduate, and non-degree level. The type of law enforcement degree a student aims for will determine his or her career ahead. It is also possible to pursue law enforcement degrees online.

Law enforcement degrees cover all major aspects of the field. As technological developments are constantly taking place, the complexity of law enforcement is increasing. It is important for students to understand the changing trends in law and how security measures are affected.

Students enrolled in a law enforcement degree will study a variety of courses. Each course covers a certain facet of the field and preps students with relevant knowledge and skill. Some of the subjects that are covered include criminology, criminal justice systems, law enforcement techniques, policing and probation, corrections, security issues, and criminal proceedings. The exact curriculum will depend upon the institute offering the program. Nowadays, many institutes are offering training along with theoretical study. This helps in allowing students to practically implement law enforcement techniques in simulated settings and acquire experience. By the end of the program, students will have a sound skill set and a broad understanding of the criminal justice system.

Career Opportunities

With a degree in law enforcement, a student can pick a career path in any law enforcement agency. Police officer jobs are among the most popular law enforcement careers. Students can apply for training in a police academy and prepare themselves for a job in this field. Apart from this, probation officer jobs are also chosen by many individuals. These are trained professionals who supervise convicts and are usually found working in state and federal prisons. According to the US Bureau of Labor Statistics, the median annual income for police officers and probation officers in 2010 was $55,010 and $47,200. The demand for law enforcement officers can vary but is generally positive as security is still a concern. The diversity and complexity of criminal activity requires such professionals to be highly skilled and trained. This career choice can be suitable for individuals who want to work in the criminal justice department and law enforcement.

Questions / Answers

Q:Law enforcement degree programs will prepare me for what kinds of jobs?

A:Law enforcement jobs are a popular career option in today's society. A degree program in this area will equip you with various skills and information need to become a law enforcement professional. You can work in the police department, border patrol agency, homeland security department, corrections and probations sector, and more.

Q:From where can I get law enforcement policing and investigation degrees?

A:Law enforcement policing degrees and investigation degrees are being offered by many schools and universities across the nation. You can search online to learn about the school rankings and program offerings. Before you do enroll in any school, check the accreditation status, the coursework being offered, and ranking of the institute.

Ask Your Question

* All Fields are Required


Managing Infertility Stress

Find a Support Group

Find a Professional

Grieving and Growing: Creative Outlets to Grieving During Infertility

By Beth Jaeger-Skigen, LCSW
Published in Resolve for the journey and beyond, Spring 2012

Grief will inevitably be a part of your infertility journey. Grieving each loss is an integral part of the process toward parenthood. I believe that in order to fully make next-step decisions with clarity, it is important to grieve losses in concrete and purposeful ways.

I believe that all people who are experiencing infertility are grieving parents. There is no avoiding grief.

Grief is never linear and grief from infertility is particularly nonlinear. People experience infertility cycles with both hope and loss. This brings high- highs and low- lows. The unique part of the infertility process is that the losses are compounding. Month after month, cycle after cycle, treatment after treatment, the losses compound and the grief can expand.

In 1969, Elisabeth Kubler Ross came up with five stages of grief in her classic book, “On Death and Dying.” She described the five stages a person goes through when they suffer a loss. Grief specialists throughout the years have found that these stages can be applicable to losses across the board. The stages aren’t linear and many grieving individuals find they move in and out of the different phases at different times during their grief.

"How will you know the difficulties of being human if you're always flying off to blue perfection? Where will you plant your grief seeds?" -- Rumi

Examples of the five stages of grieving as experienced in connection with infertility stages are:

After a diagnosis of infertility, a person might not believe it, think there was a lab error or simply, “my doctor is wrong.“ After a loss, a person might say she/he isn’t/ upset or “everything’s fine.” In this stage, often a person may continue quickly with further infertility treatment, without sitting and feeling the loss of the previous cycle.

An individual who is infertile or has experienced pregnancy loss may say “Why me? This is outrageously unfair!”  The individual experiences this stage because they realize that they cannot continue with their denial. Many people experience anger targeted towards pregnant women or people with children. The individual may be angry at their partner, doctor, or any professionals assisting them with their infertility.

