Call for Retraction of Applied Environmental Microbiology 64:4047-4052

Judge yourself whether Unilever and or the University of Amsterdam should have retracted this paper?

The former minister of justice Ernst Hirsch-Ballin, as chairman of the commission of investigation of scientific integrity at the University of Amsterdam, concluded that is not necessary to correct this type errors after more than five years!!!

My appeal that my need to mentally recover and to keep a distance to restore my balance, was the reason of the delay in raising the matter, was not granted. The subsequent unwillingness of Unilever, Applied Environmental Microbiology and the University of Amsterdam are indicative that the system and reputation of the establishment are more important than quality and integrity. The ignorant coauthor of this flawed paper Prof. Theo Verrips and former Chief Scientist of Unilever stated however, that it is never too late to correct an error. The truth will ultimately prevail!

The 'scientific'work presented in AEM 64:4047-4052 (1998) paper was proudly presented in the  inaugural public lecture of S. B. celebrating  his becoming professor in 1999. The firm conclusions were based, however, on a few hours Confocal Scanning Laser Microscopy and image analysis by his trainee Hui Zhang without proper controls and duplicates. Conclusions were based upon data manipulation without any statistical foundation. Most attention was paid to wording maximising a non-existing or minimal physiological effect. "cell rupture", "most sensitive", "most resistant", "hyper-sensitive", "prominent role", "massive", "rapidly lysed".

Coauthor prof. Theo Verrips, former Chief Scientist of Unilever Research, was not aware of the data manipulation and he fully agreed in 2015 that it should be rectified but  S. B. nor Prof. Rob Hamer of the current board of the Unilever Research Lab was willing to take responsibility for this scientific disgrace nor former minister Hirsch-Ballin and his integrity commission at the University of Amsterdam. It is, however, never too late to correct errors.

The American Society of Microbiology also did not  not seem to bother about data manipulation of a minimal effect without replication without any statistics nor that it is never too late to erase mistakes in our knowledge base.

"Thank you for sending your concern to ASM. As the Journal’s department Publishing Ethics Manager, I reviewed your allegations and have determined that this is an issue of scientific disagreement rather than a case of misconduct. Therefore, corrective action is not warranted."

Best regards,

Erika Davies,Publishing Ethics Manager

American Society for Microbiology
 

Examples of manipulative wording selected from text:

 

ABSTRACT

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture.

= Incorrect conclusion: Essential control for cell rupture was even missing. No viability check was even performed on treated yeast cells).

The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin.

= Suggestive wording after data manipulation:  The actual effect was a slight shift in  experimental noise, whilst the missing control and true Propidium Iodide-positive would have had a significant 100-1000fold higher fluorescence!

In contrast, the cells were most resistant

= Not based on any data: all cells were resistant to nisin, there was only a minor effect on membrane perturbation

to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive

= Very suggestive wording taking into account the actual data manipulation setting the thresholds to visually maximize a minimal physiological difference in membrane perturbation

to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin,

= Again, suggestive wording for a minimal effect)

and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.

INTRODUCTION: last part

Nisin has no antimicrobial effect on yeasts and filamentous fungi. These organisms each have a rigid cell wall, a complex structure consisting of glucan cross-linked with chitin and cell wall proteins (4, 18). The processing of mannoproteins is complex and has been partially characterized in yeasts (19, 21). A similar mechanism has been suggested for filamentous fungi (4). Because mannoproteins are generally considered one of the key wall components which determine cell wall porosity (8, 18), they may represent a major barrier preventing free permeation of nisin through the cell wall and thus access to the cytosolic membrane.

Initial experiments

= Data are not shown and are not present or based on an experimental artefact lacking the proper controls. The antimicrobial nisin is taken from a stock solution of diluted hydrochloric acid and if the pH is not properly controlled the measured antimicrobial effect may be solely based on differences in acidity in the actual tests.

MATERIALS AND METHODS

Analytical procedures.The confocal scanning laser microscope (CSLM) used consisted of a Zeiss Axioplan inverted microscope, a Bio-Rad model MRC-1024 system, and Lasersharp software. The objective used was a 1.5× zoom objective (magnification, ×63) with an image width of 110 μm. The software used to determine the percentage of PI-positive

= The software was manipulated to maximize the number of "PI-positive" cells. The setting of thresholds was not based on a proper control of 100% dead cells with damaged membranes. 

cells was the Leica Q500 MC software, as adapted by Aat Don (Department of Analytical & Information Sciences, Unilever, Vlaardingen, The Netherlands).

