StrVCTVRE: A supervised learning method to predict the pathogenicity of human genome structural variants.

Whole-genome sequencing resolves many clinical cases where standard diagnostic methods have failed. However, at least half of these cases remain unresolved after whole-genome sequencing. Structural variants (SVs; genomic variants larger than 50 base pairs) of uncertain significance are the genetic cause of a portion of these unresolved cases. As sequencing methods using long or linked reads become more accessible and SV detection algorithms improve, clinicians and researchers are gaining access to thousands of reliable SVs of unknown disease relevance. Methods to predict the pathogenicity of these SVs are required to realize the full diagnostic potential of long-read sequencing. To address this emerging need, we developed StrVCTVRE to distinguish pathogenic SVs from benign SVs that overlap exons. In a random forest classifier, we integrated features that capture gene importance, coding region, conservation, expression, and exon structure. We found that features such as expression and conservation are important but are absent from SV classification guidelines. We leveraged multiple resources to construct a size-matched training set of rare, putatively benign and pathogenic SVs. StrVCTVRE performs accurately across a wide SV size range on independent test sets, which will allow clinicians and researchers to eliminate about half of SVs from consideration while retaining a 90% sensitivity. We anticipate clinicians and researchers will use StrVCTVRE to prioritize SVs in probands where no SV is immediately compelling, empowering deeper investigation into novel SVs to resolve cases and understand new mechanisms of disease. StrVCTVRE runs rapidly and is publicly available.

SAM-DTA: a sequence-agnostic model for drug-target binding affinity prediction.

Drug-target binding affinity prediction is a fundamental task for drug discovery and has been studied for decades. Most methods follow the canonical paradigm that processes the inputs of the protein (target) and the ligand (drug) separately and then combines them together. In this study we demonstrate, surprisingly, that a model is able to achieve even superior performance without access to any protein-sequence-related information. Instead, a protein is characterized completely by the ligands that it interacts. Specifically, we treat different proteins separately, which are jointly trained in a multi-head manner, so as to learn a robust and universal representation of ligands that is generalizable across proteins. Empirical evidences show that the novel paradigm outperforms its competitive sequence-based counterpart, with the Mean Squared Error (MSE) of 0.4261 versus 0.7612 and the R-Square of 0.7984 versus 0.6570 compared with DeepAffinity. We also investigate the transfer learning scenario where unseen proteins are encountered after the initial training, and the cross-dataset evaluation for prospective studies. The results reveals the robustness of the proposed model in generalizing to unseen proteins as well as in predicting future data. Source codes and data are available at https://github.com/huzqatpku/SAM-DTA.

Hidden Charge Order and Multiple Electronic Instabilities in EuTe4.

The rare-earth telluride compound EuTe4 exhibits a charge density wave (CDW) and an unconventional thermal hysteresis transition. Herein, we report a comprehensive study of the CDW states in EuTe4 by using low-temperature scanning tunneling microscopy. Two types of charge orders are observed at 4 K, including a newly discovered spindle-shaped pattern and a typical stripe-like pattern. As an exotic charge order, the spindle-shaped CDW is off-axis and barely visible at 77 K, indicating that it is a hidden order developed at low temperature. Based on our first-principles calculations, we reveal the origins of the observed electronic instabilities. The spindle-shaped charge order stems from a subsequent transition based on the stripe-like CDW phase. Our work demonstrates that the competition and cooperation between multiple charge orders can generate exotic quantum phases.

DNMT1/DNMT3a-mediated promoter hypermethylation and transcription activation of ICAM5 augments thyroid carcinoma progression.

Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.

Predicting the impact of rare variants on RNA splicing in CAGI6.

