Sintilimab (anti-PD-1 antibody) combined with high-dose methotrexate, temozolomide, and rituximab (anti-CD20 antibody) in primary central nervous system lymphoma: a phase 2 study.

Primary central nervous system lymphoma (PCNSL) is a rare and frequently fatal lymphoma subtype. The programmed death-1 (PD-1) pathway has emerged as a potential therapeutic target, but the effectiveness of PD-1 antibody sintilimab in combination with immunochemotherapy as a frontline treatment for PCNSL remains to be determined. In this phase 2 trial (ChiCTR1900027433) with a safety run-in, we included patients aged 18-70 with newly diagnosed PCNSL. Participants underwent six 21-day cycles of a SMTR regimen, which includes sintilimab (200 mg, Day 0), rituximab (375 mg/m2, Day 0), methotrexate (3.0 g/m2, Day 1 or 1.0 g/m2 for patients aged ≥65 years), and temozolomide (150 mg/m2/d, Days 1-5). Among 27 evaluable patients, the overall response rate (ORR) was 96.3% (95% confidence interval: 81-99.9%), with 25 complete responses. At a median follow-up of 24.4 months, the medians for duration of response, progression-free survival (PFS), and overall survival were not reached. The most common grade 3-4 treatment-related toxicities were increased levels of alanine aminotransferase (17.9%) and aspartate aminotransferase (14.3%). Additionally, baseline levels of interferon-α and the IL10/IL6 ratio in cerebrospinal fluid emerged as potential predictors of PFS, achieving areas under the curve of 0.88 and 0.84, respectively, at 2 years. Whole-exome sequencing revealed a higher prevalence of RTK-RAS and PI3K pathway mutations in the durable clinical benefit group, while a greater frequency of Notch and Hippo pathway mutations in the no durable benefit group. These findings suggest the SMTR regimen is highly efficacious and tolerable for newly diagnosed PCNSL, warranting further investigation.

Prediction of PD-L1 and Ki-67 status in primary central nervous system diffuse large B-cell lymphoma by diffusion and perfusion MRI: a preliminary study.

OBJECTIVE: To assess whether diffusion and perfusion MRI derived parameters could non-invasively predict PD-L1 and Ki-67 status in primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL). METHODS: We retrospectively analyzed DWI, DSC-PWI, and morphological MRI (mMRI) in 88 patients with PCNS-DLBCL. The mMRI features were compared using chi-square tests or Fisher exact test. Minimum ADC (ADCmin), mean ADC(ADCmean), relative minimum ADC (rADCmin), relative mean ADC (rADCmean), and relative maximum CBV (rCBVmax) values were compared in PCNS-DLBCL with different molecular status by using the Mann-Whitney U test. The diagnostic performances were evaluated by receiver operating characteristic curves. RESULTS: PCNS-DLBCL with high PD-L1 expression demonstrated a significantly higher ADCmin value than those with low PD-L1. The ADCmean and rADCmean values were significantly lower in PCNS-DLBCL with high Ki-67 status compared with those in low Ki-67 status. Other ADC, CBV parameters, and mMRI features did not show any association with these molecular statuses The diagnostic efficacy of ADC values in assessing PD-L1 and Ki-67 status was relatively low, with area under the curves (AUCs) values less than 0.7. CONCLUSIONS: DWI-derived ADC values can provide some relevant information about PD-L1 and Ki-67 status in PCNS-DLBCL, but may not be sufficient to predict their expression due to the rather low diagnostic performance.

Evolutionary Diversity of Coxsackievirus A6 Causing Severe Hand, Foot, and Mouth Disease - China, 2012-2023.

Introduction: Coxsackievirus A6 (CVA6) has emerged as a significant pathogen responsible for severe cases of hand, foot, and mouth disease (HFMD). This study aims to delineate the demographic characteristics and analyze the viral evolution of severe HFMD associated with CVA6, thereby assisting in its surveillance and management. Methods: In this investigation, 74 strains of CVA6 were isolated from samples collected from severe HFMD cases between 2012 and 2023. The VP1 gene sequences of CVA6 were amplified and analyzed to assess population historical dynamics and evolutionary characteristics using BEAST, DnaSP6, and PopART. Results: A significant portion (94.4%) of severe CVA6-associated HFMD cases (51 out of 54, with 20 lacking age information) were children under 5 years old. Among the 74 CVA6 strains analyzed, 72 belonged to the D3a sub-genotype, while only two strains were D2 sub-genotype. The average genetic distance between VP1 sequences prior to 2015 was 0.027, which increased to 0.051 when compared to sequences post-2015. Historical population dynamics analysis indicated three significant population expansions of severe CVA6-associated HFMD during 2012-2013, 2013-2014, and 2019-2020, resulting in the formation of 65 distinct haplotypes. Consistent with the MCC tree findings, transitioning between regional haplotypes required multiple base substitutions, showcasing an increase in population diversity during the evolutionary process (from 14 haplotypes in 2013 to 55 haplotypes over the subsequent decade). Conclusions: CVA6, associated with severe HFMD, is evolving and presents a risk of outbreak occurrence. Thus, enhanced surveillance of severe HFMD is imperative.

