Category: Technology

RT-PCR is the Gold Standard for the detection of the new coronavirus (SARS-CoV-2).
A group from Mahidol University, Nakhon Pathom, Thailand etc. has reported an electrochemical detection method that has the same sensitivity and specificity as RT-PCR and do not require expensive devices.
https://www.nature.com/articles/s41467-021-21121-7

In order to detect SARS-CoV-2 with high sensitivity, of course, a gene amplification process is required. They adopted Rolling Circle Amplification (RCA). The RCA method, like the PCR and LAMP methods, has the advantage that carryover contamination has a small impact on the next amplification process and can be amplified in an isothermal bath at around room temperature (25°C to 37°C). The RCA amplicon (Padlock probe) was designed as shown below and was consist of Target gene specific to the target you want to detect (N-gene, S-gene, etc. of SARS-CoV-2), Universal capture probe-binding region, and Generic reporter probe-binding region. With this Padlock probe, RCA results in long DNA amplicons containing hundreds of tandem repeats of the Padlock DNA complementary sequence. For this RCA products, hybridize probe-conjugated magnetic bead particle (CP-MNB) and Silica methylene blue reporter probe (SiMB-RP) to obtain the final product. Methylene blue or Acridine orange was used to as a redox regent. The current obtained by the electrochemical reaction increased with copy numbers as shown below. As a result, the detection limit sensitivity reached 1 copy/uL.

A group from University of Helsinki etc. has reported on the relationship between Vitamin C and COVID-19.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848027/

In healthy people, a Vitamin C intake of about 0.1 g/day is enough to maintain the concentration of Vitamin C in the blood. However, when infected with COVID-19 and becomes severe, the concentration of Vitamin C in the blood decreases sharply, and it can be reduced to the same level as vitamin C deficiency and further to a level that is almost undetected.

Taking a large amount of Vitamin C (6-8 g/day) has the following effects:
Reduced ICU treatment duration by an average of 8%
Reduced fatality from 35% to 78%

Since there are no side effects of Vitamin C, this is also considered to be effective for the COVID-19 treatment.

A group from The First Affiliated Human of Xi’an Jiaotong University, Xi’an, China etc. has reported new biomarkers from bronchial alveoli lavage to identify adenocarcinoma, squamous carcinomas, and small cell lung cancer.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840895/

Lectin microarrays were used for the marker screening, and bronchoalveolar lavage fluid was used as a sample. The goal was to identify lung adenocarcinomas (ADC), lung squamous carcinomas (SCC), and small cell lung cancer (SCLC) from benign pulmonary diseases (BPD). The goal was also to distinguish between early stage lung cancer (LC-ES) and advanced stage long cancer (LC-AS).

From the results of the lectin microarray, significant differences were found in 15  lectins, and a logistic regression analysis was performed by combining several lectins to identify the differences between various lung cancers.

As a result
To discriminate lung cancer from good lung disease, ECA, GSL-I, and RCA120 were used (cutoff value: 0.754, ALC: 0.961, sensitivity: 0.918, specificity: 0.939),
to discriminate ADC, DBA, STL, UEA-I, BPL were used (cutoff value: 0.569, ALC: 0.619, sensitivity: 0.706, specificity: 0.586),
to discriminate SCC, PNA was used (cutoff value: 0.578, ALC: 0.693, sensitivity: 0.800, specificity: 0.667),
to discriminate SCLC, STL, BS-I, PTL-II, SBA, PSA were used (cutoff value: 0.728, ALC: 0.718, sensitivity: 0.721, specificity: 0.684),
and also
to detect early cancer, MAL-II, LTL, GSL-I, RCA120, PTL-II, PWM were used, resulting in (cutoff value: 0.668, AOC: 0.856, sensitivity: 0.829, specificity: 0.810).

In order to distinguish various diseases, it is advantageous not to rely on one variable (one lectin), but to distinguish the characteristic profile for each disease using multiple variables (multiple lectins). In my view, I think that it will become a trend to adopt AI instead of using conventional statistical analysis to improve discrimination ability of the differences in disease profiles.

A group from Columbia University Vagelos College of Physicans and Surgeons etc. has reported on the efficacy of Moderna and Pfizer vaccines for the new coronavirus (SARS-CoV-2) variants in the UK and South Africa.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852271/

In addition to the existing D614G mutation, UK and South African variants contain the following mutations: there are 8 mutations in UK variants (B.1.1.7) (abbreviated as UKΔ8), and 9 mutations in South African variants (B.1.351) (abbreviated as SAΔ9).
Neutralization titers were measured by using serum samples from volunteers vaccinated with Moderna and Pfizer referring D614G as WT.  The mean loss of neutralizing activity against UKΔ8 appeared to be small  (1.8 fold, Moderna; 2.0 fold, Pfizer), but was quite significant against SAΔ9 (8.6 fold, Moderna; 6.5 fold, Pfizer) .

In cancer, aberrant glycans are expressed on the membrane. The structures and densities, of course, vary depending on the cancer types, but in general, it would be characterized by the expression of multi-branched N-glycans, the increase of O-glycans, the truncation of O-glycans, and the changes in terminal modification (i.e., sialic acid, fucose).
A group from Univ. of Ljubljana has proposed a method of targeting to cancer cells by using geneicaly modified Lactoccus lactis (a type of Gram-positive bacteria) to express lectins on the membrane specific to the aberrant  glycans of cancer cells.
https://www.mdpi.com/2076-2607/9/2/223/htm

Specifically, they focused on two types of lectins. One is B subunit of Shiga holotoxin (Stx1B) and the other is Critocybe Nebularis lectin (CNL).
The glycan binding specificity of these lectins is as follows,
Stx1B = Gb3,
CNL = LacdiNAc.

