ga('set', 'userId', {{USER_ID}}); // Set the user ID using signed-in user_id.
shadow

A variation in the Cell Size Regulator gene bulked up early tomatoes, facilitating domestication — ScienceDaily


Farmers can grow big, juicy tomatoes thanks to a mutation in the Cell Size Regulator gene that occurred during the tomato domestication process. Esther van der Knaap of the University of Georgia, Athens and colleagues describe this gene variant in a study published in open-access journal PLOS Genetics on August 17th, 2017.

When humans first began cultivating the wild tomato in the Andean mountain regions of Ecuador and Northern Peru, they continually selected plants that produced larger fruits. Now, thousands of years later, tomatoes on the market can weigh 1,000 times more than the fruits of their ancestors. In the current study, researchers investigated a gene they named Cell Size Regulator, or CSR, that boosts fruit weight by increasing the size of the individual cells in the pericarp, which is the fleshy part of the tomato. Compared to wild tomatoes, domesticated varieties carry a mutation in the CSR genes that shortens the resulting protein in tomato cells, and that truncation likely affects its role in regulating cell differentiation and maturation in the fruit and vascular tissues. The variation originated in the cherry tomato but now appears in all large cultivated tomato varieties.

The new study expands on previous research that had identified the location of CSR at the bottom of chromosome 11 as only a small genetic contributor to tomato weight. Now with the cloning of the gene, the finding that most cultivated tomatoes carry the shortened version of the CSR gene suggests that humans selected this genetic variation extensively and that it was critical to the full domestication of tomato from its cherry tomato ancestors.

“CSR is required to create the large tomatoes that are needed for the industry. This is because large tomatoes critically raise the profit margins for farmers. The knowledge of the gene will now open up avenues of research into how fruit size can be increased further without negatively impacting other important qualities such as disease resistance and flavor,” says Dr. van der Knaap.

Story Source:
Materials provided by PLOS. Note: Content may be edited for style and length.



Source link

Mechanism of transformation in Mycobacteria using a novel shockwave assisted technique driven by in-situ generated oxyhydrogen


Design of the oxyhydrogen detonation-driven miniature shock tube for bacterial transformation

An oxyhydrogen detonation-driven miniature shock tube assembly to generate shockwaves of required strength and duration has been reported12. A similar experimental setup with slight modifications has been used for the present work (Fig. 1a). The device comprises of two main components – an oxyhydrogen generator and a miniature shock tube assembly. The oxyhydrogen generator produces the required amount of stoichiometric mixture of hydrogen and oxygen gases through alkaline electrolysis. A miniature shock tube assembly with an internal diameter of 6mm is used. The oxyhydrogen mixture is filled in the driver section of the shock tube (this is termed as initial fill pressure of oxyhydrogen henceforth) and a spark plug, placed close to the diaphragm station between the driver and the driven section, is used to ignite the mixture to produce a backward facing detonation front (Supplementary Fig. S1a). The high pressure and temperature behind the detonation front causes the instantaneous rupture of the diaphragm between the driver and driven section and produces a strong shockwave in the driven section of the shock tube. Tracing paper (95 GSM) is used as diaphragm in the shock tube which can be replaced by a quick opening solenoid valve at a later stage. A tri-clover clamp is used between the different sections of the shock tube to facilitate quicker and easier changing of diaphragm after each experiment. The biological sample is accommodated in a stainless steel sterile cavity of diameter 6mm and depth 5 mm (Fig. 1b). The optimization of the dimension of the cavity has already been reported5. In a previous study, a brass foil was suggested as a viable option for energy transfer from the shock wave to the biological sample and also to avoid contamination of the bacteria by the products of detonation10. However, brass foil needs to be replacement after every experiment and also it absorbs most of the incident shockwave energy before transmitting it to the biological sample. Therefore, in the present work, we have used a silicone rubber membrane to separate the cavity housing the biological sample from the shock tube. Silicone rubber is a biocompatible material which has a good tensile strength and is resistant to temperatures of up to 300 °C15. These properties make it ideal for our application, as there is no need for frequent replacement and the energy transfer is much better as compared to using the brass foil.

Figure
1
Figure 1

The oxyhydrogen-driven miniature shockwave device for bacterial transformation. (a) Schematic of the oxyhydrogen-driven miniature shockwave device for bacterial transformation, (b) Cross-sectional view of the end of the shock tube showing the cavity for biological sample, (c) Shock tube configurations used for the study. Different lengths of driver and driven section are depicted, (d) Pressure signal recorded for the two configurations using a sensor flush mounted to the wall of the driven section placed 12 mm from the end of the shock tube, (e) High speed Schlieren images of the blast wave recorded from the open end of the shock tube operated using the two configurations.

