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Comparative study of fungal cell disruption—scope and limitations of the methods
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Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.
KeywordsOsmotic Shock Detergent Concentration Bead Mill Cell Wall Disruption Sonication Cycle
The microbial world offers a variety of products important and useful for human life, including chemical intermediates, antibiotics, enzymes, and therapeutic agents. Some of them are produced by microbes and are excreted into the cultivation medium and easily isolated. In contradiction, isolation and canvassing of intracellular products often require complicated sequences of operations. This task usually starts from a cell disruption process. Disintegration of cells precedes the fractionating and purification of constituent of the cell structures and influences the quality of the final product (Savov et al. 2001). Various cell disruption methods have been developed to establish an efficient, low-cost, and effective release of intracellular products (Middelberg 1995; Geciova et al. 2002; Ho et al. 2008). The application of certain disintegration techniques depends on such cell properties as physical strength of the cell wall and intracellular location of the desired product (Sauer et al. 1989; Kuboi et al. 1995). Complete destruction of the microbial cell in a nonspecific manner is usually achieved by ultrasound disruption of the cell (Borthwick et al. 2005; Gogate and Kabadi 2009). This process can proceed via complete breakage of the individual cells, releasing all intracellular enzymes, or it can be shear-driven, where only the cell wall breaks and as a result only the enzyme present in the cell wall or periplasm will be released (Balasundaram and Pandit 2001). The mechanism of cell disruption is associated with the cavitation phenomena, shear stress developed by viscous dissipative eddies arising from shock waves produced by imploding cavitation bubbles (Middelberg 1995; Harrison 1991; Garcia 1993). Another mechanical method to damage microbial cell walls of different structures is bead milling, applied successfully in both laboratory and industrial scales (Doucha and Livansky 2008). Compounds are released from cells due to the action of circulating beads dispersed in the fluid (Middelberg 1995; Doucha and Livansky 2008; Heim et al. 2007). A bead mill can be operated either in batch (Ricci-Silva et al. 2000) or in a continuous recycling mode (Ho et al. 2008). For a large-scale disintegration of the biological material, the mechanical method (the high-pressure homogenizer) is usually used. High-pressure cell opening is achieved by passing them through an adjustable, restricted orifice discharge valve (Engler 1985; Hetherington et al. 1971; Keshavarz-Moore et al. 1990; Kelly and Muske 2004). Non-mechanical processes have also been designed as alternatives to mechanical techniques, but the process-scale application is a crucial limitation in this case (Yasotha et al. 2006; Joubert and Fleurence 2008; Denis et al. 2009). The advantages of the application of enzymes are gentle process conditions and their specificity (Middelberg 1995; Kitamura 1982; Taskova et al. 2006; Ablain et al. 2009). Unfortunately, enzymatic digestion of the cell wall may be too expensive when it is used in a large scale. Permeabilization or lysis of cells can be performed also by chemical treatment. Antibiotics, chelating agents, detergents, and solvents are capable of disintegrating the cells (Dean and Ward 1992; Al-Samarrai and Schmidt 2000; Corrado and Rodrigues 2004; Leuko et al. 2008; Doolan and Wilkinson 2009). These procedures rely on the selective interaction of the chosen chemicals with components of the membrane and allow proteins to seep through the cell wall. There are at least two physical procedures—osmotic shock and thermolysis—also used for cell disintegration (Chen et al. 2004; Tkac et al. 2004; Ren et al. 2007a, b). The application of two or more procedures combined with the cells wall disruption is also reported (Anand et al. 2007; Lim et al. 2008; Chaves-Lopez et al. 2009; Shynkaryk et al. 2009).
Most of the published articles were focused on the disruption of Escherichia coli (Gram-negative bacteria), Bacillus subtilis (spore-forming Gram-positive bacteria), or yeast (Saccharomyces cerevisiae or Candida utilis; Ho et al. 2008; Ren et al. 2007a; Anand et al. 2007; Lim et al. 2008; Chaves-Lopez et al. 2009; Shynkaryk et al. 2009; Fonesca and Cabral 2002; Agrawal and Pandit 2003). Literature data devoted to other microorganisms, especially fungi, are relatively scarce (Taubert et al. 2000), meaning that they are still a not fully explored source of intracellular compounds of possible industrial applications. Therefore, this report focuses on the rationally planned studies performed in order to elaborate effective disruption methods (both mechanical and non-mechanical) using Rhodotorula gracilis, Aspergillus fumigatus, and Penicillium citrinum as model objects.
Materials and methods
All chemicals were purchased from commercial suppliers: Difco, POCh, Sigma-Aldrich.
Fungal strains
R. gracilis (Rhodosporidium toruloides) DSM 4444, was from DSMZ, Germany. A. fumigatus was a kind gift from the University of Pavia, Italy. P. citrinum was a kind gift from the University of Opole, Poland.
Cultivation media
A. fumigatus and P. citrinum were cultivated on a potato dextrose broth (Difco). R. gracilis was cultivated on a malt extract peptone broth (Difco). Microbial cultivation media were prepared according to the instructions of the suppliers and autoclaved.
