Comparing the Accuracy of Patient-Specific Guide and Universal Guide for Distal Femoral Osteotomy in Dogs.

OBJECTIVE: To introduce a novel universal guide design for distal femoral osteotomy (DFO) to treat femoral deformities and other hindlimb orthopaedic diseases, and to compare its accuracy to the patient-specific guide in vitro and ex vivo on X-ray and computed tomography (CT). METHODS: Thirteen anatomic specimens of canine femurs and 13 3D-printed femoral bone models were subjected to DFO using a patient-specific and a universal guide. The anatomical lateral distal femoral angle and anteversion angle were measured on radiographic and CT images before and after surgical procedure and used to compare the accuracy of the surgical guides in treating uniplanar or biplanar deformities. Bone models were used to compare the accuracy with ex vivo models. RESULTS: The mean correction errors were -1.18° to 0.72° on X-ray and -1.91° to 0.53° on CT scan. The analysis showed no statistically significant difference in the mean error between the groups based on type of guide used, nor in the mean error between groups based on whether anteversion correction was performed based on both the CT and X-ray imaging data. CONCLUSION: Both the patient-specific and universal guides can produce accurate results in DFO surgery, and the universal guide shows potential as a useful surgical tool.

Contents of paeoniflorin and albiflorin in two Korean landraces of Paeonia lactiflora and characterization of paeoniflorin biosynthesis genes in peony.

BACKGROUND AND RESEARCH PURPOSE: Paeoniflorin and albiflorin are monoterpene glycosides that exhibit various medicinal properties in Paeonia species. This study explored the terpene biosynthesis pathway and analyzed the distribution of these compounds in different tissues of two Korean landraces of Paeonia lactiflora to gain insights into the biosynthesis of monoterpene glycosides in P. lactiflora and their potential applications. MATERIALS AND METHODS: Two Korean landraces, Hongcheon var. and Hwacheon var, of P. lactiflora were used for the analyses. Contents of the paeoniflorin and albiflorin were analyzed using HPLC. RNA was extracted, sequenced, and subjected to transcriptome analysis. Differential gene expression, KEGG, and GO analyses were performed. Paeoniflorin biosynthesis genes were isolated from the transcriptomes using the genes in Euphorbia maculata with the NBLAST program. Phylogenetic analysis of of 1-Deoxy-D-xylulose 5-phosphate synthase (DOXPS), geranyl pyrophosphate synthase (GPPS), and pinene synthase (PS) was carried out with ClustalW and MEGA v5.0. RESULTS AND DISCUSSION: Analysis of paeoniflorin and albiflorin content in different tissues of the two P. lactiflora landraces revealed significant variation. Transcriptome analysis yielded 36,602 unigenes, most of which were involved in metabolic processes. The DEG analysis revealed tissue-specific expression patterns with correlations between landraces. The isolation of biosynthetic genes identified 173 candidates. Phylogenetic analysis of the key enzymes in these pathways provides insights into their evolutionary relationships. The sequencing and analysis of DOXPS, GPPS, PS revealed distinct clades and subclades, highlighting their evolutionary divergence and functional conservation. Our findings highlight the roots as the primary sites of paeoniflorin and albiflorin accumulation in P. lactiflora, underscoring the importance of tissue-specific gene expression in their biosynthesis. CONCLUSION: this study advances our understanding of monoterpene glycoside production and distribution in Paeonia, thereby guiding further plant biochemistry investigations.

Evaluation of autoimmune liver disease antibodies in hepatitis patients.

