Tens of thousands of chimeric RNAs, i.e., RNAs with sequences of two genes, have been identified in human cells. Most of them are formed by two neighboring genes on the same chromosome and are considered to be derived via transcriptional readthrough, but a true readthrough event still awaits more evidence and trans-splicing that joins two transcripts together remains as a possible mechanism. We regard those genomic loci that are transcriptionally read through as unannotated genes, because their transcriptional and posttranscriptional regulations are the same as those of already-annotated genes, including fusion genes formed due to genetic alterations. Therefore, readthrough RNAs and fusion-gene-derived RNAs are not chimeras. Only those two-gene RNAs formed at the RNA level, likely via trans-splicing, without corresponding genes as genomic parents, should be regarded as authentic chimeric RNAs. However, since in human cells, procedural and mechanistic details of trans-splicing have never been disclosed, we doubt the existence of trans-splicing. Therefore, there are probably no authentic chimeras in humans, after readthrough and fusion-gene derived RNAs are all put back into the group of ordinary RNAs. Therefore, it should be further determined whether in human cells all two-neighboring-gene RNAs are derived from transcriptional readthrough and whether trans-splicing truly exists.

Biomedical research has advanced swiftly in recent decades, largely due to progress in biotechnology. However, this rapid spread of new, and not always-fully understood, technology has also created a lot of false or irreproducible data and artifacts, which sometimes have led to erroneous conclusions. When describing various scientific issues, scientists have developed a habit of saying “on one hand… but on the other hand…”, because discrepant data and conclusions have become omnipresent. One reason for this problematic situation is that we are not always thoughtful enough in study design, and sometimes lack enough philosophical contemplation. Another major reason is that we are too rushed in introducing new technology into our research without assimilating technical details. In this essay, we provide examples in different research realms to justify our points. To help readers test their own weaknesses, we raise questions on technical details of RNA reverse transcription, polymerase chain reactions, western blotting and immunohistochemical staining, as these methods are basic and are the base for other modern biotechnologies. Hopefully, after contemplation and reflection on these questions, readers will agree that we indeed know too little about these basic techniques, especially about the artifacts they may create, and thus many conclusions drawn from the studies using those ever-more-sophisticated techniques may be even more problematic.

Dental fluorosis, resulting from long-term environmental exposure to fluoride, is prevalent among diverse populations worldwide. Severe fluorosis not only compromises the aesthetic appeal of teeth but also impairs their functionality. This study aims to investigate the oral microbiome in dental fluorosis and the health individuals of adolescents living in the endemic fluorosis area of Guizhou, China through full-length 16S rDNA sequencing. Fourty-six individuals meet the sampling criteria, and we divided these samples into the following groups: a healthy group (H = 23) and a dental fluorosis group (F = 23), and two subgroups of Miao ethnicity: a healthy Miao group (Hm = 13) and a dental fluorosis Miao group (Fm = 15). A total of 660,389 high-quality sequences were obtained, and 12,007 Amplicon Sequence Variants (ASVs) were identified, revealing significant variations in oral microbiome between Fm and Hm groups. The composition of oral microbiota was similar between the H and F groups. At the genus level, Pseudopropionibacterium and at the species level, Streptococcus oralis_subsp.dentisani_clade_058 were less abundant in group F than in group H (P < 0.05). Further analysis revealed that the abundance of Capnocytophaga gingivalis and Kingella denitrificans was significantly lower in Fm fluorosis patients than in the Hm group (P < 0.05). Based on the LEfSe analysis, the potential core biomarkers in the oral of Fm fluorosis patients were identified at different taxonomic levels, ranging from phylum to species. These include Gammaproteobacteria, Prevotella sp_HMT_304, Gemella sanguinis, and Gracilibacteria_(GN02). Network analysis revealed that the microbiota in the fluorosis group exhibited more complex interactions with each other than the healthy group. Notably, within the Hm group, the potential biomarkers Capnocytophaga gingivalis and Kingella denitrificans exhibited a positive correlation. Finally, we employed PICRUSt2 analysis to explore the abundance clustering of the top 30 functional units in each sample, and we found that the metabolic pathway compositions of the four groups were similar. In summary, our findings suggest that the microbial composition of plaque in Hm patients with dental fluorosis is significantly altered, and we identified the potential marker microorganisms that contribute to these changes.

