Prohibitin modulates periodontium differentiation in mice development.

Introduction: Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However, its specific role in periodontium development remains less understood. This study aims to elucidate the expression pattern and function of PHB in periodontium development and its involvement in alveolar bone formation. Methods: Immunolocalization of PHB in the periodontium of postnatal (PN) mice were examined. Phb morpholino was micro-injected into the right-side mandible at PN5, corresponding to the position where the alveolar bone process forms in relation to the lower first molar. The micro-injection with a scramble control (PF-127) and the left-side mandibles were used as control groups. Five days post-micro-injection, immunohistochemical analysis and micro-CT evaluation were conducted to assess bone mass and morphological changes. Additionally, expression patterns of signaling molecules were examined following Phb downregulation using 24-h in vitro cultivation of developing dental mesenchyme at E14.5. Results: The immunostaining of PHB showed its localization in the periodontium at PN5, PN8, and PN10. The in vitro cultivation of dental mesenchyme resulted in alterations in Bmps, Runx2, and Wnt signalings after Phb knock-down. At 5 days post-micro-injection, Phb knocking down showed weak immunolocalizations of runt-related transcription factor (RUNX2) and osteocalcin (OCN). However, knocking down Phb led to histological alterations characterized by decreased bone mass and stronger localizations of Ki67 and PERIOSTIN in the periodontium compared 1 to control groups. The micro-CT evaluation showed decreased bone volume and increased PDL space in the Phb knock-down specimens, suggesting its regulatory role in bone formation. Discussion: The region-specific localization of PHB in the margin where alveolar bone forms suggests its involvement in alveolar bone formation and the differentiation of the periodontal ligament. Overall, our findings suggest that Phb plays a modulatory role in alveolar bone formation by harmoniously regulating bone-forming-related signaling molecules during periodontium development.

Nanocrystal Assemblies: Current Advances and Open Problems.

We explore the potential of nanocrystals (a term used equivalently to nanoparticles) as building blocks for nanomaterials, and the current advances and open challenges for fundamental science developments and applications. Nanocrystal assemblies are inherently multiscale, and the generation of revolutionary material properties requires a precise understanding of the relationship between structure and function, the former being determined by classical effects and the latter often by quantum effects. With an emphasis on theory and computation, we discuss challenges that hamper current assembly strategies and to what extent nanocrystal assemblies represent thermodynamic equilibrium or kinetically trapped metastable states. We also examine dynamic effects and optimization of assembly protocols. Finally, we discuss promising material functions and examples of their realization with nanocrystal assemblies.

Unraveling the structure, chemical composition, and conserved signaling in leech teeth.

Unlike vertebrates, the number of toothed taxa in invertebrates is very few, with leeches being the only tooth-bearing organisms in the phylum Annelida. Copious studies have been conducted regarding vertebrate teeth; however, studies regarding the structure and function of invertebrate teeth are limited. In this study, the tooth structure of leeches, specifically Hirudo nipponia and Haemadipsa rjukjuana, was revealed, which showed sharp and pointed teeth along the apex of three jaws. Understanding conserved signaling regulations among analogous organs is crucial for uncovering the underlying mechanisms during organogenesis. Therefore, to shed light on the evolutionary perspective of odontogenesis to some extent, we conducted de novo transcriptome analyses using embryonic mouse tooth germs, Hirudo teeth, and Helobdella proboscises to identify conserved signaling molecules involved in tooth development. The selection criteria were particularly based on the presence of tooth-related genes in mice, Hirudo teeth, and Helobdella proboscis, wherein 4113 genes were commonly expressed in all three specimens. Furthermore, the chemical nature of leech teeth was also examined via TEM-EDS to compare the chemical composition with vertebrate teeth. The examination of tissue-specific genetic information and chemical nature between leeches and mice revealed chemical similarities between leech and mice teeth, as well as conserved signaling molecules involved in tooth formation, including Ptpro, Prickle2, and Wnt16. Based on our findings, we propose that leech teeth express signaling molecules conserved in mice and these conserved tooth-specific signaling for dental hard tissue formation in mice would corresponds to the structural formation of the toothed jaw in leeches.

Chemical constituents of the marine-derived fungus Acremonium sp. AN-13.

Two new compounds named 3(S)-hydroxy-1-(2,4,5-trihydroxy-3,6- dimethylphenyl)-hex-4E-en-1-one (1) and acremonilactone (2), together with nine known compounds (3-11), were isolated from the fermentation broth of Acremonium sp. associated with marine sediments collected from South China Sea. NMR and HRESIMS spectroscopic analysis elucidated the structure of two new compounds. Compound 2 had characteristic rotary gate shape skeleton with a six-membered lactone. Compounds 1 and 9 showed DPPH radical scavenging activity with inhibition rates of 96.50 and 85.95% at the concentration of 0.5 mg/ml, respectively. Moreover, compounds 4, 6 and 11 showed definite antibacterial activity against Staphylococcus aureus ATCC 6538.

