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We determined RNA spectrum of the human RSK4 (hRSK4) gene (also called RPS6KA6) and identified 29 novel mRNA variants derived from alternative splicing, which, plus the NCBI-documented ones and the five we reported previously, totaled 50 hRSK4 RNAs that, by our bioinformatics analyses, encode 35 hRSK4 protein isoforms of 35-762 amino acids. Many of the mRNAs are bicistronic or tricistronic for hRSK4. The NCBI-normalized NM_014496.5 and the protein it encodes are designated herein as the Wt-1 mRNA and protein, respectively, whereas the NM_001330512.1 and the long protein it encodes are designated as the Wt-2 mRNA and protein, respectively. Many of the mRNA variants responded differently to different situations of stress, including serum starvation, a febrile temperature, treatment with ethanol or ethanol-extracted clove buds (an herbal medicine), whereas the same stressed situation often caused quite different alterations among different mRNA variants in different cell lines. Mosifloxacin, an antibiotics and also a functional inhibitor of hRSK4, could inhibit the expression of certain hRSK4 mRNA variants. The hRSK4 gene likely uses alternative splicing as a handy tool to adapt to different stressed situations, and the mRNA and protein multiplicities may partly explain the incongruous literature on its expression and comports., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2 ) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.

Objective: To investigate the RNA profile of synovial fluid (SF) from osteoarthritis (OA) patients and carry out cluster analysis of OA-related genes., Methods: RNA of SF from OA patients was isolated using RNA-specific Trizol. A cDNA library was built and subjected to the second-generation sequencing using HisSeq4000 with a data size of 8G. The sequencing reads were aligned to the UCSC human reference genome (hg38) using Tophat with default parameters. Gene function enrichment was generated using DAVID., Results: The minimum weight 0.096 µg RNA of SF sample was used for sequencing analysis, which produced 66,154,562 clean reads, 91.28% of which were matched to the reference with 2,682 genes identified. Some of the unmatchable reads matched RNAs of bacteria, mainly Pseudomonas . The detected human RNAs in samples fell into different categories of genes, including protein-coding ones, processed and unprocessed pseudogenes, and long noncoding, antisense and miscellaneous RNAs that mediate various biological functions. Interestingly, 80% of the expressed genes belonged to the mitochondrial genome., Conclusion: These results suggest that less than 0.1 µg RNA is sufficient for establishing a cDNA library and deep sequencing, and that the liquid fraction of SF contains a whole RNA repertoire that may reflect a history of previous microorganism infections., Competing Interests: None., (AJTR Copyright © 2022.)

Background : The currently available methods for sexing human or mouse cells have weaknesses. Therefore, it is necessary to establish new methods. Methods : We used bioinformatics approach to identify genes that have alleles on both the X and Y chromosomes of mouse and human genomes and have a region showing a significant difference between the X and Y alleles. We then used polymerase chain reactions (PCR) followed by visualization of the PCR amplicons in agarose gels to establish these genomic regions as genetic sex markers. Results : Our bioinformatics analyses identified eight mouse sex markers and 56 human sex markers that are new, i.e . are previously unreported. Six of the eight mouse markers and 14 of the 56 human markers were verified using PCR and ensuing visualization of the PCR amplicons in agarose gels. Most of the tested and untested sex markers possess significant differences in the molecular weight between the X- and Y-derived PCR amplicons and are thus much better than most, if not all, previously-reported genetic sex markers. We also established several simple and essentially cost-free methods for extraction of crude genomic DNA from cultured cells, blood samples, and tissues that could be used as template for PCR amplification. Conclusion : We have established new sex genetic markers and methods for extracting genomic DNA and for sexing human and mouse cells. Our work may also lend some methodological strategies to the identification of new genetic sex markers for other organismal species., (© 2022 Bentham Science Publishers.)