In this phase, I often see people attempting to do “everything right” to get pregnant. People will read everything on conception and change their diets or lifestyles dramatically with the bargaining/magical thinking mentality. If I work hard enough to get pregnant, then I will. I hear people say, “I promise to be happy for a friend/family member if I get pregnant” with the hope that their thoughts and actions will directly affect their pregnancy outcome.

I’m never going to have a baby so what’s the point?” In this phase the individual may become increasingly isolated, refuse visitors, often spend much of the time crying and grieving. They might not be able to enjoy previous pleasurable activities and may see changes in sleeping and eating patterns. In this phase lasts long or recurs often, it may be time to seek professional help.

It's going to be okay.” This is the phase where infertile people start to reclaim their lives. They may say that they know they will be a mother or a father at some point and accept that the baby may come to them through a different way than they originally had hoped for. This may be a time when people can begin to explore different options, ART, third party reproduction, adoption, and the option of living without children.

There is no one right way to work through your grief. Below are some suggested activities that may bring you some comfort in your infertility journey.

This can be a very good way to express feelings that people may not feel comfortable sharing with others.  Journaling can avoid holding emotions in which can often extend the grief process or even lead to other physical and emotional problems.  Journaling is a good way to express and hopefully, purge the anger and/or rage that you may be feeling.

Writing Letters to your (hoped for) Baby

Some people decide to write letters to the babies that they have miscarried. It’s often helpful to write a letter to a baby that you hoped for but have not been able to have yet. Oftentimes, people find it helpful to name the baby (even if it’s name that you don’t share with anyone else or it’s a spiritual or nature name). When you decide to move on to third party reproduction, adoption, or if you choose to live without children, it is often cathartic to write a letter to the genetic child that you had hoped and dreamed of having.

Create an Altar

When I was going through the depths of the darkness of my infertility journey, I put together an infertility altar. I collected fertility symbols, pictures, lab results, a one time positive home pregnancy test, poems, and I tended to it daily with pictures and candles. I meditated in front of this altar daily and I found a safe and balancing haven amidst all of the chaos and uncertainty of my infertility.

Plant a Garden

In my private therapy practice, I see infertile individuals and couples take to gardening during their infertility journey. Clients report that there is something healing to planting seeds, working at on one with the earth, and watching life emerge.

Additionally, you can plant a memorial garden for a miscarriage. You can plant a memorial garden for all of the babies you had hoped for month after month. You can plant a garden of hope. I have seen people plant butterfly gardens as well.

Create a Ritual or Healing Circle

Oftentimes it’s helpful to bring community together to help hold and support you in your times of both grief and hope. You may want to bring together supportive friends and family to create community to support you during a recent loss - a failed cycle or a miscarriage. One woman brought in a group of her closet friends in a time of hope-before one of her IVF cycles. Everyone brought her something for her altar, they said blessings over all of her medications, and shared stories of hardships that they had overcome in their lives. Sharing collective grief with people you trust and who love you can provide strength and support and can also reduce isolation during a particularly isolating time.

Access your own creative outlets

If you are a painter, or a sculptor, or knitter, or jewelry maker, or welder or gourmet chef, try to continue to use these creative outlets to work through your grief. Your creative outlets could be through cooking, or hiking, or yoga. If you stop your creative outlets as a result of your grief, you may want to talk with a trained therapist to assist you in finding ways to integrate these outlets back in your life.

Beth Jaeger-Skigen is a psycho-therapist in private practice in San Francisco specializing in all aspects of infertility. She provides therapy individuals, couple's, and groups and is also the consulting psycho-therapist for Acupuncture Kitchen.  Beth has spoken at RESOLVE conferences and worked on RESOLVE's infertility helpline. She currently presents webinars, teleseminars, and lectures on the psychological aspects of infertility. You can reach her at


Conferences next week!

I passed out conference scheduling forms to the students on Thursday and Friday. I am excited to meet family members as the student-led conferences can showcase the hard work they have done this year.

Looking forward to meeting everyone next week, Wednesday through Friday. Please let me know if you have any questions before your scheduled conference time.

