Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

Design, synthesis and structure-activity relationship studies on erianin analogues as pyruvate carboxylase inhibitors in hepatocellular carcinoma cells.

A series of novel erianin analogues were designed and synthesized based on the bioisosterism principle by altering the two aromatic rings of erianin, the substituents on the rings and the linker between them. The analogues were evaluated as pyruvate carboxylase (PC) inhibitors in hepatocellular carcinoma cells. It was found that compounds 35 and 36, where fluorine replaces a hydroxyl group, exhibited higher activity than erianin (IC50 value of 17.30 nM) in liver cancer cells with IC50 values of 15.15 nM and 10.05 nM, respectively. Additionally, at a concentration of 10 nM, compounds 35 and 36 inhibited PC with inhibitory rates of 39.10% and 40.15%, respectively, exhibiting nearly identical inhibitory activity to erianin (inhibitory rate of 40.07%). Additionally, a computer simulation docking study demonstrated the basis for better interactions between the receptors and ligands. The fluorine atom of 35 can not only form hydrogen bonds with Lys-1043 (NH⋯F, 2.04 Å), but also form fluorine bonds with the carbonyl groups of Lys-1043 (3.67 Å) and Glu-1046 (3.70 Å), due to the different orientations of the halogens on the B ring warhead. Conversely, the chlorine atom of 34 can only form alkyl hydrophobic interactions with the alkane chain in Lys-1043. Fluorinated compounds 35 and 36 also show better chemical stability and higher log P (clog P = 3.89 for 35 and 36) values than that of erianin (clog P = 3.07), and may be used as candidate compounds for further drug development.

Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

The Prognostic Model Established by the Differential Expression Genes Based on CD8+ T Cells to Evaluate the Prognosis and the Response to Immunotherapy in Osteosarcoma.

Osteosarcoma (OS) is a malignant tumor with an extremely poor prognosis, especially in progressive patients. Immunotherapy based on immune checkpoint inhibitors (ICIs) is considered to be a promising treatment option for OS. Due to tumor heterogeneity, only a minority of patients benefit from immunotherapy. Therefore, it is urgent to explore a model that can accurately assess the response of OS to immunotherapy. In this study, we obtained the single-cell RNA sequencing datasets of OS patients from public databases and defined 34 cell clusters by dimensional reduction and clustering analysis. PTPRC was applied to identify immune cell clusters and nonimmune cell clusters. Next, we performed clustering analysis on the immune cell clusters and obtained 25 immune cell subclusters. Immune cells were labeled with CD8A and CD8B to obtain CD8+ T cell clusters. Meanwhile, we extracted the differentially expressed genes (DEGs) of CD8+ T cell clusters and other immune cell clusters. Furthermore, we constructed a prognostic model (CD8-DEG model) based on the obtained DEGs of CD8+ T cells, and verified the excellent predictive ability of this model for the prognosis of OS. Moreover, we further investigated the value of the CD8-DEG model. The results indicated that the risk score of the CD8-DEG model was an independent risk factor for OS patients. Finally, we revealed that the risk score of the CD8-DEG model correlates with the immune profile of OS and can be used to evaluate the response of OS to immunotherapy. In conclusion, our study revealed the critical role of CD8 cells in OS. The risk score model based on CD8-DEGs can provide guidance for prognosis and immunotherapy of OS.

Tacrolimus in the treatment of childhood nephrotic syndrome: Machine learning detects novel biomarkers and predicts efficacy.