Evolution, recombination and geographic spreading of global Coxsackievirus A6.

BACKGROUND: The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6. METHODS: From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed. RESULTS: The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF-A - RF-X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1-137 and VP1-138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions. CONCLUSIONS: Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

SIRT2 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner.

Sirtuin 2 (SIRT2) regulates the maintenance of genome integrity by targeting pathways of DNA damage response and homologous recombination repair. However, whether and how SIRT2 promotes base excision repair (BER) remain to be determined. Here, we found that independent of its catalytic activity SIRT2 interacted with the critical glycosylase OGG1 to promote OGG1 recruitment to its own promoter upon oxidative stress, thereby enhancing OGG1 promoter activity and increasing BER efficiency. Further studies revealed that SIRT2 was phosphorylated on S46 and S53 by ATM/ATR upon oxidative stress, and SIRT2 phosphorylation enhanced the SIRT2-OGG1 interaction and mediated the stimulatory effect of SIRT2 on OGG1 promoter activity. We also characterized 37 cancer-derived SIRT2 mutants and found that 5 exhibited the loss of the stimulatory effects on OGG1 transcription. Together, our data reveal that SIRT2 acts as a tumor suppressor by promoting OGG1 transcription and increasing BER efficiency in an ATM/ATR-dependent manner.

Detection and complete genome sequence analysis of human adenovirus in children with acute diarrhea in Yunnan, China, 2015-2021.

The aims of this study were to determine the distribution and prevalence of gastroenteritis caused by human adenovirus (HAdV) in children in Yunnan province, China, in 2015-2021 and to identify preventive measures that can be taken to reduce morbidity and mortality in children.HAdV is a significant agent of diarrhea in children, but limited data are available regarding the epidemiology and genetic diversity of HAdV in children with diarrhea in Yunnan province, China. A total of 1754 fecal samples were subjected to real-time RT-PCR to detect and quantify HAdV. Positive samples were further analyzed using next-generation sequencing (NGS), and epidemiological data were analyzed as well.1754 patients with diarrhea were enrolled, of which 1041 were male and 713 were female (M:F ratio: 1.46). Seventy-two stool samples out of 1754 (4.10%) were positive for HAdV. The detection rates of all age groups varied from 2.50-4.78%. The highest incidence of HAdV was observed in children under 2 years of age, especially in children 12-24 months-old. From 2015-2021, the annual detection rate ranged from 1.62-12.26%. HAdV was detected throughout the year, but with marked seasonality. Children were most likely to be positive for HAdV in June and November. We detected HAdV in 15.53% (16/103) of samples collected in June and in 8.19% (14/171) of those collected in November. The entire viral genome was successfully sequenced for 13 of the 72 HAdV-positive samples, and 76.92% (10/13) of these were classified as genotype F41 and 23.08% (3/13) were classified as genotype C2.ConclusionsIn Yunnan province, children of all ages are susceptible to HAdV infection, but there has been marked variation in the yearly prevalence. The highest rate of HAdV detection was in June, followed by November. Priority should be given to disease prevention over the development of targeted antiviral therapies, and effective vaccines for preventing HAdV diarrhea are needed. It is also important to establish a surveillance system to collect relevant clinical and epidemiological data quickly in order to assess the potential risk of HAdV infection in children and to identify epidemic strains for the development of effective vaccines.

Four pairs of neolignan enantiomers with distinctive isochroman moiety from the fruits of Crataegus pinnatifida and their protective activities against H2O2-induced SH-SY5Y cells.

