Thread by @imtryam

Briefing on Gene knockdown and knockout in today's #365DaysOfScience. These two are classical genome editing tools used to study gene function and probable effects of inhibiting the target gene. These tools find relevance in molecular genetics and drug discovery.

Gene knockout is the 'knocking out' of a gene. It is attributed by the permanent deactivation or complete elimination of gene function to evaluate the functional specificity of that gene. It is achieved by homologous recombination, endonucleases, or CRISPR/Cas9.
#365DaysOfScience

Using Gene knockdown the functional expression of a gene is suppressed in a flexible way. This approach is neccesiated for the genes whose KO will have a lethal effect. Working with RNA, it uses RNAi (siRNA/shRNA) leading to abortive translation/mRNA degradation
#365DaysOfScience

I read this wonderful article in today's #365DaysOfScience. Authors aim to investigate the reactivation potential of various latency-reversing agents (LRAs) in the pure latent population of HIV.

elifesciences.org/articles/34655

Paper summarises:
👉HIV-1 preferentially targets actively transcribed regions.
👉However, non-inducible latent proviruses are observed to be integrated to a higher extent into silenced chromatin.
👉LRAs only reactivate a limited fraction of latent proviruses.

#365DaysOfScience

👉Eliminating the reservoir is critical in order to achieve a functional or sterilizing HIV cure
👉Quantifying the reservoir is still a challenge.
For a functional cure, a stably silenced, non-reactivatable provirus is preferable than to chronic active infection
#365DaysOfScience

I read about Methylmalonic acid (MMA) for a JC which will the topic of today's #365DaysOfScience. MMA is a dicarboxylic acid that is primarily produced as a by-product of propionate metabolism yielding from the catabolism of branched-chain amino acids and odd chain fatty acids.

MMA links to the coenzyme A to form methylmalonyl-CoA, which is converted to succinyl-CoA in a reaction that involves vitamin B12 as a cofactor. Succinyl-CoA subsequently enters the TCA cycle for energy production inside the cell. #365DaysOfScience

The toxic accumulation of MMA in the blood leads to metabolic disorder methylmalonic acidaemia. It results from increased flux through and/or deregulation of the enzymes in its pathway. #365DaysOfScience

Saluting the marvel of the mathematical genius who without appreciable training in pure mathematics, made pronounced contributions. The man is synonymous with anything mathematical in the country.
#365DaysOfScience
Happy Birthday, Srinivasa Ramanujan!💐

https://twitter.com/imtryam/status/1121671062922522627?s=20

This is a commentary on @lab_gomes paper in @nature. Authirs report: Ageing supports a systemic environment that supports tumour progression and aggressiveness. They find the age-induced factor and how it modulates cancer aggression. #365DaysOfScience
nature.com/articles/s4158…

Instead of FBS, authors treated NSCLC & TNBC cells with Human Serum (HS) isolated from young (<30 yo) & old (>60 yo). When treated with aged sera, cancer cells show remarkable aggression, EMT profile, chemo resistance and elevated metastatic potential (in mice). #365DaysOfScience

They hunt for this age-induced circulatory factor leading to cancer aggression. Using targetted metabolomics they find significant elevation of Methylmalonic acid (MMA) in the sera of aged men. Cells treated with MMA alone show a similar profile as aged sera.
#365DaysOfScience

MMA was found to be elevated in the aged sera at a significant level. Treatment of cells with different acids having similar structures & pKa like MMA did not have EMT. This further validates that MMA is the age-induced circulatory factor found in the aged sera.
#365DaysOfScience

Authors find some discrepancy in the intracellular MMA concentrations treated with MMA alone (with >1 mM) or with aged sera (15–80 μM). There should be some carrier in the serum transporting the MMA inside the cell.
#365DaysOfScience

To find the delivery partner, authors delipidated the serum and molecules greater than 3 kDa. Cells treated with dilipidated or size excluded serum could not induce EMT.

MMA is complexed with molecules >3 kDa in the serum that facilitate its entry into cells.

#365DaysOfScience

Next, they complexed MMA with synthetic lipids (lipofectamine) or Lipidic structures (LS) from FBS. This restored EMT at μM concentrations. Using further set of experiments they validate MMA, complexed with lipidic structures, is the circulatory factor.
#365DaysOfScience

To understand pro-aggressive transcriptional reprogramming induced by MMA authors did Global transcriptomic analysis in A549 treated with MMA and Gene set enrichment analysis on it. They found 439 altered genes out of which 11 were Transcription Factors (TF).
#365DaysOfScience

Validating the TFs using qPCR, one of the most upregulated of these was SOX4 which is involved in the regulation of embryonic development and cancer progression, and is known to be a master regulator of EMT. SOX4 mediates the pro-aggressive phenotype observed. #365DaysOfScience

shRNA induced knockdown studies in A549 cells stripped off the EMT, chemotherapeutic resistance, and metastatic properties of aged sera or MMA.
#365DaysOfScience

Next they asked how MMA alters SOX4 levels? RNA-seq analysis revealed an increase in the TGFβ signalling pathway components, including the upregulation of TGFβ-2 ligand. There is a pronounced increase of TGFβ mRNA in aged sera. #365DaysOfScience

