A comprehensive review on the recent advances for 5-aminolevulinic acid production by the engineered bacteria.

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward.

Melatonin mitigates PNMC-induced disruption of spindle assembly and mitochondrial function in mouse Oocytes.

3-methyl-4-nitrophenol (PNMC), a degradation product of organophosphorus insecticides and a byproduct of fuel combustion, exerting endocrine-disrupting effects. However, its impact on the meiotic process of oocytes remains unclear. In the present study, we investigated the effects of PNMC on meiotic maturation of mouse oocytes in vitro and related mechanisms. Morphologically, PNMC-exposure affected germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Proteomic analysis suggested that PNMC-exposure altered oocyte protein expression that are associated with cytoskeleton, mitochondrial function and oxidative stress. Further studies demonstrated that PNMC-exposure disrupted spindle assembly and chromosome alignment, caused sustained activation of spindle assembly checkpoint (SAC), and arrested meiosis in oocytes. Specifically, PNMC-exposure interfered with the function of microtubule organizing centers (MTOCs) by significantly reducing phosphorylated mitogen activated protein kinase (p-MAPK) expression and disrupting the localization of Pericentrin and p-Aurora A, leading to spindle assembly failure. Besides, PNMC-exposure also increased α-tubulin acetylation, decreased microtubule stability. Moreover, PNMC-exposure impaired mitochondrial function, evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, release of Cytochrome C into the cytoplasm, and elevated ROS levels. As a result, exposure to PNMC caused DNA damage and early apoptosis in oocytes. Fortunately, melatonin was able to promote oocyte maturation by removing the excessive ROS and enhancing mitochondrial function. These results highlight the adverse effects of PNMC on meiotic maturation, and underscore the protective role of melatonin against PNMC-induced damage.

PD-L1 dimerisation induced by biphenyl derivatives mediates anti-breast cancer activity via the non-immune PD-L1-AKT-mTOR/Bcl2 pathway.

Recent studies on biphenyl-containing compounds, a type of PD-1/PD-L1 blocker which binds to PD-L1 and induces dimerisation, have focussed on its immune function. Herein, 10 novel biphenyl derivatives were designed and synthesised. The results of the CCK-8 showed that compounds have different anti-tumour activities for tumour cells in the absence of T cells. Particularly, 12j-4 can significantly induce the apoptosis of MDA-MB-231 cells (IC50 = 2.68 ± 0.27 µM). In further studies, 12j-4 has been shown to prevent the phosphorylation of AKT by binding to cytoplasmic PD-L1, which induces apoptosis in MDA-MB-231 cells through non-immune pathways. The inhibition of AKT phosphorylation restores the activity of GSK-3ß, ultimately resulting in the degradation of PD-L1. Besides, in vivo study indicated that 12j-4 repressed tumour growth in nude mice. As these biphenyls exert their anti-tumour effects mainly through non-immune pathways, they are worthy of further study as PD-L1 inhibitors.

Bioactive dipeptides enhance the tolerance of lager yeast to ethanol-oxidation cross-stress by regulating the multilevel defense system.

Although high gravity brewing technology has been widely used for beer industries due to its economic benefits, yeast cells are subjected to multiple environmental stresses throughout the fermentation process. Eleven bioactive dipeptides (LH, HH, AY, LY, IY, AH, PW, TY, HL, VY, FC) were selected to evaluate their effects on cell proliferation, cell membrane defense system, antioxidant defense system and intracellular protective agents of lager yeast against ethanol-oxidation cross-stress. Results showed that the multiple stresses tolerance and fermentation performance of lager yeast were enhanced by bioactive dipeptides. Cell membrane integrity was improved by bioactive dipeptides through altering the structure of macromolecular compounds of the cell membrane. Intracellular reactive oxygen species (ROS) accumulation was significantly decreased by bioactive dipeptides, especially for FC, decreasing by 33.1%, compared with the control. The decrease of ROS was closely related to the increase of mitochondrial membrane potential, intracellular antioxidant enzyme activities including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and glycerol level. In addition, bioactive dipeptides could regulate the expression of key genes (GPD1, OLE1, SOD2, PEX11, CTT1, HSP12) to enhance the multilevel defense systems under ethanol-oxidation cross-stress. Therefore, bioactive dipeptides should be potentially efficient and feasible bioactive ingredients to improve the multiple stresses tolerance of lager yeast during high gravity fermentation.