BACKGROUND/AIMS: Autoimmune hepatitis (AIH) is characterized by the presence of auto-antibodies and high blood immunoglobulin G (IgG) levels. In this study, the line immunoassay (LIA) was designed to assess various autoantibodies. METHODS: In total, 1371 patients who underwent autoimmune liver disease antibody testing between July 2019 and November 2022 were enrolled. Autoantibodies including antinuclear antibody (ANA) and anti-mitochondrial antibody (AMA) were tested, and clinical data were collected. Statistical analyses were performed by categorizing the data based on diagnosis and IgG quantification separately. A scoring system was applied to identify individuals with AIH. Patients were also classified into the AIH and non-AIH groups. RESULTS: The positivity rate for ANA was 80.2% in the AIH group. The IgG-high group had a high likelihood of the presence of detectable autoantibodies, with anti-Ro-52 being the most frequently detected antibody using LIA. The "Consider AIH" and "AMA" groups had 3-4 times more patients in the IgG-high group than in the "Not Considered" group. CONCLUSIONS: Among autoantibodies, the prevalence of ANA was the highest. As per LIA results, anti-Ro-52 was the most prevalent. AIH cannot be diagnosed based on IgG levels alone and must be distinguished via autoantibody testing. Therefore, extensive testing, including autoantibodies, IgG, ANA, and liver enzyme levels, will help accurately diagnose AIH.

Effects of Biomaterials Derived from Germinated Hemp Seeds on Stressed Hair Stem Cells and Immune Cells.

Androgenetic alopecia is a genetic disorder that commonly causes progressive hair loss in men, leading to diminished self-esteem. Although cannabinoids extracted from Cannabis sativa are used in hair loss treatments, no study has evaluated the effects of germinated hemp seed extract (GHSE) and exosomes derived from the calli of germinated hemp seeds on alopecia. Therefore, this study aimed to demonstrate their preventive effects against alopecia using various methodologies, including quantitative PCR, flow cytometry, ELISA, and immunocytochemistry. Our research highlights the preventive functions of GHSE (GE2000: 2000 µg/mL) and exosomes from the calli of germinated hemp seeds (E40: 40 µg/mL) in three biochemical categories: genetic modulation in hair follicle dermal papilla stem cells (HFDPSCs), cellular differentiation, and immune system modulation. Upon exposure to dihydrotestosterone (DT), both biomaterials upregulated genes preventing alopecia (Wnt, ß-catenin, and TCF) in HFDPSCs and suppressed genes activating alopecia (STAT1, 5α-reductase type 1, IL-15R). Additionally, they suppressed alopecia-related genes (NKG2DL, IL2-Rß, JAK1, STAT1) in CD8+ T cells. Notably, E40 exhibited more pronounced effects compared to GE2000. Consequently, both E40 and GE2000 effectively mitigated DT-induced stress, activating mechanisms promoting hair formation. Given the limited research on alopecia using these materials, their pharmaceutical development promises significant economic and health benefits.

Real-World Clinical Utility of Targeted RNA Sequencing in Leukemia Diagnosis and Management.

Gene fusions are key drivers in acute leukemia, impacting diagnosis and treatment decisions. We analyzed 264 leukemia patients using targeted RNA sequencing with conventional karyotyping and reverse transcription polymerase chain reaction (RT-PCR). Leukemic fusions were detected in 127 patients (48.1%). The new guidelines introduced additional diagnostic criteria, expanding the spectrum of gene fusions. We discovered three novel fusions (RUNX1::DOPEY2, RUNX1::MACROD2, and ZCCHC7::LRP1B). We analyzed recurrent breakpoints for the KMT2A and NUP98 rearrangements. Targeted RNA sequencing showed consistent results with RT-PCR in all tested samples. However, when compared to conventional karyotyping, we observed an 83.3% concordance rate, with 29 cases found only in targeted RNA sequencing, 7 cases with discordant results, and 5 cases found only in conventional karyotyping. For the five cases where known leukemic gene rearrangements were suspected only in conventional karyotyping, we conducted additional messenger RNA sequencing in four cases and proved no pathogenic gene rearrangements. Targeted RNA sequencing proved advantageous for the rapid and accurate interpretation of gene rearrangements. The concurrent use of multiple methods was essential for a comprehensive evaluation. Comprehensive molecular analysis enhances our understanding of leukemia's genetic basis, aiding diagnosis and classification. Advanced molecular techniques improve clinical decision-making, offering potential benefits.

Evaluation of Safe Insertion Angles for Spinal Needles and Safe Intensity of the Holmium:YAG Laser during Percutaneous Laser Disc Ablations in Feline Cadavers.

