Distinct C4 sub-types and C3 bundle sheath isolation in the Paniceae grasses.

Funder: U.S. Department of Agriculture; Id: http://dx.doi.org/10.13039/100000199Funder: University of Missouri; Id: http://dx.doi.org/10.13039/100007165In C4 plants, the enzymatic machinery underpinning photosynthesis can vary, with, for example, three distinct C4 acid decarboxylases being used to release CO2 in the vicinity of RuBisCO. For decades, these decarboxylases have been used to classify C4 species into three biochemical sub-types. However, more recently, the notion that C4 species mix and match C4 acid decarboxylases has increased in popularity, and as a consequence, the validity of specific biochemical sub-types has been questioned. Using five species from the grass tribe Paniceae, we show that, although in some species transcripts and enzymes involved in multiple C4 acid decarboxylases accumulate, in others, transcript abundance and enzyme activity is almost entirely from one decarboxylase. In addition, the development of a bundle sheath isolation procedure for a close C3 species in the Paniceae enables the preliminary exploration of C4 sub-type evolution

Boiling histotripsy lesion characterization on a clinical magnetic resonance imaging-guided high intensity focused ultrasound system - Fig 12

<p><b>a</b>. T1W imaging of porcine liver using fast field echo 3D used for planning HIFU sonications <b>b</b>. T1W imaging of the liver tissue post sonication shows a hypointense signal of a tadpole-shaped lesion created using BH (red dotted circle) <b>c</b>. Subtraction of pre-sonication from post-sonication (Fig 12b minus Fig 12a) images highlights the BH lesion. <b>d</b>. T2W imaging using turbo spin echo 3D were also performed <b>e</b>. T2W imaging post sonication showing the BH lesion along the coronal plane <b>f</b>. Subtraction of pre-sonication from post-sonication images (Fig 12e minus Fig 12d) highlighting the lesion (blue box) and surrounding blood vessels (blue arrowhead).</p

Ex vivo porcine liver (top panel) and cardiac muscle tissue (bottom panel) sonicated with 600 W at 5 Hz PRF.

<p><b>Top Panel: a</b>. Gross pathology of the lesion appeared thermally denatured, with a diameter of 8 mm (blue dotted circle). This area was not structurally intact and disintegrated during pathological stain preparation process. <b>b</b>. H&E slide showing diffuse thermal effects, with little cellular structure. <b>c</b>. Magnification (40X) of the Fig 10b with parts of the lesion showing absence of cellular structure, representing homogenization of tissue. <b>Bottom Panel. d</b>. Cardiac muscle with a liquefied void in the center of the lesion with sharp boundary with negligible thermal effects (red arrowhead). <b>e</b>. H&E slide of the lesion shows that cellular structure around the void was intact, with no evidence of thermal damage. The lesion also has sharp boundaries (red arrowhead). <b>f</b>. Magnification (40X) of the lesion boundary, with most of the boundary tissue intact.</p

Effect of each of the four BH sonication parameters on significant increase in lesion volume and focal region temperature are summarized.

<p>Increasing acoustic power did not result in an increase in lesion volume, whereas temperature distribution and area of lethal thermal dose increased. While increasing number of cycles/pulse, both lesion volume and temperature distribution increased. No increase in either lesion volume or temperature distribution was observed while increasing total sonication time, but area of lethal thermal dose increased. Varying pulse repetition frequency resulted in increased lesion volume and temperature distribution and area of lethal thermal dose.</p

Diagram showing the experimental setup used to produce BH lesions on a clinical MR-HIFU system in both tissue-mimicking phantom and ex vivo tissue.

<p>The setup consists of a water bath, filled with degassed water. This water bath was placed on an acoustically hypoehoic membrane. This membrane made it possible for the HIFU beam to pass through and into the water bath. The sonication pattern is a 3 × 3 × 3 cube with 1 mm spacing between each point in either direction. The lesion in top-view, looks like a square, and appears ‘tad-pole’ shaped in any plane parallel to the HIFU beam.</p

Ex vivo porcine liver (top panel) and cardiac muscle (bottom panel) sonicated at 600 W acoustic power at 1 Hz PRF and 20,000 cycles/pulse.

<p><b>a</b>. Gross pathology of the liver tissue with the lesion in the center (red arrowhead), showing minimal thermal damage with a liquefied central void (blue dotted circle). <b>b</b>. H&E slide showing the entire lesion with sharp boundaries (red arrowhead). <b>c</b>. Magnification (4X) of the Fig 9b, presenting intact cell structures at the periphery of the lesion. <b>d</b>. Gross pathology of the cardiac tissue with a large void in the center (blue dotted oval). A concentric ring of necrosis (blue arrowheads) surrounds the central void. <b>e</b>. H&E slide, with the void outlined by the blue dotted line and the blue arrowhead pointing to the region of necrosis. <b>f</b>. Magnified image (40X) of Fig 9e show regions of both necrosis and intact cellular structures.</p

Peak positive and negative pressures measured at varying acoustic powers using a fiber optic hydrophone.

<p>Peak positive and negative pressures measured at varying acoustic powers using a fiber optic hydrophone.</p

Temperature change measured at the first coronal slice for separate liver samples.

<p>The sonication parameters for both liver samples were 650 W of acoustic power, 15,000 cycles/pulse at 1 Hz PRF. The peak temperature was reached at the end of the sonication (64°C). Both temperature curves show similar dynamics.</p

Lesion volume in tissue-mimicking phantom for varying BH sonication parameters.

<p><b>a</b>. Lesion volume did not vary significantly for acoustic power 500–650 W at constant PRF of 1 Hz (p>0.05, ANOVA). <b>b</b>. Lesion volume with varying cycles/pulse. Significant differences (p ≤ 0.05) in lesion volumes were found between 10,000 and 18,000 cycles/pulse as well as 10,000 and 20,000 cycles/pulse. <b>c</b>. Varying total sonication time between 137 to 820 seconds resulted in no significant differences (p ≤ 0.05). <b>d</b>. Pulse repetition frequency (PRF) was varied from 0.5 to 5 Hz. The lesion volumes were significantly different between 0.5 and 3, 4, and 5 Hz (p ≤ 0.05). Similar differences were found between 1 and 3, 4 and 5 Hz, as well as between 2 and 4 and 5 Hz (p ≤ 0.05).</p