Variants which disrupt splicing are a frequent cause of rare disease that have been under-ascertained clinically. Accurate and efficient methods to predict a variant's impact on splicing are needed to interpret the growing number of variants of unknown significance (VUS) identified by exome and genome sequencing. Here, we present the results of the CAGI6 Splicing VUS challenge, which invited predictions of the splicing impact of 56 variants ascertained clinically and functionally validated to determine splicing impact. The performance of 12 prediction methods, along with SpliceAI and CADD, was compared on the 56 functionally validated variants. The maximum accuracy achieved was 82% from two different approaches, one weighting SpliceAI scores by minor allele frequency, and one applying the recently published Splicing Prediction Pipeline (SPiP). SPiP performed optimally in terms of sensitivity, while an ensemble method combining multiple prediction tools and information from databases exceeded all others for specificity. Several challenge methods equalled or exceeded the performance of SpliceAI, with ultimate choice of prediction method likely to depend on experimental or clinical aims. One quarter of the variants were incorrectly predicted by at least 50% of the methods, highlighting the need for further improvements to splicing prediction methods for successful clinical application.

Reciprocal adaptation of rice and Xanthomonas oryzae pv. oryzae: cross-species 2D GWAS reveals the underlying genetics.

A 1D/2D genome-wide association study strategy was adopted to investigate the genetic systems underlying the reciprocal adaptation of rice (Oryza sativa) and its bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo) using the whole-genome sequencing and large-scale phenotyping data of 701 rice accessions and 23 diverse Xoo strains. Forty-seven Xoo virulence-related genes and 318 rice quantitative resistance genes (QR-genes) mainly located in 41 genomic regions, and genome-wide interactions between the detected virulence-related genes and QR genes were identified, including well-known resistance genes/virulence genes plus many previously uncharacterized ones. The relationship between rice and Xoo was characterized by strong differentiation among Xoo races corresponding to the subspecific differentiation of rice, by strong shifts toward increased resistance/virulence of rice/Xoo populations and by rich genetic diversity at the detected rice QR-genes and Xoo virulence genes, and by genome-wide interactions between many rice QR-genes and Xoo virulence genes in a multiple-to-multiple manner, presumably resulting either from direct protein-protein interactions or from genetic epistasis. The observed complex genetic interaction system between rice and Xoo likely exists in other crop-pathogen systems that would maintain high levels of diversity at their QR-loci/virulence-loci, resulting in dynamic coevolutionary consequences during their reciprocal adaptation.

A fluorescent probe based on cyclochalcone for detecting peroxynitrite.

A novel cyclic chalcone fluorescent probe C-PN was synthesized to detect ONOO-. After reaction with peroxynitrite, the double bond of C-PN in the cyclic chalcone structure was disconnected, which caused the change of intramolecular charge transfer (ICT) effect, emitting blue fluorescence and quenching orange red fluorescence. Visible to the naked eye, the color of the probe solution changed. The probe showed low sensitivity (detection limit = 20.2 nm), short response time (less than 60 s) at low concentration of ONOO-, good visibility, and good selectivity and stability for ONOO-.

FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells.

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer1-4. The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1+ T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

Genomic variation in 3,010 diverse accessions of Asian cultivated rice.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

Osteopontin: A Novel Therapeutic Target for Respiratory Diseases.

Osteopontin (OPN) is a multifunctional phosphorylated protein that is involved in physiological and pathological events. Emerging evidence suggests that OPN also plays a critical role in the pathogenesis of respiratory diseases. OPN can be produced and secreted by various cell types in lungs and overexpression of OPN has been found in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), pulmonary hypertension (PH), pulmonary fibrosis diseases, lung cancer, lung infection, chronic obstructive pulmonary disease (COPD), and asthma. OPN exerts diverse effects on the inflammatory response, immune cell activation, fibrosis and tissue remodeling, and tumorigenesis of these respiratory diseases, and genetic and pharmacological moudulation of OPN exerts therapeutic effects in the treatment of respiratory diseases. In this review, we summarize the recent evidence of multifaceted roles and underlying mechanisms of OPN in these respiratory diseases, and targeting OPN appears to be a potential therapeutic intervention for these diseases.

