David (Dave) Charles Fork (1929-2020): a gentle human being, a great experimenter, and a passionate researcher.

We provide here an overview of the remarkable life and outstanding research of David (Dave) Charles Fork (March 4, 1929-December 13, 2021) in oxygenic photosynthesis. In the words of the late Jack Edgar Myers, he was a top 'photosynthetiker'. His research dealt with novel findings on light absorption, excitation energy distribution, and redistribution among the two photosystems, electron transfer, and their relation to dynamic membrane change as affected by environmental changes, especially temperature. David was an attentive listener and a creative designer of experiments and instruments, and he was also great fun to work with. He is remembered here by his family, coworkers, and friends from around the world including Australia, France, Germany, Japan, Sweden, Israel, and USA.

Quantitative Comparison of Virtual Monochromatic Images of Dual Energy Computed Tomography Systems: Beam Hardening Artifact Correction and Variance in Computed Tomography Numbers: A Phantom Study.

OBJECTIVE: The aim of this study was to quantitatively compare the reduction in beam hardening artifact (BHA) and variance in computed tomography (CT) numbers of virtual monochromatic energy (VME) images obtained with 3 dual-energy computed tomography (DECT) systems at a given radiation dose. METHODS: Five different iodine concentrations were scanned using dual-energy and single-energy (120 kVp) modes. The BHA and CT number variance were evaluated. RESULTS: For higher iodine concentrations, 40 and 80 mgI/mL, BHA on VME imaging was significantly decreased when the energy was higher than 50 keV (P = 0.003) and 60 keV (P < 0.001) for GE, higher than 80 keV (P < 0.001) and 70 keV (P = 0.002) for Siemens, and higher than 40 keV (P < 0.001) and 60 keV (P < 0.001) for Toshiba, compared with single-energy CT imaging. CONCLUSIONS: Virtual monochromatic energy imaging can decrease BHA and improve CT number accuracy in different dual-energy computed tomography systems, depending on energy levels and iodine concentrations.

Ideal osmotic spaces for chlorobionts or cyanobionts are differentially realized by lichenized fungi.

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity; osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.

Low-Dose Pelvic Computed Tomography Using Adaptive Iterative Dose Reduction 3-Dimensional Algorithm: A Phantom Study.

OBJECTIVE: To evaluate the image quality and radiation dose reduction in pelvic computed tomography (CT) achieved with an adaptive iterative dose reduction 3-dimensional (AIDR 3D) algorithm using a phantom model. METHODS: Two phantoms were scanned using a 320-detector row CT scanner with 8 tube current levels, and the images were reconstructed with a standard filtered back projection (FBP) algorithm and with an AIDR 3D algorithm. RESULTS: Compared with FBP, AIDR 3D reduced image noise and improved contrast-to-noise ratios. The diagnostic performance for detection of low-contrast targets of AIDR 3D images obtained with 100 mA at 120 kVp was almost as good as that of the FBP images obtained with 200 mA. CONCLUSIONS: The AIDR 3D algorithm substantially reduced image noise and improved the image quality of pelvic CT images compared with those obtained with the FBP algorithm and can thus be considered a promising technique for low-dose pelvic CT examinations.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms.

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000µmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Arabitol provided by lichenous fungi enhances ability to dissipate excess light energy in a symbiotic green alga under desiccation.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

Spectral properties of a divinyl chlorophyll a harboring mutant of Synechocystis sp. PCC6803.

A divinyl chlorophyll (DV-Chl) a harboring mutant of Synechocystis sp. PCC 6803, in which chlorophyll species is replaced from monovinyl(normal)-Chl a to DV-Chl a, was characterized. The efficiency of light utilization for photosynthesis was decreased in the mutant. Absorption spectra at 77 K and their fourth derivative analyses revealed that peaks of each chlorophyll forms were blue-shifted by 1-2 nm, suggesting lowered stability of chlorophylls at their binding sites. This was also true both in PSI and PSII complexes. On the other hand, fluorescence emission spectra measured at 77 K were not different between wild type and the mutant. This indicates that the mode of interaction between chlorophyll and its binding pockets responsible for emitting fluorescence at 77 K is not altered in the mutant. P700 difference spectra of thylakoid membranes and PSI complexes showed that the spectrum in Soret region was red-shifted by 7 nm in the mutant. This is a characteristic feature of DV-Chl a. Microenvironments of iron-sulfur center of a terminal electron acceptor of PSI complex, P430, were practically the same as that of wild type.