Objective: A case-control study was conducted to evaluate the relationship between endothelial nitric oxide synthase (NOS3) gene polymorphism and essential hypertension in the Han, Miao, and Buyi populations in Guizhou China. Methods: DNA was collected from the blood samples of 353 essential hypertension patients and 342 healthy controls from Guizhou province of China. Eight polymorphisms of the NOS3 gene were genotyped using the Sequenom MassARRAY platform. For genetic analysis, SPSS 26.0, Haploview, SNPStats, SHEsis, and MDR were utilized. Results: All SNPs (rs11771443, rs1808593, rs753482, rs3918186, rs3918188, rs3918227, rs7830, and rs891512) satisfied the Hardy-Weinberg equilibrium test (P > 0.05). The allele and genotype frequencies of rs7830 and rs1808593 in case-control groups demonstrated significant differences (P < 0.05). Compared to the TT genotype of rs1808593, the TG or GG genotype reduced the risk of hypertension in the Miao population (OR = 0.410, 95% CI: 0.218–0.770, P = 0.006). Compared to the GG or GT genotype of rs7830, the TT genotype increased the risk of hypertension in the overall populations (OR = 1.716, 95%CI: 1.139–2.586, P = 0.010). The CATT (rs3918227-rs391818186-rs1808593-rs7830) haplotype was a risk factor for hypertension in the Miao and Han populations (OR = 1.471, 95%CI: 1.010–2.143, P = 0.044 and OR = 1.692, 95%CI: 1.124–2.545, P = 0.011). The CAGG haplotype in the Miao population was a protective factor against hypertension (OR = 0.555, 95%CI: 0.330–0.934, P = 0.025). The rs3918188, rs1808593, and rs7830 in the Miao population showed an interaction effect on hypertension (P < 0.001). The rs11771443, rs3918188, and rs7830 in the Buyi and Han populations showed an interaction effect on hypertension (P = 0.013 and P < 0.001). Conclusion: The single nucleotide polymorphisms rs1808593 and rs7830 of NOS3 gene are associated with essential hypertension in Guizhou ethnic populations.

Molecular biology research often requires extraction of DNA fragments from agarose gels. In the past decades, there have been many methods developed for this purpose. Currently most researchers, especially novices, use commercial kits for this extraction, although these kits cost money and the procedures involved are not necessarily easier than some erstwhile methods. We herein reintroduce and reassess several simple and cost-free older methods. One method involves excising a slice of the gel containing the DNA fragment, followed by a thaw-and-freeze procedure to release the DNA from the gel slice into the gel-making buffer. The second method involves a dialysis tubing and requires electroelution of the DNA from the gel slice in the tubing. The third one is to centrifuge the gel slice to release the DNA. The fourth method requires electro-transfer of the DNA from the gel into a filter paper, while the fifth one includes either allowing the DNA in the slice to be dissolved into a buffer or dissolving the DNA-containing gel slice, followed by DNA precipitation with ethanol or isopropanol. The strengths and weaknesses of these methods are discussed to assist researchers in making their choice. We also point out that some of the end uses of the DNA fragment in the agarose gel may not actually require extraction of the DNA. For instance, a tiny DNA-containing gel block or filter paper can be directly used as the template in a nested or semi-nested polymerase chain reaction to preliminarily determine the identity of the DNA fragment.

Single-nucleotide polymorphisms (SNPs) and DNA methylation are crucial regulators of essential hypertension (EH). Amyloid precursor protein (APP) mutations are implicated in hypertension development. Nonetheless, studies on the association of APP gene polymorphism and promoter methylation with hypertension are limited. Therefore, this case–control aims to evaluate the genetic association of APP gene polymorphism and promoter methylation with EH in Guizhou populations. We conducted a case–control study on 343 EH patients and 335 healthy controls (including Miao, Buyi, and Han populations) in the Guizhou province of China to analyze 11 single-nucleotide polymorphisms (rs2040273, rs63750921, rs2211772, rs2830077, rs467021, rs368196, rs466433, rs364048, rs364051, rs438031, rs463946) in the APP gene via MassARRAY SNP. The MassARRAY EpiTYPER was employed to detect the methylation levels of the promoters. In the Han population, the rs2211772 genotype distribution was significantly different between disease and control groups (χ2 = 6.343, P = 0.039). The CC genotype reduced the risk of hypertension compared to the TT or TC genotype (OR 0.105, 95%CI 0.012–0.914, P = 0.041). For rs2040273 in the Miao population, AG or GG genotype reduced the hypertension risk compared with the AA genotype (OR 0.533, 95%CI 0.294–0.965, P = 0.038). Haplotype TCC (rs364051–rs438031–rs463946) increased the risk of EH in Guizhou (OR 1.427, 95%CI 1.020–1.996, P = 0.037). Each 1% increase in CpG_19 (− 613 bp) methylation level was associated with a 4.1% increase in hypertension risk (OR 1.041, 95%CI 1.002–1.081, P = 0.039). Each 1% increase in CpG_1 (− 296 bp) methylation level was associated with an 8% decrease in hypertension risk in women (OR 0.920, 95%CI 0.860–0.984, P = 0.015). CpG_19 significantly correlated with systolic blood pressure (r = 0.2, P = 0.03). The methylation levels of CpG_19 in hypertensive patients with rs466433, rs364048, and rs364051 minor alleles were lower than that with wild-type alleles (P < 0.05). Moreover, rs467021 and rs364051 showed strong synergistic interaction with EH (χ2 = 7.633, P = 0.006). CpG_11, CpG_19, and rs364051 showed weak synergistic interaction with EH (χ2 = 19.874, P < 0.001). In summary, rs2211772 polymorphism and promoter methylation level of APP gene may be linked to EH in Guizhou populations. Our findings will provide novel insights for genetic research of hypertension and Alzheimer's disease.