Bioanalytical Studies on the Effects of Chemical Compounds on the Respiratory Chain of Candida albicans focusing on Electrochemical Methods

May combine two claims.

Biochemical assays revealed the respiratory chain's activity and the importance of particular complexes.

“By applying biochemical methods, the activity of the respiratory chain and the importance of specific complexes were elucidated.”

Classified as probably not a substantial claim.

The DCIP-mediated electron transfer method was used to assess the activity of NADH-dehydrogenase complex I.

“DCIP-mediated electron transfer approach was used to evaluate the activity of NADH-dehydrogenase complex I in particular.”

• Also stated in: § Abstract · plain

  “In the fourth chapter, the DCIP-mediated electron transfer system was exploited to identify the activity of the respiratory chain complex I (NADH-dehydrogenase).”

May combine two claims.

Pyrrolnitrin-treated C. albicans showed significant glycerol accumulation at 4 and 5 hours while ethanol production was unchanged.

“A particular feature was observed in the glycerol production of pyrrolnitrin-treated cells, where, after 4 and 5h the increase in the glycerol accumulation was significant, while the ethanol production of pyrrolnitrin-treated cells was not changed.”

May combine two claims.

Pyrrolnitrin at 12 μg/ml inhibited S. cerevisiae respiration by about 50%, and this effect decreased at lower concentrations.

“Pyrrolnitrin strongly blocks the respiration of S. cerevisiae at 12 µg/ml (about 50 % of inhibitory effect), while this effect drops out with decreasing concentrations, as shown in Figure 3 (A).”

May combine two claims.

S. cerevisiae showed similar carbon-source-dependent toxicity, but toxic effects were generally lower than in C. albicans.

“Similar were observed in S. cerevisiae, but toxic effects were generally lower than on C. albicans.”

May combine two claims.

S. cerevisiae had a very slow normal oxygen uptake rate but showed strong sensitivity to myxothiazol and antimycin.

“although the normal oxygen uptake rate of S. cerevisiae was very slow, a strong sensitivity for myxothiazol and antimycin was detected.”

May combine two claims.

ETC inhibitors had no strong effects on yeast growth rates while they did affect oxygen uptake.

“From the previous results, no strong inhibitory effects on growth rates of the chosen organisms were observed while there were effects on the oxygen uptake.”

May combine two claims. The quote only partly states this claim.

S. cerevisiae growth completely stopped when treated with antimycin, myxothiazol, or pyrrolnitrin.

“The yeast cells growth totally stopped when they were treated with antimycin, myxothiazol or pyrrolnitrin.”

May combine two claims.

Rotenone or KCN did not notably increase ethanol or glycerol production in C. albicans, while SHA and TTFA caused only slight increases.

“No remarkable increase in ethanol/glycerol production was observed for cells treated with rotenone or KCN which had no inhibitory effect on respiration and only a slight increase in ethanol/glycerol was detected after treatment of C. albicans with SHA and TTFA”

May combine two claims.

Myxothiazol and antimycin strongly inhibited oxygen uptake and significantly induced ethanol and glycerol production in C. albicans.

“i.e. myxothiazol and antimycin which had a strong inhibition effect on the oxygen uptake led to a significant induction of ethanol/glycerol production.”

May combine two claims.

KCN reduced S. cerevisiae oxygen consumption, but its inhibitory effect was weaker than that of antimycin or myxothiazol.

“When KCN was added to S. cerevisiae cultures, the oxygen consumption was reduced but its inhibitory effect was not as strong as that of antimycin or myxothiazol.”

May combine two claims.

Antimycin or myxothiazol dramatically decreased C. albicans respiration, and this effect was much stronger than in S. cerevisiae.

“Treatment of C. albicans with antimycin or myxothiazol caused a dramatic decrease in the respiration process, which was much stronger than their effect on S. cerevisiae.”

May combine two claims.

TTFA inhibited C. albicans respiration more strongly than S. cerevisiae respiration, but less strongly than antimycin or myxothiazol in both organisms.

“TTFA impacts on the respiration of C. albicans are much stronger than its effects on the respiration of S. cerevisiae, even though still less active than antimycin and myxothiazol on both organisms.”

May combine two claims.

Rotenone and KCN did not inhibit C. albicans respiration even though C. albicans contains complexes I and IV.

“For example, rotenone and KCN did not show inhibitory effects on the respiration system of C. albicans, however, this organism encloses the complexes I and IV.”

May combine two claims.

C. albicans growing on galactose produced low amounts of ethanol, with no difference between control and ETC inhibitor-treated cells.

“This is supported by the low amounts of ethanol, which were produced by C. albicans growing in galactose containing medium.There was no difference between the control and the ETCinhibitors treated cells (Figure 6).”

May combine two claims.

C. albicans suspension showed a clear anodic peak at 600-800 mV and no corresponding cathodic peak, indicating an irreversible electrochemical oxidation reaction.

“Cyclic voltammograms (CV) of Candida albicans suspension showed a clear anodic peak at potentials of 600 -800 mV and no corresponding cathodic peak (Fig. 2a), indicating an irreversible electrochemical oxidation reaction, as previously described for other microorganisms [5][6][7].”

• cites [5][6][7]Not checked yet

  Not checked in the free run.

May combine two claims.

Among the tested yeast quorum-sensing alcohols, only tyrosol and tryptophol were electrochemically oxidized, with peak potentials at 630 mV and 740 mV, respectively.

“Only tyrosol and tryptophol were electrochemically oxidized, leading to peak potentials at 630 mV and 740 mV, respectively.”

May combine two claims.

KCN inhibited S. cerevisiae viability by more than 35%, while it inhibited C. albicans viability by about 15%.

“Only KCN effects were stronger in S. cerevisiae, whereas more than 35 % of S. cerevisiae viability was inhibited by KCN, only about 15% of C. albicans viability was inhibited, (Figures 7).”

May combine two claims.

Cell viability assay results matched the growth results, with inhibitor toxicity depending on the carbon source and being dramatic on galactose.

“These results were almost identical with the results of the cell viability assay, where the degree of inhibitors toxicity was dependent on the type of carbon source utilized by the yeast cells during the cultivation process where dramatic toxicity effects were found for most of the inhibitors on C. albicans and S. cerevisiae when galactose was the carbon source.”

May combine two claims.

KCN-treated C. albicans produced less ROS than untreated C. albicans, whereas KCN caused a slight increase in ROS production in S. cerevisiae.

