Progress in the detection and quantification of collagens: a review

Collagens are an important and ubiquitous family of proteins. They have many functions in the human body and similarly have found numerous, potent applications in various industries including the manufacture of biomaterials. The ever-increasing demand for collagen has made necessary the exploration of alternative sources such as bacterial collagen-like proteins which have a triple-helical domain of Gly-X-Y amino acid repeats. Detection and quantification of native collagens have been well-established. However, collagen-like proteins differ in their composition and do not have the unique abundance of hydroxyproline and hydroxylysine found in vertebrate collagens. Thus, this poses a problem in the detection and quantification of collagen-like proteins. This paper evaluates reports on the detection and quantification of collagens and collagen-like proteins. A systematic search of the PubMed database was conducted in May 2021, to which five additional papers were added. The 310 unique search results were then subjected to a screening and elimination process, at the end of which 22 papers were included in the study. The findings were summarized and presented in a table that highlights progress in this field. While novel methods have been developed for the detection and quantitation of collagens in general, mainly using enzyme digestion, hybridization, and fluorescence, there is a need for a rapid, one-step method that selectively and sensitively detects and quantitates collagen and collagen-like protein samples with ease.

Restriction Enzyme Double Digestion v1

Enzyme digestion is to cut the DNA molecule and the carrier molecule at the sticky end to obtain the corresponding sticky end connection.

Bockenheimer Disease is Associated With a TEK Variant

Bockenheimer disease is a venous malformation involving all tissues of an extremity. Patients have significant morbidity and treatment is palliative. The purpose of this study was to identify the cause of Bockenheimer disease to develop pharmacotherapy for the condition. Paraffin-embedded tissue from 9 individuals with Bockenheimer disease obtained during a clinically-indicated operation underwent DNA extraction. Droplet digital PCR (ddPCR) was used to screen for variants most commonly associated with sporadic venous malformations [TEK (NM_000459.5:c.2740C>T; p.Leu914Phe), PIK3CA (NM_006218.4:c.1624G>A; p.Glu542Lys and NM_006218.4:c.3140A>G; p.His1047Arg)]. ddPCR detected a TEK L914F variant in all 9 patients (variant allele fraction 2%-13%). PIK3CA E542K and H1047R variants were not identified in the specimens. Sanger sequencing and restriction enzyme digestion confirmed variants identified by ddPCR. A pathogenic variant in the endothelial cell tyrosine kinase receptor TEK is associated with Bockenheimer disease. Pharmacotherapy targeting the TEK signaling pathway might benefit patients with the condition.

Multiple Biomarker-Combined Screening for Colorectal Cancer Based on Bisulfate Conversion-Free Detection of Fecal DNA Methylation

To evaluate the applicability of bisulfate conversion-free methylation assay based on enzyme digestion in fecal screening for colorectal cancer (CRC). Stool samples were collected from a total of 1142 participants with intestinal abnormalities, including 180 positive cases, 60 advanced adenomas, and 902 negative cases. DNA from reference cell lines and clinical samples was extracted and digested with an enzyme to detect the methylation of CRC markers SEPT9, SDC2, NDRG4, SFRP2, and BMP3 genes. Statistical analysis was then used to determine the ability of the markers, both individually and in combination, to detect CRC and adenoma. Our results showed that the enzyme digestion method could suitably detect DNA marker methylation in as low as 1% of the cell lines. BMP3 had a considerably low detection rate in all clinical samples, with only 6 positive cases detected out of 180 cancer samples. Our findings showed that the combination of SEPT9, SDC2, and SFRP2 had an area under the receiver operation curve of 0.937, sensitivity of 94.11%, and specificity of 89.21% for detecting CRC. Moreover, the detection sensitivity of adenoma can also reach 38.33%. After innovatively utilizing bisulfate conversion-free methylation assay for CRC screening, this study verified the potential clinical applicability of combining multiple biomarkers for CRC screening in a large number of samples.