Further, "MMA robustly induced TGFβ signalling within 24 h, during which an increase in SOX4 was observed." Treating cells with MMA and a TGFβ receptor was sufficient to block the ability of MMA to induce SOX4 expression and pro-aggressive properties.
#365DaysOfScience

They conclude that MMA relies on the activation of TGFβ signalling to induce SOX4 which consequently does transcriptional reprogramming to confer cellular plasticity to sustain tumour progression.
#365DaysOfScience

Lipids have dual roles in MMA-driven metastases:
👉Precursor of MMA
👉Carrier of MMA as large lipids

Subjects in this study with high MMA levels were cancer-free.
👉The effects of MMA are specific to cancer spread in the body, not to initiate cancer formation

#365DaysOfScience

Lacunae in the study.
👉Only male subjects were recruited.
👉MMA enhancing the TGF-β2 transcription is yet to be verified.
👉No identity of lipid structure
👉It needs validation if MMA specifically affects metastasis in humans in the same way as in mice.

#365DaysOfScience

It will be interesting to note the obesity associated MMA expression and lipid profiles in cancer progression in obese subjects. Enjoyed reading this paper. Take a bow for this wonderful piece of work @lab_gomes. @nature

#365DaysOfScience

This timeline depicts the advent of #coronavirus2, overwhelming hospitalization, global lockdowns, drug discovery and mass vaccination. With more than 200,000 papers published, this year has certainly been a roller-coaster ride. #365DaysOfScience
vis.sciencemag.org/breakthrough20…

The best gift for humankind these times are the gifts of the #COVID vaccine. With accelerated approval from @US_FDA to @pfizer and @moderna_tx vaccines to high-risk groups. Thankless job by 30k trial participants. @NIHDirector's blog here
#365DaysOfScience
directorsblog.nih.gov/2020/12/22/cel…

@US_FDA @pfizer @moderna_tx @NIHDirector A hemophilic patient injected with AAV carrying a therapeutic gene in a clinical trial has developed a liver tumor. Despite him being older and predisposing conditions, it's very crucial to rule out the possibility of LC from AAV vectors. #365DaysOfScience
sciencemag.org/news/2020/12/l…

@US_FDA @pfizer @moderna_tx @NIHDirector 🐐One of the pillar stones of medical microbiology, who discovered microorganisms behind fermentation, initiated pasteurization and developed vaccines against anthrax and rabies. Happy Birthday, Louis Pasteur. Modern dairy and alcohol industry owes you much.
#365DaysOfScience

An interesting finding by Gao et al who report that stimulation of bone marrow neurons with G-CSF or chilli peppers' components via a series of mediators drives the HSCs to move from the bone marrow into blood vessels.
#365DaysOfScience
nature.com/articles/d4158…

Drawing inspiration from the cocktail party effect where people can focus on a single voice even in noise, researchers trained a neural network to mimic the brain’s wiring. This very important leap has an immense translational impact. #365DaysOfScience
sciencemag.org/news/2020/12/t…

In the penultimate #365DaysOfScience, I am reviewing Patient-Derived Xenografts (PDX) Models serving as a valuable tool for translational research facilitating precision medicine. The tumor cells are implanted into the immunodeficient mice to mimic cancer's natural μ-environment.

The immunodeficient mice (nude/SCID) are used to counter transplant rejection. Usually, single-cell suspension of the tumor is made that is implanted into mice at the corresponding anatomical site or at a different area. Generations are F0, F1, F2...
#365DaysOfScience

The PDX models show significant advantages over cell lines. Exhibiting tumor heterogeneity, PDX maintain similar cellular/histopathological structures of the original tumors, responses to anti-cancer agents, & genetic/epigenetic abnormalities as in the patient. #365DaysOfScience

PDX models can predict effectively the efficacy of conventional and novel anti-cancer therapeutics. Using bioluminescence dyes, distant micrometastases can also be assessed using PDX models. There are several models available for different cancers available. #365DaysOfScience

On the concluding day of #365DaysOfScience challenge, I read the classic @PNASNews papers in 1951 by Linus Pauling, Corey, & Branson which proposed α-helix & β-sheets of protein structure. These papers had a similar impact as Watson-Crick DNA structure.
pnas.org/content/37/4/2…

Prof Pauling predicted that peptide groups would align in a planar position due to the resonance of electrons between the double bond of the carbonyl group and the amide C—N bond. This was verified for each of the amides studied.
#365DaysOfScience

The authors constructed the helices with planar amide groups and the precise bond dimensions to yield α-helix and the γ-helix. The notable difference between the two is C=E groups of the α-helix point in the C terminus & those of the γ-helix toward the N term. #365DaysOfScience

Next paper was on β-sheets. They reported an H-bonded layer configuration of polypeptide chains, where the peptide groups lie in the plane giving parallel/antiparallel sheets with H-bonds between protein chains, not within a chain.
#365DaysOfScience

pnas.org/content/37/5/2…

These papers had remarkable academic and political impact. Dr Pauling was defending academics (himself included) against the charges of disloyalty to the USA. Despite receiving hate mails & abhorrence from colleagues, Dr Pauling remained a world-class scientist. #365DaysOfScience