STUDY OBJECTIVE: The pharmacokinetics and pharmacodynamics of tacrolimus (TAC) vary greatly among individuals, hindering its precise utilization. Moreover, effective models for the early prediction of TAC efficacy in patients with nephrotic syndrome (NS) are lacking. We aimed to identify key factors affecting TAC efficacy and develop efficacy prediction models for childhood NS using machine learning algorithms. DESIGN: This was an observational cohort study of patients with pediatric refractory NS. SETTING: Guangzhou Women and Children's Medical Center between June 2013 and December 2018. PATIENTS: 203 patients with pediatric refractory NS were used for model generation and 35 patients were used for model validation. INTERVENTION: All patients regularly received double immunosuppressive therapy comprising TAC and low-dose prednisone or methylprednisolone. In this observational cohort study of 203 pediatric patients with refractory NS, clinical and genetic variables, including single-nucleotide polymorphism (SNPs), were identified. TAC efficacy was evaluated 3 months after administration according to two different evaluation criteria: response or non-response (Group 1) and complete remission, partial remission, or non-remission (Group 2). MEASUREMENTS: Logistic regression, extremely random trees, gradient boosting decision trees, random forest, and extreme gradient boosting algorithms were used to develop and validate the models. Prediction models were validated among a cohort of 35 patients with NS. MAIN RESULTS: The random forest models performed best in both groups, and the area under the receiver operating characteristics curve of these two models was 80.7% (Group 1) and 80.3% (Group 2). These prediction models included urine erythrocyte count before administration, steroid types, and eight SNPs (ITGB4 rs2290460, TRPC6 rs3824934, CTGF rs9399005, IL13 rs20541, NFKBIA rs8904, NFKBIA rs8016947, MAP3K11 rs7946115, and SMARCAL1 rs11886806). CONCLUSIONS: Two pre-administration models with good predictive performance for TAC response of patients with NS were developed and validated using machine learning algorithms. These accurate models could assist clinicians in predicting TAC efficacy in pediatric patients with NS before utilization to avoid treatment failure or adverse effects.

Detection of QTLs Regulating Six Agronomic Traits of Rice Based on Chromosome Segment Substitution Lines of Common Wild Rice (Oryza rufipogon Griff.) and Mapping of qPH1.1 and qLMC6.1.

Wild rice is a primary source of genes that can be utilized to generate rice cultivars with advantageous traits. Chromosome segment substitution lines (CSSLs) are consisting of a set of consecutive and overlapping donor chromosome segments in a recipient's genetic background. CSSLs are an ideal genetic population for mapping quantitative traits loci (QTLs). In this study, 59 CSSLs from the common wild rice (Oryza rufipogon Griff.) accession DP15 under the indica rice cultivar (O. sativa L. ssp. indica) variety 93-11 background were constructed through multiple backcrosses and marker-assisted selection (MAS). Through high-throughput whole genome re-sequencing (WGRS) of parental lines, 12,565 mapped InDels were identified and designed for polymorphic molecular markers. The 59 CSSLs library covered 91.72% of the genome of common wild rice accession DP15. The DP15-CSSLs displayed variation in six economic traits including grain length (GL), grain width (GW), thousand-grain weight (TGW), grain length-width ratio (GLWR), plant height (PH), and leaf margin color (LMC), which were finally attributed to 22 QTLs. A homozygous CSSL line and a purple leave margin CSSL line were selected to construct two secondary genetic populations for the QTLs mapping. Thus, the PH-controlling QTL qPH1.1 was mapped to a region of 4.31-Mb on chromosome 1, and the LMC-controlling QTL qLMC6.1 was mapped to a region of 370-kb on chromosome 6. Taken together, these identified novel QTLs/genes from common wild rice can potentially promote theoretical knowledge and genetic applications to rice breeders worldwide.

Development of Soft Rice Lines by Regulating Amylose Content via Editing the 5'UTR of the Wx Gene.

The type of soft rice with low amylose content (AC) is more and more favored by consumers for its better eating and cooking quality, as people's quality of life continuously improves in China. The Wx gene regulates the AC of rice grains, thus affecting the degree of softness of the rice. Mei Meng B (MMB), Tian Kang B (TKB), and DR462 are three indica rice maintained lines with good morphological characters, but also with undesirably high AC. Therefore, CRISPR/Cas9 technology was used to edit the Wx gene of these lines to create a batch of soft rice breeding materials. New gene-edited lines MMB-10-2, TKB-21-12, and DR462-9-9, derived from the above parental lines, respectively, were selected in the T2 generations, with an AC of 17.2%, 16.8%, and 17.8%, and gel consistency (GC) of 78.6 mm, 77.4 mm, and 79.6 mm, respectively. The rapid viscosity analysis (RVA) spectrum showed that the three edited lines had a better eating quality as compared to the corresponding wild type, and showing new characteristics, different from the high-quality soft rice popular in the market. There was no significant difference in the main agronomic traits in the three edited lines compared to the corresponding wild types. Moreover, the chalkiness of DR462-9-9 was reduced, resulting in an improved appearance of its polished rice. The present study created soft rice germplasms for breeding improved quality hybrid rice, without changing the excellent traits of their corresponding wild type varieties.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.).