--Mr. Riedl


Growing Ubuntu for cloud and IoT, rather than phone and convergence

This is a post by Mark Shuttleworth, Founder of Ubuntu and Canonical

I’d like to emphasise our ongoing passion for, investment in, and commitment to, the Ubuntu desktop that millions rely on. We will continue to produce the most usable open source desktop in the world, to maintain the existing LTS releases, to work with our commercial partners to distribute that desktop, to support our corporate customers who rely on it, and to delight the millions of IoT and cloud developers who innovate on top of it.

We care that Ubuntu is widely useful to people who use Linux every day, for personal or commercial projects. That’s why we maintain a wide range of Ubuntu flavours from both Canonical and the Ubuntu community, and why we have invested in the Ubuntu Phone.

In the community, our efforts were seen fragmentation not innovation. And industry has not rallied to the possibility, instead taking a ‘better the devil you know’ approach to those form factors, or investing in home-grown platforms. What the Unity8 team has delivered so far is beautiful, usable and solid, but I respect that markets, and community, ultimately decide which products grow and which disappear.

This has been, personally, a very difficult decision, because of the force of my conviction in the convergence future, and my personal engagement with the people and the product, both of which are amazing. We feel like a family, but this choice is shaped by commercial constraints, and those two are hard to reconcile.

The choice, ultimately, is to invest in the areas which are contributing to the growth of the company. Those are Ubuntu itself, for desktops, servers and VMs, our cloud infrastructure products (OpenStack and Kubernetes) our cloud operations capabilities (MAAS, LXD, Juju, BootStack), and our IoT story in snaps and Ubuntu Core. All of those have communities, customers, revenue and growth, the ingredients for a great and independent company, with scale and momentum. This is the time for us to ensure, across the board, that we have the fitness and rigour for that path.

Posted in:


Get Bupa now

Mon-Fri, 9am - 6pm
2517 5860

Customer Services

24 hours, 7 days a week

Individual Schemes

2517 5333

Bupa Gold Schemes

2517 5383

Bupa group members

2517 5388

Your doctor has recommended that you have a gastroscopy and this fact sheet provides some information about the procedure. If it does not answer all your questions, or if you have any worries, please do not hesitate to ask your specialist or nurse for more information.
It is natural to feel a bit nervous about having any medical procedure, but often knowing what to expect can help.

A gastroscopy is an examination of the upper part of the gastrointestinal tract - the oesophagus (gullet), stomach and duodenum (the first part of the small intestine). This is done using a flexible, tube-like telescope called an endoscope, which is about the thickness of a little finger. The endoscope is passed through the mouth and into the oesophagus. The procedure may also be simply referred to as an endoscopy.

A gastroscopy is useful for finding out what is causing your symptoms, or as a check-up for certain gastrointestinal conditions. During the procedure, the doctor may take a biopsy - a sample of the lining of the gullet, stomach or duodenum - for examination in the laboratory.

Gastroscopy is routinely performed as an outpatient or day case, requiring no overnight stay in hospital. A general anaesthetic is not generally required, but you may be given a sedative to help ensure that you are relaxed and comfortable.

A gastroscopy usually takes about 10-15 minutes. The procedure will not hurt but may feel uncomfortable to begin with. As the endoscope passes through your oesophagus, and not the trachea (windpipe), you will be able to breathe normally throughout the procedure.

Preparing for the procedure

For the doctor to get a clear view through the endoscope, the stomach needs to be empty. To achieve this, you will need to avoid eating for three to four hours before the test but can sip clear fluids for up to two hours before the test.

Sedation and pain relief

Modern endoscopes are thin and quite easy to swallow. The doctor may spray a local anaesthetic into the back of your throat or give you a lozenge to suck to numb the throat area.

In some cases, you and your doctor may decide that you need a sedative. Your doctor will explain what to expect, but it generally involves an injection into a vein in the back of the hand. Almost immediately, this will make you feel relaxed and drowsy.

Before you have an endoscopy, you will be given a consent form to sign. By doing this, you confirm that you understand what will happen during the procedure and give your permission for it to go ahead. Many patients feel uncomfortable with the idea that a procedure may involve risks, but to make an informed choice, you need to know about the possible risks before you give consent.

Gastroscopy is a very commonly performed and generally safe procedure. For most people, the benefits of having a clear diagnosis, or quick and effective treatment are much greater than any disadvantages. However, like all invasive medical procedures, there are some risks. These can be divided into the risk of side-effects and the risk of complications.