Four pairs of neolignan enantiomers (±)-1- (±)-4 with a distinctive isochroman moiety, including seven undescribed compounds, were isolated and identified from the fruits of Crataegus pinnatifida. Structural characterization of these compounds was established through comprehensive spectroscopic analyses, as well as quantum chemical calculations of ECD and NMR data. The preliminary bioassay displayed that compounds (+)-2 and (±)-3 exerted protective activities against H2O2-induced human neuroblastoma SH-SY5Y cells compared with the positive control. These bioactive compounds could be potential candidates for further pharmaceutical applications.

Circulating small extracellular vesicles microRNAs plus CA-125 for treatment stratification in advanced ovarian cancer.

BACKGROUND: No residual disease (R0 resection) after debulking surgery is the most critical independent prognostic factor for advanced ovarian cancer (AOC). There is an unmet clinical need for selecting primary or interval debulking surgery in AOC patients using existing prediction models. METHODS: RNA sequencing of circulating small extracellular vesicles (sEVs) was used to discover the differential expression microRNAs (DEMs) profile between any residual disease (R0, n = 17) and no residual disease (non-R0, n = 20) in AOC patients. We further analyzed plasma samples of AOC patients collected before surgery or neoadjuvant chemotherapy via TaqMan qRT-PCR. The combined risk model of residual disease was developed by logistic regression analysis based on the discovery-validation sets. RESULTS: Using a comprehensive plasma small extracellular vesicles (sEVs) microRNAs (miRNAs) profile in AOC, we identified and optimized a risk prediction model consisting of plasma sEVs-derived 4-miRNA and CA-125 with better performance in predicting R0 resection. Based on 360 clinical human samples, this model was constructed using least absolute shrinkage and selection operator (LASSO) and logistic regression analysis, and it has favorable calibration and discrimination ability (AUC:0.903; sensitivity:0.897; specificity:0.910; PPV:0.926; NPV:0.871). The quantitative evaluation of Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) suggested that the additional predictive power of the combined model was significantly improved contrasted with CA-125 or 4-miRNA alone (NRI = 0.471, IDI = 0.538, p < 0.001; NRI = 0.122, IDI = 0.185, p < 0.01). CONCLUSION: Overall, we established a reliable, non-invasive, and objective detection method composed of circulating tumor-derived sEVs 4-miRNA plus CA-125 to preoperatively anticipate the high-risk AOC patients of residual disease to optimize clinical therapy.

A New Assessment Method of Vitiligo by Combination of Dermoscopy and Reflectance Confocal Microscopy.

Purpose: The aim is to investigate the application value of dermoscopy combined with reflectance confocal microscopy (RCM) in assessing vitiligo disease activity and treatment response. Patients and Methods: We enrolled 279 patients with vitiligo and evaluated the disease activity by Vitiligo Disease Activity (VIDA) score, dermoscopy, RCM and dermoscopy combined with RCM respectively. The sensitivity and specificity of different assessment techniques were compared with VIDA score by the differences and consistency. The different characteristics of dermoscopy and RCM with different treatment responses were also analyzed. Results: The results showed that the sensitivity and specificity of dermoscopy combined RCM were higher than RCM or dermoscopy alone (P values less than 0.05). In the repigmentation process, leukotrichia, pigment network absent and perilesional hyperpigmentation under dermoscopy at the baseline suggested a poor treatment response, while the incompletely disappearing pigment rings under RCM and perifollicular hyperpigmentation under dermoscopy indicated a good treatment response. We also found the proportion of patients with telangiectasia, increased pigment at the lesions and around the hair follicles was significantly higher in the good treatment response group than that in the poor one by dermoscopy (χ2 = 4.423, 32.471, 4.348, P = 0.035 0.000, 0.037) and by RCM the proportion of patients with both increased pigment granules and dendritic melanocytes in the good treatment response group was higher than that in the poor one (χ2 = 38.215, 5.283, P = 0.000, 0.022, respectively). Conclusion: With the higher sensitivity and specificity than dermoscopy or RCM alone, a combination of dermoscopy and RCM may be a new more accurate measure to assess the vitiligo disease activity and the treatment response.

Improved performance of non-preloaded and high flip-angle dynamic susceptibility contrast perfusion-weighted imaging sequences in the presurgical differentiation of brain lymphoma and glioblastoma.