Since these lectin genomes have been published, they have been incorporated into plasmids, and lectococcus lactis were modified using the plasmids to express those lectins on the cell membrane.
Stx1B itself has  cytotoxicity, so it can effectively adhere to cancer cells and kill them as shown below.
Since CNL has not cytotoxicity, targeting to cancer cells is possible, but it is not cytotoxic at all.
Finally, they are proposing a method of using such bacteria modified to express lectins suitable for targeting to cancer cells carrying therapeutic agents.

In gene therapy using Adeno-associated virus (AAV), it has been suggested that there are some differences in AAV capsids in terms of tissue targeting ability, and it has been told that glycans may be involved in he AAV’s tissue targeting ability. Such arguments have been around for quite some time.
For example, the following papers are references.
https://www.nature.com/articles/gt201316
https://www.jbc.org/article/S0021-9258(20)51734-1/fulltext
https://www.jbc.org/article/S0021-9258(20)48853-2/fulltext
https://jvi.asm.org/content/80/18/9093

Specifically, it has been reported that AAV-1 and AAV-6 bind to Sialic acid, AAV-2 binds to Heparan sulfate, and AAV-9 binds to Galatose.
But, is it true?
To this blog admin., it is a kind of unbelievable thing that capsid proteins have lectin-like functions?

Toxicology tests using hepatocytes have come to be widely used as an alternative way for testing drug toxicology using animals. The problem with using hepatocytes in toxicology tests is that it is difficult to supply large quantities of hepatocytes with the same characteristics. Therefore, a group from the National Institute of Child Health and Development proposes a method to create hepatocytes from pluripotent stem cells (iPSC, ESC) and enrich highly uniform hepatocellular populations from heterogeneous cell populations using ammonia.
https://www.biorxiv.org/content/10.1101/2020.03.20.999680v1.full

After ESC is differentiated into hepatocytes, an ammonia treatment is performed for 2 days to select uniform hepatocytes that are resistant to ammonia toxicity. 70-80% will die at this stage. Surviving hepatocytes are cultured and proliferated on MEF-feeder. Hepatocytes selected in this way multiply about 30 times in 190 days. The detailed mechanism of ammonia toxicity is not known, but it is thought that ammonia ions compete with potassium ions and eventually die with intracellular and/or extracellular pH change. ALB, AFP, CYP3A4, CPS1, and OTC genes are highly expressed in ammonia-selected hepatocytes, and ALB and AFP decrease with the passage. CPS1 and OTC are genes involved in the metabolism of ammonia. CYP3A4 is one of the enzymes that metabolizes unwanted biological molecules.
The same is possible with hepatocytes made from iPSC.

The differentiated cells from iPSC and /or ECS were further cultivated and propagated in the ESTEM-HE medium (GlycoTechnica, Ltd.) .

Hepatocytes are essential for toxicology testing of pharmaceuticals in vitro. Hepatocyte strains such as HepG2, Huh7, THLE-2, PLC-PRF-5, and AML-12 are used. A group from the National Center for Child Health and Development has established a new immortalized hepatocytes  named HepaMN from from a liver associated with biliary atresia. Immortalization was performed by inoculation with CDK4, cyclin D1, and TERT.
https://www.nature.com/articles/s41598-020-73992-3

Characteristics of HepaMN strains:
Established from untransformed hepatocytes, and has normal liver function and diploid chromosomes,
Expression of albumin gene at the same level as HepaRG,
Showing the cell morphology of hepatocytes,
Induces stable cytochrome P450 3A4 (CYP3A4) and shows normal metabolic effects,
Efficiently proliferates and is stable even in very long passages,
As a result, succeeded in providing a new and useful hepatocyte strain for toxicology testing.

HepaMN can be effectively cultured by EMUKK-15

Existing Pfizer and Modelna vaccines for the new coronavirus (SARS-CoV-2) are mRNA type, but the following group is evaluating prototype vaccines using adeno-associated viral (AAV).
https://www.biorxiv.org/content/10.1101/2021.01.05.422952v3.full.pdf

Therapeutic gene transfer using AAV is recognized as a highly safe method, and Glybera, Luxturna, Zortensma etc. have already been approved by the FDA and EMA as gene therapeutics.
Vaccines using two types of AAV have been prototyped. AAVCOVID-19-1 (AC1) encodes a full-length S protein, which expresses an antigen S protein on the cell membrane of an infected cell. Another, AAVCOVID-19-3 (AC3), encodes part of the S protein, and the antigen takes the form of a secretory protein.

The evaluation was carried out with mice and nonhuman primates, and the immune response was retained for about 5 months with a single doze. There is no problem with storage at room temperature for one month, and vaccines are amenable to large scale production because the methodology has been established. As an effect, AC1 seems to be higher than AC3.

However, the blog Admin has some concerns that the antigen genes are incorporated into cells.

It has been pointed out that mutations in the new coronavirus (SARS-CoV-2) are behind infection explosions in the UK and South Africa. A number of mutations have occurred, but a mutation common to these has already been shown to be N501Y.
A group of The University of Texas Medical Branch, Galveston TX has reported on how the mutation affects the effects of Pfizer’s vaccine, Tozinameran (BNT162b2, a nucleoside modified RNA vaccine).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805448/

A comparative experiment using N501 and Y501, which is the genetic background of N501,  was done by measuring  50% plaque reduction neutralization titers for N501 and Y501 viruses using sera from 20 people collected 2 weeks and 4 weeks after administration of the vaccine. As a result, it was reported that almost the same neutralization titers were obtained.