These are the unwanted, but usually mild and temporary, effects of a successful procedure. After a gastroscopy, you may feel bloated, but this usually clears up quite quickly. If you have a sedative, it may make you feel sleepy.

Your doctor will be very experienced at performing endoscopies but, even so, a few are not successfully completed and may need to be repeated.

Other complications are uncommon, but it is possible for the oesophagus, stomach or intestine to be damaged or, in rare and extreme cases, perforated during the procedure. This can lead to bleeding and infection, which may require treatment with medicines or surgery.

The chance of complications depends on the exact type of procedure you are having and other factors such as your general health. You should ask your specialist to explain how these risks apply to you.

You may be asked to take off your shirt or top and put on a hospital gown. If you have them, you should also remove false teeth, contact lenses or jewellery, which will be kept safe for you during the procedure. To help ensure you are as comfortable as possible, and to provide reassurance, a nurse will stay with you throughout.

If a sedative has been given, the procedure will not start until it has taken effect. With you resting comfortably on your side, on a bed or examining couch, the endoscope will be carefully inserted into your mouth. The natural swallowing action will help the endoscope pass into the gullet and down towards the stomach.

Air will be passed through the tube and into the stomach to make the lining easier to see. When this happens, you may briefly feel a sensation of fullness or nausea. At the end of the endoscope, a tiny light and lens enable the doctor to see if any disease is present. If necessary, the doctor will take a small biopsy of tissue for analysis. This is a quick and painless process, using specialist instruments that can be passed inside the endoscope.

When the examination is finished, the endoscope is removed quickly and easily.

After the gastroscopy

You will rest in a full-length chair or on a bed for about half an hour, or longer if youve had a sedative. The nursing staff will be on hand to make sure you are comfortable.

Going home

The effects of any sedative may last longer than you expect, so you should not drive, operate machinery or drink alcohol for 24 hours after your examination. This means that you will have to arrange for someone to take you home and stay with you for the first day.

Once home, its sensible to take it easy for the next 24 hours. Most patients feel able to resume normal activities on the following day.

If you have a biopsy, the results will be ready several days later and will usually be sent in a report to the doctor who recommended the test. Other findings can be discussed before you leave the hospital. After having a sedative, it may be a good idea to have someone with you when you are told the results, as you may not remember them afterwards.

This leaflet describes standard treatment. Your experience may differ slightly. If you have any unanswered questions or concerns, please do feel free to raise them at any time when you come to the hospital.

You might also like

About radiotherapy Radiotherapy can be used to treat many different types of cancer. Radiotherapy works by destroying the DNA inside cancer cells, preventing them from reproducing and so shrinking cancerous tumours. Normal cells will also be affected but they are better able to repair themselves.

Read more

MRI scan

An MRI scan uses magnets and radiowaves to produce detailed images of the inside of the body. It can help to diagnose and monitor many different medical conditions.

Read more

Gallbladder removed

This factsheet is for people who are planning to have gallbladder surgery (cholecystectomy), or who would like information about it.

Read more

Talk to us

Contact our health management consultant to get details and advice.

2517 5860

Mon-Fri, 9am-6pm


New Zealand Quail

A candidate for de-extinction

Reviving the extinct New Zealand Quail

With many close living relatives the New Zealand Quail Coturnix novaezelandiae is a promising de-extinction candidate.

Meet the Researcher

Matiu Andrews-Cookson

Matiu Andrews-Cookson

Project Coordinator/Molecular Biologist

Matiu project manages construction projects by day but is also a trained molecular biologist. He's passionate about advancing the science of de-extinction and believes the New Zealand Quail is a pragmatic target species.

Why revive the New Zealand Quail?

There are exciting new scientific techniques such as interspecific germline transmission of cultured primordial germ cells that offer hints it may be possible to revive extinct bird species. New Zealand has the unfortunate distinction of having a large number of critically endangered and recently extinct species largely as a result of human colonization. What if it was possible to undo some of the ecological damage humans have inflicted upon New Zealand?