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers.

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Heritable changes in chromatin contacts linked to transgenerational obesity.

Burgeoning evidence demonstrates that effects of environmental exposures can be transmitted to subsequent generations through the germline without DNA mutations1,2. This phenomenon remains controversial because underlying mechanisms have not been identified. Therefore, understanding how effects of environmental exposures are transmitted to unexposed generations without DNA mutations is a fundamental unanswered question in biology. Here, we used an established murine model of male-specific transgenerational obesity to show that exposure to the obesogen tributyltin (TBT) elicited heritable changes in chromatin interactions (CIs) in primordial germ cells (PGCs). New CIs were formed within the Ide gene encoding Insulin Degrading Enzyme in the directly exposed PGCs, then stably maintained in PGCs of the subsequent (unexposed) two generations. Concomitantly, Ide mRNA expression was decreased in livers of male descendants from the exposed dams. These males were hyperinsulinemic and hyperglycemic, phenocopying Ide-deficient mice that are predisposed to adult-onset, diet-induced obesity. Creation of new CIs in PGCs, suppression of hepatic Ide mRNA, increased fat mass, hyperinsulinemia and hyperglycemia were male-specific. Our results provide a plausible molecular mechanism underlying transmission of the transgenerational predisposition to obesity caused by gestational exposure to an environmental obesogen. They also provide an entry point for future studies aimed at understanding how environmental exposures alter chromatin structure to influence physiology across multiple generations in mammals.

Comprehensive characterization and expression analysis of enzymatic antioxidant gene families in passion fruit (Passiflora edulis).

Passion fruit, a valuable tropical fruit, faces climate-related growth challenges. Antioxidant enzymes are vital for both stress protection and growth regulation in plants. We first provided systemic analysis of enzymatic antioxidant gene families in passion fruit, identifying 90 members including 11 PeSODs, 45 PeAPXs, 8 PeCATs, 7 PeGPXs, 6 PeMDHARs, 8 PeDHARs, and 5 PeGRs. Gene members in each gene family with same subcellular localization showed closer phylogenetic relationship. Many antioxidant genes exhibited tissue- or developmental stage-specific expression patterns during floral and fruit development, with some widely expressed. Their co-expressed genes were linked to photosynthesis and energy metabolism, suggesting roles in protecting highly proliferating tissues from oxidative damage. Potential genes for enhancing temperature stress resistance were identified. The involvement of diverse regulatory factors including miRNAs, transcription factors, and CREs might contribute to the complex roles of antioxidant genes. This study informs future research on antioxidant genes and passion fruit breeding.

Falsirhodobacter algicola sp. nov., a member of the Rhodobacteraceae isolated from the marine red algae Grateloupia sp.

A Gram-negative, pale yellow-pigmented, non-flagellated, motile, rod-shaped and aerobic bacterium, designated strain PG104T, was isolated from red algae Grateloupia sp. collected from the coastal area of Pohang, Republic of Korea. Growth of strain PG104T was observed at 15-35 °C (optimum, 30 °C), pH 6.0-10.0 (optimum, pH 7.5-8.0) and in the presence of 0-8.0 % (w/v) NaCl (optimum, 5.0 %). The predominant fatty acids included C17 : 0, C18 : 0, 11-methyl C18 : 1 ω7c and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and the major respiratory quinone was Q-10. Polar lipids included phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain PG104T formed a phylogenetic lineage with members of the genus Falsirhodobacter and exhibited 16S rRNA gene sequence similarities of 97.1 and 96.6 % to Falsirhodobacter deserti W402T and Falsirhodobacter halotolerans JA744T, respectively. The complete genome of strain PG104T consisted of a single circular chromosome of approximately 2.8 Mbp with five plasmids. Based on polyphasic taxonomic data, strain PG104T represents a novel species in the genus Falsirhodobacter, for which the name Falsirhodobacter algicola sp. nov. is proposed. The type strain of Falsirhodobacter algicola is PG104T (=KCTC 82230T=JCM 34380T).

Phase Behavior of Binary Blends of Diblock Copolymers: Progress and Opportunities.