Effect of steady time of the shock wave on bacterial transformation

It has been reported that the impulse of the shockwave (i.e. pressure integrated over time) rather than the peak pressure plays a more vital role in changing the permeability of the membrane to achieve better molecular delivery13. The impulse of the shockwave is a function of the steady time duration as well as the amplitude of the shock wave. It is calculated by isolating the first peak in the pressure vs time plot. Impulse per unit area (I) can be represented by the formula,

I=0TP(t)dt

where ‘T’ represents the steady time of the shock wave, ‘P(t)’ is the pressure experienced by the sample as a function of time. The amplitude, rise time, steady time and fall time of a typical shockwave signal is shown (Supplementary Fig. S1b). The area under the curve represented by the shaded region in the figure gives the impulse per unit area generated. The effect of steady time and amplitude of the shockwave can be easily verified using the present experimental setup as changing the length of the driver and driven sections of the shock tube is one simple way of varying these parameters16, 17. Different combinations of driver and driven lengths were chosen to arrive at two configurations of shock tube (Fig. 1c). A tri-clover assembly used to clamp the different sections of the shock tube makes diaphragm changing and length variation in the shock tube effortless. For the same initial fill pressure in the driver section (3 bar of oxyhydrogen mixture), configuration I generates an incident shock wave with a high-pressure amplitude and low steady time as compared to configuration II, which generates an incident shock wave with a low-pressure amplitude and high steady time duration (Fig. 1d). Figure 1e shows the time resolved high-speed schlieren images of blast wave evolution from the open end of the shock tube when operated using the two configurations (initial fill pressure of oxyhydrogen is 3 bar in both the cases). The stronger color gradients in the first case indicate the higher strength of the primary and secondary blast waves. When the biological sample is placed in the cavity at the end of the shock tube with the silicone rubber in between, the shockwave energy is transmitted through the silicone membrane to the biological sample. The pressure signals of the transmitted shockwave have been measured in the cavity when the shock tube is operated in the two different configurations (Supplementary Fig. S2a and b). The peak pressure, steady time duration and impulse of the transmitted shockwave have been tabulated (Supplementary Table 1). From the table, it can be seen that the peak pressure and impulse of the shockwave generated is higher for configuration-I while the steady time duration is higher for configuration-II. Experiments carried out to compare the transformation efficiencies of E.coli using the two configurations have been shown (Supplementary Fig. S2c). The transformation efficiencies using the shockwave method are significantly higher than the conventional heat shock method and also the results reveal that a higher pulse duration of the shockwave is required for efficient bacterial transformation. Therefore, all the remaining experiments reported in this paper were carried out using configuration-II of the shock tube.

Optimization of parameters for bacterial transformation

Shockwaves have been reported to show a deleterious effect on the genomic as well as extra chromosomal DNA of bacteria. It was observed that shock waves used for the experiments described do not have a significant effect on the viability of bacteria and integrity of the plasmid DNA. Only Salmonella Typhimurium shows a reduced viability,although statistically not significant, at higher fill pressures of the shock tube (Fig. S3).The dependence of transformation efficiency on the total number of bacterial cells used was also analyzed. It was observed that with an increase in the concentration of bacteria the efficiency of plasmid transfer significantly increased (Fig. S3). Different concentrations of calcium chloride ranging from 100 mM to 400 mM were used for transformation. Optimum transformation efficiency was obtained at a concentration of 200 mM CaCl2 (Fig. S3). For transformation in Mycobacteria, growth medium was supplemented with 1.5% glycine. This has been reported to aid in increasing the bacterial cell wall permeability. Bacterial transformation was also confirmed at the levels of gene expression by examining the bacteria for the expression of the mCherry red protein or green fluorescent protein using a confocal microscope (Fig. S4). Post optimizing the conditions for transformation in E.coli, the device was used to carry out transformations in other bacteria Salmonella Typhimurium, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycobacterium tuberculosis and Helicobacter pylori.

Effect of fill pressure of oxyhydrogen mixture on the transformation efficiency

Transformation in E.coli, Pseudomonas aeruginosa, Salmonella Typhimurium, Mycobacterium smegmatis, Mycobacterium tuberculosis and Helicobacter pylori was performed at different fill pressures of oxyhydrogen to achieve maximum transformation efficiency (Fig. 2a–f). E.coli required the least fill pressure for transformation while Mycobacteria required the highest fill pressure as well as shockwave pulses amongst the group of bacteria transformed. Mycobacteria were exposed to multiple shock wave pulses ranging from 1 to 5 pulses. Salmonella, Pseudomonas and Helicobacter species required intermediate shock tube fill pressures. It was observed that for every bacterial species, the conditions required for optimal transformation efficiency were different and transformation efficiencies were significantly higher than the conventional method of electroporation. The transient change in the membrane polarity was confirmed by the diBACC4 assay in the bacteria when exposed to shockwaves (Fig. 3a–d). Quantification of the membrane depolarization showed significant change after bacteria were exposed to shockwaves (Fig. 3e,f).