Cultivation methods
Fungi were cultivated in 250-mL flasks containing 50 mL of the corresponding medium until the mid-log phase (25°C, 150 rpm, RT). After 5 days for yeast and 3 days for fungi, biomass was separated by centrifugation (10 min, 4,000 rpm, 4°C). The biomass was pooled and washed twice with buffer (Tris 5 mmol/L, pH 7.5).
Disintegration procedure
Samples of washed biomass (6.5 g) were suspended in 15 mL of cold buffer (Tris 50 mmol/L, pH 7.5) and disintegrated according to the procedures described below. The efficiency of cell disruptions was monitored by measuring two parameters: concentration of the soluble proteins in cell-free extract and the activity of the glucose-6-phosphate dehydrogenase (G6PD) assignment. All the experiments were carried out independently in triplicate.
Disintegration methods
Bead mill
The microbial cells were mixed either with glass (diameter, 0.5 mm) or zirconia (diameter, 0.7 mm) beads and disintegrated using a BioSpec BeadBeater (model 1107900) under cooling on ice. The total time of disintegration was 3 min in different cycles (30 s or 1 min of disruption and 2 min of cooling or 3 min of continuous process). After the separation of beads by decantation, cell homogenates were centrifuged (6,500 rpm, 15 min, 4°C; Hettich Universal 16 Zentrifugen) and the supernatant was analyzed.
Sonication was performed using a Torbeo Ultrasonic Processor (Fisher Scientific) device to provide continuous application of acoustic waves for 11 or 30 s. The sonication was then interrupted and the suspended cells cooled in an ice bath for 2 min, and the next sonication cycle was then performed. The total time of sonication was 1, 2, 3.5, or 5 min. The homogenate was centrifuged (6,500 rpm, 15 min, 4°C; Hettich Universal 16 Zentrifugen) and the supernatant analyzed.
Chemical disintegration
The sample of biomass was suspended in an appropriate water solution of detergents (Triton X-100, CTAB, Tween 80, SDS) and incubated with shaking in room temperature. Detergent concentrations were as follows: 0.1%, 0.25%, 0.5%, and 1% for R. gracilis; 0.5%, 1%, 1.5%, and 5% for P. citrinum and A. fumigatus. After the incubation period (5 or 20 min for yeast and 20 or 40 min for molds), the mixture was clarified by centrifugation (6,500 rpm, 15 min, 4°C; Hettich Universal 16 Zentrifugen) and the supernatant analyzed.
In the case of organic solvent treatment, microbial cells were suspended in 20 mL of Tris buffer (50 mmol/L, pH 7.5) and the mixture with the addition of 2 mL of toluene was shaken for 2 h; subsequently, the cells were separated by centrifugation and the cell-free extract was analyzed.
Osmotic shock
Microbial cells were suspended in 20 mL of hypertonic solution (50 mmol/L Tris–HCl buffer, pH 7.5, containing 10 mmol/L EDTA and 30% (w/v) sucrose) and gently shaken for 20 min. After that cell suspension was centrifuged and the pellet was disintegrated by suspending in 20 mL of 10 mmol/L MgCl2 over 20 min, the cells were removed by centrifugation and the supernatant was analyzed.
Analytical methods
Protein assay
Soluble protein concentrations in clarified supernatants were estimated by the Bradford method using bovine serum albumin as the standard (Bradford 1976).
Enzyme assay
The activity of the intracellular enzyme, glucose-6-phosphate dehydrogenase (E.C. 220.127.116.11), was determined by the method described by Bergmeyer (1983). Changes in absorption at 340 nm were monitored over a period of 10 min on UV/Vis Spectrometer (Varian Cary 100 Bio). Total activity of the crude extracts was given as the specific activity of the enzyme and was expressed in units per milligram of the total amount of proteins, where "U" is defined as 1 μmol NADPH formed per minute.
To establish the most efficient method of fungal cell lysis, three morphologically different fungal species—A. fumigatus, P. citrinum, and Rhodothorula gracilis—were subjected to various disruption protocols. The applied procedures imply the following results.