“In contrast, KCNtreated C. albicans produces a lower ROS amount than the untreated-C.albicans, whereas, KCN led to slight increase in ROS production in S. cerevisiae.”

May combine two claims.

Inhibiting any classical respiratory chain component induced and activated AOX, causing electrons to pass directly from coenzyme Q to AOX and oxygen to be reduced to water.

“Inhibition of any of the classical respiratory chain led to the induction and activation of the AOX, and hence, the electrons passed directly from the coenzyme Q to the AOX and the oxygen was reduced to H 2 O.”

May combine two claims.

The anodic current increased with increasing pH up to pH 7 and decreased at higher pH.

“The anodic current increased with increasing pH up to pH 7. At higher pH the peak current decreased again (Fig. 3).”

May combine two claims.

For S. cerevisiae, adding glucose had almost no effect on the current, whereas adding galactose significantly increased the current.

“For S. cerevisiae, the addition of glucose had almost no effect, whereas the current significantly increased in the presence of galactose (Fig. 8).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cell numbers were 5.6 x 10^6/ml for S. cerevisiae WT, 5.9 x 10^6/ml for COX5a mutant, and 6.2 x 10^6/ml for COX5b mutant.

“cell numbers of 5.6 x 10 6 /ml, 5.9 x 10 6 /ml, and 6.2 x 10 6 /ml for S. cerevisiae (WT), COX5a, and COX5b, respectively.”

May combine two claims.

For C. albicans, the oxidative peak current was independent of the carbon source added, but adding carbohydrates significantly increased the current compared with plain buffer.

“For C. albicans, the oxidative peak current was independent of the carbon source added to the electrolyte solution, but the addition of the carbohydrates significantly increased the current in comparison with the plain buffer solution (Fig. 8).”

May combine two claims.

Voltammograms of the cell-free electrolyte solution showed the same oxidation peaks as the cell suspension, indicating that electron transfer was independent of the cells and attributable to a secreted compound.

“Voltammograms, which were obtained from the electrolyte solution from which the suspended yeast cells had been removed, showed the same oxidation peaks as the cell suspension.Thus, electron transfer was independent of the presence of the cells, i.e., was attributable to a secreted compound.”

May combine two claims.

For both yeasts, oxidative peak currents lay in the same potential range as described earlier for other microorganisms and correlated with cell numbers and the activity of the classical respiratory chain.

“For both yeasts, we have obtained oxidative peak currents that lie in the same potential range as described earlier for other microorganisms [6] and that correlated with cell numbers and the activity of the classical respiratory chain.”

• cites [6] 6. 9. Determination of reactive oxygen species (ROS) ....................................Not checked yet

  Not checked in the free run.

May combine two claims.

Autoclaved C. albicans cells produced no electrochemical response, indicating that the anodic current is an indicator of viable organisms.

“However, when the cells were autoclaved before they were injected into the electrochemical cell, no electrochemical response was observed.Thus, the anodic current was obviously an indicator of viable organisms.”

May combine two claims.

Most ETC inhibitors blocked O2 uptake, but rotenone and KCN increased the O2 uptake rate of C. albicans; this increase was not found in S. cerevisiae, where all respiratory chain inhibitors reduced O2 uptake.

“Concerning the effects of the ETC-inhibitors on the O 2 consumption the results showed that most inhibitors blocked the O 2 uptake.However, rotenone and KCN increased the O 2 uptake rate of C. albicans.This effect was not found in S. cerevisiae, where all of the respiratory chain inhibitors reduced the O 2 uptake.”

The quote only partly states this claim.

Inhibition of the alternative oxidase led to an increase in electrochemical signals.

“Inhibition of the alternative oxidase (AOX) led to an increase of signals.”

May combine two claims.

Rotenone, antimycin, or myxothiazol caused major ROS formation in C. albicans and S. cerevisiae, indicating NADH-dehydrogenase and cytochrome c reductase as two main mitochondrial ROS sources.

“Concerning the ROS results, the major formation of ROS by C. albicans and S. cerevisiae after addition of respiratory chain inhibitors, in particular rotenone, antimycin or myxothiazol, revealed that there were two main sources for the mitochondrial ROS production, NADH-dehydrogenase and cytochrome c reductase [28,29].”

• cites [28] 28. M. Bruatto, M. Gremmi, A. Nardacchione, and M. Amerio,Effect of glucose starvation on germ-tube production by Candida albicans. Mycopathologia, 1993. 123(2): p. 105-10.Not checked yet

  Not checked in the free run.

• cites [29] 29. G. Molero, R. Diez-Orejas, F. Navarro-Garcia, L. Monteoliva, J. Pla, C. Gil, M. Sanchez-Perez, and C. Nombela,Candida albicans: genetics, dimorphism and pathogenicity. Int Microbiol, 1998. 1(2): p. 95-106.Not checked yet

  Not checked in the free run.

May combine two claims.

In C. albicans, the impacts of ETC inhibitors on ROS production correlated with their effects on S. cerevisiae, and C. albicans generated higher amounts of ROS than S. cerevisiae.

“In C. albicans, the impacts of ETC-inhibitors on the cellular ROS production correlated to their effects on S. cerevisiae, as shown in Figure 8(B), while amounts of ROS generated by C. albicans were higher than that of S. cerevisiae.”

• AssumptionRests on an assumption: H2DCFDA is a valid measure of oxidative stress in cells.

May combine two claims.

Rotenone had no inhibitory effect on DCIP reaction with C. glabrata and S. cerevisiae regardless of glucose, while its inhibitory effect was significant for C. albicans.

“We observed no inhibitory effect of rotenone on the DCIP reaction with C. glabrata and S. cerevisiae, irrespective of the presence of glucose, while the rotenone inhibitory effect was significant for C. albicans (Figure 9).”

May combine two claims.

In C. albicans, inhibition of complex I by rotenone led to a strong increase of ROS formation, and blocking complex III by antimycin or myxothiazol also increased ROS.

“In more detail, inhibition of complex I by rotenone led to a strong increase of ROS formation, the same was observed after blocking the function of complex III by antimycin or myxothiazol.”

• AssumptionRests on an assumption: H2DCFDA is a valid measure of oxidative stress in cells.

May combine two claims.

No tryptophol was detected when cells were grown in YNB medium without tryptophan, and tryptophol concentrations increased with increasing tryptophan concentrations in the medium.

