BabcockHall

My suggestion is to rewrite the standard Michaelis-Menten equation in the form (velocity)/V_max = and so forth.  To do so is trivial from the point of view of the algebra involved.

Good Morning,

We are collaborating with a group that performs disk diffusion assays on compounds that we make.  We would like to publish some disk diffusion data in addition to reporting on the syntheses and perhaps the in vitro behavior of a set of compounds.  However, the area of whole-cell biological testing is generally unfamiliar to me, and I don't know how to present data in a manuscript.  I also don't have a good feel for what information should or should not be included in reporting these data.  Perhaps someone could suggest a model publication, for example.  Thank you.

In carbohydrate chemistry, my suggestion is generally to start with the HC-1 signal, because it verifies the configuration of this carbon.  It is also simpler in appearance.

We have tried both diethyl amine and 4-methylpiperidine in the deprotection.  Most recently we removed the white precipitate with centrifugation in Corex centrifuge tubes.  This was an improvement over having a bunch of precipitate sit on the top of the silica column.

The carbon and oxygen are still present.  What do you think happens to the hydrogen on the oxygen?

Is there any point in repeating a successful disk diffusion assay at lower starting concentration of substance?  Or would it make more sense to move to some form of minimum inhibitory concentration assays?

We tried a silica column after using diethyl amine to deprotect.  We obtained two pools, but we did not obtain our product.  We are short on mass.  It seems like time for a rethink regarding the best purification method.

We have a tertiary-butyl protected amino acid in which nitrogen is protected with FMOC.  We removed the FMOC group with diethyl amine, and we used rotary evaporation plus a toluene strip to remove the volatiles.  We attempted to purify over a short column of silica.  We were able to remove a fast-moving impurity (presumably dibenzofulvene or a derivative) using 40:60 EtOAc/hexanes, then switched to 99:1 hexanes/TEA in the hope of eluting the N-deprotected product, still bearing the carboxylate protecting group, but the product was not soluble in EtOAc or in DCM (it might have been a suspension).  We eluted with 90:10 DCM/methanol and saw two products by TLC.  They were not completely separated.  I am interested in soliciting ideas for what we should do differently next time.

In retrospect a lower percentage of methanol would have been an improvement.  I wonder whether or not the solubility of the amine would have been better in chloroform than it was in DCM.  I realize that some people do not purify at all at this stage, but instead couple the crude product with another amino acid.  We also want to couple to another amino acid. 

https://doi.org/10.1002/chem.201101163

I am working on synthetic routes to a glucoside.  The beta-trimethylsilylethoxymethyl (SEM) group has some attractive features (particularly in regards to deprotection), but I have only found one paper in which four SEM groups were used to protect the oxygen atoms at carbons 2-4 and 6 of a glycoside conjugated with a steroid.  They did not deprotect to the best of my knowledge, but I assume that one would use a standard recipe.  I did find a paper in which a glycoside was protected with several different groups, including one SEM.  Are there disadvantages to using the SEM group as the protecting group?  Are there reasons why it has been used more in carbohydrate chemistry?  Should I be favoring other protecting groups instead?

You might be able to do it using what is called the specific activity of the enzyme.  Specific activity is given as units per milligram of protein.  The denominator is a mass, not a concentration.

Does anyone know of a web-based program for estimating retention times that is simple to use?  I found one, but I struggled a bit with the output.

Our interest is in LC/MS of the peptides, and we plan to work in water/acetonitrile/formic acid.  We found a number of protocols for C-18 on line (I can provide links to these), and we are focusing on those protocols that specify formic acid (not TFA).  Our Zip Tips use C-18.  (1) Are Zip Tips necessary?  (2) Given their capacity, does one obtain enough for material for one or for several LC/MS runs?  (3) Are Zip Tips reusable? (4) Does anyone have a paper or protocol that is especially informative?  Thank you very much.

Can you explain your reasoning?

We are synthesizing the next generation of inhibitors, and they have increased water solubility.

