Category: Technology

Technology

Crowdfunding to elucidate the mechanism of the novel coronavirus （COVID-19）and develop therapeutic agents

I also participate in a project targeting elucidation of the mechanism of the sever disease of new coronavirus (COVID-19) and the development of therapeutic drugs.
We promote development of this project by making full use of our experience and wisdom in glycans and lectins.
Thank you for your continuous support on our activities.

 

 

https://readyfor.jp/projects/glycotechnica_covid19research/announcements/150515

Technology

Using CRISPR/Cas12a to detect new coronavirus (COVID-19:SARS-CoV-2): Sensitization effect adding Mn2+

CRISPR/Cas9 won the 2020 Nobel Prize in Chemistry about a week ago.
We have found several papers that used this technique as a high sensitive detection method for the new coronavirus. The following paper reports that detection using CRISPR/Cas12a could be further 13 times more sensitive by adding Mn2+.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536916/#advs2020-bib-0005

A brief description of the protocol is as follows:

  • Extract an RNA sample from a sample.
  • Make cDNA using reverse transcription, and amplify by PCR (dsDNA is made).
  • Pre-design crisper RNA (crRNA) using RNA sequences specific to the E protein of the new coronavirus.
  • Add Cas12a enzyme and crRNA to the dsDNA sample, the new coronavirus-specific sequence is cut out when it is in the sample dsDNA.
  • Thus, since the inhibitory interaction between the fluorescent probe and the quencher that have been incorporated in the PCR process is lost, the fluorescence comes out by applying the excitation light.

Of course, this fluorescence is read by a detector, but it can be confirmed by the eyes.
The addition of Mn2+ to this system increased the sensitivity by 13 times.

Nature, Technology

How to reduce cross-reactivity to other coronaviruses in the detection of new coronaviruses (SARS-CoV-2) using RT-PCR

RT-PCR is used as you know to test for suspected infection of the new coronavirus (SARS-CoV-2). Sensitivity and specificity are very important in testing, but there are some doubts as to how high the generally implemented RT-PCR could detect SARS-CoV-2 without cross-reactivity to other coronaviruses including influenza viruses. Therefore, the following groups have reported a novel methodology for improving the specificity of SARS-CoV-2 detection without sacrificing sensitivity.
https://www.mdpi.com/2075-4418/10/10/775

As the methodology, they adopted the followings, (1) in the domain of structural protein of SRAS-CoV-2, design dual-target PCR primers targeting on coding region of the accessory and envelope proteins (ORF3ab-E primers) and that of the capsid protein (N primers), and (2) use a peptide nucleic acid (PAN) designed to target on the N region as a blocker of PCR reaction.

Peptide nucleic acids are artificial compounds in which the deoxyribose phosphate backbone is replaced by a pseudo-peptide polymer to which the nucleobases are linked, and the binding affinity for target DNA and RNA is remarkably increased by nearly 1000 times, and therefore, it does not act as a primer but act as an inhibitor of PCR.

As a result, cross-reactivity to other coronaviruses and influenza viruses disappeared completely, and the detection rate of SARS-CoV-2 was 100% for ORF3ab-E and 82.6% for PNA-N.

Nature, Technology

About infection inhibitors of the new coronavirus (SARS-CoV-2) as its therapeutic drugs: Are inhibitors on furin and transmembrane protease serine 2 etc. effective?

The S protein of the new coronavirus (SARS-CoV-2) is divided into S1 sites that bind to the host receptors and S2 sites for membrane fusion. The boundary site of this S1 site and S2 site has a furin cleavage site, and there is a target domain of transmembrane protease serine 2 (TMPRSS2) in the S2 site. The following research has been reported that the use of inhibitors on those proteases may be able to reduce the infection of the new coronavirus.
https://linkinghub.elsevier.com/retrieve/pii/S2211-1247(20)31243-2

decanoyl-RVKR-chlorometylketone (CMK) for a furin inhibitor, camostat for a TMPRSS2 inhibitor, as well as naphthofluorescein which inhibits RNA replication, were studied. VeroE6 cells are used for the experiments.

