Effect of Metabolism of Carbohydrate in Mammalian Tissues and Tumors on The Level of Enzymes Involved in Direct Oxidative Pathway

Introduction: Literature review has revealed that the distribution of the enzymes of 6-phospo-gluconate dehydrogenase activities of some acetone derived tissues in animal tissue have so far not been investigated systematically. This leaves a gap for further investigation to explore the subject matter deeply. Method: Barium salts of D-Glucose 6-phosphate (0 6-P), 6-phosphogluconate (6-PO) and D-ribose 5-phosphate (R 5-P) are available and were used in our study. (TNP) triphosphopyridine was prepared and analyzed; its composition was 75% TNP without (DNP) diphosphopyridine nucleotide. Ice-cold isotone KCL (0-15M-KCL with 8ml, 0-02M-KHCO3) was disintegrated in 09 parts. It is done in a potter glass homogenizer or in a Nelco homogenizer. This is followed by centrifuging and dialysis of the supernatants. Heparinized blood 10ml was used for erythrocyte hemolysis, which was diluted with 10ml of water. 01 part of haemolysate was treated with 9 parts of isotonic KCI. Spectroscope is used to determine the dehydrogenase activity of dialyzed tissue. The method followed was of Glock and McLean. Study Design: Quantitative, cross sectional study. Settings: Institute of Biochemistry, Gulab Devi Educational Complex, Lahore Duration: 01 Year i.e. 1st July 2020 to 30th June 2021. Results: Enzymes activities of 6-PG dehydrogenase and Gluco-6- phospo dehydrogenase mentioned in table 1&2 in normal mammalian tissue and mammary glands. The results obtained on the tumour cells are given in table 3. These Values are within the limits in normal tissues whereas it becomes on higher side in lymphomas and sarcomas. Conclusion: This study shows some limitation that the maximum enzymic activities are determined, whereas in the intact cell other regulatory factors probably limit or control the activity of this pathway. Keywords: Gluco-6- phospodehydrogenase, 6-PG dehydrogenase, oxidative pathway, Ribose 5-Pentose, mammalian tissue

Novel elasticity measurement techniques reveal that the C. elegans cuticle is required for physical integrity with age

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants’ long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock (OS), and we directly perform the tensile test (TT) on isolated cuticles using microfluidic technology. Using these tools, we find that cuticle, not the muscle, is responsible for changes in ‘stretchiness’ of C. elegans, and that cuticle stiffness is highly non-linear and anisotropic. We also found that collagen mutations alter integrity of the cuticle by significantly altering elasticity. Additionally, aging stiffens the cuticle under mechanical loads beyond the cuticle’s healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.

Thioredoxin reductase controls the capacity of peroxiredoxins to limit mitochondrial H2O2 release

H2O2 performs central roles in signaling at physiological levels, while at elevated levels it causes molecular damage. Mitochondria are major producers of H2O2, which has been implied in regulating diverse processes inside and outside the organelle. However, it still remains unclear whether and how mitochondria in intact cells release H2O2. Here we employed the genetically encoded high-affinity H2O2 sensor HyPer7 in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells, and observed H2O2 released from mitochondria directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus nor on the plasma membrane, pointing to steep gradients emanating from mitochondria. These gradients are controlled by cytosolic peroxiredoxins that act redundantly and are present with a substantial reserve capacity. Furthermore, dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed cell-to-cell differences. Thus, our data indicate that H2O2-mediated signaling likely occurs close to mitochondria during specific metabolic conditions.