Figure
2
Figure 2

Efficiency of bacterial transformation using shockwaves and its comparison with electroporation. (a) E.coli, (b) P.aeruginosa, (c) Salmonella Typhimurium, (d) H. pylori, (e) Mycobacterium smegmatis and (f) Mycobacterium tuberculosis were transformed with relevant plasmids mainly by heat shock, electroporation and shock waves. After transformation LB broth was added to the cells, incubated at 37 °C for 1 h before plating. For E.coli, P.aeruginosa, Salmonella and H.pylori the shocktube fill pressure was varied. For Mycobacterial transformations, the shocktube fill pressure was maintained at 10 bar and the number of consecutive shockwave exposures were varied to achieve high efficiency transformation. Post incubation, CFU were counted and transformation efficiency was calculated. Statistical significance was defined as follows (*P < 0.05, **P < 0.005, ***P < 0.0005) (Student’s t test).

Figure
3
Figure 3

Effect of shockwaves on membrane depolarization. (a–d) diBACC4 assay was performed to assess the change in the membrane polarity during the exposure to shockwave in E.coli, Pseudomonas, Salmonella and Mycobacterium smegmatis. The bacteria were incubated with diBACC4 (1 µg/ml) prior to shockwave exposure. The bacteria were analyzed using flow cytometry for change in the membrane polarity. Results are representative of three independent experiments with similar results. (e–f) Comparison of the percent diBACC4 positive cells in E.coli, Pseudomonas, Salmonella and Mycobacterium smegmatis. Statistical significance was defined as follows (*P < 0.05, **P < 0.005, ***P < 0.0005) (Student’s t test).

Shockwaves induce increase in bacterial length and Young’s modulus facilitating high efficiency transformation

Transformation using shockwaves was highly efficient as described earlier but its mechanism was still unclear. To check the effect of shockwaves, bacteria were observed immediately under an atomic force microscope (Fig. 4a,b). Bacterial cell length was quantified in the control and shockwave exposed sample (Fig. 4c). It was observed that on an average, the cell length in the shockwave exposed sample was increased to 1.5-fold as compared to control. This observation was consistent amongst all the bacteria used for transformation wherein Mycobacteria showed the most significant increase in the cell length after shockwave exposure. Length being an important parameter in determining the stiffness and Young’s modulus of any rigid body, the Young’s modulus of bacteria pre and post-exposure to shockwaves was measured (Fig. 5a–d). Force displacement curves were generated. Hertzian model of data fit was used to compute the Young’s modulus. It was observed that upon shockwave exposure, the Young’s modulus increased significantly in all the bacterial species used (Fig. 5e–h). Next, Young’s modulus was also measured after electroporation in various bacteria. It was observed that efficient bacterial transformation was directly related to the change in Young’s modulus (Fig. 5g). In case of less efficient transformation by electroporation in Mycobacteria, it was seen that the change in Young’s modulus was not significant. Therefore, from these observations, it is concluded that a positive change in Young’s modulus of the bacteria is of utmost importance to achieve high efficiency transformation. Mathematical finite element modeling has also shown the increase in length and Young’s modulus after shockwave exposure (Fig. S6).

Figure
4
Figure 4

Topographical images of various bacterial species using Atomic force microscopy. (a) Untreated bacteria, (b) Shockwave treated bacteria. Bacteria were either exposed to shockwaves or were unexposed and were processed for imaging using noncontact mode AFM. Single bacteria were imaged and topographical analysis was done using Park systems software. A total of 10 fields were imaged and analyzed. The results are representative of the measurements. (c) Bacterial cell length in control and shockwave treated bacteria was measured and compared. A minimum of 200 bacteria per sample were analyzed.