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

1, 2-trans-Stereoselective 7-O-Glycosylation of Flavonoids with Unprotected Pyranoses by Mitsunobu Reaction.

The glycosylation of protecting-group-free pyranoses with flavonoids to generate flavonoid O-glycosides under Mitsunobu conditions was reported. The methodology allows to prepare a wide range of natural 7-flavonoid O-glycosides and their derivatives from commercially available chemicals in good to excellent yields with exclusive 1,2-trans-stereoselectivity regardless the anomeric configuration of employed pyranoses. The highly regioselective glycosylation was also achieved among different types of hydroxyl groups on the glycosyl acceptors.

Comparative transcriptome-wide identification and differential expression of genes and lncRNAs in rice near-isogenic line (KW-Bph36-NIL) in response to BPH feeding.

Brown planthopper (BPH) is the most devastating pest of rice in Asia, causing substantial yield losses and has become a challenging task to be controlled under field conditions. Although extensive measures have been taken over the past decades, which resulted in the evolution of new resistant BPH strains. Therefore, besides other possible approaches, equipping host plants with resistant genes is the most effective and environment-friendly technique for BPH control. Here, we systematically analyzed transcriptome changes in the susceptible rice variety Kangwenqingzhan (KW) and the resistant near-isogenic line (NIL) KW-Bph36-NIL, through RNA-seq, depicting the differential expression profiles of mRNAs and long non-coding RNAs (lncRNAs) in rice before and after BPH feeding. We observed a proportion of genes (1.48%) and (2.74%) were altered in KW and NIL, respectively, indicating different responses of rice strains against BPH feeding. Nevertheless, we characterized 384 differentially expressed long non-coding RNAs (DELs) that can be impacted by the two strains by alternatively changing the expression patterns of the respective coding genes, suggesting their certain involvement in response to BPH feeding. In BPH invasion, KW and NIL responded differently by modifying the synthesis, storage, and transformation of intracellular substances, adjusting the nutrient accumulation and utilization inside and outside the cells. In addition, NIL expressed stronger resistance by acutely up-regulating genes and other transcription factors related to stress resistance and plant immunity. Altogether, our study elaborates valuable insights into the genome-wide DEGs and DELs expression profiles of rice under BPH invasion by high throughput sequencing and further suggests that NILs can be utilized in BPH resistance breeding programs in developing high-resistance rice lines.

Impact of Home Parenting Environment on Cognitive and Psychomotor Development in Children Under 5 Years Old: A Meta-Analysis.

This study aims to evaluate the relationship between home parenting environment and the cognitive and psychomotor development in children under 5 years old by using meta-analysis. A systematic search of the Chinese and English databases including Pubmed, Embase, the Cochrane Library, CNKI, Weipu, Wanfang, and CBMdisc databases from January 1, 1990, to July 31, 2021, was performed. Articles concerning the relationship between home parenting environment and the cognitive and psychomotor development in children under 5 years old were included. Review Manager 5.4 was used for meta-analysis. Subgroup analysis in terms of age and region were performed. A total of 12 articles were included, including 11 in English and 1 in Chinese. Meta-analysis showed that there was significant relationship between home parenting environment and the cognitive and psychomotor development of children (r = 0.31; r = 0.21). Subgroup analysis showed that correlation between home parenting environment and the cognitive and psychomotor development of children was stronger in children over 18 months compared to those under 17 months [(r = 0.33, r = 0.21) vs. (r = 0.28, r = 0.17)]. The converted summary r value between home parenting environment and cognitive development in developing and developed countries was both 0.32. Conclusively, there is a positive correlation between the home parenting environment and the cognitive and psychomotor development of children under 5 years old. Improving the home parenting environment of children is beneficial to promote their early development.