Detection of biological signals from a live mammalian muscle using an early stage diamond quantum sensor

The ability to perform noninvasive and non-contact measurements of electric signals produced by action potentials is essential in biomedicine. A key method to do this is to remotely sense signals by the magnetic field they induce. Existing methods for magnetic field sensing of mammalian tissue, used in techniques such as magnetoencephalography of the brain, require cryogenically cooled superconducting detectors. These have many disadvantages in terms of high cost, flexibility and limited portability as well as poor spatial and temporal resolution. In this work we demonstrate an alternative technique for detecting magnetic fields generated by the current from action potentials in living tissue using nitrogen vacancy centres in diamond. With 50 pT/$$\sqrt{\text {Hz}}$$ Hz sensitivity, we show the first measurements of magnetic sensing from mammalian tissue with a diamond sensor using mouse muscle optogenetically activated with blue light. We show these proof of principle measurements can be performed in an ordinary, unshielded lab environment and that the signal can be easily recovered by digital signal processing techniques. Although as yet uncompetitive with probe electrophysiology in terms of sensitivity, we demonstrate the feasibility of sensing action potentials via magnetic field in mammals using a diamond quantum sensor, as a step towards microscopic imaging of electrical activity in a biological sample using nitrogen vacancy centres in diamond.

A far-red cyanobacteriochrome lineage specific for verdins

Cyanobacteriochromes (CBCRs) are photoswitchable linear tetrapyrrole (bilin)-based light sensors in the phytochrome superfamily with a broad spectral range from the near UV through the far red (330 to 760 nm). The recent discovery of far-red absorbing CBCRs (frCBCRs) has garnered considerable interest from the optogenetic and imaging communities because of the deep penetrance of far-red light into mammalian tissue and the small size of the CBCR protein scaffold. The present studies were undertaken to determine the structural basis for far-red absorption by JSC1_58120g3, a frCBCR from the thermophilic cyanobacteriumLeptolyngbyasp. JSC-1 that is a representative member of a phylogenetically distinct class. Unlike most CBCRs that bind phycocyanobilin (PCB), a phycobilin naturally occurring in cyanobacteria and only a few eukaryotic phototrophs, JSC1_58120g3’s far-red absorption arises from incorporation of the PCB biosynthetic intermediate 181,182-dihydrobiliverdin (181,182-DHBV) rather than the more reduced and more abundant PCB. JSC1_58120g3 can also yield a far-red–absorbing adduct with the more widespread linear tetrapyrrole biliverdin IXα (BV), thus circumventing the need to coproduce or supplement optogenetic cell lines with PCB. Using high-resolution X-ray crystal structures of 181,182-DHBV and BV adducts of JSC1_58120g3 along with structure-guided mutagenesis, we have defined residues critical for its verdin-binding preference and far-red absorption. Far-red sensing and verdin incorporation make this frCBCR lineage an attractive template for developing robust optogenetic and imaging reagents for deep tissue applications.

Cellular and molecular mechanisms that regulate mammalian digit tip regeneration

Digit tip regeneration is one of the few examples of true multi-tissue regeneration in an adult mammal. The key step in this process is the formation of the blastema, a transient proliferating cell mass that generates the different cell types of the digit to replicate the original structure. Failure to form the blastema results in a lack of regeneration and has been postulated to be the reason why mammalian limbs cannot regrow following amputation. Understanding how the blastema forms and functions will help us to determine what is required for mammalian regeneration to occur and will provide insights into potential therapies for mammalian tissue regeneration and repair. This review summarizes the cellular and molecular mechanisms that influence murine blastema formation and govern digit tip regeneration.

CRISPR-Cas12a–assisted PCR tagging of mammalian genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein–tagged genes.

A rapid and simple bead-bashing-based method for genomic DNA extraction from mammalian tissue

Conventional genomic DNA (gDNA) extraction methods can take hours to complete, may require fume hoods and represent the most time-consuming step in many gDNA-based molecular assays. We systematically optimized a bead bashing-based (BBB) approach for rapid gDNA extraction without the need for a fume hood. Human tissue specimens (n = 34) subjected to the 12-min BBB method yielded 0.40 ± 0.17 (mean ± SD) μg of gDNA per milligram of tissue, sufficient for many downstream applications, and 3- and 6-min extensions resulted in an additional 0.43 ± 0.23 μg and 0.48 ± 0.43 μg per milligram of tissue, respectively. The BBB method provides a simple and rapid method for gDNA extraction from mammalian tissue that is applicable to time-sensitive clinical applications.