The New Zealand Quail is one of many potential candidates for de-extinction. The reason it stands out as a logical starting point is that it has many genetically similar living relatives. The currently proposed avian de-extinction techniques rely on making edits to the genome of a similar species in order to change that genome to be the same as the extinct species. The fewer the number of genome edits required the higher the probability of success.

A successful de-extinction attempt for the New Zealand Quail would pave the way for more challenging de-extinction projects such as reviving the moa as well as perfecting techniques that would be equally valuable when applied to saving living critically endangered species such as the kakapo.

An introduction from the researcher

Koreke occupied a niche in NZ’s native ecosystem as a dry land seed and insect eater which is filled exclusively by exotic species today. Koreke were so important as a food and cultural resource to Maori, that early land court cases against the Crown mentioned the presence of koreke in an area of land as a delineation of ownership.

Read the full introduction

Project Status

Before embarking on an exploratory study of the scientific techniques that may enable avian de-extinction we must first have sequenced the genome of the extinct species. Matiu Andrews-Cookson is currently leading a project that aims to perform this sequencing.

Learn more

About the New Zealand Quail

The New Zealand Quail (Coturnix novaezelandiae), or koreke (the Māori name), has been extinct since 1875. The male and female were similar, except the female was lighter. The first scientist to describe it was Sir Joseph Banks when he visited New Zealand on James Cook's first voyage. Terrestrial and temperate, this species inhabited lowland tussock grassland and open fernlands. The first specimen to be obtained by a European was collected in 1827 by Jean René Constant Quoy and Joseph Paul Gaimard on Dumont D'Urville's voyage.

Research was conducted between 2007 and 2009 into whether the quails on Tiritiri Matangi Island – which was spared the worst impact of introduced predators – might be a surviving population of this species, or koreke-brown quail (Coturnix ypsilophora) hybrids. However, a genetic study showed instead that the quail on Tiritiri Matangi are Australian brown quail, Coturnix ypsilophora. Sequences were derived for all quail species within the Australian and New Zealand Coturnix sp. complex.

It has sometimes been considered conspecific with the Australian stubble quail Coturnix pectoralis, which would then be named Coturnix novaezelandiae pectoralis as the New Zealand bird was described first. However, the genetic analysis showed that they are separate though closely related species.


Tag Archives: Germany

Hitler appointed Chancellor of Germany

Inside the Nazi State – Supporters

This documentary episode presents the recollections and opinions of a number of people who supported Hitler and the Nazis.  It’s particularly interesting to see the reasons they give for why they supported them.

Part 1

Part 2

Part 3

Saar Plebiscite

The Saar plebiscite and reunion with Germany, 1935

On the 13th January 1935, the Territory of the Saar Basin voted to reunite with Germany. Having been administered by the League of Nations for 15 years following the signing of the Treaty of Versailles, the overwhelming plebiscite result of over 90% in favour of reunification surprised many observers.

In 1918 the Saar Basin was a heavily industrialised area, boasting a large number of coal mines. Following the Treaty of Versailles the area was occupied and governed by France and Britain under the auspices of the League of Nations. France was also given exclusive control of the coal mines. However the Treaty called for a plebiscite to decide the long-term future of the Saar region after a period of fifteen years.

By the time of the plebiscite Adolf Hitler had come to power in Germany, which had led a number of Nazi opponents to move to the area since it was the only part of Germany free from their rule. They were keen for the area to remain under the League’s administration, but maintaining the status quo was unpopular with ordinary Germans.

Meanwhile the Nazis began an intensive pro-Germany campaign led by propaganda chief Joseph Goebbels. As early as 1933, complaints that the Nazi campaign amounted to a “reign of terror” had been noted by American political scientists Sarah Wambaugh, one of the members of the commission overseeing the plebiscite. Although the Nazis did tone down their tactics by the end of 1934, the League of Nations provided a peacekeeping force to monitor the plebiscite.

Voter turnout plebiscite was 98%, with 90.8% voting to re-join the German Reich.

Nazi Parade

Nazi Control of Germany 1933-1945 (podcast)

This podcast examines the three broad ways through which the Nazis secured control of Germany.  These are categorised as ‘the stick’ (repression and force), ‘the carrot’ (positive social and economic effects), and propaganda.