Emerging Roles of Galectin-3 in Pulmonary Diseases.

Galectin-3 is a multifunctional protein that is involved in various physiological and pathological events. Emerging evidence suggests that galectin-3 also plays a critical role in the pathogenesis of pulmonary diseases. Galectin-3 can be produced and secreted by various cell types in the lungs, and the overexpression of galectin-3 has been found in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), pulmonary hypertension (PH), pulmonary fibrosis diseases, lung cancer, lung infection, chronic obstructive pulmonary disease (COPD), and asthma. Galectin-3 exerts diverse effects on the inflammatory response, immune cell activation, fibrosis and tissue remodeling, and tumorigenesis in these pulmonary disorders, and genetic and pharmacologic modulation of galectin-3 has therapeutic effects on the treatment of pulmonary illnesses. In this review, we summarize the structure and function of galectin-3 and the underlying mechanisms of galectin-3 in pulmonary disease pathologies; we also discuss preclinical and clinical evidence regarding the therapeutic potential of galectin-3 inhibitors in these pulmonary disorders. Additionally, targeting galectin-3 may be a very promising therapeutic approach for the treatment of pulmonary diseases.

Effects of Protection Time on Infection of Rice Panicle Blast.

Panicle blast, caused by Magnaporthe oryzae, is a destructive disease of rice worldwide. Clarifying the susceptibility of rice panicles at different stages is of great significance for effective disease management. Field experiments were conducted in two paddy fields at Wuyuan County in 2016 and 2017 to determine the effects of head covering and its timing on the infection of rice panicle blast. Results revealed that panicle blast was reduced significantly by covering rice heads with sulfuric acid paper bags, regardless of the covering time, ranging from initial heading to 15 days afterward, suggesting that rice panicles could be infected by blast pathogen even 15 days after initial heading. Panicle blast incidence was also found to be significantly influenced by plant dates, with higher panicle blast incidence observed in plots planted on early dates, suggesting adjusting plant dates could help rice panicles escape the infection by blast pathogen. The results from this study also highlighted the importance of cultivars and environmental conditions to panicle blast. In conclusion, besides planting blast-resistant cultivars, it is important to protect rice heads from the initial heading to the early dough stages, and fungicides should be applied according to infection warnings based on host, inoculum, and weather conditions.

One-Step Green Synthesis of Isoeugenol Methyl Ether from Eugenol by Dimethyl Carbonate and Phase-Transfer Catalysts.

In this paper, the green synthesis of isoeugenol methyl ether (IEME) from eugenol by O-methylation and isomerization is completed using a one-step green process. In the methylation reaction, dimethyl carbonate (DMC) was used as a green chemistry reagent instead of the traditional harmful methylation reagents, in accordance with the current concept of green chemistry. The phase transfer catalyst (PTC) polyethylene glycol 800 (PEG-800) was introduced into the isomerization reaction to break the barrier of difficult contact between solid and liquid phases and drastically reduce the reaction conditions by shortening the reaction time and reducing the alkalinity of the reaction system. The catalytic systems for the one-step green synthesis of IEME were screened, and it was shown that the catalytic system "K2CO3 + PEG-800" was the most effective. The effects of reaction temperature, n(DMC):n(eugenol) ratio, n(PEG-800):n(eugenol) ratio, and n(K2CO3):n(eugenol) ratio on eugenol conversion, IEME yield, and IEME selectivity were investigated. The results showed that the best reaction was achieved at a reaction temperature of 140 °C, a reaction time of 3 h, a DMC drip rate of 0.09 mL/min, and n(eugenol):n(DMC):n(K2CO3):n(PEG-800) = 1:3:0.09:0.08. As a result of the conversion of 93.1% of eugenol to IEME, a yield of 86.1% IEME as well as 91.6% IEME selectivity were obtained.

[Intelligent material classification of traditional Chinese medicine based on semantic analysis].