“Whereas no tryptophol could be detected when the cells were grown in YNB medium without tryptophan, increasing tryptophol concentrations were found when cells were grown in medium with increasing tryptophan concentrations (data not shown).”

May combine two claims.

The electrical signal from Δsln1 was reduced, while Δchk1 and Δcos1 behaved as wild-type.

“As shown in Figure 10 the electrical signal from Δsln1 reduced however Δchk1 and Δcos1 behaved as the wild-type.”

May combine two claims.

Inhibition of complex II had no effect on S. cerevisiae but reduced the signals from the C. albicans suspension.

“Inhibition of complex II had no effect on S. cerevisiae but reduced the signals from the C. albicans suspension.”

May combine two claims.

Galactose led to only a minor increase of signals from all three yeasts, whereas glucose increased the signals from S. cerevisiae and C. glabrata to the same level as C. albicans signals.

“The nonfermentable sugar galactose led to only a minor increase of signals from all three yeasts, whereas the addition of the fermentable sugar glucose increased the signals from S. cerevisiae and C. glabrata to the same level of C. albicans signals.”

May combine two claims.

ROS formation of C. albicans decreased after treatment with KCN by about 40%, whereas concentrations below 1.3 μg/ml KCN had no effect.

“Surprisingly, the ROS formation of C. albicans decreased after treatment with KCN by about 40 %, whereas concentrations below 1.3 µg/ml KCN had no effect.”

• AssumptionRests on an assumption: H2DCFDA is a valid measure of oxidative stress in cells.

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

Treatment with SHA increased the oxidation current of C. albicans.

“C. albicans was observed after treatment with SHA (alternative respiratory inhibitor), as shown in Fig. 9a.”

May combine two claims.

Only the complex I inhibitor rotenone decreased the DCIP signal in C. albicans, while the other inhibitors had no effect.

“As a result, only the complex I inhibitor rotenone was able to decrease the DCIP-signal in C. albicans whereas the other inhibitors had no effect.”

May combine two claims.

The sln1 deletion mutant had reduced NADH-DCIP-reductase activity, which was rotenone-resistant.

“We could show that the sln1 deletion mutant had a reduced activity of NADH-DCIP-reductase, which proved to be rotenone-resistant.”

• AssumptionRests on an assumption: A link exists between the respiratory chains of C. albicans and the HOG pathway, which comprises MAP-kinase Hog1 regulated by histidine kinases Sln1 and Canik1.

May combine two claims.

DCIP was reduced by all three yeasts and reduction was determined photometrically and voltammetrically via anodic peak current at approximately 0.6 V.

“DCIP was reduced by all three yeasts and we determined the reduction of DCIP photometrically, as the reduced form of DCIP is colorless, and voltammetrically via the anodic peak current at approximately 0.6 V.”

May combine two claims.

Most electron transport chain inhibitors reduced the electrochemical signal, but the AOX inhibitor SHA significantly increased the electrical current.

“Moreover, the addition of most electron transport chain inhibitors reduced the signal, but the inhibitor of the alternative oxidase AOX, SHA, led to a significant increase of the electrical current”

May combine two claims.

Gene expression analysis showed downregulation of genes of NADH-dehydrogenase subunits, supporting the conclusion of reduced complex I activity in the sln1 mutant.

“This conclusion is supported by the gene expression analysis showing the down regulation of genes of NADH-dehydrogenasesubunits.”

• AssumptionRests on an assumption: A link exists between the respiratory chains of C. albicans and the HOG pathway, which comprises MAP-kinase Hog1 regulated by histidine kinases Sln1 and Canik1.

May combine two claims.

The amount of tryptophol secreted into the electrolyte increases with the tryptophan concentration in the cultivation medium, and this increase correlates with increasing electrochemical signals.

“The secreted amounts of tryptophol into the electrolyte solution increased with tryptophan concentration in the cultivation medium; this effect was correlated with increasing electrochemical signals.”

May combine two claims.

Effects of KCN and pyrrolnitrin on ROS formation were concentration dependent, occurring only at high concentrations, while at lower concentrations only a slight increase was observed.

“Effects of KCN and pyrrolnitrin on the formation of ROS were also concentration dependent, occurring only at high concentrations, while, at lower concentrations only a slight increase in the ROS was observed.”

• AssumptionRests on an assumption: H2DCFDA is a valid measure of oxidative stress in cells.

May combine two claims.

Residual anodic peak currents were approximately 30% and residual absorbance changes almost 50%.

“Residual anodic peak currents of approximately 30 % and residual absorbance changes of almost 50 % were observed (Table 1).”

May combine two claims.

Signals from S. cerevisiae and C. glabrata were significantly increased by glucose, not galactose.

“The signals from S. cerevisiae and C. glabrata could significantly be increased by the addition of glucose, and not by the addition of galactose.”

May combine two claims.

Basal DCIP turnover by S. cerevisiae and C. glabrata was only 30% of that by C. albicans, but adding glucose increased it to the C. albicans level.

“Basal levels of DCIP turnover by S. cerevisiae and C. glabrata were only 30 % of those obtained from C. albicans, but could be increased to the C. albicans level by adding glucose.”

May combine two claims.

Both C. albicans and S. cerevisiae produced significant amounts of ROS after incubation with specific mitochondrial inhibitors.

“Both C. albicans and S. cerevisiae were able to produce significant amounts of ROS after incubating with specific mitochondrial inhibitors (Figure 8).”

• AssumptionRests on an assumption: H2DCFDA is a valid measure of oxidative stress in cells.

May combine two claims.

Catalyzed electron transfer from S. cerevisiae and C. glabrata to DCIP was observed at much lower rates and was rotenone-insensitive.

“We also observed the catalyzed electron transfer from S. cerevisiae and C. glabrata to DCIP, but at much lower rates, and these redox reactions were rotenone -insensitive.”

May combine two claims.

Single gene deletion mutants of COX subunits of S. cerevisiae all showed reduced electrical signals, with the strongest effect in the Δcox5a mutant.

“All of these mutants showed reduced electrical signals, the strongest effect being observed for the Δcox5a mutant.”

May combine two claims.

Results from photometric and voltammetric detection correlated very well and both signals could be used as indicators for DCIP interaction with yeast cells.

“Results from both detection methods correlated very well and both signals could be used as indicators for the interaction of DCIP with the yeast cells.”

May combine two claims.

