On 2015 Mar 26, Rahul Lall commented:

Dear Vera,

Very exciting and interesting paper. I just had a few experimental questions. How was the HA enriched from the cell media from 20 days? If you could share the protocol of the method, would be great. My email is rklall@wisc.edu.

On 2014 Nov 26, Ruchira Engel commented:

Dear Vera Gorbunova, Thank you for clarifying our doubts.

On 2014 Nov 25, Vera Gorbunova commented:

Dear Ruchira Engel, thank you for your comments. The two concerns you raised could be answered by carefully reading the paper.

1. “How much does the size actually matter?” When purified HAase is added to the cells, or when Hyal2 enzyme is overexpressed, as in Fig. 4, this does not lead to complete “disappearance” of HA, but rather to a reduced size. HAases cleave the sugar chain into smaller fragments, but these fragments actually stay around, so the experiment you are proposing with adding a shorter HA is pretty much what has been done throughout the study.

We state in the Abstract that both the size of the size of the sugar and the signaling are different in the naked mole rat: “Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells.” Clearly, both the ligand and the receptor underwent changes together over million of years of evolution; trying to separate the two is rather artificial.

1. “Is HA degradation really slower in the naked mole rat?” The time needed to accumulate the amount of HA shown on pulse field gels was 20 days (stated in the legend to Fig. 1). That was also the amount of HA used in the degradation assays. Four days used for incubation in the degradation measurements in Fig. 2c is simply not enough time to see a significant contribution of HA synthesis. Furthermore, in Fig. 2d where tissue fragments were used, the incubation time was even shorter (6 hours).

On 2014 Nov 22, Ruchira Engel commented:

An interesting study! Earlier this year we discussed this article in our journal-club and agreed that this was a well written article with experiments that support the proposed mechanism. But is there sufficient evidence to suggest that it is the size of HA and not the signalling alone in the naked mole fibroblasts that leads to the cancer free state of the naked mole rats?

Please check our review of this article.

On 2014 Nov 22, Ruchira Engel commented:

An interesting study! Earlier this year we discussed this article in our journal-club and agreed that this was a well written article with experiments that support the proposed mechanism. But is there sufficient evidence to suggest that it is the size of HA and not the signalling alone in the naked mole fibroblasts that leads to the cancer free state of the naked mole rats?

Please check our review of this article.

On 2014 Nov 25, Vera Gorbunova commented:

Dear Ruchira Engel, thank you for your comments. The two concerns you raised could be answered by carefully reading the paper.

1. “How much does the size actually matter?” When purified HAase is added to the cells, or when Hyal2 enzyme is overexpressed, as in Fig. 4, this does not lead to complete “disappearance” of HA, but rather to a reduced size. HAases cleave the sugar chain into smaller fragments, but these fragments actually stay around, so the experiment you are proposing with adding a shorter HA is pretty much what has been done throughout the study.

We state in the Abstract that both the size of the size of the sugar and the signaling are different in the naked mole rat: “Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells.” Clearly, both the ligand and the receptor underwent changes together over million of years of evolution; trying to separate the two is rather artificial.

1. “Is HA degradation really slower in the naked mole rat?” The time needed to accumulate the amount of HA shown on pulse field gels was 20 days (stated in the legend to Fig. 1). That was also the amount of HA used in the degradation assays. Four days used for incubation in the degradation measurements in Fig. 2c is simply not enough time to see a significant contribution of HA synthesis. Furthermore, in Fig. 2d where tissue fragments were used, the incubation time was even shorter (6 hours).