Cracking the DNA Code for V(D)J Recombination.

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Factors influencing psychological concerns about COVID-19 in South Korea: direct and indirect damage during the early stages of pandemic.

BACKGROUND: COVID-19 pandemic has led to psychological concerns, the distribution of which across populations may differ depending on whether pandemic-related damage is direct or indirect. This study aims to investigate concerns associated with direct and indirect damage according to population characteristics, and identify relatively vulnerable groups that are particularly affected by concerns. METHOD: This cross-sectional study used data from the 2020 Korea Community Health Survey, which collected data based on a complex sampling design. A total of 208,106 responses from individuals aged ≥ 19 were collected via in-person interviews. The items related to COVID-19 concerns were measured by Likert scales ranging from 1 to 5 and categorized into two types: direct concerns, which pertained to infection or death, and indirect concerns, which pertained to criticism, vulnerability, and economic damage, through factor analysis. We compared the means and effect size of direct concerns, indirect concerns, and overall concerns using weighted mean, ANOVA, and multiple regression analysis. RESULTS: Exploratory and confirmatory factor analyses supported a two-factor structure for psychological concerns about COVID-19 (CFI = 0.99, TLI = 0.97, SRMR = 0.02, RMSEA = 0.06), which were divided into direct and indirect concerns. Mean scores were 3.62 for direct concerns and 4.07 for indirect concerns. Direct concerns were higher in females (B = .26); the elderly (B = .15); those diagnosed with hypertension or diabetes (B = .04; B = .06); those with few assistants during quarantine (B = .15); and those whose neighbors responded inappropriately to COVID-19 (B = .07). Indirect concerns were lower among the elderly (B = -.04), and higher among young; married (B = .25); pink- or blue-collar workers (B = .08; B = .06); and those who felt that the city responded inappropriately to COVID-19 (B = .02). CONCLUSION: The prevalence of concerns regarding direct and indirect damage caused by the COVID-19 pandemic differed according to population characteristics. Some factors had a marked influence on direct and indirect concerns. Our findings could inform psychological interventions and policies for future pandemics. Customized interventions are needed to prevent negative psychological concerns and improve mental health.

Robust Long-Term Vehicle Trajectory Prediction Using Link Projection and a Situation-Aware Transformer.

The trajectory prediction of a vehicle emerges as a pivotal component in Intelligent Transportation Systems. On urban roads where external factors such as intersections and traffic control devices significantly affect driving patterns along with the driver's intrinsic habits, the prediction task becomes much more challenging. Furthermore, long-term forecasting of trajectories accumulates prediction errors, leading to substantially inaccurate predictions that may deviate from the actual road. As a solution to these challenges, we propose a long-term vehicle trajectory prediction method that is robust to error accumulation and prevents off-road predictions. In this study, the Transformer model is utilized to analyze and forecast vehicle trajectories. In addition, we propose an extra encoding network to precisely capture the effect of the external factors on the driving pattern by producing an abstract representation of the situation nearby the driver. To avoid off-road predictions, we propose a post-processing method, called link projection, which projects predictions onto the road geometry. Moreover, to overcome the limitations of Euclidean distance-based evaluation metrics in evaluating the accuracy of the entire trajectory, we propose a new metric called area-between-curves (ABC). It measures the similarity between two trajectories, and thus the accordance between the two can be effectively evaluated. Extensive evaluations are conducted using real-world datasets against widely-used methods to demonstrate the effectiveness of the proposed approach. The results show that the proposed approach outperforms the conventional deep learning models by up to 65.74% (RMSE), 60.13% (MAE) and 91.45% (ABC).

Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities.

Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.

TARGET OF RAPAMYCIN is essential for asexual vegetative reproduction in Kalanchoë.