DCIP-reducing activity could only be observed in viable C. albicans and was controlled by DCIP-NADH dehydrogenase, largely rotenone-sensitive and dependent on complex I.

“We showed that the DCIP -reducing activity could only be observed in viable C. albicans and was controlled by the catalytic activity of DCIP-NADHdehydrogenase, which was largely rotenone-sensitive, i.e. dependent on the activity of complex I of the classical respiratory chain.”

• AssumptionThe authors state this as an assumption: Respiratory chain enzymes, particularly NADH dehydrogenases, are involved in DCIP reduction.

May combine two claims.

DCIP blue color was consumed very fast and turned colorless in the presence of C. albicans, but rates were very low with C. glabrata or S. cerevisiae.

“It was observed that the blue color of DCIP (oxidized form) was consumed very fast and turned into the colorless (reduced form) in the presence of C. albicans, however the rates of blue color change were very low when DCIP was incubated with C. glabrata or with S. cerevisiae (Fig. 6A).”

May combine two claims.

When C. albicans was grown in tryptophan-free YNB medium, no oxidation peak was obtained, but adding tryptophan to the medium restored the oxidation peak.

“As a result, no oxidation peak was obtained when tryptophanfree YNB medium was used, whereas the oxidation peak was recovered where tryptophan was added to the YNB medium.”

May combine two claims.

C. albicans consumed most dissolved oxygen during the first 20 min, while more than 60% and 80% remained for C. glabrata and S. cerevisiae, indicating significantly higher respiratory activity of C. albicans.

“C. albicans consumed most of the dissolved oxygen during the first 20 min, whereas after the same incubation time still more than 60 % and 80 % dissolved oxygen was left when C. glabrata and S. cerevisiae were cultivated (Fig. 7), indicating a significantly higher respiratory activity of C. albicans.”

May combine two claims.

NADH concentrations in supernatants were higher than in cells, but DCIP reduction rates determined from yeast suspensions were higher than from supernatants, showing the catalytic effect of DCIP-NADH reductases.

“NADH concentrations in the supernatants were higher than in the cells; however, the DCIP reduction rates determined from the yeast suspensions were higher than from the supernatants, showing the catalytic effect of the DCIP -NADH reductases.”

May combine two claims.

In absence of glucose, rotenone-sensitive contribution to overall color consumption rate was 70-50%, whereas in presence of glucose it was approximately 30%.

“However, in the absence of glucose the rotenone-sensitive contribution to the overall color consumption rate was 70-50 %, whereas in the presence of glucose it was only approximately 30 % (Tab.1).”

May combine two claims.

For complex II inhibition by TTFA, oxygen uptake is inhibited, ROS production does not increase, cell viability is strongly inhibited, and growth rate is slightly inhibited.

“Complex II (TTFA): Inhibition of oxygen uptake; no increase of ROS production; a strong inhibition effect on the cell viability and a slight inhibition effect on the growth rate.”

May combine two claims.

With a 15-min reaction time of C. albicans with DCIP, the first peak became less sharp and developed into a wave whose steady-state current was almost identical to the original peak current.

“With the extension of the reaction time of C. albicans with DCIP (electrode switched off) to 15 min the first peak became less sharp and developed to a wave, of which the steady state current was almost identical to the original peak current (Fig. 2).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The details of regulation of aromatic alcohol secretion could not be elucidated by the present investigations.

“However, details of the regulation of the secretion of aromatic alcohols could not be elucidated with the present investigations.”

May combine two claims.

For the alternative oxidase inhibitor SHA, growth is not inhibited, viability is slightly affected, oxygen uptake is inhibited, and ROS formation slightly increases.

“Alternative oxidative inhibitor (SHA): No growth inhibition effect; a slight effect on viability; inhibition of the oxygen uptake rate and a slight increase of the ROS formation”

May combine two claims.

Only the fermentable carbon source glucose activated DCIP-NADH reductase activity in S. cerevisiae and C. glabrata, whereas the non-fermentable carbon source galactose had significantly smaller effects.

“However, only the presence of the fermentable carbon source glucose activated the DCIP-NADH reductase activity in the Crabtree-positive and complex I-negative yeasts S. cerevisiae and C. glabrata, whereas the non-fermentable”

• AssumptionRests on an assumption: Galactose is a non-fermentable carbon source, whereas glucose can also be consumed by fermentative pathways in S. cerevisiae.

May combine two claims.

The second DCIP oxidation peak's form did not change, and its peak current increased with reaction time at least comparably to the increase in the first oxidation step's steady-state level.

“In contrast, the peak form corresponding to the second oxidation step did not change and the peak current increased with reaction time at least comparable to the increase of the steady state level of the first oxidation step.”

May combine two claims.

The cyclic voltammogram of DCIP showed two oxidation peaks at +0.1 V and +0.5 V and a reduction peak at +0.05 V, indicating that the first oxidation step is reversible.

“The cyclic voltammogram of DCIP showed two clearly defined oxidation peaks at +0.1 V and +0.5 V, and a reduction peak at +0.05 V, indicating the reversible nature of the first oxidation step (Fig. 2a) [26].”

• cites [26] 26. F. J. Alvarez and J. B. Konopka,Identification of an N-acetylglucosamine transporter that mediates hyphal induction in Candida albicans. Molecular biology of the cell, 2007. 18(3): p. 965-75.Not checked yet

  Not checked in the free run.

May combine two claims.

For complex I inhibition by rotenone, growth rate and viability are not affected, oxygen consumption is accelerated due to induced AOX2 expression, ROS production is increased, and DCIP reduction is inhibited.

“Complex I (rotenone): growth rate and viability are not affected; oxygen consumption is accelerated due to induced expression of AOX2; ROS production is increased; DCIP reduction in inhibited.”

May combine two claims.

The wild-type was strongly affected by rotenone: rotenone decreased DCIP-reducing activity and increased ROS productivity.

“However, the wild-type was strongly affected by rotenone, whereas rotenone decreased the DCIP-reducing activity and led to increase the ROS productivity.”

May combine two claims.

The DCIP oxidation peak current increased by more than 50% when incubation with C. albicans was extended to 15 min (from about 16.5 μA to about 29 μA), while incubation with S. cerevisiae produced only a minor increase (about 10% in 15 min).

“The oxidation peak current increased by more than 50 % with increasing the incubation time of DCIP with C. albicans to 15 min (from approximately 16.5 μA to approximately 29 μA), whereas only a minor increase in the oxidation peak current of DCIP incubated with S. cerevisiae was observed (approximately 10 % in 15 min).”