S4 protein Sll1252 is necessary for energy balancing in photosynthetic electron transport in Synechocystis sp. PCC 6803.

Sll1252 was identified as a novel protein in photosystem II complexes from Synechocystis sp. PCC 6803. To investigate the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. Despite the homology of Sll1252 to YlmH, which functions in the cell division machinery in Streptococcus, the growth rate and cell morphology of the mutant were not affected in normal growth medium. Instead, it seems that cells lacking this polypeptide have increased sensitivity to Cl(-) depletion. The growth and oxygen evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells when Cl(-) and/or Ca(2+) was depleted from the medium. Recovery of photosystem II from photoinhibition was suppressed in the mutant. Despite the defects in photosystem II, in the light, the acceptor side of photosystem II was more reduced and the donor side of photosystem I was more oxidized compared with wild-type cells, suggesting that functional impairments were also present in cytochrome b(6)/f complexes. The amounts of cytochrome c(550) and cytochrome f were smaller in the mutant in the Ca(2+)- and Cl(-)-depleted medium. Furthermore, the amount of IsiA protein was increased in the mutant, especially in the Cl(-)-depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. The amount of accompanying cytochrome c(550) in purified photosystem II complexes was also smaller in the mutant. Overall, the Sll1252 protein appears to be closely related to redox sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

Multiple dissipation components of excess light energy in dry lichen revealed by ultrafast fluorescence study at 5 K.

A time-resolved fluorescence study of living lichen thalli at 5 K was conducted to clarify the dynamics and mechanism of the effective dissipation of excess light energy taking place in lichen under extreme drought conditions. The decay-associated spectra obtained from the experiment at 5 K were characterized by a drastically sharpened spectral band which could not be resolved by experiments at higher temperatures. The present results indicated the existence of two distinct dissipation components of excess light energy in desiccated lichen; one is characterized as rapid fluorescence decay with a time constant of 27 ps in the far-red region that was absent in wet lichen thalli, and the other is recognized as accelerated fluorescence decay in the 685-700 nm spectral region. The former energy-dissipation component with extremely high quenching efficiency is most probably ascribed to the emergence of a rapid quenching state in the peripheral-antenna system of photosystem II (PS II) on desiccation. This is an extremely effective protection mechanism of PS II under desiccation, which lichens have developed to survive in the severely desiccated environments. The latter, which is less efficient at 5 K, might have a supplementary role and take place either in the core antenna of PS II or aggregated peripheral antenna of PS II.

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis.

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.

Responses to desiccation stress in lichens are different from those in their photobionts.

In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to air-drying and hypertonic treatments in a green-algal lichen (a chlorolichen, Ramalina yasudae Räsänen) and its green algal photobiont (freshly released and cultured Trebouxia sp.) were studied. Responses to dehydration in the isolated Trebouxia sp. were different from those in the lichen, R. yasudae, i.e. (i) the PSII reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained partially active in isolated Trebouxia sp; (ii) dehydration-induced quenching of PSII fluorescence was less in the isolated Trebouxia sp. compared with that in R. yasudae, suggesting that a substance(s) or a mechanism(s) to dissipate absorbed light energy to heat was lost by the isolation of the photobiont; and (iii) the air-dried isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae. These results support the idea that association of the photobionts with the mycobionts increases tolerance to photoinhibition under drying conditions.

Ycf12 is a core subunit in the photosystem II complex.

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of approximately 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune.