Nisin sensitivity.

Yeast cells harvested from a synchronous culture were resuspended to an OD620 of 1.0. Then 100 μl of the suspension was incubated with 10 μg of nisin per ml at pH 4 at room temperature for 2 min.

= It is unclear how the actual experiment was performed. The concentration of nisin in the stock solution is missing. Accordingly, the final concentration of HCl in the stock solution and the actual test are unknown. I strongly doubt that the pH in the test was 4.0 for all conditions. 

In the experiments in which the nisin sensitivities of cells and protoplasts were compared, a nisin concentration of 80 μg/ml

= equivalent to 4000 ppm Nisaplin, commercial nisin,  application level in practice  is 100 – 250 ppm)

was used. In the experiments performed with cell wall mutants, a concentration range of 0 to 50 μg/ml was assessed. After the peptide was removed, the pellet was suspended in 90 μl of dH2O and incubated with 5 μl of 100 μM FUN1 at 30°C for 20 min and then with 5 μl of a 1-mg/ml PI solution for 10 min under the same conditions (5). After staining, the cells were washed twice with dH2O to remove the excess dye and were analyzed with the CSLM system.

 

RESULTS

Yeast wall forms a barrier for small antifungal peptides.In order to determine whether the yeast cell wall forms a barrier to nisin, we first incubated cells with EDTA and dithiothreitol as described by de Nobel et al. (11) in order to increase the wall permeability. We observed that treatment of log-phase yeast cells with EDTA and/or dithiothreitol made them significantly more sensitive (by a factor of 2 to 4) to treatment with small antimicrobial peptides, such as nisin, as measured by the increase in the percentage of PI-positive cells.

 = The authors have defined PI-positive by setting a subjective value and subsequent data manipulation. They did not consult the experts Joerg Ueckert PhD, Ad Bos or Pieter ter Steeg PhD. Practice in Unilever Research Vlaardingen was to heat cells (e.g. the routine protocol in Vlaardingen of lactobacilli for 10 minutes at 80dC yielding 100% PI-positive cells with a fluorescent intensity of 100-1000x) which can be confirmed by Ad Bos (retired) and Joerg Ueckert)

Ueckert, J., P. Breeuwer, T. Abee, P. Stephens, G. Nebe von Caron, and P.F. ter Steeg (1995) Flow cytometry applications in physiological study and detection of foodborne microorganisms. Int. J. Food Microbiol. 28: 317-326.

Ueckert, J., G. Nebe von Caron, A.P. Bos, and P.F. ter Steeg (1997) Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury. Letters Appl. Microbiol. 25:295-299

To study the inferred barrier function of the yeast wall for nisin in more detail, we removed the cell wall by incubation with a wall-lytic enzyme preparation and incubated the spheroplasts with nisin. Spheroplasts did not stain with Calcofluor White but were stained when they were incubated with the viability dye FUN1. Cells with damaged membranes were stained red due to uptake of PI. Yeast spheroplasts rapidly lysed when they were incubated in the presence of nisin at concentrations which hardly affected intact cells (10 to 80 μg/ml).

= No results are shown + claimed lysis  was not observed and was negligible considering the small difference in PI-uptake  actually measured by CSLM.

Apparently, the cell wall normally forms a barrier for nisin. As the composition of the cell wall varies during the cell cycle, we set out to analyze the nisin sensitivity of yeast cells during the cycle.

Nisin sensitivity during the yeast cell cycle.After incubation of a yeast culture with α-factor, the synchronous growth of this culture was checked (Fig. 1). Three synchronous consecutive cell cycles were observed. Confirmation of the cell cycle progression was obtained by measuring the fluctuation in H2A mRNA levels. The level of the mRNA of H2A, a prominent cell cycle marker gene which is actively transcribed in the S phase, peaked at 60, 150, and 240 min after the transfer to fresh medium without α-factor. This is consistent with the observation that cells had small buds at these time points.

 

Fig. 1.

S. cerevisiae synchronized by α-factor. Line A, nonbudding cells; line B, cells with small buds; line C, budding cells with migrating nuclei; line D, large budded binuclear cells.

The nisin sensitivity of the synchronous culture is shown in Fig.2a. In the first cycle high percentages of PI-positive cells were recorded, and the culture was dominated by cells with small buds. In the second and third generations, however, cells with migrated nuclei seemed to be most sensitive to nisin. Perhaps the cells in the first cycle still suffered from direct effects of α-factor on the structure of their cell walls.