I don't know the answer to your question, but I am inclined to say that we will try to optimize the compounds.  With regard to these compounds, we have in vitro data against a validated target enzyme.  They are irreversible inhibitors with ligand binding efficiencies greater than 0.3.  The second generation inhibitors that we are currently synthesizing were designed with transport across membranes as a consideration.

Our collaborators used only volatile organic solvents to dissolve the compounds prior to the preparation of the disks.  I am not sure whether or not this information is useful.

I asked my institution to file a provisional patent, but I have not yet heard back.

The enzyme has been shown to be essential in some pathogenic bacterial strains and also in Candida albicans.  We are in the process of revising our first manuscript on our inhibitors.  For the best compounds in this series, the ratios of binding energies to molecular weights are promising, among other possibly favorable properties.  We envision either an antifungal or antibacterial compound, although at the moment we are more fungal-focused.

What sort of near-term commercial applications do you mean?

That is a helpful reference; thank you.  We synthesized a family of compounds that are good inhibitors of an essential microbial enzyme.  As a whole they are not very water-soluble, although there is variation in the group.  One of the more hydrophilic ones is one of two that showed growth inhibition.  Therefore, we are synthesizing the next generations of inhibitors that might be more water-soluble and thinking about what are the meanings of various test results.

One other issue occurs to me.  As the compound diffuses, the microorganisms are growing.  So it is possible that the compound arrives at a certain place with a particular concentration only after growth has ceased for other reasons, such as buildup of waste or consumption of nutrients.

"VAERS cannot prove that a vaccine caused a problem. Specifically, a report to VAERS does not mean that a vaccine caused an adverse event. But VAERS can give CDC and FDA important information. If it looks as though a vaccine might be causing a problem, FDA and CDC will investigate further and take action if needed."

"In some cases, multiple reports are submitted for the same adverse event."

"VAERS data alone cannot determine if the vaccine caused the reported adverse event.

These three quotes about the vaccine adverse events reporting system come from the CDC.  

https://www.cdc.gov/vaccinesafety/ensuringsafety/monitoring/vaers/index.html

Politifact or some other fact-checking organization also had an article about this kind of erroneous interpretation, if I recall correctly.

The Economist magazine calculated an estimate of worldwide excess deaths, of 10 million people.  The CDC has a table broken down by age at this link.  https://www.cdc.gov/coronavirus/2019-ncov/covid-data/investigations-discovery/hospitalization-death-by-age.html

"Sample interpretation: Compared with 5—17-year-olds, the rate of death is 45 times higher in 30—39-year-olds and 8,700 times higher in 85+-year-olds."

One more thing for protein helices.  I would ask what are the values of the dihedral angles, phi and psi, and whether or not they fall into the allowed region of the Ramachandran diagram.

I don't know the answer to this question, but the kinds of questions I would ask about any helix would be handedness, hydrogen bonding patterns, and translation of the helix per unit residue.

"Two doses of BNT162b2 are highly effective across all age groups (≥16 years, including older adults aged ≥85 years) in preventing symptomatic and asymptomatic SARS-CoV-2 infections and COVID-19-related hospitalisations, severe disease, and death, including those caused by the B.1.1.7 SARS-CoV-2 variant. There were marked and sustained declines in SARS-CoV-2 incidence corresponding to increasing vaccine coverage. These findings suggest that COVID-19 vaccination can help to control the pandemic."  From a study of the  Pfizer vaccine in Israel, published earlier this month in The Lancet.

"This study in a nationwide mass vaccination setting suggests that the BNT162b2 mRNA vaccine is effective for a wide range of Covid-19–related outcomes, a finding consistent with that of the randomized trial."  From a study published in April, in the New England Journal of Medicine.

There was an article at CNN that quoted some hematologists, all of whom recommended that people get the vaccine.

Hello Everyone, I am making enzyme inhibitors and turning them over to a microbiology laboratory for testing against standard strains of bacteria and fungi.  They are performing disk diffusion assays, and seeing a few results that look positive.  I would like better to understand the meaning and limitations of this experiment.  I would hazard a guess that the compound's concentration falls off at greater distances from the disk, but I don't know how to interpret the diameter of the dead zone in a quantitative way, or even if that is possible.  Perhaps there is a good textbook treatment of this subject, for example.