A desiccation-tolerant cyanobacterium, Nostoc commune, shows unique responses to dehydration. These responses are: (i) loss of PSII activity in parallel with the loss of photosynthesis; (ii) loss of PSI activity; and (iii) dissipation of light energy absorbed by pigment-protein complexes. In this study, the deactivation of PSII is shown to be important in avoiding photoinhibition when the Calvin-Benson cycle is repressed by dehydration. Furthermore, our evidence suggests that dissipation of light energy absorbed by PSII blocks photoinhibition under strong light in dehydrated states.

The expression pattern of NAD(P)H oxidases and the cyclic electron transport pathway around photosystem I of Synechocystis sp. PCC6803 depend on growth conditions.

Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and high-light (300 microE m(-2) s(-1))-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl(2), but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO(2) (0.03%) or normal (100 microE m(-2) s(-1), 3% CO(2)) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl(2). In high-salt (0.55 M NaCl)-grown cells, the amount of ferredoxin-NADP(+) oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl(2) were strong inhibitors of the FNR route.

Effects of reconstruction technique on the quality of abdominal CT angiography: A comparison between forward projected model-based iterative reconstruction solution (FIRST) and conventional reconstruction methods.

PURPOSE: This study aimed to compare the quality of abdominal CT angiography (CTA) images obtained using conventional reconstruction algorithms with those obtained using a novel iterative algorithm (forward projected model-based iterative reconstruction solution, FIRST) for evaluating arteries in the abdomen. MATERIALS AND METHODS: Abdominal CTA images from 60 patients (M:F = 27:33; mean age, 62.4 ± 16.7 years) were reconstructed using three algorithms - filtered back projection (FBP), adaptive iterative dose reduction 3D (AIDR 3D), and FIRST. Image noise, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) in the abdominal aorta, celiac trunk, superior mesenteric, renal, and right hepatic arteries were objectively evaluated via region-of-interest analysis and compared using the one-way analysis of variance test. Two radiologists independently scored and selected the best (score 3), second best (score 2), and the worst (score 1) images based on the visual image quality of peripheral arteries. Wilcoxon signed rank test was used for comparing image quality scores. RESULTS: FIRST and AIDR 3D significantly reduced image noise compared with FBP (P < 0.001). SNR and CNR were significantly higher with AIDR 3D and FIRST than with FBP reconstruction (P < 0.001), with FIRST displaying the highest CNR (14.31 ± 4.17) for the right hepatic artery than the other two methods (P < 0.05). Both radiologists scored FIRST images as having the best image quality among the three methods for peripheral abdominal artery evaluation (3.0 ± 0.0, P < 0.001). CONCLUSION: FIRST reconstruction yielded superior abdominal CTA images as compared with FBP or AIDR 3D reconstruction.

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis.

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-beta-D-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.

Jaw muscle functional anatomy in northern grasshopper mouse, Onychomys leucogaster, a carnivorous murid.

The jaw muscle anatomy of the northern grasshopper mouse, Onychomys leucogaster, was observed and the mechanical basis of the insectivorous/carnivorous adaptations were examined. Compared with Peromyscus maniculatus, a granivorous relative of Onychomys, there is a reduction of some aponeuroses within the masseter deep layer. This characteristic indicates that shearing meat or crushing arthropod exoskeletons requires less occlusal pressure than does grinding plant material. In Onychomys both the anterior and posterior portions of the masseter deep layer are more anterodorsally inclined, so that the line of action of the masseter lies further from the jaw joint than in Peromyscus. A strong incisal bite for killing vertebrates such as other rodents can be produced by a jaw mechanism with the high lever advantage of this muscle, which compensates for the decline in muscle mass. Our quantitative analysis suggests that the disappearance of an aponeurosis along the zygomatic plate in Onychomys decreases the stretch of the corresponding muscle, i.e., the anterior fibers of the masseter deep layer, accompanying jaw opening, and increases the maximum gape necessary for hunting large prey.

Internal architecture, origin-insertion site, and mass of jaw muscles in Old World hamsters.