= Speculation based on subjective data manipulation. Again, PI-positive control was not included in this experiment and the actual effects are only signs of minor membrane perturbation. The spectacuar differences in red and green are no real fluorescent differences but just based on careful selecting a "threshold"-value in datamanipulation to maximise a minimal "effect" without replication,

The maximal levels of CWP1 and SED1 mRNA in the second and third generations were followed by maximal

= suggestive wording as cells were hardly sensitive to nisin and the actual levels of nisin were unrealistically high: >>commercial application

cell sensitivity to nisin. In contrast, peaks in CWP2 transcription were followed by maximal cell resistance to nisin. No clear correlation ofTIP1 expression with nisin sensitivity was observed, although a tendency towards a high level of expression being followed by a high level of resistance to nisin was noted. Nor was there a correlation between the levels of expression of FKS1 andCHS3 and the cyclic sensitivity to nisin. Thus, we concluded that glucan and chitin levels as such were not important in the protection of yeast cells from nisin, whereas Cwp2p seemed to be very important in conferring resistance to nisin upon yeast cells. These inferred roles were further substantiated by an analysis of nisin sensitivity in which various knockout yeast mutants were used. We analyzed a cwp1Δ mutant yeast strain, a cwp2Δ mutant yeast strain, a cwp1cwp2Δ mutant yeast strain, apmt1Δ mutant yeast strain, and a cwh53(fks1)Δ mutant yeast strain.

Sensitivity of yeast cell wall mutants to nisin.Figure3 shows that logarithmically growncwp1Δ cells were clearly more sensitive to the peptide than wild-type yeast cells were.

= Again, the authors have selected the threshold value for giving the cells a green or red color maximizing the differences whilst the actual levels had nothing to do cell viability. A proper control would have led to 0 % PI-positive for all treatments.

cwp2Δ cells were even more sensitive to nisin. Finally, almost all of the cells of the double mutant were sensitive to nisin, as shown by their massive

= the actual trainee report of Hui Zhang did not mention massive, The last and  responsible author S.B. is using very suggestive wording to sell the results.

 uptake of PI. Figure 3E shows the results in a bar diagram for nisin concentrations up to 50 μg/ml. A similar analysis was subsequently performed with apmt1Δ strain. Bourdineaud et al. have recently shown that this strain contains exceptionally low amounts of glucanase-extractable mannoproteins in its cell wall and accordingly has increased wall permeability, as shown by its hypersensitivity to Zymolyase treatment (3). However, pmt1Δ cells were not hypersensitive

= again, hypersensitive: a statement without proper control and data manipulation

to nisin (Fig. 4). As Cwp2p does occur in the walls of pmt1Δ cells, although at low levels, we concluded that Cwp2p plays a more crucial role than other glucanase-extractable mannoproteins in structuring the cell wall in such a way that the cell is protected against nisin. Fks1p-deficient cells, whose walls have a much lower β-1,3-glucan level, did not exhibit increased nisin sensitivity, confirming that glucan layers as such do not play a major role in the prevention of nisin permeation through the cell wall (data not shown).

 

Fig. 3.

Nisin sensitivity of wild-type and cell wall protein-deficient yeast strains at an OD620 of 1.0. (A through D) Images obtained with the CSLM of membrane integrity and cell viability

= Incorrect, no cell viability check was done, it claims only that “PI-positive” is dead,  which is not true in this study

after treatment with nisin (30 μg/ml). (A) Parent. (B)cwp1Δ. (C) cwp2Δ. (D) cwp1cwp2Δ. (E) Percentages of cells affected by nisin (0 to 50 μg/ml), as inferred from the images shown in panels A through D.

 

Fig. 4.

Nisin sensitivity ??????? of pmt1Δ and its parent, SEY6210. The values were deduced from images similar to those shown in Fig. 3A through D.

DISCUSSION

Our studies show that specific glucanase-extractable cell wall proteins with no known physiological function are crucial

= crucial after data manipulation and without proper controls

) in conferring resistance to the antimicrobial peptide nisin upon yeast cells. As the yeast cell wall mannoproteins are heavily glycosylated and therefore determining their specific levels immunologically is very difficult, we chose to measure the level of transcription of the genes involved in the expression of cell wall proteins rather than work with the proteins themselves. Although transcription levels do not necessarily correlate with the presence of the components in the cell wall, we know from preliminary studies performed with green fluorescent protein fusions that Cwp1p and Cwp2p appear in the cell wall at distinct points in the cell cycle, in agreement with Northern analysis data (17b). High levels of transcription of Cwp2p just before the stage in the cell cycle when the cells were very resistant

= again suggestive wording, without any thorough experimental evidence)  

to nisin suggested that this protein protects the cell from nisin and similar peptides. Upon depletion of both Cwp2p and Cwp1p, the cells were very sensitive (not based on proper studies) to nisin, as demonstrated by the high percentage of PI-positive cells.