May combine two claims.

For the complex III inhibitor, oxygen uptake is strongly inhibited, ROS production significantly increases, cell viability and growth rate are very strongly inhibited, and AOX2 is strongly induced.

“A strong inhibition of oxygen uptake; a significant increase in ROS production; very strong inhibition effect on the cell viability growth rate; a strong induction of AOX2.”

May combine two claims.

After adding C. albicans, the first DCIP peak became broader and the second peak shifted by almost 0.1 V to +0.6 V.

“Additionally, the first peak became broader, and the second peak shifted by almost 0.1 V to +0.6 V (Fig. 2b).”

May combine two claims.

Incubation of the Δsln1 mutant with rotenone did not inhibit DCIP-reducing activity or increase ROS production.

“Incubation of Δsln1 with rotenone did not inhibit the DCIPreducing activity (Figure 11A) or increase the ROS production (Figure 11B).”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

The respiratory chain's complexity requires sensitive and mechanistic assays to evaluate its status.

“In order to evaluate the respiratory chain status, the complexity of this structure necessities sensitive and mechanistic assays.”

• AssumptionRests on an assumption: The electron transport chain of Candida albicans is composed of three respiratory pathways: the classical, alternative oxidative, and parallel pathways.

Probably not a substantial claim: may not pass S3 (load-bearing).

Candida albicans is the most common human fungal pathogen.

“Candida albicans is the most prevalent human fungal pathogen.”

• Also stated in: § Candida albicans · plain

  “C. albicans is the major fungal pathogen of humans.”

• cites [5] 5. 6. Effects of electron transfer chain inhibitors ............................................. 2.7. Metabolic activation.................................................................................Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Candida albicans colonizes the mucosa and skin of roughly half of healthy people.

“This fungus colonizes mucosa and skin in approximately 50% of healthy individuals.”

• Also stated in: § Candida albicans · plain

  “C. albicans can colonize skin and mucosal surfaces of healthy people”

• cites [8] 8. 2. Peak current correlated with cell numbers and growth rate ..................... 3.3. Assay specificity ......................................................................................Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Candida albicans can cause opportunistic infections in immunocompromised patients, ranging from superficial to systemic.

“However, C. albicans causes opportunistic infections in immunocompromised patients that range from superficial to systemic infection.”

• Also stated in: § Candida albicans · plain

  “Candida infections are a serious problem, especially in individuals whose immune defense mechanisms have been weakened [6,7].”

• cites [6] 6. 9. Determination of reactive oxygen species (ROS) ....................................Not checked yet

  Not checked in the free run.

• cites [7] 7. Results and discussion ............................................................................................ 3.1. Electrochemical reaction of DCIP with yeasts ........................................Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

The electron transport chain is important in oxidative phosphorylation and aerobic metabolism.

“The electron transport chain plays a key role in processes such as oxidative phosphorylation, aerobic metabolism, and in the expression of virulence factors of C. albicans.”

• cites [47] 47. L. A. Walker, N. A. Gow, and C. A. Munro,Fungal echinocandin resistance. Fungal Genet Biol, 2010. 47(2): p. 117-26.Not checked yet

  Not checked in the free run.

• cites [48] 48. M. J. Dagley, I. E. Gentle, T. H. Beilharz, F. A. Pettolino, J. T. Djordjevic, T. L. Lo, N. Uwamahoro, T. Rupasinghe, D. L. Tull, M. McConville, C. Beaurepaire, A. Nantel, T. Lithgow, A. P. Mitchell, and A. Traven,Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2. Mol Microbiol, 2011. 79(4): p. 968- 89.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In S. cerevisiae ETC, electron transport to UQ by internal and external NADH dehydrogenases is not coupled to proton translocation, so only complex III and IV pump protons.

“The major functional difference of the internal and external NADH-dehydrogenases from complex I is that electron transport to UQ is not coupled to proton translocation and therefore, there are only two protons pumping sites (complex III and IV) in S. cerevisiae electron transport chain.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

This DCIP reaction is catalyzed by NAD(P)H dehydrogenases, and DCIP is used as an indicator of NADH dehydrogenase activities, particularly complex I.

“This reaction is catalyzed by NAD(P)H dehydrogenases and DCIP is used as an indicator of NADH dehydrogenase activities, in particular the activity of complex I (Figure 13).”

Probably not a substantial claim: may not pass S3 (load-bearing).

In MFCs, microbial activity is exploited to generate electricity from organic substrates such as glucose or other carbon sources.

“exploited to generate electricity from various organic substrates (such as glucose or other carbon sources) [86].”

• cites [86] 86. D. M. Kuhn, M. Balkis, J. Chandra, P. K. Mukherjee, and M. A. Ghannoum,Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol, 2003. 41(1): p. 506-8.Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

A mediator's reduced state should easily escape from the cell through the cell membrane after reduction.

“(b) The reduced state of the mediator should easily escape from the cell (after reduction) through the cell membrane.”

• cites [101] 101. Z. D. Liu, Z. W. Du, J. Lian, X. Y. Zhu, S. H. Li, and H. R. Li,Improving energy accumulation of microbial fuel cells by metabolism regulation using Rhodoferax ferrireducens as biocatalyst. Lett Appl Microbiol, 2007. 44(4): p. 393-8. Chapter 1: Background and Overview of the Study 94. K. T. Finneran, C. V. Johnsen, and D. R. Lovley,Rhodoferax ferrireducens sp. nov., a psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the reduction of Fe(III). Int J Syst Evol Microbiol, 2003. 53(Pt 3): p. 669-73.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

ROS are generated in mitochondria as a normal byproduct of aerobic metabolism; 0.2-2% of O2 consumption is estimated lost as superoxide under normal conditions.

“ROS are generated in mitochondria as a normal byproduct of aerobic metabolism; 0.2-2% of O 2 consumption is estimated to be lost as superoxide under normal conditions [71].”

• cites [71] 71. N. Minagawa, S. Koga, M. Nakano, S. Sakajo, and A. Yoshimoto,Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala. FEBS Lett, 1992. 302(3): p. 217-9.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

The oxidized form of DCIP easily penetrates cell membranes and reacts with the intracellular NAD(P)H pool.

“The oxidized form of DCIP easily penetrates cell membranes and reacts with the intracellular NAD(P)H pool.”

May combine two claims.