In the context of veterinary medicine, minimally invasive techniques for feline spinal surgery remain underexplored, particularly for percutaneous laser disc ablation (PLDA) when using the Holmium:YAG (Ho:YAG) laser. This study aimed to refine the application of the Ho:YAG laser in PLDA by determining the optimal laser intensity and safe insertion angles for the thoracic and lumbar intervertebral discs (IVDs) in cats. Through utilizing computed tomography (CT) for precise guidance, this research involved a cadaveric study of 10 cats to evaluate the spatial configurations that allow for safe needle insertions and effective laser ablation. Various energy settings of the Ho:YAG laser (20 J, 40 J, and 60 J) were tested to ascertain the balance between adequate disc vaporization and minimal adjacent tissue damage. The results demonstrate that a 40 J setting is the most effective in achieving significant disc decompression without compromising surrounding tissue integrity. Additionally, the CT scans proved crucial in confirming the accuracy of the needle placement and the safety of the laser application angles. This study established that the 40 J setting on the Ho:YAG laser, combined with CT-guided insertion techniques, offers a reliable method for PLDA, thus enhancing the safety and efficacy of feline spinal surgeries.

Comprehensive insights into AML relapse: genetic mutations, clonal evolution, and clinical outcomes.

INTRODUCTION: Acute myeloid leukemia (AML) is a complex hematologic malignancy characterized by uncontrolled proliferation of myeloid precursor cells within bone marrow. Despite advances in understanding of its molecular underpinnings, AML remains a therapeutic challenge due to its high relapse rate and clonal evolution. METHODS: In this retrospective study, we analyzed data from 24 AML patients diagnosed at a single institution between January 2017 and August 2023. Comprehensive genetic analyses, including chromosomal karyotyping, next-generation sequencing, and gene fusion assays, were performed on bone marrow samples obtained at initial diagnosis and relapse. Clinical data, treatment regimens, and patient outcomes were also documented. RESULTS: Mutations in core genes of FLT3, NPM1, DNMT3A, and IDH2 were frequently discovered in diagnostic sample and remained in relapse sample. FLT3-ITD, TP53, KIT, RUNX1, and WT1 mutation were acquired at relapse in one patient each. Gene fusion assays revealed stable patterns, while chromosomal karyotype analyses indicated a greater diversity of mutations in relapsed patients. Clonal evolution patterns varied, with some cases showing linear or branching evolution and others exhibiting no substantial change in core mutations between diagnosis and relapse. CONCLUSIONS: Our study integrates karyotype, gene rearrangements, and gene mutation results to provide a further understanding of AML heterogeneity and evolution. We demonstrate the clinical relevance of specific mutations and clonal evolution patterns, emphasizing the need for personalized therapies and measurable residual disease monitoring in AML management. By bridging the gap between genetics and clinical outcome, we move closer to tailored AML therapies and improved patient prognoses.

Morphometric Evaluation of Thoracolumbar Spinal Canal and Cord by Magnetic Resonance Imaging in Normal Small-Breed Dogs.

Thoracolumbar intervertebral disc disease (IVDD) is the most common cause of spinal injury in dogs. MRI has been considered the gold standard for neurologic diagnosis, but studies focusing on the thoracolumbar spinal canal and spinal cord using MRI in small-breed dogs are limited. Therefore, this study aimed to establish an MRI reference range for the spinal cord and canal measurements (height, width, cord-to-canal ratio of height, width, cross-sectional area (CSA)) of each intervertebral disc level from T11 to L5 (total of seven levels) on transverse T2-weighted images in normal small-breed dogs. We hypothesized that the spinal cord and spinal canal measurements might vary according to the body weight and age. The width and height of the spinal cord and canal increased as the body weight increased at all levels (p < 0.05). The cord-to-canal ratio of the width showed a negative correlation to the body weight at all levels. The cord-to-canal ratio of the height did not show any correlation to the body weight at all levels. All measurements (height, width, cord-to-canal ratio of height, width, CSA) did not show any statistical correlation between the groups subdivided by age. These measurements could serve as a morphometric baseline for thoracolumbar spinal diseases and clinical research in small-breed dogs.

Evaluation of a 3D-Printed Reduction Guide for Minimally Invasive Plate Osteosynthesis of Short Oblique Radial Diaphyseal Fracture in Dogs: A Cadaveric Study.