 

 

 

 

 
Obtained efficacy and toxicity were as follows; the 50% inhibitory concentration (IC50) was 0.057 μM for CMK, 9.025 μM for naphthofluorescein, and 0.025 μM for camostat. The 50% cytotoxic concentration (CC50) was 318.2 μM for CMK, 57.44 μM for naphthofluorescein, and 2,000 μM for camostat. The resulting selection index is 5,567 for CMK, 6.36 for naphthofluorescein, and 81,004 for camostat.

Note that there is a difference among these inhibitors, CMK and camostat prevent the initial infection of the virus, and naphthofluorescein prevents the replication of the virus. We look forward to further consideration as a lead compound for the development of therapeutic drugs in the future.

Nature, Technology

To suppress infection with the new coronavirus (SARS-CoV-2), pineapple intake will be GOOD.

The Univ. of Nebraska Medical Center group has reported a research finding that bromelain extracted from pineapples (enzyme classified as a cysteine protease in proteolytic enzymes) is effective in suppressing infection with the new coronavirus (SARS-Co-2).
https://www.biorxiv.org/content/10.1101/2020.09.16.297366v1

 

 

 

 

 

Bromelain targets angiotensin-converting enzyme 2 (ACE2), type II membrane-penetrating serine protease (TMPRSS2) and SARS-CoV-2 S-protein, and thereby suppresses SAS-CoV-2 infection. Because bromelain is well absorbed through digestive organs and maintains its biochemical activity in the body, it is said that ingesting pineapples rich in bromelain will suppress infection with the new coronavirus (SARS-CoV-2).

Nature, Technology

Biomarker research for pancreatic duct adenocarcinoma using its organoids

A group of Harvard Medical Schools has reported potential biomarkers for pancreatic duct adenocarcinoma using pancreatic duct adenocarcinoma derived organoids.

https://insight.jci.org/articles/view/135544

(1) From the viewpoint of glycans: High mannose and Lewis X epitope structures increase in pancreatic duct adenocarcinoma.

(2) From the viewpoint of extracellular vesicles (EV) proteins: ANXA11 (anexine A11: calcium-dependent phospholipid binding protein mobilized in the budding region of the transport endoplasmic reticulum COPII of the endoplasmic reticulum) is increased.

Let’s look forward to further research progress.

Technology

Diagnosis of chest X-ray imaging by Deep Learning greatly improves the diagnostic accuracy of the new coronavirus (COVID-19)

Chest X-rays and CT are used as a diagnosis of the new coronavirus (COVID-19). The Univ. of Oklahoma group has succeeded in improved diagnostic accuracy by using Deep Learning techniques to determine whether or not the breast X-ray images are from COVID-19-derived pneumonia.
https://www.sciencedirect.com/science/article/pii/S138650562030959X?via%3Dihub

Deep Learning uses a six-layer Convolutional Neural Network (CNN), with a chest X-ray image resized into 224 x 224 x 3, and a Convolution of 3 x 3. The x3 in the input image indicates that it is three colored (R, G, and B). Since the X-ray image is black and white gray, the three colors of R, G, and B are pre-processed for the image as shown in the figure below. In the figure below, (Ip) is an image with the diaphragm removed, (Ieq) is an image processing method that adjusts the contrast using the intensity histogram of the image, and (Ib) is an image obtained by bilateral filtering on (Ieq). R, G, and B images are simulated by using these three (Ip), (Ib), and (Ieq) images.

 

 

 

 

 

The results of Deep Learning are,
with the simple model using the X-ray image as it is, 88% of the accuracy was obtained, and the accuracy was improved to 94.5% by adding the pre-processing of the above mentioned image editing method.

Wouldn’t it be time for diagnosis using Deep Learning to be used more and more in the medical field?