Although the concept that cancer is caused by mutations has been widely accepted, there still are ample data deprecating it. For example, embryonic cells displaced in non-embryonic environments may develop to cancer, whereas cancer cells placed in embryonic environments may be reverted to phenotypic normal. Although many intracellular or extracellular aberrations are known to be able to initiate a lengthy tumorigenesis, the molecular or cellular alterations that directly establish a neoplastic state, namely cellular immortality and autonomy, still remain unknown. Hereditary traits are encoded not only by gene sequences but also by karyotype and DNA or chromosomal structures that may be altered via non-mutational mechanisms, such as post-translational modifications of nuclear proteins, to initiate tumorigenesis. However, the immortal and autonomous nature of neoplasms makes them "new" organisms, meaning that neoplasms should have mutations to distinguish themselves from their host patients in the genome. Neoplasms are malignant if they bear epigenetic or genetic alterations in mutator genes, i.e. the genes whose alterations accelerate other genes to mutate, whereas neoplasms are benign if their epigenetic or genetic aberrations occur only in non-mutator genes. Future mechanistic research should be focused on identifying the alterations that directly establish cellular immortality and autonomy. Benign tumors may have many fewer alterations and thus be much better models than cancers for such research. Future translational research should be aimed at identifying the cellular factors that control cancer cells' phenotypes and at establishing approaches of directing cancer cells towards differentiation, which should be a promising therapeutic tactic., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The small nucleolar RNA host gene 16 (SNHG16) has recently been shown to be a putative oncogene in gastric cancer (GC) and other cancer types, but how its four lncRNA variants are expressed in any physiological and pathological situation remains unknown. To investigate the expression and function of the four lncRNA variants of SNHG16, mainly the variant 1, in GC, we performed quantitative PCR to determine the RNA levels of the four variants in 60 GC tissue samples and several cell lines. We also studied how knocking down of SNHG16 with siRNA affected proliferation, apoptosis, cell cycle progression, as well as migration and invasion of GC cells. Our results showed that variants 1 and 4 were overexpressed in GC tissues compared with adjacent uninvolved tissues. Knockdown of the four variants, mainly the variant 1, enhanced apoptosis and inhibited cell cycle progression of a GC cell line by arresting the cells at the G1 phase. These cellular effects were associated not only with decreased protein levels of c-Myc, PCNA, cyclins D1, E1, A2 and B, as well as CDKs 2 and 6, but also with increased protein levels of the p21, p27 and p53. Knockdown of total SNHG16 lncRNAs also inhibited invasion and migration of the GC cells in vitro. These results collectively suggest that SNHG16 may be oncogenic in GC by regulating cell cycle progression and may serve as a GC biomarker.

We performed polyacrylamide gel electrophoresis of human proteins with sodium dodecyl sulfate, isolated proteins at multiple positions, and then used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 41.7 and 36 kDa proteins, respectively, LC-MS/MS identified their peptides at all the positions studied. The National Center for Biotechnology Information (USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one noncoding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatics analysis identified a 17.6 kDa isoform encoded by the noncoding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with significant similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to the ACTB. These data lead us to conclude that GAPDH and ACTB are poor reference genes for determining the protein loading in such techniques as Western blotting, a leading role these two genes have been playing for decades in biomedical research., Competing Interests: Conflict of interest: The authors state no conflict of interest., (© 2021 Keyin Zhang et al., published by De Gruyter.)

Rice blast is a serious threat to global rice production. Large-scale and long-term cultivation of rice varieties with a single blast resistance gene usually leads to breakdown of resistance. To effectively control rice blast in Taiwan, marker-assisted backcrossing was conducted to develop monogenic lines carrying different blast resistance genes in the genetic background of an elite japonica rice cultivar, Kaohsiung 145 (KH145). Eleven International Rice Research Institute (IRRI)-bred blast-resistant lines (IRBLs) showing broad-spectrum resistance to local Pyricularia oryzae isolates were used as resistance donors. Sequencing analysis revealed that the recurrent parent, KH145, does not carry known resistance alleles at the target Pi2 / 9 , Pik , Pita , and Ptr loci. For each IRBL × KH145 cross, we screened 21 to 370 (average of 108) plants per generation from the BC 1 F 1 to BC 3 F 1 /BC 4 F 1 generation. A total of 1,499 BC 3 F 2 /BC 4 F 2 lines carrying homozygous resistance alleles were selected and self-crossed for four to six successive generations. The derived lines were also evaluated for background genotype using genotyping by sequencing, for blast resistance under artificial inoculation and natural infection conditions, and for agronomic performance in multiple field trials. In Chiayi and Taitung blast nurseries in 2018 to 2020, Pi2 , Pi9 , and Ptr conferred high resistance, Pi20 and Pik-h moderate resistance, and Pi1 , Pi7 , Pik-p , and Pik susceptibility to leaf blast; only Pi2 , Pi9 , and Ptr conferred effective resistance against panicle blast. The monogenic lines showed agronomic traits, yield, and grain quality similar to those of KH145, suggesting the potential of growing a mixture of lines to achieve durable resistance in the field.