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

Biochemical and molecular characterization of a novel porphobilinogen synthase from Corynebacterium glutamicum.

Corynebacterium glutamicum porphobilinogen synthase (PBGS) is a metal enzyme with a hybrid active site metal binding sequence. In this study, the porphobilinogen synthase gene of C. glutamicum was cloned and heterogeneously expressed in Escherichia coli. C. glutamicum PBGS was purified, and its enzymatic characteristics were analyzed. The results showed that C. glutamicum PBGS is a Zn2+-dependent enzyme, and Mg2+ has allosteric regulation. The allosteric Mg2+ plays a vital role in forming the quaternary structure of C. glutamicum PBGS. Based on the ab initio predictive structure modeling of the enzyme and the molecular docking model of 5-aminolevulinic acid (5-ALA), 11 sites were selected for site-directed mutagenesis. When the hybrid active site metal binding site of C. glutamicum PBGS is converted into a cysteine-rich motif (Zn2+-dependent) or an aspartic acid-rich motif (Mg2+/K+-dependent), the enzyme activity is basically lost. Four residues, D128, C130, D132, and C140, in the metal binding site, were the binding sites of Zn2+ and the active center of the enzyme. The band migration, from the native PAGE, of five variants with mutations in the center of enzyme activity was the same as that of the variant enzymes as purified, individually adding two metal ion chelating agents. Their Zn2+ active center structures were abnormal, and the quaternary structure equilibrium was altered. The destroyed active center affects the construction of its quaternary structure. The quaternary structural balance between octamer and hexamer through dimers was regulated by the allosteric regulation of C. glutamicum PBGS. The enzyme activity was also affected by the change of the active site lid structure and (α ß)8-barrel structure caused by mutation. Structural changes in the variants were analyzed to understand C. glutamicum PBGS better.

Fine-tuning OsCPK18/OsCPK4 activity via genome editing of phosphorylation motif improves rice yield and immunity.

Plants have evolved complex signalling networks to regulate growth and defence responses under an ever-changing environment. However, the molecular mechanisms underlying the growth-defence tradeoff are largely unclear. We previously reported that rice CALCIUM-DEPENDENT PROTEIN KINASE 18 (OsCPK18) and MITOGEN-ACTIVATED PROTEIN KINASE 5 (OsMPK5) mutually phosphorylate each other and that OsCPK18 phosphorylates and positively regulates OsMPK5 to suppress rice immunity. In this study, we found that OsCPK18 and its paralog OsCPK4 positively regulate plant height and yield-related traits. Further analysis reveals that OsCPK18 and OsMPK5 synergistically regulate defence-related genes but differentially regulate development-related genes. In vitro and in vivo kinase assays demonstrated that OsMPK5 phosphorylates C-terminal threonine (T505) and serine (S512) residues of OsCPK18 and OsCPK4, respectively. The kinase activity of OsCPK18T505D , in which T505 was replaced by aspartic acid to mimic T505 phosphorylation, displayed less calcium sensitivity than that of wild-type OsCPK18. Interestingly, editing the MAPK phosphorylation motif in OsCPK18 and its paralog OsCPK4, which deprives OsMPK5-mediated phosphorylation but retains calcium-dependent activation of kinase activity, simultaneously increases rice yields and immunity. This editing event also changed the last seven amino acid residues of OsCPK18 and attenuated its binding with OsMPK5. This study presents a new regulatory circuit that fine tunes the growth-defence tradeoff by modulating OsCPK18/4 activity and suggests that CRISPR/Cas9-mediated engineering phosphorylation pathways could simultaneously improve crop yield and immunity.

A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants.

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.

Functional reorganization of brain regions into a network in childhood absence epilepsy: A magnetoencephalography study.