Comparison of the effectiveness of the mechanical cell disruption
Assayed values
Penicillium citrinum
Aspergillus fumigatus
Rhodotorula gracillis
Protein concentration (μg/mL)
Specific enzyme activity of G6PDa (U/mg, ×10−3)
Protein concentration (μg/mL)
Specific enzyme activity of G6PDa (U/mg, ×10−3)
Protein concentration (μg/mL)
Specific enzyme activity of G6PDa (U/mg, ×10−3)
Total timeb, 1 min (cycle: 11-s sonications, 2-min cooling)
1,300 ± 187
1.90 ± 0.13
258 ± 75
9.50 ± 1.20
3,121 ± 298
16.20 ± 1.70
Total timeb, 2 min (cycle: 11-s sonications, 2-min cooling)
890 ± 108
2.90 ± 0.20
285 ± 86
13.50 ± 0.87
5,212 ± 234
12.50 ± 3.60
Total timeb, 3.5 min (cycle: 11-s sonications, 2-min cooling)
975 ± 103
2.80 ± 0.24
300 ± 54
17 ± 0.98
5,166 ± 324
56.70 ± 2.90
Total timeb, 5 min (cycle: 11-s sonications, 2-min cooling)
900 ± 115
6 ± 0.87
278 ± 32
15 ± 0.85
8,444 ± 356
4.90 ± 0.78
Total timeb, 1 min (cycle: 30-s sonications, 2-min cooling)
320 ± 52
5 ± 0.76
87 ± 8.50
1.10 ± 0.13
3,638 ± 167
13.30 ± 1.70
Total timeb, 2 min (cycle: 30-s sonications, 2-min cooling)
220 ± 23
10.50 ± 0.89
35 ± 4.70
2.50 ± 0.17
2,950 ± 134
14.9 ± 1.50
Total timeb, 3.5 min (cycle: 30-s sonications, 2-min cooling)
370 ± 89
8 ± 0.87
41 ± 4.30
0.8 ± 0.10
4,921 ± 198
10.70 ± 1.80
Total timeb, 5 min (cycle: 30-s sonications, 2-min cooling)
250 ± 32
13.300 ± 0.65
9 ± 0.80
2.0 ± 0.20
4,254 ± 223
13.80 ± 2.80
Bead mill
Total timec, 3 min (glass beads; cycle: 30-s milling, 2-min cooling)
127 ± 13
15.40 ± 2.30
110 ± 12
5.50 ± 0.50
3,180 ± 98
22.30 ± 1.80
Total timec, 3 min (glass beads; cycle: 1-min milling, 2-min cooling)
90 ± 5
13.50 ± 4.50
121 ± 3
1 ± 0.10
3,611 ± 78
19.30 ± 2.50
Total timec, 3 min (glass beads, continuous process)
175 ± 23
12.40 ± 1.60
129 ± 15
1.40 ± 0.30
2,919 ± 36
20.50 ± 1.89
Total timec, 3 min (zirconium beads; cycle: 30-s milling, 2-min cooling)
134 ± 14
20 ± 1.97
141 ± 12
1.500 ± 0.25
134 ± 12
23 ± 1.98
Total timec, 3 min (zirconium beads; cycle: 1-min milling, 2-min cooling)
144 ± 23
18 ± 0.67
172 ± 13
1.55 ± 0.34
144 ± 12
23.70 ± 2.80
Total timec, 3 min (zirconium beads, continuous process)
157 ± 11
16.50 ± 0.98
140 ± 13
1.80 ± 0.24
159 ± 16
27 ± 1.68
Bead mill
The amount of proteins released from the cells of the Aspergillus strain was comparable to the values obtained by the sonication procedures. The short time of disintegration (30 s) and the use of smaller glass beads seemed to be optimal for allotted aims (Table 1). The most effective protocol for the disintegration of P. citrinum biomass seemed to be the procedure using a 30-s operating time and the use of 0.7-mm zirconium beads. In the case of Rhodotorula biomass, the level of obtained soluble proteins in the crude extracts disintegrated with zirconia beads was very low compared with the procedures in which glass beads of a smaller size were used. In turn, the highest specific activity of G6P dehydrogenase in the crude extract was achieved with the application of zirconia beads within the overall operating time of 3 min (Table 1).
Chemical treatment
The application of Triton X-100, which is a non-ionic type of detergent recommended for permeabilization and/or lysis of cell walls, was found to be ineffective for fungi (data not shown). In the case of P. citrinum, the activity of G6P dehydrogenase in the cell-free extracts was not found. The application of this detergent at a 5% concentration for the disintegration of A. fumigatus led to a very low level of specific activity of the model enzyme (1.46 ± 0.13 × 10−5 U/mg of proteins). The procedure based on Tween 80 was more effective for the tested molds. The best results were obtained for P. citrinum (7.33 ± 0.27 × 10−5 U/mg of proteins) when using 1.5% of Tween 80 for 40 min of treatment. On the other hand, for A. fumigatus, the G6P dehydrogenase activity was 3.8 ± 0.17 × 10−4 U/mg of protein, with 0.5% of detergent concentration and 40 min of the process. Both non-ionic detergents were useless for R. gracilis. Increasing the detergent concentration (up to 5%) resulted in the release of very high levels of total proteins from the cells of the Penicillium and Aspergillus strains (up to 7.5 and 12.3 mg/mL, respectively). Unfortunately, only in the case of P. citrinum did the cell-free extract show the required specific activities of G6P dehydrogenase—3.44 ± 0.32 × 10−5 U/mg of proteins (0.5%, 40 min) and 1.4 ± 0.09 × 10−5 U/mg of proteins (1.5%, 40 min). CTAB disintegration of yeast cells resulted in a low level of total proteins released (data not shown), and the most effective procedure appeared to be with 0.1% of detergent solution. Extending the time of incubation of the cells with detergents (from 5 to 20 min) allowed increasing the value of specific activities of the model enzyme from 1.62 ± 0.56 × 10−3 up to 2.23 ± 0.13 × 10−3 U/mg of proteins. The effects of anionic detergents on the microorganisms were also investigated using SDS as a model. The procedures based on the application of 0.5% SDS solution for molds and 0.1% for yeasts allowed detecting the specific activities of the model dehydrogenase in crude extracts. In the case of P. citrinum, after 40 min of incubation, the value of specific activity was 2.5 ± 0.06 × 10−4 U/mg of proteins, for A. fumigatus was 1.2 ± 0.08 × 10−4 U/mg of proteins (20 min), and for R. gracilis was 4.04 ± 0.23 × 10−3 U/mg of proteins (20 min). Also, the influence of toluene on the cohesion of the cell wall of the microorganisms was examined. The release of a very low amount of the proteins from the cells was observed, and there was no detectable activity of the model dehydrogenase in crude fungal extracts.
