Bacteria such as Escherichia coli are frequently studied during exponential- and stationary-phase growth. However, many strains can survive in long-term stationary phase (LTSP), without the addition of nutrients, from days to several years. During LTSP, cells experience a variety of stressors, including reactive oxidative species, nutrient depletion, and metabolic toxin buildup, that lead to physiological responses and changes in genetic stability. In this study, we monitored survival during LTSP, as well as reporters of genetic and physiological change, to determine how the physical environment affects E. coli during long-term batch culture. We demonstrate differences in yield during LTSP in cells incubated in LB medium in test tubes versus Erlenmeyer flasks, as well as growth in different volumes of medium. We determined that these differences are only partially due to differences in oxygen levels by incubating the cells in different volumes of media under anaerobic conditions. Since we hypothesized that differences in long-term survival are the result of changes in physiological outputs during the late log and early stationary phases, we monitored alkalization, mutation frequency, oxidative stress response, and glycation. Although initial cell yields are essentially equivalent under each condition tested, physiological responses vary greatly in response to culture environment. Incubation in lower-volume cultures leads to higher oxyR expression but lower mutation frequency and glycation levels, whereas incubation in high-volume cultures has the opposite effect. We show here that even under commonly used experimental conditions that are frequently treated as equivalent, the stresses experienced by cells can differ greatly, suggesting that culture vessel and incubation conditions should be carefully considered in the planning or analysis of experiments.
Escherichia coli can survive in batch culture for days, weeks, or even years without the addition of nutrients (1–4). While cells grown in laboratory culture are frequently examined during the first three phases of the bacterial life cycle (lag phase, exponential or logarithmic growth phase, and stationary phase), two additional phases can be observed when cells are incubated in long-term batch culture. After stationary phase, cells can enter the fourth phase, termed death phase, in which approximately 99% to 99.9% of the cells in the culture lose viability, depending on the strain and culture conditions. Cells that survive death phase may then enter long-term stationary phase (LTSP), in which the total number of cells remains roughly constant, accompanied by significant shifts in subpopulations within the culture (1, 3, 5, 6). Survival of cells better suited to their particular environment during LTSP leads to selection of beneficial alleles, called GASP (growth advantage in stationary phase) mutations, and therefore evolution of these populations (2–4, 6, 7).
Although the mechanisms controlling entry into death phase are not well understood, several stressors may contribute to cell death in long-term batch cultures. A major factor contributing to death phase is likely endogenous oxidative stress from normal cellular metabolism of growing cells (8–13). During the transition into stationary phase, oxidative stress response genes are often induced (11, 14). One mechanism by which oxidative stress contributes to cell death is the promotion of glycation, or nonenzymatic glycosylation, of proteins and nucleic acids in cells (15–18). It has been shown that death due to glycation can be abrogated by addition of the antiglycation agents carnosine and aminoguanidine (15, 19–21). Another factor modulating the survival of cells in batch culture is changes in pH (22–25). Typically, when E. coli is grown in Luria-Bertani (Lennox) medium (LB), cultures become slightly alkaline during log phase growth, with pH further increasing during early stationary phase. This increase in pH contributes to cell death and affects the timing of entry into long-term stationary phase (25–27).
Commonly, laboratory experiments may necessitate incubation of different volumes of cultures depending on the desired cell, protein, or plasmid yield, etc. In fact, growing cells in different culture volumes is routinely suggested as interchangeable in several commonly used reference handbooks (28–30). For example, in section 1.25 of Molecular Cloning: a Laboratory Manual, by Sambrook and Russell (28), it is suggested that for small-scale preparations of DNA, 2-ml cultures of E. coli should be grown in 15-ml test tubes (∼1/10 volume), while for large-scale preparations, it is suggested in section 1.33 that cells be cultured in 525 ml of media in a 2-liter flask (∼1/4 volume). In these cases, cultures may be scaled up from incubation in test tubes to flasks or even up to larger-scale bioreactors or fermentors. In fact, in common practice, the final cell yield (CFU/ml) appears to depend exclusively on the growth medium rather than the type of vessel used (e.g., 5 ml of cells grown in a test tube and 12.5 ml grown in a flask reach the same final density of ∼5 ×109 CFU/ml). However, in different vessels, cells likely experience different levels of oxidative or other stresses, which may lead to differences in survival and mutation frequency. We hypothesized that these differences can be measured and signatures of growth in specific environments could be revealed. Here we show that by measuring survival and physiological responses of cells grown in different vessels, while controlling for oxygen availability by incubating cells both in different culture volumes and anaerobically, we can identify factors affecting long-term survival.
Bacterial strains and growth conditions.An E. coli K-12 lineage strain, MG1655 derivative PFM2 (31), was used in this study. Unless otherwise stated, cultures were initiated by transferring cells directly from a frozen 20% glycerol stock into 5 ml of Luria-Bertani (Lennox) medium (LB) (Difco) in an 18- by 150-mm borosilicate test tube and incubated overnight with aeration in a TC-7 rolling drum (New Brunswick Scientific, Edison, NJ) at 37°C. Cells were then diluted into LB, 1:1,000 (vol/vol), to initiate experiments. Cultures were incubated either in test tubes as described above or in 125-ml Erlenmeyer flasks on a shaking platform (200 rpm). For inoculation of cultures in multiple vessels at once, one large volume of LB was inoculated initially and then distributed into individual culture vessels.
For anaerobic growth experiments, different volumes of LB were distributed into screw-top 125-ml flasks, sealed with polypropanol caps with rubber septa (Qorpak), and then autoclaved. After sterilization, the air in the headspace of each flask was replaced with sterile nitrogen. Cells from an overnight culture were then inoculated 1:1,000 (vol/vol) by a needle through the septum. Samples for viable counts and pH analysis were removed by needle through the septum.
Monitoring of cell growth, survival, culture pH, and mutation frequency.To monitor cell growth and survival, viable cell counts were determined by serial dilution of cells sampled periodically from the cultures, followed by plating on LB agar (32). The limit of detection in all experiments was >1,000 CFU/ml (32). pH was monitored using pH paper with a range of 6.0 to 10.0 and increments of 0.3 to 0.5 pH unit (EMD Chemicals, La Jolla, CA).
Mutation frequency, as monitored by spontaneous resistance to rifampin (Rifr), was determined by plating ∼109 cells on LB agar supplemented with 100 μg/ml of rifampin (Sigma-Aldrich) (33). To determine mutation frequency, the number of Rifr colonies was divided by the total number of CFU in each culture. This frequency is expressed as number of Rifr colonies per 107 CFU in the culture.
β-Galactosidase assays.A plasmid expressing lacZ from the E. coli oxyR promoter, pAQ23 (31) (gift of B. Demple, Stony Brook University School of Medicine), was introduced into PFM2 by electroporation as described previously (28) and selected on LB agar plates supplemented with 150 μg/ml of ampicillin (Sigma-Aldrich). The plasmid was maintained by supplementing liquid cultures with ampicillin as appropriate. PFM2/pAQ23 (strain SF2562) was grown as described above, and cultures were sampled periodically during 1 to 4 days of incubation. β-Galactosidase activity was determined as described previously (29), but without the addition of chloroform. Briefly, 1.0 ml of cells was pelleted and resuspended in 1.0 ml of Z buffer (29). Then 100 μl of cells was mixed with 900 μl of Z buffer containing SDS at room temperature in order to lyse cells. Disrupted cells were mixed with 4 mg/ml of o-nitrophenyl-β-d-galactopyranoside (ONPG), incubated at room temperature for approximately 15 min, and centrifuged to remove debris. A420 and A550 readings were determined and modified Miller units were calculated based on the cell titer (CFU/ml) of each culture, rather than the optical density at 600 nm (OD600); since some samples were taken post-death phase, the OD600 does not fully reflect the viable cell counts. Endogenous activity of LacZ in PFM2 is virtually undetectable (data not shown).
Isolation of E. coli protein.LB cultures of different volumes were grown for 16 to 20 h, and 1 ml from each flask was processed for protein isolation. Cells were pelleted by centrifugation at 13,000 rpm for 1 min, followed by resuspension in 10 mM Tris-HCl (pH 7.5) and 0.15 M NaCl. Cells were then sonicated, on ice, twice at the maximum setting at 1-min intervals (Bronson Scientific), followed by enzymatic digestion with DNase I (50 μg/ml), RNase A (50 μg/ml), and lysozyme (20 μg/ml) (Sigma-Aldrich) at 37°C for 1 h. Protein levels in the whole-cell lysate were quantified using the Quick Start Bradford dye reagent, as per the manufacturer's instructions (Bio-Rad).
Enzyme-linked immunosorbent assay (ELISA) to determine glycation levels.Fifty microliters of whole-cell lysate (protein concentration, 0.5 to 1.0 mg/ml) was loaded into standard polystyrene 96-well plates (Corning, Inc.) with 100 μl of sodium carbonate binding buffer and incubated overnight at 4°C. After 16 to 20 h, wells were washed twice with phosphate-buffered saline (PBS)–Tween 20 (0.05%), blocked with 200 μl of blocking solution (5% bovine serum albumin in PBS) for 1 h at 37°C, washed again three times in PBS–Tween 20, and incubated (1 h at 37°C) with 100 μl of horseradish peroxidase (HRP)-conjugated anti-carboxymethyl lysine (anti-CML) antibody (diluted 1:1,000 [vol/vol] in blocking solution) obtained from Cosmobio, Japan. After three additional washes (PBS–Tween 20) to remove excess antibody, 100 μl of color reagent (G Biosciences) was added for 10 min at room temperature. The color developed in the wells was quantified at 630 nm using a plate reader (BioTek ELx808) as specified by the manufacturer.
For antiglycation activity analysis, carnosine (Sigma-Aldrich) was added to LB at a final concentration of 50 mM (15).
The type of culture vessel affects long-term survival.With an interest in identifying how laboratory growth environments might affect the dynamics of long-term survival, we examined long-term batch culture under two typical laboratory conditions: 5-ml cultures grown in test tubes and 12.5-ml cultures grown in 125-ml Erlenmeyer flasks. Generation time during exponential growth and cell yields after overnight growth were the same in both culture vessels (data not shown and Fig. 1A). In tubes, PFM2 entered death phase before day 2, with more than 90% of cells losing viability. PFM2 titers continued to decrease to ∼106 to 107 CFU/ml by day 4, where the viable cell counts "bottomed out." Between days 4 and 5, cell titers increased, stabilizing at ∼1 × 108 CFU/ml. We refer to the extended death phase and recovery experienced by PFM2 as a "dip." In flasks, however, once cells entered death phase, viable counts decreased more rapidly and more severely than with cultures incubated in tubes (Fig. 1A). On day 3, the viable counts in flasks dropped more than 3 orders of magnitude, 10-fold more than cells cultured in test tubes. However, the cells cultured in flasks began to recover from death phase a day earlier than cells in tubes, and the viable counts were the same in both vessels by 4 days of incubation.
(A) Survival dynamics of PFM2 in tubes and flasks. Shown are growth curves of PFM2 in 5 ml in tubes (squares) and 12.5 ml in flasks (circles). Average data shown are from a representative experiment of three biological replicates. Error bars represent standard deviations. (B) pH of the same samples as in panel A.
We monitored pH levels of the medium in these cultures. While the LTSP profiles of cultures in tubes and flasks varied in both the severity of death phase and the timing of recovery, pH did not differ between the two conditions at any time (Fig. 1B). Alkalization of bacterial cultures is frequently correlated with entry into, but not necessarily severity of, death phase (12, 25, 26).
Oxygen availability affects survival dynamics.The shape of the culture vessel can affect the dynamics of gas exchange between the medium and its environment, including the availability of oxygen (34). One difference between media in tubes and media in flasks is the surface area of the gas-liquid interface. This can affect the oxygen transfer rate (OTR), which correlates with the growth phase of a bacterial culture (35). We hypothesized that differences in OTR might also contribute to the dynamics of death phase. To determine if oxygen availability is a potential factor contributing to the observed differences in survival, we inoculated bacteria into tubes or flasks with different volumes of media (2, 5, or 12.5 ml in tubes and 5, 12.5, 25, or 50 ml in flasks). We found that the culture volume affected survival during LTSP in both tubes (Fig. 2A) and flasks (Fig. 2C).
Survival dynamics of PFM2 in cultures of different volumes. Growth curves (A) and pH (B) of PFM2 incubated in 2 ml (squares), 5 ml (circles), or 12.5 ml (triangles) of media in test tubes are shown. Growth curves (C) and pH (D) of PFM2 cultured in 5 ml (squares), 12.5 ml (circles), 25 ml (triangles), or 50 ml (diamonds) of media in flasks are also shown. Data are the averages of three biological replicates; error bars represent standard deviations.
In both tubes and flasks, cells incubated in the smallest tested volumes (2 ml in tubes and 5 ml in flasks) entered death phase between the first and second days of incubation but did not display the dip that is normally observed for PFM2 in standard 5-ml test tube long-term cultures. Instead, the counts remained stable, at ∼107 to 108 CFU/ml, for the remainder of the experiment. In contrast, cells cultured in larger volumes (12.5 ml in tubes and 50 ml in flasks) showed a slower death phase (on the second day of culture, viable counts were 10 times higher than in other cultures), but ultimately death phase was just as severe. The slower death phase in higher-volume cultures correlated with a lower rate of alkalization (Fig. 2B and D). While smaller-volume cultures had a pH of ∼9 after overnight incubation, the pH in larger-volume cultures was below 8.5. In contrast, the smaller volumes alkalized as quickly as the cultures with more standard volumes (5 ml in tubes and 12.5 ml in flasks; Fig. 2B and D). Twenty-five-milliliter flask cultures also had a slight delay in death phase compared to that of 12.5-ml flask cultures, correlating with a delay in pH increase (note the 10-fold-higher cell yield on the third day of incubation), but the extent of death phase was more severe than in any other volume, with minimum viable counts 1,000-fold lower than in 12.5-ml cultures. A comparison of the days at which the cultures reach their lowest viable counts (troughs) prior to recovery is summarized in Table 1.
Oxygen is not the only factor responsible for differences in survival under various culture conditions.To determine if differing oxygen levels were solely responsible for influencing the variation in LTSP survival, flask cultures with different volumes of media were incubated under anaerobic conditions (Fig. 3A). Cell growth dynamics in 5-, 12.5-, 25-, and 50-ml anaerobic cultures were much more similar to each other; the severe dip seen in aerobic cultures on days 2 to 5 was gone in all cultures, and viable counts remained above 1 × 108 CFU/ml throughout the experiment. However, there were still significant differences in the viable counts of cells at various points during incubation, particularly for cells incubated in 5 ml of culture media. These cells grew to a 10-fold-higher density than cells incubated in the other culture volumes after overnight incubation and remained 10-fold higher after 2 days of incubation. These results indicate that oxygen was not the only factor affecting LTSP survival in these cultures.
Survival dynamics of PFM2 in anaerobic cultures. Growth curves (A) and pH (B) of PFM2 in anaerobic cultures in flasks are shown for the following volumes: 5 ml (squares), 12.5 ml (circles), 25 ml (triangles), and 50 ml (diamonds). Data are the averages of three biological replicates; error bars represent standard deviations.
Differences among the culture conditions were more obvious when observing pH changes over time (Fig. 3B). While none of the cultures alkalized to the extent of aerobic cultures, pH values increased most rapidly in 5-ml cultures, while 50-ml cultures were the slowest to alkalize, similar to what was observed in aerobic cultures (Fig. 2D). Since oxygen was not available in any of these cultures, the differences in pH must have been due to another factor.
Survival in LTSP correlates with mutation frequency.The observed differences in the effect of oxygen tension led us to determine if the culture conditions described above led to differences in mutation frequency. By monitoring mutation frequency using spontaneous rifampin resistance as a reporter after 1 or 2 days under aerobic long-term culture conditions, we observed environment-specific differences in mutation frequency (Fig. 4). The median mutation frequencies on the first day of incubation were lowest in flasks with 5-ml and 12.5-ml cultures and highest in 5-ml cultures in tubes and 50-ml cultures in flasks (Fig. 4).
Spontaneous rifampin resistance mutations in different culture volumes. Median values (n = 15) for the number of rifampin-resistant mutants in different culture conditions are shown per 107 CFU/ml for 5 ml in tubes (hatched bar), 5 ml in flasks (black bar), 12.5 ml in flasks (white bar), 25 ml in flasks (dark gray bar), and 50 ml in flasks (light gray bar).
Compared with the mutation frequencies on day 1, all of the mutation frequencies increased at least 2-fold after 2 days of incubation, except in 50-ml cultures. For 50-ml cultures, the delay in change of mutation frequency correlated with the delay of entry into death phase (Fig. 2C).
Oxygen stress varies under different culture conditions.We hypothesized that there would be a higher mutation frequency in the cells experiencing the highest oxygen tension, but in fact the opposite was true. To determine whether cells were experiencing oxidative stress that correlated with the volume of media under the different culture conditions, we directly monitored oxyR expression using a transcriptional lacZ reporter plasmid (pAQ23) (36). β-Galactosidase levels were measured for cultures of strain SF2562 incubated in different volumes of LB in flasks during LTSP incubation over 4 days (Fig. 5). After 1 day of incubation, PoxyR expression was highest for cells incubated in 5-ml and 12.5-ml cultures. Cells in 25-ml cultures had ∼2-fold-lower activity and cells in 50-ml cultures had ∼4-fold lower activity than smaller-volume cultures. These data confirm that cells in the different culture volumes were experiencing different levels of oxidative stress.
Expression of PoxyR in different culture volumes in flasks. β-Galactosidase assays were performed on PFM2/pAQ23 (SF2562) during long-term growth in 5 ml (black bar), 12.5 ml (white bar), 25 ml (dark gray bar), and 50 ml (light gray bar). Bars represent average modified Miller units (see Materials and Methods) of duplicate samples. Error bars represent standard deviations. *, P < 0.05 using Student's t test.