The unique mechanism by which leaf margin cells regain potency and then form a plantlet in Kalanchoë spp. remains elusive but involves organogenesis and embryogenesis in response to age, day length, nutrient availability, and drought stress. In light of this, we investigated whether TARGET OF RAPAMYCIN (TOR), a conserved protein kinase in eukaryotes that controls cell growth and metabolism in response to nutrient and energy availability, may regulate plantlet formation. Kalanchoë daigremontiana TOR (KdTOR) was expressed in the leaf margin at the site of plantlet initiation, in the early plantlet cotyledons, and in the root tip of the developed plantlet. Both chemical and genetic inhibition of TOR Kinase activity in Kalanchoë daigremontiana leaves disrupted plantlet formation. Furthermore, downregulation of KdTOR in transgenic plants led to wide-ranging transcriptional changes, including decreased K. daigremontiana SHOOTMERISTEMLESS and K. daigremontiana LEAFYCOTYLEDON1 expression, whereas auxin treatments induced KdTOR expression in the plantlet roots. These results suggest that the KdTOR pathway controls plantlet development in cooperation with auxin, organogenesis, and embryogenesis pathways. The ancient and highly conserved TOR Kinase therefore controls diverse and unique developmental pathways, such as asexual reproduction within the land plant lineage.

Fully Automated MRI Segmentation and Volumetric Measurement of Intracranial Meningioma Using Deep Learning.

BACKGROUND: Accurate and rapid measurement of the MRI volume of meningiomas is essential in clinical practice to determine the growth rate of the tumor. Imperfect automation and disappointing performance for small meningiomas of previous automated volumetric tools limit their use in routine clinical practice. PURPOSE: To develop and validate a computational model for fully automated meningioma segmentation and volume measurement on contrast-enhanced MRI scans using deep learning. STUDY TYPE: Retrospective. POPULATION: A total of 659 intracranial meningioma patients (median age, 59.0 years; interquartile range: 53.0-66.0 years) including 554 women and 105 men. FIELD STRENGTH/SEQUENCE: The 1.0 T, 1.5 T, and 3.0 T; three-dimensional, T1 -weighted gradient-echo imaging with contrast enhancement. ASSESSMENT: The tumors were manually segmented by two neurosurgeons, H.K. and C.-K.P., with 10 and 26 years of clinical experience, respectively, for use as the ground truth. Deep learning models based on U-Net and nnU-Net were trained using 459 subjects and tested for 100 patients from a single institution (internal validation set [IVS]) and 100 patients from other 24 institutions (external validation set [EVS]), respectively. The performance of each model was evaluated with the Sørensen-Dice similarity coefficient (DSC) compared with the ground truth. STATISTICAL TESTS: According to the normality of the data distribution verified by the Shapiro-Wilk test, variables with three or more categories were compared by the Kruskal-Wallis test with Dunn's post hoc analysis. RESULTS: A two-dimensional (2D) nnU-Net showed the highest median DSCs of 0.922 and 0.893 for the IVS and EVS, respectively. The nnU-Nets achieved superior performance in meningioma segmentation than the U-Nets. The DSCs of the 2D nnU-Net for small meningiomas less than 1 cm3 were 0.769 and 0.780 with the IVS and EVS, respectively. DATA CONCLUSION: A fully automated and accurate volumetric measurement tool for meningioma with clinically applicable performance for small meningioma using nnU-Net was developed. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 2.

Oncologic relevance of genetic alterations in sporadic synchronous and solitary colorectal cancer: a retrospective multicenter study.

BACKGROUND: Oncologic impact of genetic alteration across synchronous colorectal cancer (CRC) still remains unclear. This study aimed to compare the oncologic relevance according to genetic alteration between synchronous and solitary CRC with performing systematic review. METHODS: Multicenter retrospective analysis was performed for CRC patients with curative resection. Genetic profiling was consisted of microsatellite instability (MSI) testing, RAS (K-ras, and N-ras), and BRAF (v-Raf murine sarcoma viral oncogene homolog B1) V600E mutation. Multivariate analyses were conducted using logistic regression for synchronicity, and Cox proportional hazard model with stage-adjusting for overall survival (OS) and disease-free survival (DFS). RESULTS: It was identified synchronous (n = 36) and solitary (n = 579) CRC with similar base line characteristics. RAS mutation was associated to synchronous CRC with no relations of MSI and BRAF. During median follow up of 77.8 month, Kaplan-meier curves showed significant differences according to MSI-high for OS, and in RAS, and BRAF mutation for DFS, respectively. In multivariable analyses, RAS and BRAF mutation were independent factors (RAS, HR = 1.808, 95% CI = 1.18-2.77, p = 0.007; BRAF, HR = 2.417, 95% CI = 1.32-4.41, p = 0.004). Old age was independent factor for OS (HR = 3.626, 95% CI = 1.09-12.00, p = 0.035). CONCLUSION: This study showed that oncologic outcomes might differ according to mutation burden characterized by RAS, BRAF, and MSI between synchronous CRC and solitary CRC. In addition, our systematic review highlighted a lack of data and much heterogeneity in genetic characteristics and survival outcomes of synchronous CRC relative to that of solitary CRC.