A method for material classification of traditional Chinese medicines based on the physical properties of powder has been established by our research group. This method involves pre-treatment of traditional Chinese medicine decoction pieces, powder preparation, and determination of physical properties, being cumbersome. In this study, the word segmentation logic of semantic analysis was adopted to establish the thesaurus and local standardized semantic word segmentation database with the macroscopic and microscopic characteristics of 36 model traditional Chinese medicines as the basic data. The physical properties of these medicines have been determined and the classification of these medicines is clear in the cluster analysis. A total of 55 keywords for powdery, fibrous, sugary, oily, and brittle materials were screened by association rules and the set inclusion and exclusion criteria, and the weights of the keywords were calculated. Furthermore, the algorithms of the keyword matching scores and the computation rules of the single or multiple material classification were established for building the intelligent model of semantic analysis for the material classification. The semantic classification results of the other 35 TCMs except Pseudostellariae Radix(multi-material medicine) agreed with the clustering results based on the physical properties of the powder, with an agreement rate of 97.22%. In model validation, the prediction results of semantic classification of traditional Chinese medicines were consistent with the clustering results based on the physical properties of powder, with an agreement rate of 83.33%. The results showed that the method of material classification based on semantic analysis was feasible, which laid a foundation for the development of intelligent decision-making technology for personalized traditional Chinese medicine preparations.

UHRF1 regulates alternative splicing by interacting with splicing factors and U snRNAs in a H3R2me involved manner.

The well-established functions of UHRF1 converge to DNA biological processes, as exemplified by DNA methylation maintenance and DNA damage repair during cell cycles. However, the potential effect of UHRF1 on RNA metabolism is largely unexplored. Here, we revealed that UHRF1 serves as a novel alternative RNA splicing regulator. The protein interactome of UHRF1 identified various splicing factors. Among them, SF3B3 could interact with UHRF1 directly and participate in UHRF1-regulated alternative splicing events. Furthermore, we interrogated the RNA interactome of UHRF1, and surprisingly, we identified U snRNAs, the canonical spliceosome components, in the purified UHRF1 complex. Unexpectedly, we found H3R2 methylation status determines the binding preference of U snRNAs, especially U2 snRNAs. The involvement of U snRNAs in UHRF1-containing complex and their binding preference to specific chromatin configuration imply a finely orchestrated mechanism at play. Our results provided the resources and pinpointed the molecular basis of UHRF1-mediated alternative RNA splicing, which will help us better our understanding of the physiological and pathological roles of UHRF1 in disease development.

Tumor-derived exosomes induce immunosuppressive macrophages to foster intrahepatic cholangiocarcinoma progression.

BACKGROUND AND AIMS: Macrophages are prominent components of solid tumors and exhibit distinct functions in different tumor microenvironments. Exosomes are emerging as necessary mediators of the cross-talk between tumor cells and the microenvironment. However, the underlying mechanisms of exosomes involving into crosstalk between tumor cells and macrophages during disease progression of intrahepatic cholangiocarcinoma (ICC) have not been yet fully realized. APPROACH AND RESULTS: We found that the macrophages of ICC tumor tissues up-regulated the expression levels of immunosuppressive molecule programmed death-ligand 1 (PD-L1). Increased PD-L1+ macrophages in tumor tissues effectively suppressed T-cell immunity and correlated with poor survival rates in patients with ICC. High-throughput RNA-sequencing analysis that was performed to identify differential levels of microRNAs (miRNAs) between exosomes derived from ICC cells and primary human intrahepatic biliary epithelial cells revealed that miR-183-5p was increased in ICC cell-derived exosomes. Exosomal miR-183-5p inhibited phosphatase and tensin homolog (PTEN) expression, to subsequently affect the elevations on both phosphorylated AKT and PD-L1 expression in macrophages. Furthermore, macrophages that treated with ICC cell-derived exosomes significantly suppressed T-cell immunity in vitro and contributed to the growth and progression of ICC in vivo, which were reversible through blockages on PD-L1 of these macrophages. Finally, clinical data showed that up-regulated levels of plasma exosomal miR-183-5p correlated with poor prognosis of patients with ICC after curative resection. CONCLUSIONS: Tumor-derived exosomal miR-183-5p up-regulates PD-L1-expressing macrophages to foster immune suppression and disease progression in ICC through the miR-183-5p/PTEN/AKT/PD-L1 pathway. Exosomal miR-183-5p is a potential predictive biomarker for ICC progression and a potential target for development of therapeutic strategies against immune tolerance feature of ICC.