Figure
5
Figure 5

Determination of Young’s modulus of bacteria after shockwave exposure. (a) E.coli, (b) Salmonella, (c) M. smegmatis and (d) M. smegmatis grown in presence of 1.5% Glycine were either exposed to shockwaves or were unexposed prior to atomic force microscopy for measuring the Young’s modulus, a measure of elasticity. Uniformly spaced points for indentation were selected on the bacterial surface and contact mode indentation was performed. The force displacement curves were analyzed using the Hertzian model. A total of 10 bacteria were analyzed per sample. The images are representative of the experiment. (e–h) Comparison of Young’s modulus after shockwave exposure. (i) Comparison of Young’s modulus after electroporation. Statistical significance was defined as follows (*P < 0.05, **P < 0.005, ***P < 0.0005) (Student’s t test).

Mechanism of enhanced bacterial transformation using the shockwave device

The incident shock wave, which is the driving force for transformation, undergoes many changes upon hitting the silicone rubber membrane at the end of the shock tube. A major portion of the incident energy is transmitted through the rubber to the bacterial culture in the cavity. A part of the incident energy is absorbed by the rubber membrane while another portion of the energy is reflected back into the shock tube. The pressure measurement inside the cavity gives some useful insight into the possible mechanism of enhanced transformation in the bacteria using the device. Figure 6a shows the pressure experienced by the bacterial culture in the cavity during operation of the device. It can be seen that although the incident shockwave has a pressure jump of only about 7 bar (See Fig. 1d Configuration II), the pressure inside the cavity reaches a pressure of about 90 bar. A closer look at the pressure signal shows the presence of two distinct pressure peaks. The first peak reaches a pressure of about 90 bar and the time period from rise to fall is about 40 microseconds. This time duration can be easily related to the steady time duration of the incident shockwave (~50 microseconds) seen in Fig. 1d Configuration II. The rise in pressure inside the cavity to a pressure of up to 90 bar could be due to the combined effect of the transmitted shockwave and the pressure rise in the liquid due to the bulging of the rubber membrane. The drop in the pressure after the peak is because of the reflected pressure waves from the bottom of the cavity causing the rubber to go back to its original shape and hence creating a pressure relieving effect in the cavity. In Fig. 6a, it can be seen that the second peak reaches up to a pressure of about 28 bar and the time period is about 30 microseconds. Although, this time duration corresponds to a continuous drop in the pressure of the incident shockwave after the steady time, a second pressure peak is still observed in the cavity. This could be because of the fact that the high pressure in the shock tube is still persistent during this time duration but the pressure value is lower in amplitude as compared to the initial pressure jump. Therefore, the rubber bulges again to give the second pressure peak of lower amplitude in the cavity as compared to the first pressure peak.

Figure
6
Figure 6

Proposed Mechanism of enhanced bacterial transformation. (a) Pressure signal indicating the location of the stages shown in the schematic diagram (Only E.coli has been considered) (b) Schematic diagram showing the different stages during the bacterial transformation experiment. The pressure pulse due to the vibration of rubber membrane is indicated in orange while the transmitted shock wave is shown in brown.

Also, some high frequency oscillations can be observed in the fall time duration after the first pressure peak. These high frequency oscillations slowly dampen out with time. The vibration of the silicone membrane or the multiple reflections in the cavity could be a reason for these oscillations. The time period for silicone rubber vibrating at its natural frequency lies in the range of 1.2–6.2 milliseconds (see Supplementary Note S1). Therefore, the high frequency oscillations cannot be due to the vibrations of silicone rubber. A simple calculation reveals that the time taken for stress waves to travel inside the cavity is about 3.33 microseconds (see Supplementary Note S2), which is of the same order as the time period of the high frequency oscillations observed in the Fig. 6a. Therefore, the transmitted shock wave undergoes a series of reflections against the bottom of the cavity and the face of the rubber membrane causing high frequency oscillations in the pressure inside the cavity and also continuously attenuating in intensity. Keeping all these points in mind and correlating the pressure signals shown in Fig. 1d Configuration II and Fig. 6a, illustrations of the possible mechanism of bacterial transformation using the shockwave assisted device have been shown in Fig. 6b. The corresponding time instants of the illustrations have been indicated in Fig. 6a.



Source link

The Causes and Treatment of Toothache (Odontalgia)


According to the British Dental Health Foundation “about 5 million people visit their dentist with toothache every year”. Toothache is a common problem that can be prevented with good oral hygiene.

Toothache occurs because the pulp of the tooth is exposed, disturbed or infected. The pulp is the inner layer of the tooth which is engulfed in a layer of dentin and then by the hard layer that we see called the enamel, which is packed full of minerals. Toothache can also occur if just the outer enamel layer is damaged exposing only the dentin.

It is very important that you go to a dentist if you have toothache so that they can find a cause and apply appropriate treatment to ease your discomfort.