Facile Achievement of Complementary Resistive Switching in Block Copolymer Micelle-Based Resistive Memories.

Interest in resistive random access memory (RRAM) has grown rapidly in recent years for realizing ultrahigh density data storage devices. However, sneak currents in these devices can result in misreading of the data, thus limiting the applicability of RRAM. Complementary resistive switching (CRS) memory consisting of two antiserial RRAMs can considerably reduce sneak currents; however, complicated device architectures and manufacturing processes still remain as challenges. Herein, an effective and simple approach for fabricating CRS memory devices using self-assembled block copolymer micelles is reported. Cu ions are selectively placed in the core of polystyrene-block-poly(2-vinylpyridine) spherical micelles, and a hexagonally packed micelle monolayer is prepared through spin-coating. The micelle monolayer can be a symmetrical resistive switching layer, because the micelles and Cu act as dielectric and active metals in memory devices, respectively. The locally enhanced electric field and Joule heating achieved by the structured Cu atoms inside the micelles promote metal ionization and ion migration in a controlled manner, thus allowing for position selectivity during resistive switching. The micelle-based memory device exhibits stable and reliable CRS behavior, with a nonoverlapping and narrow distribution of threshold voltages. Therefore, this approach is promising for fabricating CRS memory devices for high-performance and ultrahigh-density RRAM applications.

Safety of endoscopic endonasal biopsy for the pituitary stalk-hypothalamic lesions.

PURPOSE: The indications for and the optimal biopsy approach in pituitary stalk-hypothalamic (PsH) lesions are controversial. Biopsies through an endoscopic endonasal approach (EEA) for PsH lesions have often been considered to cause the infundibulo-tuberal syndrome. The purpose of this study was to analyze the surgical and endocrinological safety of EEA biopsies for PsH lesions. METHODS: A total of 39 consecutive patients who underwent an EEA biopsy between June 2011 and August 2020 in a single institute were retrospectively analyzed. The ophthalmological and endocrinological outcomes were assessed before and after surgery. RESULTS: PsH lesions were confirmed to be diverse pathological diagnoses, ranging from lymphocytic hypophysitis to diffuse midline glioma, and the most common pathologic diagnosis was a germinoma (18 patients, 46.2%). No patients developed visual deterioration after the biopsy. In patients without preoperative panhypopituitarism, 13 out of 28 patients (46.4%) developed new anterior pituitary hormonal deficiencies after the biopsy. When the tissue was collected from the stalk, the endocrinological deterioration rate was 100% (6 of 6 patients), while the rate was 31.8% (7 of 22 patients) when tissue could be harvested from an extra-stalk lesion. The rate of newly developed permanent diabetes insipidus after surgery was 40.9% (9 of 22 patients). The median surgery time was 125 min, and there was no postoperative CSF leakage or infections noted. CONCLUSIONS: An EEA biopsy for PsH lesions is a safe and efficient surgical method unless the tissue is collected from the stalk.

Patient-Derived Tumor Organoids for Guidance of Personalized Drug Therapies in Recurrent Glioblastoma.

An obstacle to effective uniform treatment of glioblastoma, especially at recurrence, is genetic and cellular intertumoral heterogeneity. Hence, personalized strategies are necessary, as are means to stratify potential targeted therapies in a clinically relevant timeframe. Functional profiling of drug candidates against patient-derived glioblastoma organoids (PD-GBO) holds promise as an empirical method to preclinically discover potentially effective treatments of individual tumors. Here, we describe our establishment of a PD-GBO-based functional profiling platform and the results of its application to four patient tumors. We show that our PD-GBO model system preserves key features of individual patient glioblastomas in vivo. As proof of concept, we tested a panel of 41 FDA-approved drugs and were able to identify potential treatment options for three out of four patients; the turnaround from tumor resection to discovery of treatment option was 13, 14, and 15 days, respectively. These results demonstrate that this approach is a complement and, potentially, an alternative to current molecular profiling efforts in the pursuit of effective personalized treatment discovery in a clinically relevant time period. Furthermore, these results warrant the use of PD-GBO platforms for preclinical identification of new drugs against defined morphological glioblastoma features.