Genomic evolution and the impact of SLIT2 mutation in relapsed intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. APPROACH AND RESULTS: We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. CONCLUSIONS: We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Creating a near-perfect circularly polarized terahertz beam through the nonreciprocity of a magnetoplasma.

Compared to other parts of the electromagnetic spectrum, the terahertz frequency range lacks efficient polarization manipulation techniques, which is impeding the proliferation of terahertz technology. In this work, we demonstrate a tunable and broadband linear-to-circular polarization converter based on an InSb plate containing a free-carrier magnetoplasma. In a wide spectral region (∼ 0.45 THz), the magnetoplasma selectively absorbs one circularly polarized mode due to electron cyclotron resonance and also reflects it at the edges of the absorption band. Both effects are nonreciprocal and contribute to form a near-zero transmission band with a high isolation of -36 dB, resulting in the output of a near-perfect circularly polarized terahertz wave for an incident linearly polarized beam. The near-zero transmission band is tunable with magnetic field to cover a wide frequency range from 0.3 to 4.8 THz.

Intramuscular mitochondrial and lipid metabolic changes of rats after regular high-intensity interval training (HIIT) of different training periods.

BACKGROUND: High-intensity Interval Training (HIIT) is a time-efficient form of exercise and has gained popularity in recent years. However, at molecular level, the understanding about the effects of HIIT is not comprehensive, and even less is elucidated about HIIT of different training duration cycles, although different durations always lead to different post-training consequences. METHOD: In this study, by training SD rats using HIIT protocols lasting for different training duration cycles, we investigated the adaptive response of intramuscular triglyceride abundance as well as mitochondrial and lipid metabolic changes after HIIT training (2, 4, 6, 8, and 10 weeks). We selected 72 h after the last session of training as the time point of sacrifice. RESULTS: The suppressed activation of the cAMP-PKA pathway indicates that skeletal muscle was in the recovery phase at this time point. Intramuscular triglyceride abundance was significantly elevated after 2, 4, and 10 weeks of HIIT. However, the lipid metabolism-related proteins inconsistently changed in a chaotic trend (see Table 1). The expression levels of PGC1-α and COX IV decreased after 2 and 4 weeks of training and raised after 6 and 8 weeks of training. The expression level of citrate synthase (CS) decreased after 2, 4, 8, and 10 weeks of training, and showed an upward trend after 6 weeks of training. While the activity of CS decreased after 2 and 8 weeks of training and showed an upward trend after 6 weeks of HIIT. CONCLUSION: Given the abovementioned changing trends, we propose two speculations: (A) the damaged mitochondria oxidation capacity might be one of the causes of IMTG accumulation observed after 2 and 4 weeks of HIIT. This phase might be similar to the condition of type 2 diabetes. (B) after 6-week HIIT, mitochondria function and biogenesis might be improved and the IMTG contents declined to baseline. This might be explained as: mitochondrial enhancement increased the capacity of lipid oxidation and then offset the increase in IMTG achieved during the first 4 weeks. For HIIT Rat Modelling, if the aim is to observe HIIT-induced positive effects, caution should be exercised when considering 2 and 4 weeks of training under our HIIT frame. Also, implementing six-week training is at least effective for mitochondrial enhancement when using similar HIIT frame of this study.