The first method of control outlined in the podcast is repression, which began following the Reichstag Fire in 1933.  The four key areas of repression are explained: Nazi control of the police and courts, the SS and the use of concentration camps, and the Gestapo.

Secondly, the episode explains how Nazi achievements were used to maintain support for the party.  Particularly achievements that ensured support included such things as assistance to farming communities (e.g. the Reich Entailed Farm Law) and more jobs and improved working conditions for industrial workers.  Middle-class support was secured through the removal of the threat of Communism.  Big-business was also very supportive due to being able to secure large government contracts, and the fact that trade unions had been banned by the Nazis in 1933.  Remilitarisation and large-scale public works projects also secured wide public support for the Nazis.

Thirdly, the podcast assesses the impact of propaganda on the German population.  An explanation is given of the role of the Reich Chamber of Culture, before describing specific propaganda achievements such as the effect of the radio, the use of films, and the 1936 Olympic Games in Berlin.

The podcast ends with some tips on how to answer a question about Nazi control in Germany by explaining how the three factors worked together to support each other.

The SS

The foundation of the SS: an overview

The SS was established on the orders of Adolf Hitler to act as his personal bodyguard. He had been released from prison the previous December, having been found guilty of treason following the failed Munich Putsch, and was keen to ensure his safety when attending party functions and events.

The SS was technically a division of the longer-established SA, but the group’s loyalty to Hitler meant that over time it grew to be the dominant organisation. This came about under the leadership of Heinrich Himmler, who was appointed Reichsfuhrer-SS in January 1929. He transformed the SS from a small bodyguard of less than 300 members into a private army containing over 50,000 men by the time Hitler became Chancellor of Germany in January 1933.

Membership of the SS was reserved for those men who would loyally and unquestioningly serve Hitler and the Nazi Party, and who met the SS’s strict racial policy. Through racial selection of both SS members and their spouses, the Nazis hoped to create an ‘elite’ community of people with an ‘Aryan-Nordic bloodline’.

The Night of the Long Knives in June 1934 removed any remaining authority the SA once had over the SS. A month later Hitler formally separated the two organisations, meaning that the SS became answerable to him alone. As the organisation grew even further under the Nazi dictatorship, separate SS subdivisions were established within a sprawling bureaucracy.

By the end of the Second World War the SS had responsibilities that stretched from policing and the collection of intelligence to running Nazi concentration and death camps.

Speeches by Goebbels

This video presents an edited collection of speeches given by Josef Goebbels, the Nazi propaganda chief, in which he presents his views on the role of propaganda in the Nazi regime.

Nazi Book Burning

Hitler’s Germany 1933-36 (Home Policy)

Nazi Propaganda

Life in Nazi Germany 1933-1945 (podcast)

The private Hitler

Inside the Nazi State – Knowing Hitler

Diary of Anne Frank

Anne Frank received a diary on her 13th birthday 12.06.1942

On the 12th June 1942, Anne Frank received a diary as a thirteenth birthday present from her father. Barely three weeks later, Anne and her family went into hiding in Nazi-occupied Amsterdam.

Her diary, which chronicled her experiences over the next two years, was published posthumously after the war under the title The Diary of a Young Girl and became one of the twentieth century’s most celebrated books.

The Frank family originated in Frankfurt, Germany, but moved away after the Nazi party won local elections in 1933. Anne’s father, Otto, was a businessman who chose to move the family to Amsterdam after receiving an offer to start a company there. However, when the German army invaded the Netherlands in 1940 the family found themselves trapped in a country subjected to anti-Semitic laws.

When, in July 1942, Anne’s older sister Margot was ordered by the Nazi authorities to go to a labour camp, their father instead arranged for the family to go into hiding in a so-called ‘Secret Annexe’ above his office building. It was here that Anne wrote her diary, which she addressed as Kitty. Over three volumes she recorded the relationships between the Frank family, the Van Pels family, and her father’s friend Fritz Pfeffer with whom they shared their confined hiding place.

An anonymous tip-off led to the discovery and arrest of the eight inhabitants on the 4th August 1944. They were deported to the Auschwitz concentration camp a month later. Anne died of typhus in early 1945 after being transferred to Bergen-Belsen. She was fifteen years old.