OBJECTIVE: Epilepsy is considered as a network disorder. However, it is unknown how normal brain activity develops into the highly synchronized discharging activity seen in disordered networks. This study aimed to explore the epilepsy brain network and the significant re-combined brain areas in childhood absence epilepsy (CAE). METHODS: Twenty-two children with CAE were recruited to study the neural source activity during ictal-onset and interictal periods at frequency bands of 1-30 Hz and 30-80 Hz with magnetoencephalography (MEG) scanning. Accumulated source imaging (ASI) was used to analyze the locations of neural source activity and peak source strength. RESULTS: Most of the participants had more active source activity locations in the ictal-onset period rather than in the interictal period, both at 1-30 Hz and 30-80 Hz. The frontal lobe (FL), the temporo-parietal junction (T-P), and the parietal lobe (PL) became the main active areas of source activity during the ictal period, while the precuneus (PC), cuneus, and thalamus were relatively inactive. CONCLUSIONS: Some brain areas become more excited and have increased source activity during seizures. These significant brain regions might be re-combined to form an epilepsy network that regulates the process of absence seizures. SIGNIFICANCE: The study confirmed that important brain regions are reorganized in an epilepsy network, which provides a basis for exploring the network mechanism of CAE development. Imaging findings may provide a reference for clinical characteristics.

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm.

INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Quantitative Dynamic Thoracic MRI: Application to Thoracic Insufficiency Syndrome in Pediatric Patients.

Background Available methods to quantify regional dynamic thoracic function in thoracic insufficiency syndrome (TIS) are limited. Purpose To evaluate the use of quantitative dynamic MRI to depict changes in regional dynamic thoracic function before and after surgical correction of TIS. Materials and Methods Images from free-breathing dynamic MRI in pediatric patients with TIS (July 2009-August 2015) were retrospectively evaluated before and after surgical correction by using vertical expandable prosthetic titanium rib (VEPTR). Eleven volumetric parameters were derived from lung, chest wall, and diaphragm segmentations, and parameter changes before versus after operation were correlated with changes in clinical parameters. Paired analysis from Student t test on MRI parameters and clinical parameters was performed to detect if changes (from preoperative to postoperative condition) were statistically significant. Results Left and right lung volumes at end inspiration and end expiration increased substantially after operation in pediatric patients with thoracic insufficiency syndrome, especially right lung volume with 22.9% and 26.3% volume increase at end expiration (P = .001) and end inspiration (P = .002), respectively. The average lung tidal volumes increased after operation for TIS; there was a 43.8% and 55.3% increase for left lung tidal volume and right lung tidal volume (P < .001 for both), respectively. However, clinical parameters did not show significant changes from pre- to posttreatment states. Thoracic and lumbar Cobb angle were poor predictors of MRI tidal volumes (chest wall, diaphragm, and left and right separately), but assisted ventilation rating and forced vital capacity showed moderate correlations with tidal volumes (chest wall, diaphragm, and left and right separately). Conclusion Vertical expandable prosthetic titanium rib operation was associated with postoperative increases in all components of tidal volume (left and right chest wall and diaphragm, and left and right lung tidal volumes) measured at MRI. Clinical parameters did not demonstrate improvements in postoperative tidal volumes. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Paltiel in this issue.

Ictal Source Locations and Cortico-Thalamic Connectivity in Childhood Absence Epilepsy: Associations with Treatment Response.