The electron transport chain of Candida albicans is vital for its survival, especially when the growth medium contains a non-fermentable sugar such as galactose.

“Therefore, the electron transport chain of C. albicans is a vital unit for its survival, particularly if the growth medium contains a non-fermentable sugar (galactose).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The density of electrical current from microorganisms depends on concentrations of electron transfer agents and efficiency of electron transfer.

“The density of the electrical current coming from the microorganisms is dependent on the concentrations of the electron transfer agents and on the efficiency of the electron transfer.”

Probably not a substantial claim: may not pass S3 (load-bearing).

C. glabrata respiratory chain structure is similar to S. cerevisiae.

“It's worth mentioning here that the respiratory chain structure of C. glabrata is similar to the respiratory chain structure of S. cerevisiae [68].”

• cites [68] 68. X. Roucou, I. M. Artika, R. J. Devenish, and P. Nagley,Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal Chapter 1: Background and Overview of the Study activity and structural stability of the enzyme complex. Eur. J. of biochem./ FEBS, 1999. 261(2): p. 444-51.Not checked yet

  Not checked in the free run.

May combine two claims.

The polarographic method for monitoring oxygen uptake requires large culture volume, consumes large amounts of expensive inhibitors, and has limited sample number per assay.

“However, this method required a large volume of culture which leads to consume large amounts of the expensive inhibitors, moreover, the limited sample number per assay.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

In BES, microorganisms catalyze electrochemical reactions through interactions with electrodes.

“In the bioelectrochemical system (BES), microorganisms catalyze electrochemical reactions through interactions with electrodes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Mitochondrial complexes I and III are generally regarded as the main sources of ROS in the respiratory chain.

“Concerning the nature of the superoxide generators in the respiratory chain, mitochondrial complexes I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase) are generally regarded as the main sources of ROS (Fig. 11) [72].”

• cites [72] 72. M. G. Shepherd, C. M. Chin, and P. A. Sullivan,The alternate respiratory pathway of Candida albicans. Arch Microbiol, 1978. 116(1): p. 61-7.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

DCIP was also used in a toxicity assay based on complex I-negative yeast S. cerevisiae when glucose was used as carbon source.

“However, DCIP was also used in a toxicity assay based on the complex Inegative yeast S. cerevisiae, when glucose was used as a carbon source [81,85].”

• cites [81] 81. S. Drose and U. Brandt,The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. J Biol Chem, 2008. 283(31): p. 21649-54.Not checked yet

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• cites [85] 85. a. K. N. Paul Held. Using BioTek's Synergy™ HT Reader to Measure Reactive Oxygen Species (ROS) Generation in Stimulated Cells. 2008.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Disruption of NDE2 had no effect.

“However, disruption of NDE2 had no effect [66,67].”

• cites [66] 66. J. P. Collman, L. Fu, P. C. Herrmann, and X. Zhang,A functional model related to cytochrome c oxidase and its electrocatalytic four-electron reduction of O2. Science, 1997. 275(5302): p. 949-51.Not checked yet

  Not checked in the free run.

• cites [67] 67. P. V. Burke and R. O. Poyton,Structure/function of oxygen-regulated isoforms in cytochrome c oxidase. J Exp Biol, 1998. 201(Pt 8): p. 1163-75.Not checked yet

  Not checked in the free run.

May combine two claims.

Respiratory chain activity is linked to multiple virulence factors of Candida albicans, including hyphal formation, biofilm formation, cell wall integrity, adhesion, and exoenzyme production.

“The hyphal formation of C. albicans can be suppressed by blocking the enzymatic functions of the respiratory chains [31,32].”

• Also stated in: § 1.3.2. Biofilm formation · plain

  “Interestingly, blocking the respiratory chain leads to prevention of biofilm formation in C. albicans [35].”

• Also stated in: § 1.3.4. Adhesion of C. albicans · plain

  “It has been shown that a deficiency in the respiratory system led to a reduction in the adhesion properties of C. albicans to the host cells [39].”

• cites [31] 31. S. P. Hawser, G. S. Baillie, and L. J. Douglas,Production of extracellular matrix by Candida albicans biofilms. J Med Microbiol, 1998. 47(3): p. 253-6.Not checked yet

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• cites [32] 32. G. Ramage, S. P. Saville, D. P. Thomas, and J. L. Lopez-Ribot,Candida biofilms: an update. Eukaryotic cell, 2005. 4(4): p. 633-8.Not checked yet

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• cites [35] 35. W. L. Chaffin, J. L. Lopez-Ribot, M. Casanova, D. Gozalbo, and J. P. Martinez,Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev, 1998. 62(1): p. 130-80. Chapter 1: Background and Overview of the Study 25. Y. Fukazawa and K. Kagaya,Molecular bases of adhesion of Candida albicans. J Med Vet Mycol, 1997. 35(2): p. 87-99.Not checked yet

  Not checked in the free run.

• cites [39] 39. B. Hube,Possible role of secreted proteinases in Candida albicans infections. Rev Iberoam Micol, 1998. 15(2): p. 65-8.Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Organisms such as Shewanella putrefaciens, Geobacter sulfurreducens, Geobacter metallicreducens and Rhodoferax ferrireducens generate electricity in mediatorless MFC systems.

“Organisms such as Shewanella putrefaciens [90], Geobacter sulfurreducens [91,92], Geobacter metallicreducens and Rhodoferax ferrireducens [93,94] have been shown to generate electricity in mediatorless MFC systems.”