Osmotic shock
The disintegration of fungi with this method failed (data are not shown).
The overriding purpose of this study was to set up unique, effective, and repetitive disruption protocols for particular fungal species. The tested fungi have been chosen because of their industrial and/or medical importance. Rhodotorula strains are known for the intracellular production of useful amino acid oxidases (Kuan et al. 2008) and β-carotene (Vijayalakshmi et al. 2001). Secondary metabolites produced inside the cells of A. fumigatus and P. citrinum are also of commercial interest (Du et al. 2010; Chen et al. 2011; Malmstrom et al. 2000; Lim et al. 2007; Singh et al. 2011). Additionally, molds like A. fumigatus are widely considered as a hazard contaminant of food and human pathogen (Debeaupuis et al. 1997; Denning 1998). Molecular methods of detecting fungal pathogen in clinical or food samples require simple and fast methods that sufficiently lyse fungal cells.
The fungal wall structure is highly dynamic, changing constantly during cell division, growth, and morphogenesis. The specific composition and mechanical properties of the cell wall are varied not only between genera but also within closely related species. Moreover, this protective layer could change with growth conditions and genetic modifications (Firon et al. 2004; Klis et al. 2006). Thus, in the case of the sonication methods, most of the ultrasound energy, which is absorbed by cell suspension, appeared as heat; hence, temperature control is crucial and obligatory. Besides the cell types, the appropriate time of exposure of the biological material to the ultrasound is also a critical parameter influencing the activity of the intracellular compounds. In the case of P. citrinum strain, the prolongation of sonication cycles from 11 to 30 s admittedly resulted in the decrease of the amount of released proteins, but allowed obtaining a higher level of specific activity of G6P dehydrogenase in the crude extract (Table 1), suggesting the existence of a lower thermal stress. In contradiction, for the A. fumigatus cells, the short period of sonication was appropriate (11-s cycles and overall operation time of 3.5 min), giving as well the extract with a high level of released proteins and a high specific activity of G6P dehydrogenase. The level of proteins released under variable conditions of ultrasound disruption procedures is much higher in the case of yeast cells than molds. This can be simply explained by the different strengths and structures of the fungal cell walls which comprised glycoproteins and polysaccharides, mainly glucan and chitin. Chitin has an enormous tensile strength and significantly contributes to the overall integrity of the cell wall. Chitin microfibrils account for only 1–2% of the yeast cell walls by dry weight, whereas the cell walls of filamentous fungi, such as Aspergillus, are reported to contain 10–20% chitin (Bowman and Free 2006). Additionally, G6P dehydrogenases derived from A. fumigatus and R. gracilis seemed to be more sensitive to increasing temperature, which made prolongation of the disruption time useless because it caused the significant decrease of the specific activity of the examined model enzyme in the crude extract. The observed inactivation of dehydrogenase might also derive from ionization and subsequent free radical formation (Gogate and Kabadi 2009). Application of the bead mill turned out to be effective in the case of the destruction of Penicillium cells. Although the amount of the released proteins was lower, the specific activities of the model enzyme were surprisingly high (up to 20 ± 1.97 × 10−3 U/mg of proteins). However, in the case of Rhodotorula biomass, the kind of beads and their size were critical parameters, but the results were still unsatisfactory compared with those discussed previously, the sonication procedure. The bead mill is reported to be an efficient technique for rupturing yeast and mold cells compared with its effectiveness toward bacteria (Middelberg 1995). Many operating parameters of milling were used, the most important of which is the design of the stirrer, geometry of the grinding chamber, size of beads, and the temperature. In our case, the disruption method based on the application of BeadBeater® was effective only in the case of P. citrinum. Both A. fumigatus and R. gracilis cells were found to be resistant to milling disruption; thus, the sonication procedures are recommended in these cases. Finally, the chemical treatment led to unsatisfactory results in every experimental attempt. Admittedly, procedures based on detergent treatment, applied for the disintegration of Aspergillus and Penicillum cells, resulted in a high level of released proteins with low or no detectable activity of G6P dehydrogenase in cell-free extracts. The examined detergents inhibited the activity of G6P dehydrogenase and/or forced the conformational changes in the enzyme structure, which influenced the stability of fungal dehydrogenase. In the case of charged detergents, there is also the possibility of a destructive impact which comes from the ionic interaction between enzyme and detergent residues.