Absolute quantification of tumor-infiltrating immune cells in high-grade glioma identifies prognostic and radiomics values.

PURPOSE: To understand the tumor immune microenvironment precisely, it is important to secure the quantified data of tumor-infiltrating immune cells, since the immune cells are true working unit. We analyzed unit immune cell number per unit volume of core tumor tissue of high-grade gliomas (HGG) to correlate their immune microenvironment characteristics with clinical prognosis and radiomic signatures. METHODS: The number of tumor-infiltrating immune cells from 64 HGG core tissue were analyzed using flow cytometry and standardized. After sorting out patient groups according to diverse immune characteristics, the groups were tested if they have any clinical prognostic relevance and specific radiomic signature relationships. Sparse partial least square with discriminant analysis using multimodal magnetic resonance images was employed for all radiomic classifications. RESULTS: The median number of CD45 + cells per one gram of HGG core tissue counted 865,770 cells which was equivalent to 8.0% of total cells including tumor cells. There was heterogeneity in the distribution of immune cell subpopulations among patients. Overall survival was significantly better in T cell-deficient group than T cell-enriched group (p = 0.019), and T8 dominant group than T4 dominant group (p = 0.023). The number of tumor-associated macrophages (TAM) and M2-TAM was significantly decreased in isocitrate dehydrogenase mutated HGG. Radiomic signature classification showed good performance in predicting immune phenotypes especially with features extracted from apparent diffusion coefficient maps. CONCLUSIONS: Absolute quantification of tumor-infiltrating immune cells confirmed the heterogeneity of immune microenvironment in HGG which harbors prognostic impact. This immune microenvironment could be predicted by radiomic signatures non-invasively.

In vitro osteogenic activities of sulfated derivative of polysaccharide extracted from Tamarindus indica L.

Osteoporosis, one of the most serious public health concerns caused by an imbalance between bone resorption and bone formation, has a major impact on the population. Therefore, finding the effective osteogenic compounds for the treatment of osteoporosis is a promising research approach. In our study, tamarind (Tamarindus indica L.) seed polysaccharide (TSP) extracted from tamarind seed was subjected to synthesize its sulfate derivatives. The 1H NMR, FT-IR, SEM, monosaccharide compositions and elemental analysis data revealed that tamarind seed polysaccharide sulfate (TSPS) was successfully prepared. As the result, TSPS showed potent effects on inducing osteoblast differentiation via increasing alkaline phosphatase (ALP) activity up to 20% after 10 days and bone mineralization approximately 58% after four weeks at concentration of 20 µg/mL, whereas no statistically increase for both ALP activity and bone mineralization was observed in TSP treatment. Furthermore, TSPS enhanced expression of several marker genes in bone formation. Overall, the obtained data provided novelty on osteogenic compounds originated from TSP of T. indica, as well as scientific fundamentals on drug development and bone tissue engineering for the treatment of osteoporosis and other bone-related diseases.

Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens.

ABCG subfamily proteins are highly enriched in terrestrial plants. Many of these proteins secrete secondary metabolites that repel or inhibit pathogens. To establish why the ABCG subfamily proteins proliferated extensively during evolution, we constructed phylogenetic trees from a broad range of eukaryotic organisms. ABCG proteins were massively duplicated in land plants and in oomycetes, a group of agronomically important plant pathogens, which prompted us to hypothesize that plant and pathogen ABCGs coevolved. Supporting this hypothesis, full-size ABCGs in host plants (Arabidopsis thaliana and Glycine max) and their pathogens (Hyaloperonospora arabidopsidis and Phytophthora sojae, respectively) had similar divergence times and patterns. Furthermore, generalist pathogens with broad ranges of host plants have diversified more ABCGs than their specialist counterparts. The hypothesis was further tested using an example pair of ABCGs that first diverged during multiplication in a host plant and its pathogen: AtABCG31 of A. thaliana and HpaP802307 of H. arabidopsidis. AtABCG31 expression was activated following infection with H. arabidopsidis, and disrupting AtABCG31 led to increased susceptibility to H. arabidopsidis. Together, our results suggest that ABCG genes in plants and their oomycete pathogens coevolved in an arms race, to extrude secondary metabolites involved in the plant's defense response against pathogens.