• cites [90] 90. H. Nakamura, Y. Hirata, Y. Mogi, S. Kobayashi, K. Suzuki, T. Hirayama, and I. Karube,A simple and highly repeatable colorimetric toxicity assay method using 2,6- dichlorophenolindophenol as the redox color indicator and whole eukaryote cells. Anal Bioanal Chem, 2007. 389(3): p. 835-40.Not checked yet

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• cites [91] 91. A. J. Janssen, F. J. Trijbels, R. C. Sengers, J. A. Smeitink, L. P. van den Heuvel, L. T. Wintjes, B. J. Stoltenborg-Hogenkamp, and R. J. Rodenburg,Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin Chem, 2007. 53(4): p. 729-34. Chapter 1: Background and Overview of the Study 83. R. Naumann, D. Mayer, and P. Bannasch,Investigation of the carbohydrate metabolism of normal and neoplastic hepatocytes using 2,6-dichlorophenolindophenol as a probe for NAD(P)H production measured by voltammetry. Biochim Biophys Acta, 1985. 847(1): p. Not checked yet

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• cites [92] 92. G. G. a. R. Narayanaswamy,Effect of pH on the redox equilibria of immobilised 2,6- dichloroindophenol Analytica Chimica Acta, 1993. 279: p. 6.Not checked yet

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• cites [93] 93. H. Nakamura, S. Kobayashi, Y. Hirata, K. Suzuki, Y. Mogi, and I. Karube,A spectrophotometric biochemical oxygen demand determination method using 2,6- dichlorophenolindophenol as the redox color indicator and the eukaryote Saccharomyces cerevisiae. Anal Biochem, 2007. 369(2): p. 168-74.Not checked yet

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• cites [94] 94. T. H. Pham, P. Aelterman, and W. Verstraete,Bioanode performance in bioelectrochemical systems: recent improvements and prospects. Trends Biotechnol, 2009. 27(3): p. 168-78.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

ROS generation by ETC is critically dependent on ETC redox state, and disturbance of normal redox state leads to ROS production that can damage living system components.

“ROS generation by the electron transport chain (ETC) is critically dependent upon ETC redox state, and the disturbance of the normal redox state of mitochondria leads to a production of ROS that can cause damage in components of the living system.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The use of yeasts to generate electrical current has been suggested because they can grow on cheap substrates and do not require anaerobic conditions.

“However, the use of yeasts to generate electrical current has also been suggested [88] as they can grow on cheap substrates and do not require anaerobic conditions.”

• cites [88] 88. P. W. Sylvester,Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods Mol Biol, 2011. 716: p. 157-68.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Inhibitors are known for each major respiratory complex: rotenone for complex I, TTFA for complex II, antimycin and myxothiazol for complex III, cyanide, azide, and carbon monoxide for complex IV, and oligomycin for complex V.

“Inhibitors are known for each of the major enzyme complexes; SHA: salicylhydroxamic acid; AA: antimycin A; KCN: cyanide; TTFA: Thenoyltrifluoroacetone.”

• Also stated in: § 3.1.1.1. Complex I · plain

  “Rotenone inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone, which leads to preventing the oxidation of NADH.”

• Also stated in: § 3.1.1.5. Complex V (ATP Synthase) · plain

  “Oligomycin is the specific inhibitor of complex V [59].”

• cites [59] 59. J. E. Walker,The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains. Q Rev Biophys, 1992. 25(3): p. 253-324.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In S. cerevisiae, most elements of classical respiratory chain are present, but complex I is absent.

“In S. cerevisiae, most elements of the classical respiratory chain are present, but complex I is absent (Figure 9).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Electron transfer to extracellular solid materials such as anodes and metal oxides occurs either directly or indirectly via electron mediators.

“These studies have shown that electron transfer to extracellular solid materials, such as anodes and metal oxides, occurs either directly or indirectly via electron mediators (natural mediators or artificial mediators) [89].”

• cites [89] 89. J. Hoper,Spectrophotometric in vivo determination of local mitochondrial metabolism by use of a tetrazolium salt. Physiol Meas, 1997. 18(1): p. 61-6.Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

To measure ROS in living cells, reagents must cross intact cell membranes to react with superoxides.

“However, to measure the ROS in the living cells the used reagents must cross the intact cell membranes to be able to react with the superoxides.”

Probably not a substantial claim: may not pass S4 (based).

Electrochemical detection methods allow direct communication with the cellular electron transfer system.

“Electrochemical detection methods were of particular interest, because they offer the possibility of direct communication with the cellular electron transfer system.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

External and internal NADH dehydrogenases are alternatives for complex I, which is missing in S. cerevisiae.

“External and internal NADH dehydrogenases are alternatives for the complex I which is missing in S. cerevisiae.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Under specific conditions such as biofilm formation, some bacteria (Geobacter and Shewanella) produce conductive nano-wires (Pili) as main connector between microorganisms and electrode surface.

“Under specific conditions (e.g.biofilm formation), some bacteria (Geobacter and Shewanella) are able to produce conductive nano-wires (Pili) as a main connector between the microorganisms and the electrode surface [97][98][99].”

• cites [97][98][99]Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The bacterial electron transport chain is more easily accessible than that of eukaryotes, and most reports of MFCs deal with bacterial bioanodes.

“Thus, the bacterial electron transport chain is more easily accessible than that of eukaryotes, and most reports of microbial fuel cells deal with the development of bacterial bioanodes [87].”

• cites [87] 87. T. Mosmann,Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): p. 55-63.Not checked yet

  Not checked in the free run.

May combine two claims.

Candida albicans is Crabtree-negative and obtains energy mainly through aerobic respiration via the mitochondrial TCA cycle even when glucose is present.

“C. albicans is a Crabtree-negative organism and obtaining its energy relies mainly upon the (aerobic respiration) oxidation of substrates via the mitochondrial tricarboxylic acid cycle to generate ATP even in the presence of glucose [34].”

• cites [34] 34. M. Fukayama and R. A. Calderone,Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants. Infect Immun, 1991. 59(4): p. 1341-5.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

The complex and flexible electron transport system of Candida albicans lets it adapt to inhibition of individual complexes or pathways by redirecting metabolism or expressing additional genes.

“The complex and flexible structure of the electron transport system in C. albicans allows the organism to adapt to the inhibition of single complexes or pathways of the respiratory chain by the redirection of metabolic pathways or the expression of additional genes.”

• AssumptionRests on an assumption: The electron transport chain of Candida albicans is composed of three respiratory pathways: the classical, alternative oxidative, and parallel pathways.

Probably not a substantial claim: may not pass S3 (load-bearing).

In complex I, superoxide is primarily produced at the bound flavine facing the matrix side.

“In complex I, superoxide is primarily produced at the bound flavine facing the matrix side.”

• cites [72] 72. M. G. Shepherd, C. M. Chin, and P. A. Sullivan,The alternate respiratory pathway of Candida albicans. Arch Microbiol, 1978. 116(1): p. 61-7.Not checked yet

  Not checked in the free run.

May combine two claims.

The classical respiratory chain of Candida albicans produces the largest amount of cellular ATP because it is the main source of proton pumping across the mitochondrial inner membrane.

“The CRC of C. albicans provides the largest amount of cellular ATP because it is the major source of pumping protons across the mitochondrial inter-membrane space [49].”