Non-mechanical cell disruption methods require less energy, although some of them resulted in the release of side products. However, their application is often limited to processes of a small scale, owing to restrictions of process economics or efficiency. Consequently, they have found limited commercial application.
To sum up, the undertaken efforts allowed elaborating protocols of the cells wall disruption for the examined fungi. The mechanical methods appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to enzymatically active cell-free extracts, which can be a starting point for the isolation and purification of intracellular components of interest.
This work was financed from Project "Biotransformations for pharmaceutical and cosmetics industry" no. POIG.01.03.01-00-158/09-05, part-financed by the European Union within the European Regional Development Fund for the Innovative Economy.
Open Access
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There's no secret that the console launch of Bluehole Studio's PUBG has been a little bit shitty, what with all the bugs, glitches and performance issues – fans were a bit saddened that they got the experience of an unpolished PlayStation 2 game on a current gen console. That being said, the team behind it has been extremely hard at work to iron out all the problems, and so far the horizon has been looking brighter and brighter.
Month: February 2018
Dragon Ball FighterZ the side-view fighter title from Bandai Namco has just received a new update. Update 1.04 is now available and introduces a host of new changes to the title.
Starting today, Lienzo's indigenous 3D action-adventure Mulaka will be descending upon multiple platforms. In it, you inherit the spirit of Sukurúame, a magically gifted being who's sniffed out a malevolent presence brewing in the Mexican sierras.
Last year, Funbakers treated digital snoopers worldwide to an augmented reality hunt in Silent Streets. Set within the mysterious, monochrome hush of the Victorian era, it challenged players with questioning murder suspects and tracking down a missing person. Episode 2 "Mockingbird" promises to deliver more of the same delicious duplicity.
As the highly anticipated March 6 release of Final Fantaasy XV Windows Edition draws closer, NVIDIA is celebrating the launch with its brand new Game Ready Driver.
Cybercriminals have figured out a way to abuse widely-used Memcached servers to launch over 51,000 times powerful DDoS attacks than their original strength, which could result in knocking down of major websites and Internet infrastructure.
kicking off season 3 of Fortnite: Battle Royale, a new timed mode has been introduced. The mode is available to PC, PS4 and Xbox One, and is called Solid Gold. Essentially the mode has switched out all regular loot with Legendary weapons, denoted by their Gold background and tier. Players will be able to find the legendary weapons everywhere on the map, chests, houses, supply drops.
Xenolinguistics, anyone? Heaven's Vault is a 3D adventure wrapped in antiquated puzzles and alien language decryption, and it's scheduled for release on PlayStation 4 this year.
At the beginning of the Warhammer 40K universe, there was a big bang. It was the sound of shots being fired by Brother Legions of Space Marines, the sound of betrayal when the treacherous Word Bearers ambushed the Ultramarines. The Horus Heresy: Betrayal at Calth formally invites you to join an epic battle of 31st millenium strategy.

subset: commoncrawl


text: I'm not sure if this counts, but my favorites have so far been "Shards of Honor" and "Barrayar". Miles is nice, but I loved Cordelia... Jill Wadsworth ----- Original Message ----- From: Courtney M Eckhardt <cme at MIT.EDU> To: <dwj at suberic.net> Sent: Thursday, August 24, 2000 10:40 AM Subject: Re: Lois McMaster Bujold > In message <126.96.36.199.20000824233245.00945f00 at pop.corplink.com.au>, Robyn Starke > y writes: > >Just to add my 2cents worth (in Australian currency worth about 1 cent), I > >read the Miles books in the order I could get my hands on them when I first > >got LMB addicted thanks to the comments on this list, and I think that the > >books suffer a lot less than many series from this treatment because they > >are all pretty complete stories in themselves. So if you can't acquire them > >in order quickly enough, you won't really wreck anything by reading them > >out of order. > > I agree with this... the first one I ever read was Brothers in Arms, and I > read the others in an order that could only be called random. I've reread > them in many different orders, and I learn new things about the characters > and the world every time. > > >Also, I must say, I never would have coped with MIrror Dance > >if I hadn't read a later book first. > > Really? I managed okay with Mirror Dance... possibly because I didn't > have much confidence in Mark but I did have confidence in the other > characters being able to fix things... (and I *really* liked the end scene > in the ballroom! :) The one I wouldn't have got through without > encouragement was Memory; I kept giving it up near the beginning with all > of the voices in my head screaming in concert at Miles: "No! No that's > stupid that's dumb you're gonna... when are you going to learn to listen > to Ellie!" :P > > In my case, the encouragement was that I had promised myself I wouldn't > read Komarr until I finished Memory, and I was *dying* to know who the > woman on the front cover looking at Miles was... ;) > > While we're on the subject (and off the topic... :), I was wondering if > other people had favorite books in that series, and what they were and > why. > > Courtney > -- > To unsubscribe, email dwj-request at suberic.net with the body "unsubscribe". > Visit the archives at http://suberic.net/dwj/list/ > -- To unsubscribe, email dwj-request at suberic.net with the body "unsubscribe". Visit the archives at http://suberic.net/dwj/list/


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The Columbus High basketball teams host Grand Island Northwest tonight. The girls game tips off at 5:45, with the boys contest to start at 7:30 p.m. The Discoverers wrestling team is on the road to face Elkhorn in a dual.