Genome-wide screening of SARS-CoV-2 infection-related genes based on the blood leukocytes sequencing data set of patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is a global epidemic disease caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing serious adverse effects on human health. In this study, we obtained a blood leukocytes sequencing data set of COVID-19 patients from the GEO database and obtained differentially expressed genes (DEGs). We further analyzed these DEGs by protein-protein interaction analysis and Gene Ontology enrichment analysis and identified the DEGs closely related to SARS-CoV-2 infection. Then, we constructed a six-gene model (comprising IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1) by logistic regression analysis and calculated the area under the ROC curve (AUC) for the diagnosis of COVID-19. The AUC values of the training group, testing group, and entire group were 0.930, 0.914, and 0.921, respectively. The six genes were highly expressed in patients with COVID-19 and positively correlated with the expression of SARS-CoV-2 invasion-related genes (ACE2, TMPRSS2, CTSB, and CTSL). The risk score calculated by this model was also positively correlated with the expression of TMPRSS2, CTSB, and CTSL, indicating that the six genes were closely related to SARS-CoV-2 infection. In conclusion, we comprehensively analyzed the functions of DEGs in the blood leukocytes of patients with COVID-19 and constructed a six-gene model that may contribute to the development of new diagnostic and therapeutic ideas for COVID-19. Moreover, these six genes may be therapeutic targets for COVID-19.

Hepatic Polarization Accelerated by Mechanical Compaction Involves HNF4α Activation.

There remain few data about the role of homeostatic compaction in hepatic polarization. A previous study has found that mechanical compaction can accelerate hepatocyte polarization; however, the cellular mechanism underlying the effect is mostly unclear. Hepatocyte nuclear factor 4 alpha (HNF4α) is crucial for hepatic polarization in liver morphogenesis. Therefore, we sought to identify any possible involvement of HNF4α in the process of hepatocyte polarization accelerated by mechanical compaction. We first verified in the nonhepatic cell model HEK-293T, and the hepatic cell model primary hepatocytes that the mechanical compaction on cell aggregates simulated by using transient centrifugation can directly activate the expression of HNF4α promoters. Moreover, data using primary hepatocytes showed that the HNF4α expression is positively associated with the levels of compaction force: 2.1-folds higher at the mRNA level and 2.1-folds higher at the protein level for 500 g vs. 0 g. Furthermore, activated HNF4α expression is associated with the enhanced biliary canalicular formation and the increased production of albumin and urea. Pretreatment with Latrunculin B, an inhibitor of F-actin, and SHE78-7, an inhibitor of E-cadherin, which both interrupt the pathway of mechanical transduction, partially but significantly reduced the HNF4α expression and production of albumin and urea. In conclusion, HNF4α can be actively involved in the hepatic polarization in the context of environmental mechanical compaction.

Knockdown of CDCA8 inhibits the proliferation and enhances the apoptosis of bladder cancer cells.