After 2 days of incubation, expression from PoxyR increased ∼2-fold in 5- and 12.5-ml cultures, more than 3-fold in 25-ml cultures, and ∼6-fold in volumes of 50 ml. After 4 days of incubation, expression levels in all cultures were equivalent; however, while this represents only a small increase of expression in the 25-ml cultures, expression was almost doubled in the 50-ml cultures.
Culture volume affects glycation.Since cells experienced different amounts of oxidative stress as a function of culture volume, as well as differences in timing and degree of loss of viability, we wished to determine if glycation was affected. It has previously been shown that advanced glycation end products (AGEs) accumulate in E. coli during LTSP (15). We quantified the amount of carboxymethyl lysine (CML), a common glycation end product, using an ELISA with an anti-CML antibody for cells cultured in different volumes (Fig. 6). Glycation levels in 5-ml and 12.5-ml cultures were ∼8-fold lower than in 50-ml cultures; cells cultured in 25 ml showed an intermediate amount of glycation, about three times higher than that observed for the smaller volumes.
ELISA showing differences in glycation after overnight incubation using an anti-CML antibody. Assays were performed on total cellular protein from PFM2 during long-term growth in 5-ml (black bar), 12.5-ml (white bar), 25-ml (dark gray bar), or 50-ml (light gray bar) flask cultures. Bars represent average glycation/mg of protein of duplicate samples. Error bars represent standard deviations. *, P < 0.05 using Student's t test.
Pepper et al. (15) demonstrated that the addition of the antiglycation agent carnosine to the culture medium significantly reduces the amount of CML while also increasing viability. Because cells cultured in different volumes experience different levels of glycation, we determined whether carnosine affects these cultures differentially. In fact, carnosine did little to change viable counts of cells in 5-ml cultures (Fig. 7A), which experienced the least viability loss overall, but dramatically changed survival in 12.5- and 25-ml cultures (Fig. 7B and C) and, especially, 50-ml cultures, in which the loss of viability was the greatest (Fig. 7D). Addition of carnosine to cultures did not alter pH (data not shown).
Carnosine partially rescues cells from death phase. Growth without (solid lines) or with (dotted lines) 50 mg/ml of carnosine in 5-ml (A; squares), 12.5-ml (B; circles), 25-ml (C; triangles), or 50-ml (D; diamonds) cultures in flasks is shown. Lines represent average of four biological replicates over two separate experiments; error bars represent standard deviations.
The type of culture vessel used to incubate cells affects long-term survival in E. coli. In E. coli K-12 strain PFM2, we see differences in both the timing of death phase and overall cell yields throughout the bacterial life cycle dependent on these factors. We chose to compare two very common laboratory conditions: growth in 5 ml of medium in test tubes and growth in 12.5 ml of culture medium in 125-ml flasks. The differences in survival between these two conditions, which are often considered interchangeable, were striking. The death phase of cultures in flasks was much more severe than in tubes, suggesting that these cells were experiencing a much greater level of stress in flasks.
We show that PFM2 causes an increase in culture pH during exponential-phase growth. Cultures alkalize during growth when amino acids are used as a carbon source because the catabolism of amino acids leads to excess nitrogen, which is converted to ammonia or other nitrogenous compounds and released. Therefore, in unbuffered media (such as LB), the pH of the culture increases; the pH in minimal medium cultures that are naturally buffered (such as M9 or M63) remains ∼7.5 for days or even weeks in LTSP (25; S. Finkel, unpublished data). However, pH profiles are the same under both conditions, indicating that pH is not primarily responsible for the more severe death phase in cells cultured in flasks.
Since survival dynamics of the cells under these culture conditions are so different, what are the stressors experienced by the cells? It is important to understand what factors contribute to these differences in survival; therefore, we initially addressed the role of oxygen tension. We tested this in two ways: (i) by changing the headspace in the flask to affect mixing at the air-medium interface and (ii) by growing cells under anaerobic conditions. We found that oxygen is an important factor in survival during LTSP.
At smaller culture volumes (2 ml in tubes and 5 ml in flasks) cells enter death phase on the second day of incubation, as do cells under more standard culture conditions (5 ml in tubes and 12.5 ml in flasks), but then immediately level off and do not experience the dip seen under other culture conditions. We hypothesized that the number of cells that experience high oxygen tension at any given time is the greatest under these lower-volume conditions due to maximal mixing. Perhaps it is this exposure to oxygen, allowing more efficient respiration, which enables cells to recover from death phase so quickly. Another possibility is that volatile metabolic products are more efficiently released into the gaseous environment of the flask, therefore slowing the buildup of potential toxic products in the medium. This also suggests that the prolonged death phase observed under other culture conditions could be an effect of toxic by-products. One possible toxic by-product is ammonia, which we know accumulates in the medium and increases the pH. It has been shown that alkali stress can lead to induction of oxidative stress response proteins, as well as amino acid catabolism proteins that remove ammonia and generate acids (37). It is possible that increased exposure to oxygen in the medium from the headspace, due to the larger gas-liquid interface, may further induce alkali stress-responsive genes, which may be beneficial to the bacteria.
Cells cultured in larger volumes (12.5 ml in tubes and 50 ml in flasks) showed a delay of entry into death phase, which correlates with a delay in alkalization compared to that for cells cultured in intermediate volumes. In contrast, the pH in smaller-volume cultures (2 ml in tubes and 5 ml in flasks) does not correlate with survival, in that these cultures lack the dip seen in other cultures but do not have a difference in alkalization. These data suggest that while a change in pH affects the timing of entry into death phase, it does not determine the extent of survival during LTSP. In larger-culture-volume flasks, 50-ml cultures experience a less severe death phase than 25-ml cultures. This may reflect a less harsh environment in 50-ml cultures, since alkalization occurs more slowly. Alternatively, the delay in alkalization and entry into death phase may give cells more time to acclimate to their environment and therefore recover more quickly post-death phase.
While it is clear that oxygen availability is a major factor in survival, it is not the only important factor, as demonstrated by incubating cultures in different volumes of media in flasks under anaerobic conditions. The cells incubated anaerobically in 5-ml culture volumes still have higher viable counts than cells incubated anaerobically in larger culture volumes, even though the pH shows the greatest increase in 5-ml cultures. These data also support a hypothesis where the larger surface-to-headspace ratio allows these cells to better release a volatile toxic substance(s).
We hypothesized that the differences in survival seen during death phase are due to differences the cells experience as early as during the exponential growth and early stationary phases, even though cell counts at those times are equivalent. We tested this hypothesis by determining mutation frequencies in overnight and early-death-phase cultures. We expected that cells grown in 5-ml cultures in flasks would have the highest mutation frequency, because we expected there to be more oxidative stress. However, we observed that cells in 50-ml cultures had the highest mutation frequency in overnight cultures. These data suggest either that the cells were not experiencing oxidative stress as we hypothesized or that oxidative stress does not directly correlate with mutation frequency. Shu et al. (38) observed an increase in mutation frequency at very low oxygen levels (<1%) compared to that at ambient levels (∼20%) and suggested that this may be due to oxidative species generated under hypoxic conditions. Perhaps the cells in larger-volume cultures experience similar environments. Since specific types of mutations are more likely to occur due to oxidative agents (11), it would be interesting to determine if the spectrum of mutations occurring under the different culture conditions reveals the role oxygen plays in modulating the mutation frequency in these different cultures.
We also observed a difference in mutation frequency between cells cultured overnight in tubes versus flasks, the "interchangeable" conditions we first examined. From these data, it is clear that incubation under different culture conditions results in different genetic effects. Since basic science laboratories, as well as biotechnology researchers, frequently use larger culture vessels to scale up experiments, it is important to consider the possibility that the physiology of the cells is actually quite different in these environments. As these types of stresses may affect gene expression, protein production, growth, etc., all of which may affect the phenotype(s) being studied, it appears that these environments may not be interchangeable at all.
When we examined the mutation frequency on the second day of culture, we observed an increase in mutation frequency under all conditions studied, except in 50-ml flask cultures, in which there was also a delay in entrance to death phase.
Given these results, we wanted to determine if cells under different culture conditions were actually experiencing different levels of oxidative stress. We monitored expression of oxyR in these cells during long-term growth using a lacZ-based transcriptional reporter. OxyR is a major oxidative stress transcription factor and so is a good indicator of the level of oxidative stress cells may be experiencing. We hypothesized that there would be more mixing at the air-medium interface in smaller-volume cultures and that cells in those cultures would experience higher oxygen tension leading to increased levels of oxidative stress. In this study, our results were as hypothesized: during overnight growth, cells in 5-ml and 12.5-ml cultures in flasks had the highest level of PoxyR expression, while expression by cells incubated in 25-ml and 50-ml cultures was lower. For cells grown in 50-ml cultures, PoxyR expression was still low after 2 days of incubation. Levels of PoxyR expression correlate with both pH and entry into death phase, and therefore, oxidative stress very likely plays a role in those phenotypes.
Given that PoxyR expression is highest in 5-ml and 12.5-ml cultures, slightly lower in 25-ml cultures, and significantly lower in 50-ml cultures, it appears that the oxyR level in flasks is inversely correlated with mutation frequency. This may explain why the mutation frequency in 50-ml cultures did not increase after 2 days of incubation, as these cells experienced less oxidative stress than those in cultures of lower volumes. In fact, PoxyR expression in all culture volumes correlated with the amount of death observed on day 2, with 5-ml and 12.5-ml cultures experiencing the greatest loss of viability, followed by an intermediate loss in 25-ml cultures and almost no loss of viability in 50-ml cultures (Fig. 2C).
Glycation is a nonenzymatic process in which proteins and nucleic acids are modified by reactive electrophiles generated during normal metabolism. Given that oxidative damage can lead to glycation of proteins, which, in turn, can lead to cell death (15–17), we monitored glycation levels under different culture conditions. We found that glycation levels correlated not with oxidative stress but instead with mutation frequency, with cells in 50-ml cultures having the most glycation end products.
Because mutation frequency and glycation correlate negatively with PoxyR expression, we hypothesize that there may be a threshold of expression needed in order to protect against oxidative stress. It is possible that in 50-ml cultures, the amount of OxyR is too low during the first 2 days of incubation to activate the protective responses needed to lower glycation and mutation frequency, while cells in lower-volume cultures induce more OxyR earlier during incubation and therefore more of the protective responses it regulates. OxyR may also respond to, or protect against, pH stress, explaining why cells in 5-ml cultures survive better than those in larger volumes even though alkalization occurs more quickly.
To determine if glycation is a factor contributing directly to death, we added carnosine, which blocks glycation, to the different cultures. We found that addition of carnosine does in fact protect against death and is even more protective in the larger-volume cultures, correlating with higher glycation levels. Perhaps these cells have less protection against glycation from OxyR expression, which means that carnosine can have more of a protective effect. However, carnosine does not completely rescue cells from entering death phase, which indicates that glycation is not the only factor contributing to death in these cultures.
We have shown that there are significant changes of physiological responses and mutational responses due to culture conditions that are frequently used interchangeably in laboratory experiments. It is important to consider these factors when planning experiments or analyzing data. This is consistent with an analysis made by Fang (39) stating that results from experiments determining the effect of oxygen on antibiotic activity may diverge due to the differences in experimental conditions, namely, that some experiments were performed in 1/10 volume in flasks and some in 1/6 volumes in bottles; we have shown here definitively that oxidative stress can vary greatly with culture volume and vessel, which would likely lead to differential results in experiments depending on oxidative stress, It has also previously been shown that differences in culture volume can lead to changes in DNA supercoiling (40), which can lead to transcriptional and mutational changes. We know that differences in mutational and stress responses can lead to adaptive evolutionary events. Therefore, very simple changes in growth conditions may lead to complex evolutionary change and may leave specific genetic signatures of the environments in which the cells were adapted.
We thank Lacey Westphal, Christopher Corzett, and Kavita Chavan for helpful suggestions and comments on the manuscript. We also thank Micheline Strand and Robert Kokoska for helpful suggestions regarding this project. We thank Patricia Foster and Ellen Popodi for providing strains used in this work.
This work was supported by U.S. Army Research Office grants W911NF1010444 and W911NF1210321.
Accepted manuscript posted online 27 December 2013.
. 2006. Long-term survival during stationary phase: evolution and the GASP phenotype. Nat. Rev. Microbiol. 4:113–120. doi:10.1038/nrmicro1340.
. 1993. Microbial competition: Escherichia coli mutants that take over stationary phase cultures. Science 259:1757–1760. doi:10.1126/science.7681219.
. 1999. Evolution of microbial diversity during prolonged starvation. Proc. Natl. Acad. Sci. U. S. A. 96:4023–4027. doi:10.1073/pnas.96.7.4023.
. 2004. Escherichia coli evolution during stationary phase. Res. Microbiol. 155:328–336. doi:10.1016/j.resmic.2004.01.014.
. 1935. Bacteria growth and reproduction, p 1–38. In Gay FP, Bachman GW, Benham RH, Buchbinder L (ed), Agents of disease and host resistance. Charles C Thomas, Springfield, IL.
. 2000. Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12. J. Bacteriol. 182:4361–4365. doi:10.1128/JB.182.15.4361-4365.2000.
. 2003. Bacterial evolution through the selective loss of beneficial genes. Trade-offs in expression involving two loci. Genetics 164:1271–1277.
. 1988. Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli. J. Bacteriol. 170:3910–3914.
. 1993. The stationary phase of the bacterial life cycle. Annu. Rev. Microbiol. 47:855–874. doi:10.1146/annurev.mi.47.100193.004231.
. 2001. Not quite dead enough: on bacterial life, culturability, senescence, and death. Arch. Microbiol. 176:159–164. doi:10.1007/s002030100314.
. 2013. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat. Rev. Microbiol. 11:443–454. doi:10.1038/nrmicro3032.
. 2004. Stationary-phase physiology. Annu. Rev. Microbiol. 58:161–181. doi:10.1146/annurev.micro.58.030603.123818.
. 2006. Conditional and replicative senescence in Escherichia coli. Curr. Opin. Microbiol. 9:612–618. doi:10.1016/j.mib.2006.10.010.
. 1988. Transcriptional regulation of katE in Escherichia coli K-12. J. Bacteriol. 170:4286–4292.
. 2010. Antiglycation effects of carnosine and other compounds on the long-term survival of Escherichia coli. Appl. Environ. Microbiol. 76:7925–7930. doi:10.1128/AEM.01369-10.
. 2001. Evidence for non-enzymatic glycosylation in Escherichia coli. Mol. Microbiol. 39:1061–1068. doi:10.1046/j.1365-2958.2001.02304.x.
. 2005. Evidence for non-enzymatic glycosylation of Escherichia coli chromosomal DNA. Mol. Microbiol. 55:1801–1811. doi:10.1111/j.1365-2958.2005.04504.x.
. 2004. Glycation of proteins in Escherichia coli: effect of nutrient broth ingredients on glycation. Biotechnol. Biotechnol. Equip. 18:99–103. http://www.diagnosisnet.com/bbeq/article/e5acd52e-2501-40b8-a75c-dd9f52cdcc0c.
. 2004. Anti-crosslinking properties of carnosine: significance of histidine. Life Sci. 75:1379–1389. doi:10.1016/j.lfs.2004.05.002.
. 2000. Carnosine reacts with protein carbonyl groups: another possible role for the anti-ageing peptide? Biogerontology 1:217–223. doi:10.1023/A:1010057412184.
. 2001. Pharmacological inhibition of diabetic retinopathy: aminoguanidine and aspirin. Diabetes 50:1636–1642. doi:10.2337/diabetes.50.7.1636.
. 1984. Escherichia coli intracellular pH, membrane potential, and cell growth. J. Bacteriol. 158:246–252.
. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Mol. Biol. Rev. 49:359–378.
. 1994. Role of an Escherichia coli stress-response operon in stationary-phase survival. Proc. Natl. Acad. Sci. U. S. A. 91:2191–2195. doi:10.1073/pnas.91.6.2191.
. 2003. The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment. J. Bacteriol. 185:7044–7052. doi:10.1128/JB.185.24.7044-7052.2003.
. 1981. pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate. Proc. Natl. Acad. Sci. U. S. A. 78:6271–6275. doi:10.1073/pnas.78.10.6271.
. 2005. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J. Bacteriol. 187:304–319. doi:10.1128/JB.187.1.304-319.2005.
. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
. 2002. Growth in liquid media. Curr. Protoc. Mol. Biol. 59:1.2.1–1.2.2. doi:10.1002/0471142727.mb0102s59.
. 2012. Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. Proc. Natl. Acad. Sci. U. S. A. 109:2774–2783. doi:10.1073/pnas.1210309109.
. 2009. Adaptive evolution in single species bacterial biofilms. FEMS Microbiol. Lett. 293:135–140. doi:10.1111/j.1574-6968.2009.01526.x.
. 2013. Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli. Genetics 194:409–420. doi:10.1534/genetics.113.151837.
. 2007. Microtiter plates as mini-bioreactors: miniaturization of fermentation methods. Trends Microbiol. 15:469–475. doi:10.1016/j.tim.2007.09.004.
. 2013. Oxygen consumption rates of bacteria under nutrient-limited conditions. Appl. Environ. Microbiol. 79:4921–4931. doi:10.1128/AEM.00756-13.
. 1989. OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. Proc. Natl. Acad. Sci. U. S. A. 86:3484–3488. doi:10.1073/pnas.86.10.3484.
. 2002. pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli. J. Bacteriol. 184:4246–4258. doi:10.1128/JB.184.15.4246-4258.2002.
. 2006. Stationary phase-induction of G→T mutations in Escherichia coli. Mutat. Res. 596:106–112. doi:10.1016/j.mrfmmm.2005.12.015.
. 2013. Antibiotic and ROS linkage questioned. Nat. Biotechnol. 31:415–416. doi:10.1038/nbt.2574.
. 2011. DNA supercoiling is differentially regulated by environmental factors and FIS in Escherichia coli and Salmonella enterica. Mol. Microbiol. 80:85–101. doi:10.1111/j.1365-2958.2011.07560.x.