This study aims to evaluate the clinical application of three-dimensional (3D)-printed custom reduction guides (3DRG) for minimally invasive plate osteosynthesis (MIPO) of short oblique radial diaphyseal fractures. Canine forelimb specimens (n = 24) were prepared and a diaphyseal short oblique fracture was simulated in the distal radius and ulna. Bone fragments were stabilized with the MIPO technique using a 3DRG (Group A), open reduction (Group B), or closed reduction with circular external skeletal fixation (ESF) (Group C). The diaphyseal short oblique fractures were created in each radius at one-third of the radial length from the distal radial articular surface. Surgical stabilization of the fractures was performed in each group. Pre and postoperative radiographic images were obtained to measure frontal angulation (FA), sagittal angulation (SA), frontal joint reference line angulation (fJRLA), sagittal joint reference line angulation (sJRLA), translational malalignment and fracture gap width. Surgical time was also measured. In the homogeneity test, differences in SA, sJRLA, craniocaudal translation and fracture gap before and after surgery had no significant difference among the three groups. On the other hand, differences in FA, fJRLA, mediolateral translation and surgical time before and after surgery had significant differences among the three groups. In the post hoc test, only surgical time showed a significant difference between the three groups, and group A showed the shortest surgical time. The use of 3DRG for MIPO of short oblique radial diaphyseal fractures in dogs is reliable for the alignment and apposition of fractures and reduces surgical time.

Unraveling trajectories from aplastic anemia to hematologic malignancies: genetic and molecular insights.

Background: Aplastic anemia (AA), characterized by hematopoietic stem cell deficiency, can evolve into different hematologic malignancies. Our understanding of the genetic basis and mechanisms of this progression remains limited. Methods: We retrospectively studied 9 acquired AA patients who later developed hematologic malignancies. Data encompassed clinical, laboratory, karyotype, and next-generation sequencing (NGS) information. We explored chromosomal alterations and mutation profiles to uncover genetic changes underlying the transition. Results: Nine AA patients developed myelodysplastic syndrome (seven patients), acute myeloid leukemia (one patient), or chronic myelomonocytic leukemia (one patient). Among eight patients with karyotype results at secondary malignancy diagnosis, monosomy 7 was detected in three. Trisomy 1, der(1;7), del(6q), trisomy 8, and del(12p) were detected in one patient each. Among three patients with NGS results at secondary malignancy diagnosis, KMT2C mutation was detected in two patients. Acquisition of a PTPN11 mutation was observed in one patient who underwent follow-up NGS testing during progression from chronic myelomonocytic leukemia to acute myeloid leukemia. Conclusion: This study highlights the genetic dynamics in the progression from AA to hematologic malignancy. Monosomy 7's prevalence and the occurrence of PTPN11 mutations suggest predictive and prognostic significance. Clonal evolution underscores the complexity of disease progression.

O- and N-Methyltransferases in benzylisoquinoline alkaloid producing plants.

BACKGROUND: Secondary metabolites such as benzylisoquinoline alkaloids (BIA) have attracted considerable attention because of their pharmacological properties and potential therapeutic applications. Methyltransferases (MTs) can add methyl groups to alkaloid molecules, altering their physicochemical properties and bioactivity, stability, solubility, and recognition by other cellular components. Five types of O-methyltransferases and two types of N-methyltransferases are involved in BIA biosynthesis. OBJECTIVE: Since MTs may be the source for the discovery and development of novel biomedical, agricultural, and industrial compounds, we performed extensive molecular and phylogenetic analyses of O- and N-methyltransferases in BIA-producing plants. METHODS: MTs involved in BIA biosynthesis were isolated from transcriptomes of Berberis koreana and Caulophyllum robustum. We also mined the methyltransferases of Coptis japonica, Papaver somniferum, and Nelumbo nucifera from the National Center for Biotechnology Information protein database. Then, we analyzed the functional motifs and phylogenetic analysis. RESULT: We mined 42 O-methyltransferases and 8 N-methyltransferases from the five BIA-producing plants. Functional motifs for S-adenosyl-L-methionine-dependent methyltransferases were retained in most methyltransferases, except for the three O-methyltransferases from N. nucifera. Phylogenetic analysis revealed that the methyltransferases were grouped into four clades, I, II, III and IV. The clustering patterns in the phylogenetic analysis suggested a monophyletic origin of methyltransferases and gene duplication within species. The coexistence of different O-methyltransferases in the deep branch subclade might support some cases of substrate promiscuity. CONCLUSIONS: Methyltransferases may be a source for the discovery and development of novel biomedical, agricultural, and industrial compounds. Our results contribute to further understanding of their structure and reaction mechanisms, which will require future functional studies.