The Lakeview basketball squads play at home tonight against Logan View/Scribner-Snyder. The girls action starts at 6:15, with the boys game to start at 8 p.m.
And the Scotus basketball teams will travel to Omaha to face Concordia.


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I am searching to make use of either Firebird or Postgres during my next development project ... largely because both of them are available within BSD-like license.
I discovered an excellent comparison of these two database at http://www.amsoftwaredesign.com/pg_vs_fb
But this comparison is a great 2+ years of age and both databases came a lengthy ways since.
Does anybody mind upgrading the comparison table to become relevant for that current versions of both Firebird and Postgres ... or possess a connect to a website that does a great recent comparison between your two database?
You need to choose technology like RDBMS not (only) on evaluating listing of features but on responding to questions such as these:
- will it reliable enough to your requirements?
- will it suit all of your needs?
- what exactly are perspectives/support/developmet/documentation?
For me personally Facebook isn't a sensible choice. I'm able to point two tales:
- I authored constraint on table. Customers then add strings to table. Before long user is attempting to include yet another string, but Facebook gripes on constraint. Wrong? This new string is definitely under constraint. Issue is inside a last string which was added before it! That string was added a while ago, it had been wrong, and to date it's in DB, and you never know the number of wrong data have reached DB. Very bad. Facebook implements constraints inside a wrong manner.
- I authored a Saved Procedure (SP). Facebook saved it in DB, Facebook can run it, and delay pills work - choose data not surprisingly. Nobody warning. Then backup is performed without errors. And just on restore Facebook gripes that something is wrong with SP and ... what ... you've got no restorable backup copy now.
I'm able to point 2 or 3 more however these are sufficient that i can say: no, Facebook isn't my choice, it isn't RDBMS, it is a toy.
And they've bad documentation. Actually there's no actual references with every release. When individuals request in forums: exactly what is a full listing of functions in release XX? The conventional response is: take a listing from last official Interbase release and add (remove) functions all later releases's "release notes".
And they've strange support/development strategy. They're focusing on what exactly are interesting on their behalf, on deep intricacies, this is not on what exactly are really critical or annoying for customers.


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Court of Criminal Appeals of Tennessee, Nashville
November 14, 2017
STATE OF TENNESSEEv.JOHN DAVID SMARTT
Session February 15, 2017
from the Circuit Court for Warren County No. F-14190 Larry B.
Stanley, Jr., Judge
John David Smartt, was indicted for five counts of rape of a
child, (Counts 1, 2, 3, 6, and 9); one count of rape, (Count
12); three counts of especially aggravated sexual
exploitation of a child, (Counts 4, 7, and 10); and three
counts of aggravated sexual exploitation of a child (Counts
5, 8, and 11). Following a second jury trial, after the first
trial ended in a mistrial, Defendant was convicted as charged
on all twelve counts. The trial court imposed an effective
sentence of 153 years' incarceration. In this appeal as
of right, Defendant contends that: 1) the trial court erred
by allowing testimony regarding a recorded phone call by the
victim to Defendant; 2) the trial court erred by allowing
into evidence Defendant's wife's journal; 3) the
evidence was insufficient to support Defendant's
convictions; 4) this court should overturn prior case law, so
that the testimony of a victim regarding the content of
sexually explicit material must be corroborated; and 5) his
sentence is excessive. Having reviewed the entire record and
the arguments of the parties, we affirm the judgments of the
trial court.
R. App. P. 3 Appeal as of Right; Judgments of the Circuit
Court Affirmed
T. Crain, Murfreesboro, Tennessee, for the appellant, John
David Smartt.
Herbert H. Slatery III, Attorney General and Reporter; Brent
C. Cherry, Senior Counsel; Lisa Zavogiannis, District
Attorney General; and Thomas Miner, Assistant District
Attorney General, for the appellee, State of Tennessee.
T. Woodall, P.J., delivered the opinion of the court, in
which Robert W. Wedemeyer and Timothy L. Easter, JJ., joined.
victim in this case, whom we will identify by her initials,
J.S., was 20 years old at the time of trial. Defendant is her
father. She testified that Defendant had not worked since
2001, due to his degenerative disc disease and
osteoarthritis. J.S.'s mother "always had a
job" and worked as a certified nursing assistant. J.S.
testified that her mother typically left the house at 5:30
a.m. to go to work and left work to come home at 4:30 p.m.