The jaw muscle (i.e., masticatory, suprahyoid, and extrinsic tongue) anatomy and mass were examined in four genera of Old World hamsters (cricetine murids), Mesocricetus, Cricetulus, Tscherskia, and Phodopus. The masseter was the largest and most complicated of the muscles examined. In the superficial layer, a few ventral fibers form a small medially turned portion with an insertion site more similar to those of sciurids than of other murids. In Mesocricetus, the superficial layer has a discrete anteroventral portion that has not been reported for other murid rodents. Examination of the fiber attachment sites indicated that the deep layer contains four parts and the medial layer contains three parts. The deep layer originates from two aponeuroses that are firmly connected to each other at their anterior ends and lie along the zygomatic arch. The aponeurosis of insertion for the deep layer is situated along the masseteric ridge and the dorsal border of the angular process, but is absent in its middle part, consistent with reports in two relatives, sigmodontine and arvicoline murids. In cricetine murids, unlike in other rodents, fibers insert on the dorsal narrow strip of the posterior mandibular aponeurosis, not on its broad medial aspect. The relative mass of some masticatory and suprahyoid muscles is related to body mass. Small species (Cricetulus and Phodopus) have relatively larger masseter and mylohyoid muscles and smaller temporalis and geniohyoid muscles than large species (Mesocricetus and Tscherskia).

Short piston-cylinder pressure cells based on Ni-Cr-Al cylinders and their application to fragile materials.

In this decade, the performance of piston-cylinder pressure cells has been drastically improved by using robust materials such as nickel-chromium-aluminum and cobalt-nickel-chromium-molybdenum alloys to construct the inner cylinders. In this article, we present several experimental techniques for carrying out resistivity measurements under high pressure by applying the piston-cylinder devices based on the nickel-chromium-aluminum cylinders to fragile materials such as organics. These techniques are, in principle, applicable to measurements on any solid-state conductor. First, we introduce the construction of our piston-cylinder cells including two kinds of wired platforms for transport measurements. Second, we describe the construction of the platforms and the method of introducing the samples. After reporting test results for conventional materials such as ammonium fluoride, bismuth, and tellurium, lastly, we present examples of the successful application of our method to organic materials.

Shallow sink in an antenna pigment system of photosystem I of a marine centric diatom, Chaetoceros gracilis, revealed by ultrafast fluorescence spectroscopy at 17 K.

The ultrafast fluorescence dynamics of photosystem I (PS I) purified from a marine centric diatom, Chaetoceros gracilis, at 17 K was studied using fluorescence up-conversion and streak-camera setups. The experiments were done for two kinds of sample preparations containing different amounts of the peripheral antenna proteins, the fucoxanthin-chlorophyll (Chl) binding proteins associated with PS I (FCPI). Upon excitation at 430 nm, which selectively excites Chl a mainly contained in the core complex, the fluorescence dynamics of both samples was roughly expressed by four decay-associated spectra (DASs) with time constants of ca. 5, ca. 22, ca. 100, and ca. 400 ps. These DAS components have corresponding counterparts in the results of a previous study of Thermosynechococcus elongatus PS I (Shibata et al. J. Phys. Chem. B 2010, 114, 2954) except for that with a time constant of ca. 22 ps. The similar distribution of the time constants suggests a shared light-harvesting pathway by PS I of these two organisms. The DAS with a ca. 400 ps time constant has its peak wavelength at around 710 nm, suggesting the presence of antenna pigment states with slightly lower excitation energy than that of P700. This antenna state acts as a shallow sink in the core complex of the diatom PS I and causes a specific temperature dependence of its fluorescence spectrum below 77 K. Excitation energy funneling into the shallow-sink state seems to take place within 0.2 ps, suggesting an extremely efficient energy transfer. Upon the selective excitation of Chl c in FCPI by a 460 nm laser, three DAS components suggesting excitation energy transfers were obtained. The 0.2 ps DAS shows the energy transfer from Chl c to Chl a within FCPI, while the 0.7 and 40 ps DASs suggest the energy transfer from FCPI to the core complex. The excitation energy seems to be effectively transferred from FCPI to the core complex in diatom PS I because the selective excitation of Chl c in FCPI does not induce a severe retardation of the overall light-harvesting kinetics.