The most common dental causes of toothache are:

  • Tooth Root Sensitivities – over-sensitivity when consuming hot or cold, sweet or sour food and beverages.
  • Tooth Decay – also known as tooth ‘cavities’ or tooth ‘caries’.
  • Tooth Abscess – a complication of tooth decay.
  • Gum Disease – also known as gingivitis and in severe cases periodontal disease.
  • Jaw Disease – also known as TMJ (Temporo-Mandibular Joint) dysfunction.
  • A Cracked Tooth.

Tooth Root Sensitivities occur when bacterial toxins get to work and dissolve the bone around the root of the tooth, the gum and bone recede exposing the root of the tooth causing the sensitivity and toothache. This is then likely to lead to chronic gum disease.

Treatment: Visit your dentist. Fluoride gel and sensitivity toothpastes that contain fluoride will both help the root to become stronger and in turn reduce the toothache. If the root sensitivity causes the inner pulp to die a root canal procedure or tooth extraction will need to be carried out to stop the toothache.

Tooth Decay occurs when the minerals of the enamel are dissolved by acid created by bacteria in our mouths (a build up of this bacteria is known as plaque). This demineralisation of the enamel forms a hole in the tooth exposing the dentin causing the toothache. If the toothache is severe then the hole has most likely exposed the inner pulp as well.

The obvious prevention for tooth decay is to eat as little sugar as possible because the acid that causes the enamel to decay is created by the bacteria eating the sugar and starch left in our mouths. So brush your teeth preferably after every meal or snack with fluoride toothpaste. Flossing will also help a lot. Being thorough with your brushing and flossing will stop any build of plaque forming.

Treatment: Your dentist will in most cases apply a filling to the tooth cavity, large cavities may need a crown. If the cavity damages the inner pulp then a root canal procedure or extraction of the tooth may be necessary to stop the toothache.

Tooth Abscesses occur when a dental cavity has been left untreated. The bacteria has infected the tooth from the inner pulp all the way up to the bone tissue at the end of the root causing severe toothache.

Treatment: Your dentist will have to carry out a root canal procedure where the pulp of the tooth is removed and then filled and sealed with an inert material. If this is unsuccessful then the tooth will have to be removed.

Gum Disease occurs when the soft tissue in our mouths becomes infected due to a build up of plaque or tartar along the gum line. It is highly likely that your toothache will be accompanied with bleeding gums if you have gum disease.

Treatment: In mild cases of gum disease your dentist will help you become more informed in order to improve your oral hygiene, they will also remove any build up of plaque. Root planning may need to be done which is the removal of plaque and tartar from the exposed roots. In more severe cases the surface of the inflamed gum tissue will have to be removed which is known as subgingival curettage. Oral antibiotics will also need to be taken alongside these procedures.

Jaw Disease usually occurs when there has been an impact or injury to the head such as whiplash. Bruxism (grinding of the teeth) often leads to TMJ as well as arthritis and having an over-bite. Jaw disease is often characterised by pain in the muscles around the jaw and limitations in jaw movement.

Treatment: Your dentist will fit a special intraoral splint for you to wear. If your bite needs to be fixed then crowns and orthodontic treatment are likely as well as medication to relieve the toothache.

Cracked Tooth can occur for many reasons such as an injury to the mouth, bruxism, chewing on hard objects or extreme changes in temperature on your teeth (such as eating hot food immediately followed by an iced drink) can all cause a tooth to crack and expose the dentin or inner pulp. The toothache may occur when the crack closes after releasing the pressure of a bite. The toothache gets worse over time if left untreated as the inner pulp can become infected.

If you have visible a crack in your teeth that is not accompanied by toothache then it is known as a ‘craze’ line and is considered to be part of the natural anatomy of the tooth, they usually occur as we age.

Treatment: Your dentist will evaluate the treatment needed depending on the severity of the crack. This can involve bonding for a small crack or a root canal treatment for a large crack where the inner pulp of the tooth has been damaged. In severe cases the tooth may need to be removed to stop the toothache.

In very rare cases toothache can also be caused by the following:

  • Angina – a disease of the throat marked by spasmodic attacks of intense suffocating pain
  • Heart Disease.
  • Myocardial Infarction – destruction of heart tissue resulting from obstruction of the blood supply to the heart muscle.

Whatever the cause of your toothache it is important you see a dentist so that they can determine the cause and apply the appropriate treatment or refer you to a doctor. If you have to wait for your appointment then to soothe the pain you can apply a cold compress to the area of the cheek where the toothache is. Rinsing your mouth with warm salt water and taking aspirin will also help. A good oral hygiene routine will prevent any toothache occurring.



Source by Peter Bradford

%d bloggers like this:
Skip to toolbar