Bladder cancer is a tumour of the urinary system with high mortality, and there is also a great lack of therapeutic targets in the clinic. Cell division cycle associated 8 (CDCA8), an important component of the vertebrate chromosomal passenger complex, is highly expressed in various tumours and promotes tumour development. However, the role of CDCA8 in bladder cancer is not fully understood. This study aimed to reveal the function of CDCA8 in bladder cancer by determining the relationship between CDCA8 expression and proliferation, metastasis and apoptosis of bladder cancer cells. Firstly, we studied the mRNA expression of CDCA8 through the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases and analysed the correlation between CDCA8 expression and prognosis of patients with bladder cancer. We also verified CDCA8 expression in bladder cancer tissues by immunohistochemistry. In addition, CDCA8 expression was inhibited in bladder cancer T24 and 5637 cells, and the effects of CDCA8 on the proliferation, migration and invasion of bladder cancer cell lines were investigated using cell counting kit-8, colony formation, cell cycle, apoptosis, wound healing and Transwell invasion assays. Results showed that CDCA8 was highly expressed in bladder cancer compared with normal tissues, and the high CDCA8 expression was significantly correlated with the poor prognosis of patients. Inhibiting CDCA8 expression inhibited the proliferation, migration and invasion of T24 and 5637 cells and induced the apoptosis of bladder cancer cells. CDCA8 was involved in the regulation of the growth cycle of bladder cancer cells. Bioinformatics-based mechanism analysis revealed that high CDCA8 expression may affect the cell cycle and P53 signalling pathways. In conclusion, our results suggest that CDCA8 is highly expressed in bladder cancer and can promote tumour development. Hence, CDCA8 may serve as an effective therapeutic target for treatment of bladder cancer.

Identification of a four immune-related genes signature based on an immunogenomic landscape analysis of clear cell renal cell carcinoma.

Renal clear cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which has strong immunogenicity. A comprehensive study of the role of immune-related genes (IRGs) in ccRCC is of great significance in finding ccRCC treatment targets and improving patient prognosis. In this study, we comprehensively analyzed the expression of IRGs in ccRCC based on The Cancer Genome Atlas datasets. The mechanism of differentially expressed IRGs in ccRCC was analyzed by bioinformatics. In addition, Cox regression analysis was used to screen prognostic related IRGs from differentially expressed IRGs. We also identified a four IRGs signature consisting of four IRGs (CXCL2, SEMA3G, PDGFD, and UCN) through lasso regression and multivariate Cox regression analysis. Further analysis results showed that the four IRGs signature could effectively predict the prognosis of patients with ccRCC, and its predictive power is independent of other clinical factors. In addition, the correlation analysis of immune cell infiltration showed that this four IRGs signature could effectively reflect the level of immune cell infiltration of ccRCC. We also found that the expression of immune checkpoint genes CTLA-4, LAG3, and PD-1 in the high-risk group was higher than that in the low-risk group. Our research revealed the role of IRGs in ccRCC, and developed a four IRGs signature that could be used to evaluate the prognosis of patients with ccRCC, which will help to develop personalized treatment strategies for patients with ccRCC and improve their prognosis. In addition, these four IRGs may be effective therapeutic targets for ccRCC.

Quantifying and monitoring fibrosis in non-alcoholic fatty liver disease using dual-photon microscopy.

OBJECTIVE: Fibrosis stage is strongly associated with liver-related outcomes and is a key surrogate endpoint in drug trials for non-alcoholic steatohepatitis. Dual-photon microscopy allows automated quantification of fibrosis-related parameters (q-FPs) and may facilitate large-scale histological studies. We aim to validate the performance of q-FPs in a large histological cohort. DESIGN: 344 patients with non-alcoholic fatty liver disease (NAFLD) underwent 428 liver biopsies (240 had paired transient elastography examination). Fibrosis stage was scored using the NASH Clinical Research Network system, and q-FPs were measured by dual-photon microscopy using unstained slides. Patients were randomly assigned to the training and validation cohorts to test the performance of individual q-FPs and derive optimal cut-offs. RESULTS: Over 25 q-FPs had area under the receiver-operating characteristics curves >0.90 for different fibrosis stages. Among them, the perimeter of collagen fibres and number of long collagen fibres had the highest accuracy. At the best cut-offs, the two q-FPs had 88.3%-96.2% sensitivity and 78.1%-91.1% specificity for different fibrosis stages in the validation cohort. q-FPs and histological scoring had nearly identical correlations with liver stiffness measurement, suggesting that the accuracy of q-FPs approached that of histological assessment. Among patients with paired liver biopsies, changes in the same q-FPs were associated with changes in fibrosis stage. At a median follow-up of 5.6 years, baseline q-FPs predicted liver-related events. CONCLUSION: q-FP is highly accurate in the assessment of fibrosis in NAFLD patients. This automated platform can be used in future studies as objective and reliable evaluation of histological fibrosis.