J.S. testified that her mother also had second and third jobs
and worked "late into the night." J.S. and her
brothers were left in the care of Defendant.
testified that on an occasion when she was four years old,
she was alone with Defendant in the living room, and
Defendant turned on a pornographic movie. She described the
movie and testified, "it went to a shot of a woman
giving a man a blow job and [Defendant] kind of explained to
me, you know, that she was enjoying it, he was enjoying
it." She testified that Defendant then took her into his
bedroom, and "it was the first time that I remember he
told me to give him a blow job and when I wasn't
understanding what I was supposed to be doing he just kept
saying, suck it like a straw . . . ." J.S. testified
that she "couldn't do what he wanted [her] to do and
he stopped" and told her not to tell anyone about the
incident. She testified that Defendant "put his penis in
[her] mouth and wanted [her] to suck on it."
J.S. described another occasion when Defendant showed her a
There was a home video of my mom that he showed to me and
told me, you know, that she liked what she was doing and it
like started out with her playing with herself with a toy and
the camera was up close and you could hear him talking and
then later on in the video it backs up and she is on the bed
and then he gets onto the bed with her and they have sex and
he showed it to me. I only remember seeing it one time but it
was when we lived in Morrison.
testified that she did not remember how old she was at the
time of the incident, but she estimated that she was eight or
nine years old. She described another incident when Defendant
showed her a sexually explicit video:
On one other occasion that he showed me a video on the
computer. It was like of a young girl and a man where she was
playing to be a young girl. In the video she was playing like
his daughter and he was getting on to her because her room
was dirty and told her that he needed to punish her and like
she runs upstairs and then the video starts out with her
giving him a blow job and then proceeds to go on and they
have sex.
. . . .
She looked young. I mean I guess she was an adult playing
like a teenager but she looked young.
testified that Defendant told her, "that's what
happens to bad girls." She testified that Defendant
asked her to perform oral sex "basically . . . on a
daily basis. It happened so frequently that I knew when he
cracked the door open and poked his head in and said, [J.S.],
come here, I knew what was going to happen."
She described an incident when she was in kindergarten:
I was in kindergarten. We were living in a house on Bybee
Drive here in McMinnville and he brought me into his bedroom,
my parents' bedroom, and he had me lay on the bed and try
like while I was laying down trying to do it upside down
while he would stand up on the side of the bed and try to
give him a blow job and he would lean over me and I would be
laying down and he had me do that until he ejaculated and
when he came on my body he asked me what it felt like and I
didn't know. The only thing I associated it with was like
ketchup so I told him that it felt like hot ketchup.
testified that she did not know that Defendant's behavior
was wrong at the time. She testified that Defendant told her,
"that's what daddies do to show their little girls
that they love them . . . so I thought that if I did that
that he would be nice to me."
testified about an incident that occurred in the family's
minivan when she was in kindergarten. She testified that
Defendant picked her up from school, and stopped the car and
"had [her] come up to the front of the van and he pulled
out his penis and he told [her] to suck it and [they] sat at
the stop sign and [she] gave him a blow job." She
testified that "[i]t was the first and only time that
that had ever happened."
also testified about an incident when Defendant took
photographs of her. She testified that Defendant picked her
up from basketball camp and took her home. She testified,
[H]e brought me to my parents' bedroom and it was just me
and him in there. My mom had just gotten him a digital camera
that he had been wanting for a long time and he got a white
towel and laid it over the foot of their bed and told me to
get undressed except for the shirt and he wanted me to get on
my knees so that he could take pictures from the angle
looking straight on to my vagina from that angle and he would
have me hold my hair up and look up and my face was not in
the pictures. He took a bunch of pictures and when he was
finished he was like, look, and after I got dressed he showed
me - he pulled them up on the screen and showed me the
pictures that he had just taken.
J.S. was embarrassed and concerned that Defendant would show
the photos to other people. She remembered that the incident
occurred when she was in fourth or fifth grade because
that's when she attended basketball camp.
testified that when she was in fifth grade, she was taken out
of class one day and asked questions about her family by a
lady she did not know. The lady asked J.S. if her father had
ever harshly disciplined her, whether she had ever seen her
parents or siblings without their clothing, and whether she
had ever seen a movie that showed people not wearing clothes.
J.S. denied that any of those things had happened. She
testified, "I didn't know who she was or why she was
asking me those questions and my parents had always said,
'what happens under this roof stays under this roof. . .
.'" She testified that Defendant imposed
"ridiculously horrible punishments for things that
shouldn't have been an issue and so I was afraid of
telling . . . ."