 

PS

Publish or perish? What is more important? Quantity or quality? One excellent idea can already be sufficient for a Nobel Price. I am very concerned about the quality of our future students, if an ambitious person puts objective science on a second or even third place and can even become director of a research school at the University of Amsterdam. He became professor whilst almost religiously stating that genomics made (sound) scientific hypotheses redundant. Or that al major hygiene incidents of last century could have been prevented by genomics (95-99% were solely due to human error). Or if he deliberately ignored sound arguments and had the tunnel vision that he could use DNA methods for food product sterility testing. In essence, absence of survivors testing in > 10,000 liters by a method which needs at least 1000/ml. More than 10,000,000,0000 off! The wish to detect not even the needle but the very absent needle in a haystack. The flawed scientific base of the Unilever-TNO EET-Genomics proposal in 2002. Senior management at TNO was made to belief that Unilever supported the idea and vice versa and millions of government euros were wasted. His intellectual contributions to the field are minimal or even negative. He was mostly focused on extending his publication list. Even in 2014 he lifts as coauthor with negligible intellectual input on  a mediocre review (Critical Reviews in Food Science and Nutrition, 54:1371–1385 (2014))

For the record as I stated before I became responsible for the quality and vision of the science base in Preservation Microbiology at Unilever Research end of 2001. Besides dubious science I was subsequently confronted with a series of misleading subsidy proposals of  S.B. and his partner H.L, Our boss L.W.  bonus happened to be dependent in 2002 (I did not know till 2005) on the amount of subsidy acquired by the lab and I assume his personal target was cascaded down into the personal targets of my colleagues Brul and Lelieveld.  My objections against their poor quality and misleading proposals conflicted with their financial interests.  L.W.  put me out of my job in September 2002 when I objected against the deception and science fiction. In the summer of 2002 I overheard that H.L. had even arranged jobs for his son and son in law at his loyal subsidy partner ATO (the current AFGS of Wageningen University).  Unilever Research managed to get millions of subsidy twice on the same equipment. First via a national grant, secondly via the EU. It did not matter that the equipment was not even designed properly. The non-functioning Pulsed Electric Fields apparatus of Unilever had even to be moved from Vlaardingen to ATO at Wageningen to cash the EU subsidy. At ATO it was cynically called "The Horse of Troy" (source; Wouter de Heij) because the walls of the pilot plant litterally had to be broken down to get it in and the equipment was never used.  H.L. had also deceived the global R&D director of Unilever Foods, Ed Veltkamp, to put $10,000 per month in a Pulsed Pressure Sterilization (PPS) patent of Rich Meyer. The temperature in the vessel during the pressurization was misleadingly presented too low (see figure below), suggesting very mild sterilization conditions.

In 1999 I already suggested that the whole killing effect on bacterial spores by PPS was no magic but likely based on the high temperature during pressure pulses.  In 2003 I had to write a decision paper to allow Veltkamp an honourable exit strategy to end the consultancy of Rich Meyer before Veltkamps retirement. In 2003 poor Ed Veltkamp had already spent $480,000.on a worthless patent. Early 2003 H.L.  had already been discretely sent on early retirement because of his nepotism. Veltkamp was also not clean being a great supporter of an entrepreneurial climate at Vlaardingen. Creative (fraudulous) book keeping was stimulated to generate "matching funds"  for subsidy apllications lacking internal commitment.

 In those years Human Resources had also collaborated in facilitating an entrepreneurial climate by removing Objective Analytical Power and Self-Confident Integrity as core competencies for Unilever scientists. The introduction of a ranking system of dubious competencies was a sufficient instrument to eliminate too strict scientists. Senior managers no longer were capable to distinguish facts from opinions. Unilever was world leading in food microbiology but the science base was effectively destroyed by first putting me out of my job, chasing away the competent ones and subsequently appointing a successor Balkumar Marthi, who could not discriminate even protein precipitation from Staphylococcus growth in microtitre plate wells at extremely low pH and high salt concentration.

 

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