• cites [49] 49. Z. R. Arie, Z. Altboum, H. Sandovsky-Losica, and E. Segal,Adhesion of Candida albicans mutant strains to host tissue. FEMS Microbiol Lett, 1998. 163(2): p. 121-7.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Further studies showed pyrrolnitrin not only acts as ETC-inhibitor but also interferes with osmotic signal transduction pathways in Neurospora crassa.

“Further studies showed that pyrrolnitrin not only acts as ETC-inhibitor but also interferes with the osmotic signal transduction pathways in Neurospora crassa [21].”

• cites [21] 21. J. Chandra, D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick, and M. A. Ghannoum,Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol, 2001. 183(18): p. 5385- 94.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The CRC comprises four enzymatic complexes.

“The CRC comprises the four enzymatic complexes as described before [10].”

• cites [10] 10. 5. Effect of rotenone after metabolic activation ...........................................Not checked yet

  Not checked in the free run.

May combine two claims.

Myxothiazol was extracted from Myxococcus fulvus and used to inhibit mitochondrial complex III.

“Myxothiazol was extracted from the myxobacterium, Myxococcus fulvus, and used to inhibit the mitochondrial complex III [17,18].”

• cites [17] 17. E. Segal,Candida, still number one--what do we know and where are we going from there? Mycoses, 2005. 48 Suppl 1: p. 3-11.Not checked yet

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• cites [18] 18. F. C. Odds, A. D. Davidson, M. D. Jacobsen, A. Tavanti, J. A. Whyte, C. C. Kibbler, D. H. Ellis, M. C. Maiden, D. J. Shaw, and N. A. Gow,Candida albicans strain maintenance, replacement, and microvariation demonstrated by multilocus sequence typing. J Clin Microbiol, 2006. 44(10): p. 3647-58.Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Metabolite quantification of ethanol and glycerol is used as indicator of activity of respiratory vs fermentation pathways.

“The metabolites quantification of ethanol and glycerol is used as indicator of the activity of the respiratory vs the fermentation metabolic pathways.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cyclic voltammetry can rapidly provide information on thermodynamics of redox processes, kinetics of electron transfer reactions, and coupled chemical reactions or adsorption processes.

“The power of cyclic voltammetry results from its ability to rapidly provide considerable information on the thermodynamics of redox processes and the kinetics of electron transfer reactions and on coupled chemical reactions or adsorption processes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

These alternative dehydrogenases are usually rotenone-insensitive.

“These alternative dehydrogenases are usually rotenone-insensitive.”

May combine two claims.

In other fungi such as C. glabrata and S. cerevisiae, mitochondrial complex I is absent and external and internal NADH dehydrogenases catalyze oxidation of NADH without generating trans-membrane proton gradient.

“In other fungi, such as the pathogenic yeast Candida glabrata and the non-pathogenic yeast Saccharomyces cerevisiae, the mitochondrial complex I is absent and the so-called external and internal NADH dehydrogenases catalyze the oxidation of NADH without generation of a trans-membrane proton gradient [10,11,13,14].”

• cites [10] 10. 5. Effect of rotenone after metabolic activation ...........................................Not checked yet

  Not checked in the free run.

• cites [11] 11. 6. Correlations to NADH concentrations in cells and supernatants .............Not checked yet

  Not checked in the free run.

• cites [13] 13. A. T. Sangamwar, U. D. Deshpande, and S. S. Pekamwar,Antifungals: need to search for a new molecular target. Indian J Pharm Sci, 2008. 70(4): p. 423-30.Not checked yet

  Not checked in the free run.

• cites [14] 14. J. A. van Burik and P. T. Magee,Aspects of fungal pathogenesis in humans. Annu Rev Microbiol, 2001. 55: p. 743-72.Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

These oxidases catalyze direct oxidation of ubiquinol by oxygen without proton translocation, enabling respiration even in presence of CRC inhibitors.

“These oxidases catalyze the direct oxidation of ubiquinol by oxygen without proton translocation and, thus, enable respiration even in the presence of CRC inhibitors.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Pyrrolnitrin treatment activates the HOG pathway in Neurospora crassa.

“It was reported that the HOG pathway of Neurospora crassa treated with pyrrolnitrin is activated which may indicate that pyrrolnitrin has an additional function beside its effect on the respiratory chain [21].”

• cites [21] 21. J. Chandra, D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick, and M. A. Ghannoum,Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol, 2001. 183(18): p. 5385- 94.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

Determination of oxygen consumption rates shows overall activity of terminal oxidases.

“Determination of oxygen consumption rates shows the overall activity of terminal oxidases.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Better potential control is achieved using a potentiostat and three-electrode system.

“Better potential control is achieved using a potentiostat and the three electrode system (Figure 16), in which the potential of one electrode (the working electrode) is controlled relative to the reference electrode, and the current passes between the working electrode and the third electrode (the auxiliary electrode) [108].”

• cites [108] 108. A. J. Carrillo-Munoz, G. Giusiano, P. A. Ezkurra, and G. Quindos,Antifungal agents: mode of action in yeast cells. Rev Esp Quimioter, 2006. 19(2): p. 130-9.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The paste composition strongly affects electrode reactivity; increasing pasting liquid content decreases electron transfer rates and background current contributions.

“The paste composition strongly affects the electrode reactivity, with the increase in pasting liquid content decreasing the electron transfer rates, as well as the background current contributions [110].”

• cites [110] 110. J. A. van Burik and P. T. Magee,Aspects of fungal pathogenesis in humans. Annu Rev Microbiol, 2001. 55: p. 743-72.Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

Analysis of mitochondrial complex I and IV activities without isolation of mitochondria and enzyme purification is difficult using classical biochemical assays.

“Analysis of the mitochondrial complex I and IV activities, without the isolation of mitochondria and enzyme purification, is difficult using classical biochemical assays.”

May combine two claims.

The effect of respiratory chain inhibitors on yeast cells can be easily detected by measuring oxygen uptake as growth is not affected in rich media.

“We have reported that the effect of respiratory chain inhibitors on yeast cells can be easily detected by measuring oxygen uptake as growth is not affected in rich media [16].”

• cites [16] 16. D. Gozalbo, P. Roig, E. Villamon, and M. L. Gil,Candida and candidiasis: the cell wall as a potential molecular target for antifungal therapy. Curr Drug Targets Infect Disord, 2004. 4(2): p. 117-35.Not checked yet

  Not checked in the free run.