Crystal structure of the V(D)J recombinase RAG1-RAG2.

V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 Å resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.

Characteristics and Prognosis of Colorectal Cancer after Liver or Kidney Transplantation.

BACKGROUND: The purpose of this study was to evaluate the characteristics and prognosis of de novo CRC patients who underwent liver or kidney transplantation. METHODS: We retrospectively reviewed the medical records of 66 de novo CRC patients selected from 8,734 liver transplant (LT) or kidney transplant (KT) recipients. We analyzed characteristics and survival outcomes of de novo CRC patients and sporadic CRC patients who underwent radical surgery with stage I-III in Asan Medical Center between 2005 and 2016. Survival outcomes were analyzed via the 1:4 matching method. RESULTS: The standard incidence ratio (SIR) of de novo CRC in KT recipients is 1.67 in men and 2.54 in women. That in LT recipients is 3.10 in men and 2.25 in women. Compared with sporadic CRC patients, de novo CRC patients had more colon cancer than rectal cancer (p=0.041). In 9 patients (13.6%), CRC was diagnosed within one year after transplantation, 21 patients (31.8%) were diagnosed between 1-5 years, and the remaining 36 patients (54.6%) were diagnosed thereafter. There were no significant differences in recurrence-free survival and overall survival between the two groups (p=0.211 and p=0.324, respectively). CONCLUSIONS: The risk of developing de novo CRC in transplant recipients was higher than in the general population. The survival outcome of de novo CRC was no different compared with the sporadic CRC. Therefore, regular surveillance is essential for timely diagnosis and treatment for transplantation patients. A large prospective study for an intense CRC surveillance program in transplantation patients is needed.

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid.

HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.

Development of an inside-out augmented reality technique for neurosurgical navigation.

OBJECTIVE: With the advancement of 3D modeling techniques and visualization devices, augmented reality (AR)-based navigation (AR navigation) is being developed actively. The authors developed a pilot model of their newly developed inside-out tracking AR navigation system. METHODS: The inside-out AR navigation technique was developed based on the visual inertial odometry (VIO) algorithm. The Quick Response (QR) marker was created and used for the image feature-detection algorithm. Inside-out AR navigation works through the steps of visualization device recognition, marker recognition, AR implementation, and registration within the running environment. A virtual 3D patient model for AR rendering and a 3D-printed patient model for validating registration accuracy were created. Inside-out tracking was used for the registration. The registration accuracy was validated by using intuitive, visualization, and quantitative methods for identifying coordinates by matching errors. Fine-tuning and opacity-adjustment functions were developed. RESULTS: ARKit-based inside-out AR navigation was developed. The fiducial marker of the AR model and those of the 3D-printed patient model were correctly overlapped at all locations without errors. The tumor and anatomical structures of AR navigation and the tumors and structures placed in the intracranial space of the 3D-printed patient model precisely overlapped. The registration accuracy was quantified using coordinates, and the average moving errors of the x-axis and y-axis were 0.52 ± 0.35 and 0.05 ± 0.16 mm, respectively. The gradients from the x-axis and y-axis were 0.35° and 1.02°, respectively. Application of the fine-tuning and opacity-adjustment functions was proven by the videos. CONCLUSIONS: The authors developed a novel inside-out tracking-based AR navigation system and validated its registration accuracy. This technical system could be applied in the novel navigation system for patient-specific neurosurgery.

CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae.

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4-6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

Re"CYC"ling molecular regulators in the evolution and development of flower symmetry.

Flower forms are both highly diverse and multifaceted. As well as varying in colour, size, organ number, and much more, flowers show different types of symmetry. Floral symmetry can be grouped into three main categories: asymmetry, bilateral symmetry and radial symmetry, characterised by zero, one, and multiple planes of symmetry, respectively. This review will first explore floral symmetry from a classical morphological view, then from a modern molecular perspective. The recent molecular work on symmetry in monocots and eudicots will be discussed, followed by an in-depth discussion into the evolution of CYC genes, particularly in the capitulum of the sunflower family (Asteraceae). Whilst recent studies on non-model species are helping to bring new light to this field, more species coverage is required to understand how traits such as bilateral symmetry have evolved so many times, and whether the same molecular regulators were recruited for this function.