OBJECTIVE: This study aimed to compare the accuracy of relative cerebral blood volume (rCBV) and percentage signal recovery (PSR) obtained from high flip-angle dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI) sequences with and without contrast agent (CA) preload for presurgical discrimination of brain glioblastoma and lymphoma. METHODS: Consecutive 336 patients (glioblastoma, 236; PCNSL, 100) were included. All the patients underwent DSC-PWI on 3.0-T magnetic resonance units before surgery. The rCBV and PSR with preloaded and non-preloaded CA were measured. The means of the continuous variables were compared using Welch's t-test. The diagnostic accuracies of the individual parameters were compared using the receiver operating characteristic curve analysis. RESULTS: The rCBV was higher with preloaded CA than with non-preloaded CA (glioblastoma, 10.20 vs. 8.90, p = 0.020; PCNSL, 3.88 vs. 3.27, p = 0.020). The PSR was lower with preloaded CA than with non-preloaded CA (glioblastoma, 0.59 vs. 0.90; PCNSL, 0.70 vs. 1.63; all p < 0.001). Regarding the differentiation of glioblastoma and PCNSL, the AUC of rCBV with preloaded CA was indistinguishable from that of non-preloaded CA (0.940 vs. 0.949, p = 0.703), whereas the area under the curve of PSR with preloaded CA was lower than non-preloaded CA (0.529 vs. 0.884, p < 0.001). CONCLUSION: With preloaded CA, diagnostic performance in differentiating glioblastoma and PCNSL did not improve for rCBV and it was decreased for PSR. Therefore, high flip-angle non-preload DSC-PWI sequences offer excellent accuracy and may be of choice sequence for presurgical discrimination of brain lymphoma and glioblastoma. CLINICAL RELEVANCE STATEMENT: High flip-angle DSC-PWI using non-preloaded CA may be an excellent diagnostic method for distinguishing glioblastoma from PCNSL. KEY POINTS: • Differentiating primary central nervous system lymphoma and glioblastoma accurately is critical for their management. • DSC-PWI sequences optimised for the most accurate CBV calculations may not be the optimal sequences for presurgical brain tumour diagnosis as they could be masquerading leakage phenomena that may provide interesting information in terms of differential diagnosis. • High flip-angle non-preloaded DSC-PWI sequences render the best accuracy in the presurgical differentiation of brain lymphoma and glioblastoma.

Probiotics improve symptoms of patients with COVID-19 through gut-lung axis: a systematic review and meta-analysis.

Background: Multi system symptoms such as gastrointestinal tract and respiratory tract exist in coronavirus disease 2019 (COVID-19) patients. There is a lack of reliable evidence to prove that probiotics are effective in improving these symptoms. In this study, we aimed to evaluate the efficacy of probiotics in meta-analysis. Methods: We systematically searched PubMed, Embase, Web of Science, and Cochrane Library up to February 15, 2023. Randomized controlled trials or high quality retrospective studies comparing the efficacy of probiotics as supplementation with non-probiotics in improving symptoms for patients with COVID-19 were included. This meta-analysis assessed endpoints using Review Manager 5.3. Result: Ten citations comprising 1198 patients with COVID-19 were included. The results showed that probiotics could increase the number of people with overall symptom improvement (RR = 1.62, 95% CI [1.10, 2.38], P = 0.01) and shorten the duration (days) of overall symptoms (MD = -1.26, 95% CI [-2.36, -0.16], P = 0.02). For the duration (days) of specific symptoms, probiotics could improve diarrhea (MD = -2.12, 95% CI [-2.41, -1.83], P < 0.00001), cough (MD = -2.21, 95% CI [-4.56, 0.13], P = 0.06) and shortness of breath (MD = -1.37, 95% CI [-2.22, -0.53], P = 0.001). Probiotics had no obvious effect on fever, headache and weakness. For inflammation, probiotics could effectively reduce C-reactive Protein (CRP) serum level (mg/L) (MD = -4.03, 95% CI [-5.12, -2.93], P < 0.00001). Regarding hospital stay (days), probiotics group was shorter than non-probiotics group (MD = -0.98, 95% CI [-1.95, -0.01], P = 0.05). Conclusion: To some extent probiotics could improve the overall symptoms, inflammatory reaction and shorten hospital stay of patients with COVID-19. Probiotics may improve gastrointestinal symptoms (such as improving intestinal flora and reducing the duration of diarrhea) and further improve respiratory symptoms through the gut-lung axis. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=398309, identifier: CRD42023398309.

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway.

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.

Advances in Oral Drug Delivery Systems: Challenges and Opportunities.