Childhood absence epilepsy (CAE), the most common pediatric epilepsy syndrome, is usually treated with valproic acid (VPA) and lamotrigine (LTG) in China. This study aimed to investigate the ictal source locations and functional connectivity (FC) networks between the cortices and thalamus that are related to treatment response. Magnetoencephalography (MEG) data from 25 patients with CAE were recorded at 300 Hz and analyzed in 1-30 Hz frequency bands. Neuromagnetic sources were volumetrically scanned with accumulated source imaging. The FC networks between the cortices and thalamus were evaluated at the source level through a connectivity analysis. Treatment outcome was assessed after 36-66 months following MEG recording. The children with CAE were divided into LTG responder, LTG non-responder, VPA responder and VPA non-responder groups. The ictal source locations and cortico-thalamic FC networks were compared to the treatment response. The ictal source locations in the post-dorsal medial frontal cortex (post-DMFC, including the medial primary motor cortex and the supplementary sensorimotor area) were observed in all LTG non-responders but in all LTG responders. At 1-7 Hz, patients with fronto-thalamo-parietal/occipital (F-T-P/O) networks were older than those with fronto-thalamic (F-T) networks or other cortico-thalamic networks (p = 0.000). The duration of seizures in patients with F-T-P/O networks at 1-7 Hz was longer than that in patients with F-T networks or other cortico-thalamic networks (p = 0.001). The ictal post-DMFC source localizations suggest that children with CAE might experience initial LTG monotherapy failure. Moreover, the cortico-thalamo-cortical network is associated with age. Finally, the cortico-thalamo-cortical network consists of anterior and posterior cortices and might contribute to the maintenance of discharges.

Altered Effective Connectivity Network in Childhood Absence Epilepsy: A Multi-frequency MEG Study.

Using multi-frequency magnetoencephalography (MEG) data, we investigated whether the effective connectivity (EC) network of patients with childhood absence epilepsy (CAE) is altered during the inter-ictal period in comparison with healthy controls. MEG data from 13 untreated CAE patients and 10 healthy controls were recorded. Correlation analysis and Granger causality analysis were used to construct an EC network at the source level in eight frequency bands. Alterations in the spatial pattern and topology of the network in CAE were investigated by comparing the patients with the controls. The network pattern was altered mainly in 1-4 Hz, showing strong connections within the frontal cortex and weak connections in the anterior-posterior pathways. The EC involving the precuneus/posterior cingulate cortex (PC/PCC) significantly decreased in low-frequency bands. In addition, the parameters of graph theory were significantly altered in several low- and high-frequency bands. CAE patients display frequency-specific abnormalities in the network pattern even during the inter-ictal period, and the frontal cortex and PC/PCC might play crucial roles in the pathophysiology of CAE. The EC network of CAE patients was over-connective and random during the inter-ictal period. This study is the first to reveal the frequency-specific alteration in the EC network during the inter-ictal period in CAE patients. Multiple-frequency MEG data are useful in investigating the pathophysiology of CAE, which can serve as new biomarkers of this disorder.

Decreased XY recombination and disturbed meiotic prophase I progression in an infertile 48, XYY, +sSMC man.

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal rare chromosomes, difficult to characterize by karyotyping, and have been associated with minor dysmorphic features, azoospermia, and recurrent miscarriages. However, sSMC with a gonosomal trisomy has never been reported. Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis, and recombination. In all the analyzed spermatocytes of the patient, the extra Y chromosome was not detected while the sSMC was present. The recombination frequency on autosomes was not affected, while the recombination frequencies on XY chromosome was significantly lower in the patient than in the controls. The meiotic prophase I progression was disturbed with significantly increased proportion of zygotene and decreased pachytene spermatocytes in the patients as compared with the controls. These findings highlight the importance of studies on meiotic behaviors in patients with an abnormal chromosomal constitution and provide an important framework for future studies, which may elucidate the impairment caused by sSMC in mammalian meiosis and fertility.

Microfluidic fabrication of core-shell fucoxanthin nanofibers with improved environmental stability for reducing lipid accumulation in vitro.

Fucoxanthin is a xanthophyll carotenoid that possesses potent antioxidant, anti-obesity, and anti-tumor properties. However, its limited solubility in water and susceptibility to degradation create challenges for its application. In this study, a microfluidic coaxial electrospinning technique was used to produce core-shell zein-gelatin nanofibers for encapsulating fucoxanthin, enhancing its bioavailability, and improving its stability. In comparison to uniaxially-loaded fucoxanthin nanofibers, the encapsulation efficiency of fucoxanthin reached 98.58 % at a core-shell flow rate ratio of 0.26:1, representing a 14.29 % improvement. The photostability of the nanofibers increased by 74.59 % after three days, UV stability increased by 38.82 % after 2 h, and temperature stability also significantly improved, demonstrating a protective effect under harsh environmental conditions (P < 0.05). Additionally, nanofibers effectively alleviated oleic acid-induced reactive oxygen species production and reduced fluorescence intensity by 54.76 %. MTT experiments indicated great biocompatibility of the nanofibers, effectively mitigating mitochondrial membrane potential polarization and lipid accumulation in HepG2 cells. Overall, the microfluidic coaxial electrospinning technique enables promising applications of fucoxanthin delivery in the food industry.