Whole Blood Viscosity Reference Intervals and Its Correlation with Hematology and Serum Chemistry in Cats Using Scanning Capillary Method.

Whole blood viscosity, a hemorheological factor, is currently used for diagnosis, as it is correlated with various vascular diseases that are difficult to diagnose early with a general blood test. It was determined that it was necessary to set reference intervals for further studies and utilization of whole blood viscosity in cats, a representative companion animal, and this study was conducted. Fifty healthy cats were recruited for the study, and whole blood viscosity, complete blood count, and serum chemistry tests were performed. The reference intervals of whole blood viscosity were 15.169 to 43.684 cP at a shear rate of 1 s-1 reflecting diastole, and 3.524 to 5.544 cP at a shear rate of 300 s-1 reflecting systole. Red blood cells, hematocrit, hemoglobin, white blood cells, and neutrophils in the complete blood count, and total protein, albumin, globulin, and cholesterol in the serum chemistry were significantly correlated with whole blood viscosity. The results of this study set the reference intervals of whole blood viscosity for healthy cats in a wide shear rate range that has not yet been fully established, and its correlation with other blood indicators investigated.

Training-induced circuit-specific excitatory synaptogenesis in mice is required for effort control.

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC→DMS). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC→DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC→DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion.

In-situ δ18O and 87Sr/86Sr proxies in an unconformable clastic unit at the Ordovician-Silurian transition.

Clastic successions found in the carbonate platform of continental margin during the Ordovician-Silurian Transition (OST) period are archives for interpreting paleo-depositional systems. Here, we report in-situ δ18Oquartz and 87Sr/86Srcarbonate isotope chemo-stratigraphy for an unconformable clastic unit from the Cathaysia terrane that rifted off the Gondwana Supercontinent in the Early Paleozoic Era. Our results suggest a depositional proxy and model for geological events attributed to rapid changes in the sedimentary environment during the OST period. Importantly, these results present crucial clues that infer the influence of Paleo-Tethys Sea opening, global eustatic regression, and rapid sedimentary provenance change. Our study provides insight into paleo-tracer that could be a key method for interpreting depositional system of carbonate platform based on in-situ mineral isotope chemo-stratigraphy that preserves the original value of provenance and geochemical condition.

Comparison of patellofemoral contact pressure after semi-cylindrical recession trochleoplasty and trochlear block recession in feline cadavers.

Introduction: The purpose of this study was to compare the changes in the patellofemoral joint (PFJ) contact mechanisms of the normal state, trochlear hypoplasia model and after performing trochleoplasty on the hypoplasia model in feline cadavers. Methods: Twenty normal pelvic limbs were acquired from the 10 feline cadavers. First, the PFJ contact mechanisms were measured in normal state, then trochlear hypoplasia models were created using customized trochlear ridge cutting guides. After measuring PFJ contact mechanisms in the trochlear hypoplasia models, they were divided into two groups and performed semi-cylindrical recession trochleoplasty (SCRT) and trochlear block recession (TBR) were performed, respectively. After that, the PFJ contact mechanisms were measured and the values of the 4 groups (normal state, trochlear hypoplasia, SCRT, TBR) were compared. Results: The trochlear hypoplasia group showed increased contact pressure and decreased contact areas compared to the normal state group. In the groups that underwent tracheoplasty (SCRT and TBR), PFJ contact mechanisms were recovered similarly to that of the normal state group. The PFJ of the SCRP group was measured similar to that of the normal group than that of the TBR group. Discussion: Tracheoplasty can be useful in restoring PFJ contact mechanisms and SCRT can be considered as a good alternative to the conventional methods of trochleoplasty.