Your nervous system controls and coordinates every cell, tissue and organ in your body. Because chiropractic care focuses on the health of the nervous system, it can have a dramatic impact on your overall wellness and performance.
While some people seek chiropractic care for pain or other obvious symptoms, others seek chiropractic care to maintain a healthy lifestyle and optimum performance in daily life, work and play. Whatever your health concern, Dr. Korchok and his team can provide professional chiropractic care to suit your needs and wellness goals.
We are specialists in athletic performance, nutritional counseling, wellness consultation and complete family care. Learn more about our programs and services, and find out about designing your blueprint for total health and wellness.


New Faces & New Products - Meyer Service & Supply Ltd.
It has been an eventful couple of months since our last blog post!
The CheckPoint 3 has been very well received so far and people have been commenting on how versatile it is compared to the older CheckPoints and handheld Pac Checks. We're also seeing more people who are interested in the LeakPointer II and LeakMatic II rapid leak detection systems.
We have gotten to welcome Stephanie Bota and Nicholas Tardif to our Meyer team. Some of you might have already spoken with them; Steph is on the front line of making sure your orders are received, processed, and shipped out on time and Nick is starting in our sales department as an Inside Sales Representative. He'll be spending the next little while reaching out to our customers to see how things are going with their instruments.
Besides two new faces here at the Long Sault office, we're happy to announce that we've acquired the distribution rights for OPSIS Liquidline in Canada. Liquidline is a Swedish manufacturer of Kjeldahl protein testing equipment and Soxhlet/Randall extraction systems for fat, oil, and other extractable matter. We're just gearing up now, but we'll soon have a full line of Kjeldahl tubes, tablets, and parts in-stock and ready to ship, and we should be receiving our KjelROC Analyzer demonstration system soon as well, so stay tuned!