Investigation of the Basic Steps in the Chromosome Conformation Capture Procedure

A constellation of chromosome conformation capture methods (С-methods) are an important tool for biochemical analysis of the spatial interactions between DNA regions that are separated in the primary sequence. All these methods are based on the long sequence of basic steps of treating cells, nuclei, chromatin, and finally DNA, thus representing a significant technical challenge. Here, we present an in-depth study of the basic steps in the chromatin conformation capture procedure (3С), which was performed using Drosophila Schneider 2 cells as a model. We investigated the steps of cell lysis, nuclei washing, nucleoplasm extraction, chromatin treatment with SDS/Triton X-100, restriction enzyme digestion, chromatin ligation, reversion of cross-links, DNA extraction, treatment of a 3C library with RNases, and purification of the 3C library. Several options were studied, and optimal conditions were found. Our work contributes to the understanding of the 3C basic steps and provides a useful guide to the 3C procedure.

An Efficient Genetic Manipulation Protocol for Dark Septate Endophyte Falciphora Oryzae

Falciphora oryzae is a dark septate endophyte (DSE) isolated from wild rice roots (Oryza sativa L.). It was classified as a non-clavicitaceous endophyte. The fungus colonizes rice roots, showing a significant increase in agronomic parameters with plant biomass, rice blast resistance, yield, and quality. The construction of the genetic manipulation system is critical to study the relationship between F. oryzae and O. sativa. In the present study, the protoplast preparation and transformation system of F. oryzae was investigated. The key parameters affecting the efficiency of protoplast production, such as osmotic pressure stabilizer, enzyme digestion conditions, and fungal age, were studied. The results showed that F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9mol L-1 KCl solution and 10 mg mL−1 glucanase at 30℃ with shaking 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar (RgA) medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired GFP-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.The genetic manipulation system of endophytic fungus facilitates the further exploration the interaction between the endophytic fungus and their hosts.

Different molecular markers to identify the fertility of sugar beet monogerm germplasm resources

Experiments were conducted to validate different molecular markers associated with sugar beet breeding using available sugar beet monogerm germplasm resources to explore their effectiveness in different sugar beet populations. We used multiple primer pairs to amplify sugar beet monogerm sterile and maintainer lines in order to verify their polymorphism. For the nucleus Rf1 locus genotype enzyme digestion was also required to verify. The results showed that three pairs of primers, TR1, s17 and 11E8M4S, produced polymorphism when amplifying sugar beet sterile and maintainer lines; primers o7 and AB-18, although polymorphic, did not correlate significantly with sugar beet fertility; primer cpSSR-2 did not produce significant band differences when amplifying sterile and maintainer lines, however, the number of single nucleotide sequence repeats of base A needed to be further verified as a basis for differentiating sugar beet fertility. The polymorphism of 15 pairs of primers related to sugar beet fertility identification was verified and TR1, s17 and 11E8M4S could be used to differentiate sugar beet sterile lines from the maintainer lines and used for subsequent Marker-Assisted Selection (MAS).

DNA translocase repositions a nucleosome by the lane-switch mechanism

ABSTRACTTranslocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone core complexes and forms nucleosomes, translocases inevitably encounter nucleosomes. Previous studies have shown that a histone core complex repositions upstream (downstream) when SP6RNA or T7 RNA polymerase (bacterial exonuclease, RecBCD) partially unwraps nucleosomal DNA. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identify the lane-shift mechanism for downstream nucleosome repositioning via coarse-grained molecular dynamics simulations, which we validated by restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding region (lane) to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have crucial implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.SIGNIFICANCEEukaryotic chromosomes are composed of repeating subunits termed nucleosomes. Thus, proteins that translocate along the chromosome, DNA translocases, inevitably collide with nucleosomes. Previous studies revealed that a translocase repositions a nucleosome upstream or downstream upon their collision. Though the molecular mechanisms of the upstream repositioning have been extensively studied, that of downstream repositioning remains elusive. In this study, we performed coarse-grained molecular dynamics simulations, proposed the lane-shift mechanism for downstream repositioning, and validated this mechanism by restriction enzyme digestion assays and deep sequencing assays. This mechanism has broad implications for how translocases deal with nucleosomes for their functions.