Count 6 of the indictment, J.S. testified that when she was
12 years old, she began menstruating. Defendant told her,
"[w]hen you start your period you will be a woman and we
can have sex and we can make a baby and he would ask me if I
had his baby if he would be the baby's dad or the
baby's grandpa . . . ." J.S. testified that shortly
after she started having periods,
[Defendant] took me into his room he took off all my clothes
and he was wearing his pants but he had pulled his penis out
of the zipper hole and he pulled me up to the very edge of
the bed and tried to wrap my legs around his waist and was
trying to like thrust into me but I kept squeezing my legs so
that my body would move as he tried to push forward that I
would just slide up the bed. I didn't want that to happen
and then he started trying to grab my shoulder and just
pushing me into him or himself into me just very rough and
like he was rubbing himself up and down my vagina with his
penis and trying to spit on it and he was pushing in and he -
I could feel that he was like trying to push into my hole and
then I hollered out that it had hurt. He told me to go to the
bathroom and wash myself out.
Counts 7 and 8 of the indictment, J.S. testified that
Defendant showed her photographs on his digital camera of her
mother in their bedroom. J.S. described the photographs
Defendant showed her:
Some of them like he was holding her vagina lips open or had
his finger inside of her or she was doing the same thing,
holding herself open and like he would - he showed me there
was a picture of her vagina and there was like white, creamy
stuff and he asked me if I knew what that was and then
explained to me that he had made her have an orgasm.
Defendant showed J.S. the photographs, he told her take off
her clothes "and pose the same way that [her mother]
had." She testified that Defendant "took the exact
same pictures of [her] with the exclusion of [her] finger
inside of [her] or his finger inside of [her] vagina and the
one where she had cream on her." Defendant then showed
J.S. the photographs he took of her. J.S. was around 13 years
old at the time of the incident. She testified that, unlike
when she was younger, she understood that Defendant's
actions were wrong. She testified, "I wanted him to be
nice to me. I didn't want him to hurt me."
Count 9 of the indictment, J.S. testified that her brothers
were all spending the night at her grandparents' houses,
and she was alone with Defendant. She was nine or ten years
old at the time of the incident. She testified,
It was an awful, awful day because from the moment that I
woke up until soon before my mom came home I had to spend the
entire day naked. [Defendant] fixed a breakfast for us and he
was completely naked wanting us to eat breakfast but I just
felt so uncomfortable that I couldn't eat and we were in
the dining room and he was telling me how sexy that I was and
like he was pulling my leg up on to his side and I remember
looking over in the reflection of the fish tank and I could
see myself and he was telling me that I'm sexy and I
remember just thinking I don't look anything like my mom.
I wasn't developed in the same way. I was just a little
girl and I was confused really, mainly just confused and
disgusted and throughout the day it was just he would - he
would put his mouth on my vagina and the very end of the day
what finally led him to let me put my clothes back on is he
had me give him a blow job and he like ejaculated in my mouth
but I wouldn't swallow it and I ran to the bathroom and
spit it out and he got mad at me and was telling me that
there were thousands of girls in this world that would kill
to be in my position and that I should have swallowed it and
be grateful for what I had and he let me put my clothes back
on and hooked up the Playstation and just went out in the
living room and acted like nothing ever happened when my mom
came home.
to Counts 10 and 11 of the indictment, J.S. testified that on
one occasion, when she was 11 or 12 years old, Defendant made
a video with his cell phone of her performing oral sex on
him. She testified,
[H]e pulled out his penis and he told me to suck it and he
had his phone out taking a video of it and then afterwards he
showed me the video of it and that was the last time I ever
saw it and I didn't even see the whole thing. He just
showed me a little bit of it.
testified, "I saw my face and I was sucking his
to Count 12 of the indictment, J.S. testified that when she
was a freshman in high school, Defendant again had vaginal
intercourse with her:
It was very similar to the first time as far as the place and
the side of the bed and exactly what he was doing. He was
rubbing his penis up and down my vagina and he was trying to
push himself into me really hard and I kept trying to avoid
it by stiffening up and moving with him but this time he
actually - I felt him push himself inside my vagina and it
was horrible and painful and I screamed out that it was
painful and [ ] he stopped instantly and was telling me that
he was sorry and that he didn't mean to hurt me but he
testified that the first time she told anyone about
Defendant's sexual abuse, she was 11 or 12 years old. She
told her older brother. She testified, "I told him
everything. I mean we sat and talked for a while and he was
upset and crying and he told me that - like everything that
with what he's been through and what I've been
through, we need to tell Mom." J.S. and her brother
devised a plan that she would scream and run away from
Defendant the next time he attempted to abuse her, and they
would call her brother's friend to drive them to their
mother's workplace. J.S. testified, "a couple of
days later [Defendant] pulled me downstairs and he tried to
force my hand down his pants and when I yanked away I yelled
no and I ran up as fast as I could. We wedged the door. He
didn't chase me . . . but we wedged the door anyway . . .
." She testified that they did not have time to call her
brother's friend and drive to her mother's workplace
before her mother got off work, "[s]o instead we just
took the chair away from the door and waited for my mom to
come home."
evening, J.S.'s brother told their mother that Defendant
had been molesting her since she was four years old. J.S.
testified that her mother left the room and came back into
the room with Defendant. J.S.'s mother told her,
"you're ...
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