text:
Electronic discovery about more than unplugging the shredders
The new rules of eDiscovery require more thought and action than simply turning off the office shredder. In this fourth and final part of WTN's series on electronic document retention, attorney Joe Olson addresses the how and when of eDiscovery, including effective litigation hold -...
17 July, 2007 / 0 Comments
Platypus Technologies appoints Jeff Williams as CEO
Platypus Technologies has announced the appointment of Dr. Jeff Williams as cocmpany CEO. Williams, a graduate of the University of Wisconsin-Madison, began his career with Promega Corp. He will succeed co-founder Barb Israel, who will serve the organization as a board member and as a...
Biotech association chief applauds Kohl's work on FDA budget
The executive vice president of the Wisconsin Biotechnology and Medical Device Association is throwing his support behind the efforts of U.S. Sen. Herb Kohl to speed the transfer of medical research into clinical practice and to improve the drug-approval process. Jim Leonhart said additional spending...
Open source software: Is it really and truly free?
For anyone interested in open source software but operating under the assumption that it's "free," attorney and columnist Deborah Wilcox has some upsetting news. In this edition of IP Online, she explains why open source is anyting but free, and she advises companies to develop...
Third Wave Technologies to collaborate on personalized heart care
Madison's Third Wave Technologies will collaborate with a North Carolina clinical laboratory on the development of a companion diagnostic test to personalize the treatment of heart failure. The diagnostic would be a companion to a new pharma product being developed by the Denver-based ARCA Discovery....
City Wi-Fi challenges are no surprise to broadband executives
To the chagrin of municipal officials across Wisconsin, more doubts are emerging about whether Wireless Fidelity or Wi-Fi service really is a disruptive technology that can bring affordable broadband Internet access to the masses. With cities struggling to establish their own Wi-Fi "clouds," WTN sought...
California group turns up heat on WARF stem cell patents
The fate of three stem cell patents held by the Wisconsin Alumni Research Foundation hangs in the balance before the U.S. Patent and Trademark Office, and opinions vary wildly as to whether they will be upheld, weakened, or overturned. In this WTN guest column, the...
Crazy like a Google? With GE-Abbott deal scrapped, could Google be next buyer?
To many, the thought of Google getting into the pharmaceutical diagnostics business is absurd, but upon closer inspection, Dr. Ogan Gurel doesn't believe it's too far fetched. In this brief edition of MedTech Futures, Gurel speculates about what torpedoed the GE Healthcare-Abbott deal, and why...
Musicnotes metrics: Madison dotcom passes $5M in royalties paid
At a time when the music industry is struggling to find new revenue streams, the pay-for-content digital delivery model of a Madison business is gaining traction. The Madison-based company has announced that royalty payments to music publishers and the songwriters they represent recently passed the...
Green is the color of speed in ranking broadband infrastructure
Establishing a state ranking system for broadband connectivity, columnist James Carlini has made the color green signify not only environmentally friendly status, but broadband rich as well. Under his system, green stands for fiber to the premise and high speed (50Mbps or more) connections,...