Dual-photon microscopy-based quantitation of fibrosis-related parameters (q-FP) to model disease progression in steatohepatitis.

There is a need for further refinement of current histological systems for assessment of hepatic fibrosis in nonalcoholic fatty liver disease (NAFLD). This study evaluated hepatic fibrosis in NAFLD using dual-photon microscopy-based quantitation of fibrosis-related parameters (q-FPs). Fifty test cohort subjects and 42 validation cohort subjects with NAFLD and the full spectrum of fibrosis were studied. q-FPs were measured in specific predefined regions of interest (general, vessel, perisinusoid, and vascular septa). Seventy q-FPs had inter- and intraobserver concordance ≥0.8 and were related to the NASH Clinical Research Network fibrosis staging. Of these, 16 q-FPs with the strongest correlations (P < 0.001 for all) were entered in a principal component analysis model (odds ratio [OR] 7.8, P < 0.001), which separated any stage of fibrosis versus no fibrosis, and cirrhosis versus earlier stages with the areas under the receiver operating characteristic curves of 0.88 and 0.93 (P ≤ 0.01 for both), respectively. In an independent multivariable analysis, four q-FPs-the number of collagen strands (OR 8.5, P = 0.004), strand length (OR 12.0, P = 0.02), strand eccentricity (OR 8.3, P = 0.004), and strand solidity (OR 8.0, P = 0.003)-were independently associated with fibrosis stages and were used to model fibrosis along a continuous linear scale using desirability functions; this linear scale of fibrosis measurement was also related to fibrosis stage (P < 0.0001). The robustness of both the multivariable model and the linear scale of measurement was confirmed in the validation cohort. CONCLUSION: The q-FP model provides an accurate reproducible method to evaluate fibrosis in NAFLD along a quantitative and continuous scale. (Hepatology 2017;65:1891-1903).

Systematic quantification of histological patterns shows accuracy in reflecting cirrhotic remodeling.

BACKGROUND AND AIM: There still lacks a tool for precisely evaluating cirrhotic remodeling. Histologic distortion characterized in cirrhosis (i.e. cirrhotic patterns) has a validated pathophysiological meaning and potential relevance to clinical complications. We aimed to establish a new tool to quantify the cirrhotic patterns and test it for reflecting the cirrhotic remodeling. METHODS: We designed a computerized algorithm, named qCP, dedicated for the analysis of liver images acquired by second harmonic microscopy. We evaluated its measurement by using a cohort of 95 biopsies (Ishak staging F4/5/6 = 33/35/27) of chronic hepatitis B and a carbon tetrachloride-intoxicated rat model for simulating the bidirectional cirrhotic change. RESULTS: QCP can characterize 14 histological cirrhosis parameters involving the nodules, septa, sinusoid, and vessels. For chronic hepatitis B biopsies, the mean overall intra-observer and inter-observer agreement was 0.94 ± 0.08 and 0.93 ± 0.09, respectively. The robustness in resisting sample adequacy-related scoring error was demonstrated. The proportionate areas of total (collagen proportionate area), septal (septal collagen proportionate area [SPA]), sinusoidal, and vessel collagen, nodule area, and nodule density (ND) were associated with Ishak staging (P < 0.01 for all). But only ND and SPA were independently associated (P ≤ 0.001 for both). A histological cirrhosis parameters-composed qCP-index demonstrated an excellent accuracy in quantitatively diagnosing evolving cirrhosis (areas under receiver operating characteristic curves 0.95-0.92; sensitivity 0.93-0.82; specificity 0.94-0.85). In the rat model, changes in collagen proportionate area, SPA, and ND had strong correlations with both cirrhosis progression and regression and faithfully characterized the histological evolution. CONCLUSIONS: QCP preliminarily demonstrates potential for quantitating cirrhotic remodeling with high resolution and accuracy. Further validation with in-study cohorts and multiple-etiologies is warranted.