The oral route is the most preferred route for systemic and local drug delivery. However, the oral drug delivery system faces the harsh physiological and physicochemical environment of the gastrointestinal tract, which limits the bioavailability and targeted design of oral drug delivery system. Innovative pharmaceutical approaches including nanoparticulate formulations, biomimetic drug formulations, and microfabricated devices have been explored to optimize drug targeting and bioavailability. In this review, the anatomical factors, biochemical factors, and physiology factors that influence delivering drug via oral route are discussed and recent advance in conventional and novel oral drug delivery approaches for improving drug bioavailability and targeting ability are highlighted. We also address the challenges and opportunities of oral drug delivery systems in future.

A glucose-blue light AND gate-controlled chemi-optogenetic cell-implanted therapy for treating type-1 diabetes in mice.

Exogenous insulin therapy is the mainstay treatment for Type-1 diabetes (T1D) caused by insulin deficiency. A fine-tuned insulin supply system is important to maintain the glucose homeostasis. In this study, we present a designed cell system that produces insulin under an AND gate control, which is triggered only in the presence of both high glucose and blue light illumination. The glucose-sensitive GIP promoter induces the expression of GI-Gal4 protein, which forms a complex with LOV-VP16 in the presence of blue light. The GI-Gal4:LOV-VP16 complex then promotes the expression of UAS-promoter-driven insulin. We transfected these components into HEK293T cells, and demonstrated the insulin was secreted under the AND gate control. Furthermore, we showed the capacity of the engineered cells to improve the blood glucose homeostasis through implantation subcutaneously into Type-1 diabetes mice.

Rapid screening of diarylpentanoids from Daphne bholua.

Fractionation motivated by biological activity screening and NMR characteristic signals analysis led to the isolation of seventeen diarylpentanoids from the whole plant of Daphne bholua Buch.-Ham. ex D. Don, among which nine compounds were undescribed. Their structures and stereochemistry were determined by comprehensive spectroscopic data, J-based configurational analysis, and quantum chemical calculations. The inhibitory potentials of all isolates against acetylcholinesterase were evaluated in vitro and in silico.

Dermoscopy and reflection confocal microscope features of pigmented prurigo.

BACKGROUND: Pigmented prurigo (PP) is a chronic and recurrent inflammatory skin disease. PP is not common clinically, but it is easily misdiagnosed because of its diversified clinical manifestations in different stages. MATERIALS AND METHODS: We retrospectively analyzed the clinical, histopathological, dermoscopy, and reflectance confocal microscopy (RCM) features of 20 patients diagnosed as PP. RESULTS: The female predominance ratio was revealed with male to female of 1:4. Seven female patients were on a diet (without staple food) and one patient had a history of diabetes. Eight cases were suffered in spring, six cases in winter, three cases in summer, and three cases in autumn. Multiple sites were involved in 13 cases. Four patients had urticarial papules and plaques. Nineteen patients had erythematous papules with reticular distribution, of which 14 cases accompanied reticulate hyperpigmentation, four cases with papulovesicle, and two cases accompanied with pustules. One patient only showed reticulate hyperpigmentation. In the early lesions, dermatoscopy showed pink oval lesions, punctate or linear vessels, and pale yellow rings around the skin lesions. RCM is characterized by spongiosis, spongy vesicle, neutrophils scattered in the epidermis, which was consistent with epidermis spongiosis, neutrophils infiltrating into the upper epidermis and necrotic keratinocytes in histopathology. In the fully developed lesions, dermatoscopy showed pink lesions with brown pigment granules in the center and linear vessels in the edge. RCM showed that demarcation of epidermis and dermis is not clear, and inflammatory cells can be seen in the upper dermis and histopathologically lesions assumed a patchy lichenoid pattern, and the inflammatory cells infiltrating the dermis were dominated by lymphocytes. In the late lesions, dermatoscopy showed grainy grayish-brown or yellowish-brown pigmentation surrounding the hair follicle merging with each other. RCM showed that pigment granules were increased on the ring of basal cells, inflammatory cells were sparsely infiltrated in the dermal papilla and superficial layer, and epidermis slightly hyperplastic, with melanophages and a few lymphocytes infiltrating the superficial dermis in histopathology. CONCLUSION: PP is easily misdiagnosed and not always occurs in those on a restrictive diet. A combination of dermatoscopy and RCM is helpful for its diagnosis of PP.

The chromosome-scale assembly of the Salvia rosmarinus genome provides insight into carnosic acid biosynthesis.

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.