redpajama_set_name: RedPajamaCommonCrawl


Mormon Station is located in the town of Genoa, and is Nevada's first permanent non-native settlement. Originally constructed in 1851, a replicated fort lies on the grounds today and houses a museum with pioneer-era artifacts.
Originally the discovery of gold at Sutter's Mill in 1848 initiated a frantic scramble to the West. After facing many challenges on their journey west, many pioneers were not prepared to conquer their final obstacle: the treacherous Sierra Nevada range. As many were travel weary and likely out of supplies, many settlers made the lush and fertile Carson Valley their final destination.
In June of 1850, Abner Blackburn and Hampton Beatie, members of the Mormon Battalion, established a temporary trading post on the west side of Carson Valley. They documented many compelling stories of this beautiful region, intriguing their Salt Lake employer John Reese and his nephew Stephan Kinsey. In fact, their journals were so interesting that it encouraged Reese and Kinsey to relocate to the west side of Carson Valley in 1851.
The Reese Company then built a permanent trading post nearly one mile south of the temporary trading post established by Blackburn and Beatie. The new station featured a blacksmith shop, livery stable and even included flour and saw mills nearby.
During that same year, residents formed a squatter's government, and later in 1854, Utah Governor Brigham Young appointed Mormon Apostle Orson Hyde to serve as probate judge and spiritual head of the community.
Hyde eventually changed the name of the community to Genoa (after Genoa, Italy) and made it the official county seat. Mormon pioneers made their way to the area in 1857, and in 1861 Congress created the Territory of Nevada. James Nye assumed the role of Governor and designated Carson City as the capital of this new territory.
Then, in 1864, Nevada became the 36th state in the Union. Because statehood occurred during the Civil War, Nevada became known as the "Battle Born" state. As the Comstock silver and gold mine exploded with success, Genoa remained a key-player in economic activity both in freight and passenger activity.
The park grounds includes a museum, stockade and wagon shed, park and group pavilion area. Admission to the museum is $1.00, while the stockade and wagon shed can be enjoyed for free. The parks' mature trees and large grassy area is the perfect place to have a picnic. The State Park also boasts clean flushing restrooms and is dog-friendly! Located 12 miles south of Carson City via US 395 and State Route 206, the museum and group use area are open May through mid-October. Visit today!


text:
9 Xiaomi Devices That Will Be Getting MIUI 14!
By Jay / October 25, 2022 / 2022 Android
MIUI 14 is a major update for Xiaomi smartphones that has not yet been launched; those of you interested in purchasing a new smartphone may wish to consider it. This version, which is based on Android 13, will deliver a new user interface to Xiaomi smartphones in 2023; we will explore this today.
The following devices will receive the MIUI 14 update upon its initial release. Please be advised that MIUI 14 will not be distributed after the MIUI 13 update, and will only be available for certain devices. Here is detailed information about the MIUI 14 Update, including which devices will receive it and when it will be made available.
When will MIUI 14 be Released for its Users?
Several Alpha and Beta versions of MIUI 14 have been issued to several Chinese brand users. One of them is, of course, accessible exclusively to beta test users exclusively in the countries.; it has not yet been made available to global users. Multiple leakers have indicated that the release of MIUI 14 would occur in December.
The bad news is that this December version of MIUI 14 will not be released officially, but it will be announced publicly. After testing on multiple models, a global release will occur the following year.
Which Xiaomi Devices Get the Update:
The following is a list of Xiaomi smartphone models that have gotten the MIUI 14 beta, with a total of nine smartphones that will receive MIUI 14 first.
Xiaomi-12S Pro
Redmi-K50 Pro
You must utilize a high-end device to receive the MIUI 14 upgrade based on Android 13 first. Nonetheless, an updated list of Xiaomi devices that will receive the update can be seen on the official Xiaomi website. Our website will be updated with new content in the future.
androidMIUI 14Newsxiaomi


text:
use kernel::*;
use variable::concept::*;
use variable::ops::*;
use variable::memory::copy_memory::*;
use variable::memory::ops::*;
use gcollections::kind::*;
use gcollections::ops::*;
use gcollections::ops::sequence::ordering::*;
use std::slice;
use std::ops::{Index, DerefMut};
use std::fmt::{Formatter, Display, Error, Debug};

#[derive(Clone, Debug, PartialEq, Eq)]
pub struct TrailMemory<Trail, Domain>
{
  variables: CopyMemory<Domain>,
  trail: Trail
}

impl<Trail, Domain> MemoryConcept for TrailMemory<Trail, Domain> where
 Trail: TrailRestoration + AssociativeCollection<Location=usize, Item=Domain> + TrailVariable + Empty + Debug,
 Domain: Clone + Display + Debug
{}

impl<Trail, Domain> ImmutableMemoryConcept for TrailMemory<Trail, Domain> where
 Trail: TrailRestoration + AssociativeCollection<Location=usize, Item=Domain> + Empty + Debug,
 Domain: Clone + Display + Debug
{}

impl<Trail, Domain> Collection for TrailMemory<Trail, Domain>
{
  type Item = Domain;
}

impl<Trail, Domain> AssociativeCollection for TrailMemory<Trail, Domain>
{
  type Location = usize;
}

impl<Trail, Domain> Empty for TrailMemory<Trail, Domain> where
 Trail: Empty
{
  fn empty() -> TrailMemory<Trail, Domain> {
    TrailMemory {
      variables: CopyMemory::empty(),
      trail: Trail::empty()
    }
  }
}

impl<Trail, Domain> Cardinality for TrailMemory<Trail, Domain>
{
  type Size = usize;
  fn size(&self) -> usize {
    self.variables.size()
  }
}

impl<Trail, Domain> Iterable for TrailMemory<Trail, Domain>
{
  fn iter<'a>(&'a self) -> slice::Iter<'a, Domain> {
    self.variables.iter()
  }
}

impl<Trail, Domain> Push<Back> for TrailMemory<Trail, Domain> where
 Trail: TrailVariable + AssociativeCollection<Location=usize, Item=Domain>,
 Domain: Clone
{
  fn push(&mut self, dom: Domain) {
    self.variables.push(dom);
  }
}

impl<Trail, Domain> Replace for TrailMemory<Trail, Domain> where
 Trail: TrailVariable + AssociativeCollection<Location=usize, Item=Domain>,
 Domain: Clone
{
  fn replace(&mut self, key: usize, dom: Domain) -> Domain
  {
    let dom = self.variables.replace(key, dom);
    self.trail.trail_variable(key, dom.clone());
    dom
  }
}

impl<Trail, Domain> Index<usize> for TrailMemory<Trail, Domain>
{
  type Output = Domain;
  fn index<'a>(&'a self, index: usize) -> &'a Domain {
    &self.variables[index]
  }
}

impl<Trail, Domain> Display for TrailMemory<Trail, Domain> where
 Domain: Display
{
  fn fmt(&self, formatter: &mut Formatter) -> Result<(), Error> {
    self.variables.fmt(formatter)
  }
}

impl<Trail, Domain> Freeze for TrailMemory<Trail, Domain> where
 Trail: TrailRestoration + Collection<Item=Domain>
{
  type FrozenState = FrozenTrailMemory<Trail, Domain>;
  fn freeze(mut self) -> Self::FrozenState
  {
    self.trail.commit();
    FrozenTrailMemory::new(self)
  }
}

pub struct FrozenTrailMemory<Trail, Domain>
{
  store: TrailMemory<Trail, Domain>
}

impl<Trail, Domain> FrozenTrailMemory<Trail, Domain>
{
  fn new(store: TrailMemory<Trail, Domain>) -> FrozenTrailMemory<Trail, Domain> {
    FrozenTrailMemory {
      store: store
    }
  }
}

impl<Trail, Domain> Snapshot for FrozenTrailMemory<Trail, Domain> where
 Trail: TrailRestoration + Collection<Item=Domain>
{
  type Label = Trail::Mark;
  type State = TrailMemory<Trail, Domain>;

  fn label(&mut self) -> Self::Label {
    self.store.trail.mark()
  }

  fn restore(mut self, label: Self::Label) -> Self::State {
    self.store.trail.undo(label, self.store.variables.deref_mut());
    self.store
  }
}

redpajama_set_name: RedPajamaGithub


• They anchor the plant firmly to the ground.
• They absorb water and mineral salts.
the root cap which were damaged.
which absorb water and mineral salts.
the xylem of the stele.
while at the same time reducing water loss through transpiration.
stem and helps keep it upright.
• The parenchyma of the cortex stores food.
transport of water and gases.
• The endodermal cells stores starch.
the inside and secondary phloem on the outside.
• Every year a new ring of secondary xylem is formed.
water and mineral salts. They become heartwood.
• The cork cambium divides to form cork cells on its outside.
• The cork cells have suberin laid down on their cell walls.
called lenticles which allow gaseous exchange.
Xylem vessels and xylem tracheids are highly specialised cells.
Xylem vessels are dead cells. They are long, cylindrical cells.
continuous tubes from roots to leaves.
Xylem vessels have thick walls made up of lignin.
Sieve tubes are living cells with long cylindrical cells.
BECAUSE OF HIGH CONCENTRATION OF SALTS.
reduces the rate of transpiration.
also trap water vapour, restricting water loss.
increasing water vapour pressure gradient.
Then the plant is called wilting.
hydathodes on margin of leaf.


Here comes a little update!
I'm sitting in an internet cafe now and right now the internet connection seem to work fine but it's slow on and off here pretty much every day and so when I'm finally online I wanted to write you and tell you that after the hacking of my site I have implemented more security around it in order to prevent another hacking from happening.
The thing is that there's so many different plugins out there so it takes time to investigate and do research on which one to buy so in the meantime I'm testing a plugin that is one of the best (supposedly) but I haven't had time to work out the settings just yet.
I haven't been able to be online since my tablet stopped working due to some electric conflict stuff (other people with European chargers has had the same problem) and my phone is not letting me in on my site (and other sites as well) via my phone so I have simply not been able to work with the settings yet.
And doing it via my phone would had been a real hassle as anyone can imagine!
To not have been able to work with the settings of this particular plugin has even caused me to be blocked out from my own site once (!), so if you are experiencing any issues with logging in (this applies only for paying members) then please let me know so that I can unlock you as soon as possible.
It helps me to know if you're experiencing any issues at all for that matter so please contact me should there be any and I look into it as soon as I can, but the security plugin should be the only "problem" right now, but I can't remove it either at this point because that would leave my site vulnerable.
I will be traveling the whole Sunday (going to Delhi) and then from there I'll be doing a 26 hour long train journey down to Goa, where I'll arrive on Wednesday around noon.
So after Wednesday sometime I'm hoping to be back on track again with everything, so please bear with me and have patience with me (I'm exercising it like crazy here on my end! 🙂 ) as it's very challenging at the moment.
PS: I was just thinking the other day; is there anything you'd like to know about India traveling..?
Comment below here on my blog or on my Facebook, Twitter etc, and I reply whenever I have the chance!
I know I had a lot of questions myself before I got here and I'm learning something new every day.