The gelling properties of fish gelatin as improved by ultrasound-assisted phosphorylation.

This study investigated the effects of ultrasound-assisted phosphorylation on gelling properties of fish gelatin (FG). Ultrasound-assisted phosphorylation (UP) for 60, 90, and 120 min resulted in >6.54% increase of phosphorylation degree and decreased zeta potential of FG. Atomic force microscopy revealed that UP-FGs showed larger aggregates than P-FGs (normal phosphorylation FGs). Low frequent-NMR and microstructure analysis revealed that phosphorylation enhanced water-binding capability of FG and improved the gel networks. However, UP60 had the highest gel strength (340 g), gelling (17.96 °C) and melting (26.54 °C) temperature while UP90 and UP120 showed slightly lower of them. FTIR analysis indicated thatß-sheet and triple helix content increased but random coil content decreased in phosphorylated FGs. Mass spectrometry demonstrated phosphate groups mainly bound to serine, threonine and tyrosine residues of FG and UP-FG exhibited more phosphorylation sites. The study showed that mild phosphorylation (UP60) could be applied to improve FG gel properties.

Sirtuin 5-driven meiotic spindle assembly and actin-based migration in mouse oocyte meiosis.

Sirtuin 5 (Sirt5), a member of the Sirtuin family, is involved in various intracellular biological processes. However, the function of Sirt5 in oocyte maturation has not been clearly elucidated. In this study, we observed that Sirt5 was persistently expressed during the meiotic division of mouse oocytes, with a notable decline in expression in aging oocytes. Sirt5 inhibition led to the failure of the first polar body extrusion and induced cell cycle arrest, indicative of unsuccessful oocyte maturation. Furthermore, Sirt5 inhibition was associated with the extrusion of abnormally large polar bodies, suggesting disrupted asymmetric oocyte division. Mechanistically, the inhibition of Sirt5 resulted in aberrant spindle assembly and disordered chromosome alignment in oocytes. Moreover, Sirt5 inhibition caused the spindle to be centrally located in the oocyte without migrating to the cortical region, consequently preventing the formation of the actin cap. Further investigation revealed that Sirt5 inhibition notably diminished the expression of phosphorylated cofilin and profilin1, while increasing cytoplasmic F-actin levels. These findings suggest that Sirt5 inhibition during oocyte maturation adversely affects spindle assembly and chromosome alignment and disrupts actin dynamics impairing spindle migration and contributing to the failure of symmetric oocyte division and maturation.

Optimal strategies for modeling anatomy in a hybrid intelligence framework for auto-segmentation of organs.

Organ segmentation is a fundamental requirement in medical image analysis. Many methods have been proposed over the past 6 decades for segmentation. A unique feature of medical images is the anatomical information hidden within the image itself. To bring natural intelligence (NI) in the form of anatomical information accumulated over centuries into deep learning (DL) AI methods effectively, we have recently introduced the idea of hybrid intelligence (HI) that combines NI and AI and a system based on HI to perform medical image segmentation. This HI system has shown remarkable robustness to image artifacts, pathology, deformations, etc. in segmenting organs in the Thorax body region in a multicenter clinical study. The HI system utilizes an anatomy modeling strategy to encode NI and to identify a rough container region in the shape of each object via a non-DL-based approach so that DL training and execution are applied only to the fuzzy container region. In this paper, we introduce several advances related to modeling of the NI component so that it becomes substantially more efficient computationally, and at the same time, is well integrated with the DL portion (AI component) of the system. We demonstrate a 9-40 fold computational improvement in the auto-segmentation task for radiation therapy (RT) planning via clinical studies obtained from 4 different RT centers, while retaining state-of-the-art accuracy of the previous system in segmenting 11 objects in the Thorax body region.