Application of topical rocuronium bromide dosing by ocular size in four species of wild birds.

BACKGROUND: Rocuronium bromide has been evaluated as a mydriatic agent in birds, but the species applied were limited and the dose and effect were variable. OBJECTIVE: This study aims to evaluate the efficacy of topical rocuronium bromide as mydriatics in 4 species according to horizontal palpebral fissure length: Feral pigeon (Columba livia), Common kestrel (Falco tinnunculus), Northern boobook (Ninox japonica), and Eurasian eagle owl (Bubo bubo). METHODS: A total of 32 birds (8 for each species) were included as pre-releasing examination. Rocuronium bromide was instilled in one randomly selected eye of each bird based on palpebral fissure length criteria (0.5 mg/50 µL for pigeons, 1 mg/100 µL for kestrels and boobook owls, and 2 mg/200 µL for eagle owls). The contralateral eye was used as control and treated with normal saline. After instillation of the drug, pupil diameter, pupillary light reflex, intraocular pressure, heart rate, and respiratory rate were evaluated at 10 min intervals up to 180 min and at 30 min intervals up to 360 min. RESULTS: Statistically significant mydriasis was obtained in all birds (p < 0.001). However, in boobook and eagle owls, marked mydriasis persisted until 360 min. Side effects including corneal erosion and lower eyelid paralysis were common, which was observed in 26/32 birds. Blepharospasm was also noted during this study. No systemic adverse signs were observed. CONCLUSIONS: Rocuronium bromide could be a good mydriatics option for 4 species of birds, however, further studies are needed to find lowest effective dose to reduce drug-related side effects.

The role of the parafascicular thalamic nucleus in action initiation and steering.

The parafascicular (Pf) nucleus of the thalamus has been implicated in arousal and attention, but its contributions to behavior remain poorly characterized. Here, using in vivo and in vitro electrophysiology, optogenetics, and 3D motion capture, we studied the role of the Pf nucleus in behavior using a continuous reward-tracking task in freely moving mice. We found that many Pf neurons precisely represent vector components of velocity, with a strong preference for ipsiversive movements. Their activity usually leads velocity, suggesting that Pf output is critical for self-initiated orienting behavior. To test this hypothesis, we expressed excitatory or inhibitory opsins in VGlut2+ Pf neurons to manipulate neural activity bidirectionally. We found that selective optogenetic stimulation of these neurons consistently produced ipsiversive head turning, whereas inhibition stopped turning and produced downward movements. Taken together, our results suggest that the Pf nucleus can send continuous top-down commands that specify detailed action parameters (e.g., direction and speed of the head), thus providing guidance for orienting and steering during behavior.

Ex Vivo Biomechanical Assessment of Various Repair Techniques in a Rabbit Calcaneal Tendon Avulsion Model: Application of Polycaprolactone Plate.

This study was aimed at evaluating the biomechanical properties and gapping characteristics of tendon repair methods using a combination of a three-loop pulley (3LP) pattern, a titanium plate, and a polycaprolactone (PCL) plate in a rabbit gastrocnemius tendon (GT) model (n = 50). GTs were randomly assigned to five groups (n = 10/group). Transected GTs were repaired with a 3LP pattern alone or in conjunction with an epitendinous suture (ES), a 5-hole 1 mm PCL plate, a 5-hole 2 mm PCL plate, or a 5-hole 1.5 mm titanium plate. The yield, peak, and failure force, as well as the occurrence and force of 1-mm and 3-mm gapping were examined. The mean yield, peak, and failure force of the 3LP + titanium plate group were higher than that of other groups. The biomechanical properties of a 3LP + a 2 mm PCL plate were similar to 3LP + ES constructs in this model. In all specimens in all groups, 1 mm gap formation was observed. The frequency of 3 mm gap formation was 70% and 90% in the 3LP + 2 mm PCL plate group and the 3LP + 1.5 mm titanium plate group, respectively. Additional studies evaluating PCL plates to determine the effect on the healing and blood supply of tendon are needed.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.