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text:
Acromatopsia, cunoscută și sub denumirea de orbire totală colorată, este un sindrom medical care prezintă simptome legate de cel puțin cinci afecțiuni. Termenul se poate referi la afecțiuni dobândite, cum ar fi acromatopsia cerebrală, dar, în mod special,  se referă la afecțiunea autozomală recesivă congenitală a vederii în culorii, la incapacitatea de a percepe culoarea și de a obține o acuitate vizuală satisfăcătoare la niveluri de lumină ridicate, de obicei lumina exterioară. Sindromul este de asemenea prezent într-o formă mai puțin severă numită discromatopsie. Se estimează, că, afectează 1 din 30.000 de nașteri vii în întreaga lume.

Sunt unele discuții despre acromații în sensul că aceștia pot vedea culoarea sau nu. După cum este ilustrat în Insula culorii de Oliver Sacks, unii acromați nu pot vedea culoare, doar negru, alb și nuanțe de gri. Cu cinci gene diferite cunoscute în prezent care cauzează simptome similare, se poate ca unii să vadă niveluri marginale de diferențiere a culorii datorită caracteristicilor genice diferite. Cu dimensiuni de eșantion atât de mici și rate de răspuns scăzute, este dificil de diagnosticat cu exactitate "condițiile achromatice tipice". Dacă nivelul de lumină în timpul testării este optimizat pentru acestea, acestea pot atinge o acuitate vizuală corectată de la 20/100 la 20/150 la niveluri mai mici de lumină, indiferent de absența culorii.

O trăsătură comună este hemeralopia sau orbirea la soare. La pacienții cu acromatopsie, sistemul conului și fibrele purtătoare de informații despre culoare rămân intacte. Acest lucru indică faptul că mecanismul utilizat pentru construirea culorilor este defect.

Semne și simptome
Sindromul este frecvent observat mai întâi la copii în jurul vârstei de șase luni prin activitatea fotofobă sau prin nistagmusul lor. Nistagmusul devine mai puțin vizibil odată cu vârsta, dar celelalte simptome ale sindromului devin mai relevante pe măsură ce vârsta școlară se apropie. Acuitatea vizuală și stabilitatea mișcărilor oculare se îmbunătățesc, în general, în primii șase-șapte ani de viață (dar rămân aproape 20/200). Formele congenitale ale afecțiunii sunt considerate staționare și nu se agravează odată cu vârsta.

Cele cinci simptome asociate cu acromatopsia sau discromatopsia sunt:

 acromatopsia
 ambliopia (acuitate vizuală redusă)
 hemeralopia (subiectul prezintă fotofobie)
 nistagmus
 anomalii de funcționare ale irisului

Sindromul de acromatopsie sau discromatopsie este slab descris în textele medicale și neurooftalmologice actuale. A devenit un termen comun în urma lansării cărții neuroștiințifice Oliver Sacks, Insula culorii, în 1997. Până atunci, cei mai mulți subiecți au fost descriși ca acromați sau acromatopi. Cei cu un grad mai mic de anomalii de percepție a culorii au fost descriși drept protanopi, deuteranopi sau tetartanopi (istoric tritanopi).

Acromatopsia  a fost, de asemenea, numită monocromație de tijă și orbire totală congenitală a culorii. Persoanele cu forma congenitală a acestei afecțiuni, prezintă absența completă a activității celulelor cu conuri evidentiață prin electroretinografie la niveluri ridicate de lumină. Există cel puțin patru cauze genetice ale acromatopsiei congenitale, dintre care două implică canale ionice cu nucleotide ciclice (ACHM2, ACHM3), o a treia implică transducina fotoreceptorului conului (GNAT2, ACHM4), iar ultima rămâne necunoscută.

Acromatopsia completă
În afară de o incapacitate completă de a vedea culoarea, persoanele cu acromatopsie completă au o serie de alte aberații oftalmologice. Printre aceste aberații optice sunt incluse scăderea acutății vizuale (<0,1 sau 20/200) în lumina zilei, hemeralopia, nistagmus și fotofobie severă. Fundul ochiului pare complet normal.

Acromatopsie incompletă
În general, simptomele acromatopsiei incomplete (discromatopsia) sunt similare cu cele ale acromatopsiei complete, cu excepția unei forme diminuate. Persoanele cu acromatopsie incompletă au o acuitate vizuală redusă cu sau fără nistagmus sau fotofobie. Mai mult decât atât, acești indivizi au doar o afectare parțială a funcției celulelor cu conuri, însă celulele cu tije și-au păstrat funcția.

Cauze

Moștenite
Acromatopsia sau discromatopsia dobândită este o afecțiune asociată cu deteriorarea diencefalului - în special atalamusul din creierul mijlociu - sau cortexul cerebral (neocortex), în special în patra zonă de asociere vizuală, V4, care primește informații de pe calea parvocelulară implicată în prelucrarea  culorilor.

Acromatopsia sau discromatopsia talamică este cauzată de deteriorarea talamusului; este cel mai frecvent cauzată de apariția și dezvoltarea tumorilor, fiind singurele care îl pot afecta, acesta fiind bine protejat de deteriorarea externă.

Acromatopsia cerebrală este o formă de orbire dobândită, care este cauzată de deteriorarea cortexului cerebral al creierului, mai degrabă decât de anomalii în celulele retinei ochiului. Acesta este cel mai frecvent cauzat de traume fizice, hemoragie sau creșterea țesutului tumoral.

Congenitale
Cauzele cunoscute ale formelor congenitale de achromatopsie se datorează tuturor funcționării defectuoase a căii fototransductiei retiniene. Mai exact, această formă de achromatopsie pare să rezulte din incapacitatea celulelor conului de a răspunde corect la intrarea luminii prin hiperpolarizare. Cauzele genetice cunoscute sunt mutațiile din canalele ionice cu nucleotide ciclice cu celule conice CNGA3 (ACHM2) și CNGB3 (ACHM3), precum și transducina cu celule conice, GNAT2 (ACHM4).

O a patra cauză genetică (ACHM5, OMIM 613093) a fost descoperită în 2009.  Este o mutație a genei PDE6C, localizată pe locusul cromozomului  10, 10q24. Se estimează că mai puțin de două procente din acromatopsii sunt cauzate de o mutație a acestei gene.

Fiziopatologie
Aspectul hemeralopic al acromatopsiei poate fi diagnosticat neinvaziv folosind electroretinografie. Răspunsul la nivel scăzut (scotopic) și median (mezotopic) va fi normal, dar răspunsul în condiții de nivel luminos ridicat (fotopic) va fi absent. Nivelul mezotopic este de aproximativ 100 de ori mai mic decât nivelul clinic utilizat pentru electroretinograma de nivel înalt tipic. Când este descris, starea se datorează unei saturații în porțiunea neurală a retinei și nu datorită absenței fotoreceptorilor în sine.

În general, patomecanismul molecular al acromatopsiei este fie incapacitatea de a controla corect sau de a răspunde la niveluri modificate de cGMP; deosebit de importantă în percepția vizuală, deoarece nivelul său controlează deschiderea canalelor ionice cu nucleotide ciclice (CNGs). Scăderea concentrației de cGMP determină închiderea CNG-urilor și rezultă hiperpolarizarea și încetarea eliberării glutamatului.

CNG-urile retiniene native sunt compuse din 2   α- și 2   Subunități β, care sunt CNGA3 și, respectiv, CNGB3, în celule conice. Când este exprimat singur, CNGB3 nu poate produce canale funcționale, în timp ce nu este cazul CNGA3. Asamblarea CNGA3 și CNGB3 produce canale cu expresie de membrană alterată, permeabilitate ionică ( Na + vs. K + și Ca 2+ ), eficacitatea relativă a activării cAMP / cGMP, reducerea rectificării exterioare, pâlpâirea curentului și sensibilitatea la blocare prin L-cis-diltiazem.

Mutațiile tind să conducă la pierderea funcției CNGB3 sau câștigul funcției (adesea crește afinitatea pentru cGMP) de CNGA3. Nivelurile de cGMP sunt controlate de activitatea transducinei cu celule conice, GNAT2. Mutațiile din GNAT2 tind să conducă la o proteină trunchiată și, probabil, nefuncțională, prevenind astfel modificarea nivelurilor de cGMP de fotoni. Există o corelație pozitivă între severitatea mutațiilor din aceste proteine și completitudinea fenotipului de achromatopsie.

Diagnosticul molecular poate fi stabilit prin identificarea variantelor bialelice din genele cauzale. Abordările de testare genetică moleculară utilizate în achromatopsie pot include analiza țintită pentru varianta comună CNGB3 c.1148delC   (p. Thr383IlefsTer13), utilizarea unui panou multigenerațional sau testări genomice complete.

ACHM2
În timp ce unele mutații din CNGA3 au ca rezultat canale trunchiate și, probabil, nefuncționale, acesta nu este în mare parte cazul. În timp ce puține mutații au primit studii aprofundate, cel puțin o mutație are ca rezultat canale funcționale. Curios, această mutație, T369S, produce modificări profunde atunci când este exprimată fără CNGB3. O astfel de modificare este scăderea afinității pentru monofosfat de guanozină ciclică. Altele includ introducerea unei sub-conductanțe, modificarea cineticii de canale cu un singur canal și permeabilitatea crescută a calciului.

Când canalele T369S mutante se reunesc cu CNGB3, totuși, singura aberație rămasă este permeabilitatea crescută a calciului.  Deși nu este clar imediat cum această creștere a Ca 2+ duce la acromatopsie, o ipoteză este că acest curent crescut scade raportul semnal-zgomot. Alte mutații caracterizate, cum ar fi Y181C și celelalte S1   mutații din regiune, au ca rezultat o scădere a densității curente datorită incapacității canalului de a circula la suprafață.  O astfel de pierdere a funcției va anula, fără îndoială, capacitatea celulei conului de a răspunde la intrarea vizuală și de a produce achromatopsie. Cel puțin o altă mutație missense în afara S1   regiunea, T224R, duce de asemenea la pierderea funcției.

ACHM3
Deși au fost caracterizate foarte puține mutații în CNGB3, marea majoritate a acestora au ca rezultat canale trunchiate care, probabil, sunt nefuncționale. Acest lucru va duce în mare măsură la haploinsuficiență, deși în unele cazuri, proteinele trunchiate pot fi capabile să se adune cu canale de tip sălbatic într-o manieră negativă dominantă. Cea mai răspândită mutație ACHM3, T383IfsX12, are ca rezultat o proteină trunchiată non-funcțională care nu circulă în mod corespunzător către membrana celulară.

Cele trei mutații missense care au primit studiu suplimentar arată o serie de proprietăți aberante, cu o temă de bază. R403Q   mutația, care se află în regiunea porilor a canalului, are ca rezultat o creștere a rectificării curentului exterior, față de relația curent-tensiune liniară în mare parte a canalelor de tip sălbatic, concomitent cu o creștere a afinității cGMP.  Celelalte mutații arată fie o expresie de suprafață crescută (S435F), fie o scădere (F525N), dar și o afinitate crescută pentru cAMP și cGMP.   Este o afinitate crescută pentru cGMP și cAMP la acești mutanți care este probabil modificarea cauzatoare de tulburări. O astfel de afinitate crescută va duce la canale care sunt insensibile la ușor modificări ale concentrației de cGMP din cauza intrării de lumină în retină.

ACHM4
La activarea prin lumină, con-opsina determină schimbul de PIB pentru GTP în proteina de legare a nucleotidelor de guanină (proteina G ) , activitate transducătoare a polipeptidei   2 (GNAT2). Aceasta provoacă eliberarea subunității α activate din subunitățile β / γ inhibitoare. Această subunitate α activează apoi o fosfodiesterază care catalizează conversia cGMP în GMP, reducând astfel curentul prin CNG3   canale.

Deoarece acest proces este absolut vital pentru procesarea corectă a culorilor, nu este surprinzător că mutațiile din GNAT2 duc la achromatopsie. Mutațiile cunoscute din această genă, toate conduc la proteine trunchiate. Probabil, atunci, aceste proteine sunt nefuncționale și, în consecință, con-opsina care a fost activată de lumină nu duce la modificarea nivelurilor de cGMP sau la hiperpolarizarea membranei fotoreceptorilor.

Administrare
În general nu există tratament pentru a vindeca achromatopsia. Cu toate acestea, filtrele de culoare roșu închis sau prune sunt de mare ajutor în controlul sensibilității la lumină.

Din 2003, există un dispozitiv cibernetic numit eyeborg care permite oamenilor să perceapă culoarea prin undele sonore.  Artistul cu acromatopsie Neil Harbisson a fost primul care a folosit un astfel de dispozitiv la începutul anului 2004, ochiul i-a permis să înceapă să picteze în culori memorând sunetul fiecărei culori.

Mai mult, există unele cercetări asupra terapiei genice pentru animale cu achromatopsie, cu rezultate pozitive la șoareci și câini tineri, dar o eficiență mai mică la câinii mai în vârstă. Cu toate acestea, nu s-au făcut experimente pe oameni. Există multe provocări pentru efectuarea terapiei genice pentru orbirea culorii la om.

Epidemiologie
Acromatopsia este o afecțiune rară având o prevalență de 1 la 30 000 de oameni .

Cu toate acestea, pe micul atol micronesian din Pingelap, aproximativ cinci procente din 3.000 de locuitori ai atolului sunt afectați.   Acesta este rezultatul unui blocaj al populației cauzat de un tifon și de foamete care a decedat în anii 1770, care a ucis pe toți, în afară de aproximativ douăzeci de insulari, inclusiv unul care a fost heterozigot pentru acromatopsie.

Oamenii din această regiune au denumit achromatopsia "maskun", ceea ce înseamnă literal "a nu vedea" în Pingelapese.  Această populație neobișnuită l-a atras pe insula neurologul Oliver Sacks pentru care a scris cartea sa din 1997, Insula coloratului.

Terminologie

Referințe

Note

Legături externe

 Achromatopsia at MedicineNet
 Achromatopsia at Merriam-Webster
 Achromatopsia at NCBI
 Achromatopsia at ScienceDirect

Sistem vizual
Percepție vizuală
Afecțiuni oftalmologice
Boli rare
Boli umane

redpajama_set_name: RedPajamaWikipedia


text:
Tight Necktie May Boost Glaucoma Risk
July 28, 2003 — Depending on your tastes, wearing neckties may be an ambush to some folks' vision. According to a modern think about, wearing them as well tightly may hurt your own.
Researchers say that a too-tight necktie may increment the risk of glaucoma by boosting blood pressure interior the eyes to perilous levels. Particularly, a tight bowtie contracts neck veins and raises the pressure within the eyes. Glaucoma, which torments at least 3 million Americans and is the leading cause of visual impairment in the U.S., more often than not occurs when weight within the eyes increments to dangerous levels.
In their ponder, distributed within the Eminent issue of the British Journal of Ophthalmology, analysts noticed a little but critical increase in this intraocular weight in men who wore tight neckties. The 40 men examined — half of them glaucoma patients and the others with no obvious vision-threatening condition — were observed beneath three scenarios. When their neckties were fixed for three minutes, intraocular pressure spiked in 60% of the glaucoma patients and 70% of the sound men. There were no such increments when they extricated their ties for three minutes or while wearing open-collared shirts.
Loosen Up
Does this mean that neckwear will before long join a list of established hazard components for glaucoma that incorporates being more seasoned than age 40; of African-American, Irish, Russian, Japanese, Hispanic, or Scandinavian plummet; or having diabetes, hypertension, poor vision, or a family history of glaucoma?
Maybe not, but it does suggest that when it comes to neckwear, tight isn't right — at least when it comes to ensuring your vision.
"No one says you've got to strangle yourself," says think about author Robert Ritch, MD, chief of glaucoma administrations and specialist chief at the New York Eye and Ear Clinic. "On the off chance that you can't get your finger in between your neck and your collar effectively, it's as well tight."
Ritch tells WebMD that he conducted the consider with other Unused York researchers because he taken note that in his claim patients — particularly those with thick necks — their intraocular pressure would decrease when he had them extricate their neckties during an eye exam.
"I have been telling my patients for years that on the off chance that they have glaucoma, they should not wear tight neckties." Still, although eye weight increases can be noted after just a few minutes of a wearing a tight necktie, Ritch says that in all probability, it would have to be compelled to be "visit and delayed" to likely make tight neckwear a real threat to vision.
Other experts not involved in Ritch's ponder say that whereas it copies what has long been known — that a short-term boost in neck pressure produces a sudden and gentle increment in eye pressure — there's no reason to believe that neckties can cause glaucoma.
"Since the study endured as it were three minutes, it is inconceivable to know how long it would take to return to the original eye pressure," says glaucoma pro Harry A. Quigley, MD, of the Wilmer Eye Founded at Johns Hopkins School of Medicine.
"This investigate does not display any real evidence that this calculate increases chance of glaucoma," he tells WebMD. "It is basically common sense not to wear collars that are so tight that they cut off blood return from the brain. How many men tie their ties to the point of distress and leave them that way for prolonged periods?"
Steven J. Gedde, MD, of Bascom Palmer Eye Organized, lauded the consider — and its research group — for reminding ophthalmologists that a tight necktie may be along the other "outside" factors, such as holding one's breath or having the hiccups, which can temporarily raise intraocular weight and conceivably produce untrue readings.
"When I degree someone's intraocular weight, I tell patients not to hold their breath because we know that can cause a (short-term) rise in intraocular pressure. Which might impact how these levels are examined and the chosen course of treatment," Gedde says. "Wearing a tie seems to drop beneath that same category."
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July 14, 2018 .Reading time 7 minutes.
It can be difficult to find a balance between spending time and money on improving and your home versus actually enjoying it! Here are a few small ways that you can renovate your home to boost its kerb appeal, while still taking pleasure in the process..
September 29, 2017 .Reading time 8 minutes.


text:
Hundreds of Nigerian schoolboys released after six-day kidnap ordeal
Bullet holes in a locker at the at the secondary school in Kankara from where hundreds of schoolboys were seized
Buses carrying the freed children were given a military escort
Ordeal: Some of the children were clearly distressed by the nightmare experience
Safe: Newly-released students were brought to a government building in Katsina after their release on Thursday
17 December 2020, 5:29 pm ·3-min read
More than 300 Nigerian schoolboys seized in a mass abduction claimed by Boko Haram experienced their first full day of freedom on Friday after a nearly week-long ordeal.
But relief at their survival mingled with concern that many others could still be captive, and the circumstances of their release remained unclear.
Looking dirty, worn-out and distraught, and most of them without shoes, the boys were brought to the governor's offices in Katsina, the capital of Katsina state in northwestern Nigeria, after being released late Thursday.
The assault last Friday on a rural school in Kankara, carried out by gunmen arriving on motorbikes, was initially blamed on criminal gangs who have terrorised the region for years.
But on Tuesday Boko Haram, the brutal jihadist group behind the abduction of 276 schoolgirls in Chibok in 2014, claimed responsibility.
Local officials announced late Thursday that the boys had been released and would spend the night in the protection of security agents.
A security source, speaking on condition of anonymity, said they had been left in the forest after negotiations between the authorities and the abductors, but gave no further details.
State governor Aminu Bello Masari said "344 are now with the security agencies" and were being taken to Katsina. They would be given medical care before reunited with their families, he said.
"This is a huge relief to the entire country & international community," President Muhammadu Buhari said on Twitter.
It remained unclear, though, if all the abducted schoolboys had been released, amid ongoing uncertainty over the number taken in the first place.
In an interview with state channel NTA, the governor added: "I think we have recovered most of the boys, it's not all of them."
In a video released by Boko Haram Thursday, a distressed teenager said he was among 520 students kidnapped.
The security source said late Thursday that the exact number would only be known after a head count in Katsina.
- Alliance of 'bandits and terrorists' -
Sources had previously told AFP that the raid was carried out by a well known criminal in the region, Awwalun Daudawa, in collaboration with Idi Minorti and Dankarami, two other crime chiefs with strong local followings, acting on behalf of Boko Haram.
Experts recently warned that jihadists -- operating in the northeast of the country, hundreds of kilometres (miles) from where Friday's attack occurred -- were attempting to forge an alliance with criminal gangs in the northwest.
President Buhari's official spokesman Garba Shehu said on Twitter "the northwest now presents a challenge which his administration is determined to deal with."
"It is unfortunate that the bandits and terrorists continue to get weapons even under the circumstances of the border closure. We are going to dare them."
Many parents of the missing students in Kankara said they had long feared an attack, given escalating violence in the region.
"Our children told us armed men would come up to the school fence but they never breached the fence... until last Friday," Hauwa'u Isah, mother of an abducted child said.
Around 8,000 people have been killed in the northwest since 2011, according to the International Crisis Group (ICG) think tank.
#BringBackOurBoys started trending on social media earlier this week, in reference to a similar hashtag after the Chibok kidnappings.
Small protests to push for the boys' release took place in Katsina on Thursday as Buhari was visiting the state.
"Why we are here today is because we want to tell the federal government that what they are doing is not enough," protester Jamilu Aliyu Turanci said. "Mr President has failed us."
abu/ri


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On the merits of indie gaming
(This was originally posted in response to the thread Indie > AAA on NeoGAF)
When I first posted here in July, I had only recently gotten into PC gaming. Now almost seven months later, I wanted to share my thoughts on the debate again from a more informed perspective. In those months, I've found myself immersed in the indie gaming scene. I rarely touch my consoles anymore, I check r/IndieGaming and TIGForum everyday, and have become one of the primary contributors to the NeoGAF monthly Indie Games threads, as well as the author of my own blog focused on indie gaming.
To say it was a radical shift, and an unexpected one at that, is probably an understatement. A year ago, even less than a year ago, I would have never imagined myself to be such an advocate for indie gaming. While I had dabbled in freeware and flash games over the years, I was always primarily a console gamer, mainly Playstation, since I was a kid. Between Uncharted, God of War, pretty much anything by Rockstar, I always loved games with the best visuals, crazy set pieces, bombastic action, etc....you know, the typical AAA experience.
However, I found myself less enamored by the wonders of AAA gaming as I got older. Now don't get me wrong, I'm still impressed by and enjoy big-budget console gaming as much as the next guy. The Last of Us is probably one of my favorite games of 2013, maybe even all time. But as games shifted more towards online focused, and many just seeming rote and uninspired, I was looking for original and unique experiences to play. And I found them in indie games
Personally I feel that gaming is the most innovative art medium out there. It's definitely the youngest, and definitely has to grow and develop more before being appreciated on the same level as literature and film, but I still feel that gaming is the one form of art that can truly immerse you in an experience, through a multi-faceted array of the visual, audio, narrative, and of course the interactive element. While many AAA games seems to only represent gaming as entertainment, the indie side of gaming has embraced the medium as an art form and in this way, continue to deliver unique original experiences that just can't be found anywhere else and certainly wouldn't be attempted by AAA publishers.
There are several reasons why I feel indie gaming is so much more rewarding than its AAA counterpart. Perhaps the most evident is the closeness between developer and community. While Ubisoft, Rockstar, Square Enix may communicate with its fanbase through community managers and the like, more often than not, indie developers have a direct line of communication between themselves and their fans, through devlogs, Tumblrs, Twitters, and forums. It's this closeness and transparency that makes indie development, in many ways, feel like a conversation between creator and community, more intimate than the relationship between fanbase and publisher.
The other reason and probably the one that intrigues me the most is the seemingly endless depth of originality and diversity that indie games offer. Put aside the notions and biases, look beyond those common cries of "It's all 2D pixel art platformers" and "Everything's inspired by Dark Souls", and you'll find a wide expanse of unique titles of all genres, styles, and themes.
In 2013 alone, I played the bleak hopeless tale of Gods Will Be Watching, delved into twisting meta-rabbit hole that was The Stanley Parable, fought for desperate survival and died under the cold rain in the roguelike Neo Scavenger. I explored an abstract frontier in Mirrormoon EP and discovered unexplored worlds in Outer Wilds. I watched my plans fall apart in blood and anarchy in Prison Architect, dropped tangos with timing and precision in Door Kickers, endured bullet hell mayhem in Assault Android Cactus. I become the lynchpin in fates both big and small in Papers Please, I admired the fluid artistry of Dust: An Elysian Tail, and honed my skills in the frantic Samurai Gunn.
That was only 2013. 2014 is even more promising, between the free-form rocket-propelled platforming of Cloudbuilt, the Greek mythos given life in Apotheon, the hyper-kinetic high-speed Distance, from the atmospheric worlds of Hyper Light Drifter and Rain World to the first-person adventure awaiting in The Witness and Routine. And that's only a fraction of what indie gaming in 2014 has to offer. And the fact that each and every day I discover new promising experiences that I had never heard of before, speaks volumes about the diversity and potential of independent gaming
However there is a final element that makes indie gaming worthwhile and worthy of your time and attention and it's an element that's easily summed up in one word: "independent." Indie developers are free to express ideas, values, narratives that just wouldn't be profitable or perhaps be deemed unfit for mainstream gamers. Would Rockstar or Ubisoft seek to tackle the subject of cancer and its effect on family (That Dragon, Cancer) or lead a player through a world crafted from the history of typography (Type:Rider)? Would a game about a troubled girl's childhood (Journal) focus test well enough to see release or would an experience about building massive self-sustaining autonomous factories on alien worlds (Factorio) be considered too complex and complicated to be released without hand-holding and myriad tutorials and tips? Indie developers are able to breathe life into their wildest concepts and most personal experiences, unfettered by the restrictions and barriers of AAA development, able to tackle concepts and themes that big mainstream developers can't or won't.
But in the end, it doesn't matter if a game has pixel graphics, or is inspired by Dark Souls, or is on IOS, or is an mobile-to-PC port, or is indie or AAA or whatever. What matters, what should always come first, is the gameplay, the experience itself. Is the game good, is it worth playing, is it fun, is it enjoyable? Doesn't matter the platform, or budget, or team size; a good game is a good game, period. Perhaps that's what we should strive for, not to enjoy or dismiss games because they're labelled as indie or AAA,, but to judge a game by the depth of its experience and the wonder of its artistry, by the quality of its gameplay,
Labels: Articles, Ramblings


Chapter 12. Methods of Evidence-Based Medicine and Decision Analysis. In: Dawson B, Trapp RG. Dawson B, Trapp R.G. Eds. Beth Dawson, and Robert G. Trapp.eds. Basic & Clinical Biostatistics, 4e New York, NY: McGraw-Hill; 2004. http://accessmedicine.mhmedical.com/content.aspx?bookid=356&sectionid=40086291. Accessed April 22, 2019.
. "Chapter 12. Methods of Evidence-Based Medicine and Decision Analysis." Basic & Clinical Biostatistics, 4e Dawson B, Trapp RG. Dawson B, Trapp R.G. Eds. Beth Dawson, and Robert G. Trapp. New York, NY: McGraw-Hill, 2004, http://accessmedicine.mhmedical.com/content.aspx?bookid=356&sectionid=40086291.


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European Film Awards
The European Film Awards are presented in 15 categories and take place in Berlin every second year.
Every year, the various activities of the European Film Academy culminate in the ceremony of the European Film Awards which are presented jointly with EFA Productions GmbH. In a total of 18 categories, among them European Film, European Director, European Actress and European Actor, the European Film Awards annually honour the greatest achievements in European cinema.
The awards ceremony takes place in Berlin every second year. In the other years, the Awards travel: London, Paris, Rome, Barcelona, Warsaw, Copenhagen, Tallinn and Malta.
@EuroFilmAwards #efa2018 #efa europeanfilmawards.eu
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EURIMAGES Co-Production Award for Kontovrakis & Karnavas
This year's EURIMAGES CO-PRODUCTION AWARD, an award acknowledging the decisive role of co-productions in fostering international exchange, goes to two fellow producers: Konstantinos Kontovrakis and Giorgos Karnavas.
Konstantinos Kontovrakis was born and raised in Athens, Greece. He obtained a BA in Film Studies and English Literature and an MA in Arts Management in London. He has published film reviews and articles in various Greek media and edited publications while collaborating with the Thessaloniki International Film Festival, for which he worked for ten years mainly as a programmer.
In 2010, Konstantinos turned to production as an executive for the film WASTED YOUTH by Argyris Papadimitropoulos & Jan Vogel. The film was the official opening film of Rotterdam's Tiger Award Competition and became an international arthouse and festival hit. This is where he met with Giorgos Karnavas, who produced the film and together they worked on various features as independent producers. Konstantinos was "Producer on the Move" for Greece in 2014 and he is the national representative of EAVE in Greece.
Giorgos Karnavas was born in 1973 in Athens, studied Political Sciences at the University of Athens and Finance at the Hellenic Management Association while taking courses on scriptwriting. In 1998 he joined Stefi Productions and was the General Manager and Head of Events from 2007 to September 2012.
He is the founder and producer of Synch Festival (2006 Best European Electronic Music Festival). During his career as event producer, he has promoted shows by artists such as Pet Shop Boys, Antony and the Johnsons, Nine Inch Nails, Einstuerzende Neubauten and Róisin Murphy.
Giorgos Karnavas entered film production in 2010 and is a member of ACE and the European and Greek Film Academies. He was a "Producer on the Move" in 2013 when he joined forces with Konstantinos Kontovrakis in order to establish the Athens-based production company Heretic (http://www.heretic.gr).
Heretic is focusing on quality-driven films and international co-productions, dedicated to working with filmmakers with a natural talent for storytelling and bringing like-minded people together.
Holding strong ties with the international film world, Heretic looks for projects with an international perspective and ensures their position in the world-wide market from development till completion.
The company produced the Eurimages-supported films SON OF SOFIA (EFA Feature Film Selection 2017) by Elina Psykou, which won the top prize of the International Narrative Feature Competition at the last edition of the Tribeca Film Festival, and PARI by Siamak Etemadi, which was selected for the prestigious L'atelier of the Cinefondation of the Cannes Film Festival in 2016. It also co-produced THE HARVESTERS by Etienne Kallos, supported by Eurimages, which was nominated in the section Un Certain Regard of the last Cannes Film Festival and SUMENDIA by Miguel Angel Jimenez, also supported by Eurimages, which is currently in post-production.
In 2014 Heretic launched Heretic Outreach, the international sales arm of the company that handles internationally outstanding films such as Ralitza Petrova's GODLESS (Golden Leopard, Locarno IFF; EFA Discovery Nominee 2017, Isabella Eklöf's HOLIDAY (Sundance World Cinema Competition) and Muayad Alayan's THE REPORTS ON SARAH AND SALEEM (Special Jury Award, Rotterdam IFF).
The EURIMAGES CO-PRODUCTION AWARD will be presented during the European Film Awards Ceremony on Saturday, 15 December, in Seville (Spain).
22.11.2018 | European Film Awards's blog
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The 2013 edition returns to Berlin this December.
After Paris, Glasgow, London, Rome, Barcelona, Warsaw, Copenhagen, Bochum, Tallinn and Valletta the European Film Awards 2014 will take place in Riga, Saturday, 13 December 2014
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