The phase behavior of binary blends of diblock copolymers has been examined extensively in the past decades. Experimental and theoretical studies have demonstrated that mixing two different block copolymers provides an efficient and versatile route to regulate their self-assembled morphologies. A good understanding of the principles governing the self-assembly of block copolymer blends has been obtained from the study of A1B1/A2B2 diblock copolymer blends. The second (A2B2) diblocks could act synergistically as fillers and cosurfactants to regulate the domain size and interfacial properties, resulting in the formation of ordered phases not found in the parent (A1B1 or A2B2) diblock copolymer melts. The study of A1B1/A2B2 block copolymer blends further provides a solid foundation for future research on more complex block copolymer blends.

The role of O-GlcNAcylation mediated by OGT during tooth development.

To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of ß-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.

Carbon quantum dot fluorescent probe for labeling and imaging of stellate cell on liver frozen section below freezing point.

The targeted labeling imaging of stellate cells on liver frozen section by immunofluorescence is a very promising visualization technique to study the distribution of stellate cells in the liver. In this study, water soluble carbon quantum dots that can emit blue, green and yellow fluorescence are synthesized by the hydrothermal method, and their sizes are 3.2, 3.7, and 4.3 nm, respectively. The three carbon quantum dots have good fluorescence stability, and the quantum yields are 36.1%, 26.3% and 21%, respectively. When the mass fraction of KCl in the blue carbon quantum dot dispersion system is 13%, it still maintains the liquid state at -30 °C. The final fluorescent probe is obtained after the carbon quantum dots are coupled with the secondary antibody, spectral characterizations confirm that the conjugate probe still maintains protein immunoactivity and has good stability. Cell experiments prove that the probe has good biocompatibility, the rabbit anti-mouse Desmin antibody is used as the primary antibody, the results of cellular immunofluorescence imaging and flow cytometry show that the probe can specifically label hepatic stellate cell at -20 °C. The results of liver frozen section experiments show that hepatic stellate cell can be specifically targeted and labeled by the fluorescent probe. This labeling technology provides an important technical means for elucidating the structure and function of the liver at the cellular level, exploring the liver pathological change, and designing and developing drug.

Rotational Dynamics of Discoid Colloidal Particles in Attractive Quasi-Two-Dimensional Plastic Crystals.

Plastic crystals formed from anisotropic molecules or particles are an important state of matter characterized by the presence of long-range positional order and the lack of long-range orientational order. The rotational motion of molecules or particles in plastic crystals is the most attractive characteristic of the system. Here the rotational dynamics of the discoid particles in quasi-two-dimensional colloidal plastic crystals stabilized via depletion interactions are quantitatively studied using time-resolved confocal microscopy. The measured probability distribution of particle orientation reveals the existence of a strong coupling between the lattice symmetry and particle rotation, resulting in anisotropic rotational dynamics modes resembling the underlying hexagonal crystalline symmetry. Furthermore, the orientational distribution function provides information about the potential surface of rotational dynamics. The observed slow rotational diffusion can be attributed to the presence of orientational minima and potential barriers on the potential surface. Our findings with a real experimental system provide important insights into the role of attraction in the phase behaviors of plastic crystals.

Simultaneous labeling and multicolor fluorescence imaging of multiple immune cells on liver frozen section by polychromatic quantum dots below freezing points.

A method for simultaneous labeling and multicolor fluorescence imaging of different hepatic immune cells below freezing point is established based on quantum dots. In the experiment, carbon quantum dots with emission wavelength of 435 nm, CdTe@CdS quantum dots at 542 nm and CdSe@ZnS quantum dots at 604 nm are synthesized respectively, it is found that when the mass fractions of KCl (as antifreeze) are 12 %, 14 %, and 12 %, respectively, the three quantum dot dispersion systems remain liquid state at -20 °C. After they are conjugated with the corresponding secondary antibodies, agarose gel electrophoresis, circular dichroism and capillary electrophoresis confirm the effectiveness of conjugation. By indirect immunofluorescence method, the above three quantum dot fluorescent probes are used to simultaneously and specifically target a variety of liver immune cells, and the multi-color simultaneous imaging of different liver immune cells is realized under the same excitation wavelength, it is found that hepatic macrophages are arranged radially in the liver, hepatic stellate cells present punctate distribution, and hepatic sinusoidal endothelial cells present circular distribution, which is consistent with the results of H&E staining and ultrathin section TEM. This study provides an important technical means for elucidating the structure and function of the liver.

Genome-wide identification and expression analysis of the bHLH gene family in passion fruit (Passiflora edulis) and its response to abiotic stress.

Passion fruit is a tropical fruit crop with significant agricultural, economic and ornamental values. The growth and development of passion fruit are greatly affected by climatic conditions. In plants, the basic helix-loop-helix (bHLH) gene family plays essential roles in the floral organ and fruit development, as well as stress response. However, the characteristics and functions of the bHLH genes of passion fruit remain unclear. Here, 138 passion fruit bHLH members were identified and classified into 20 subfamilies. The structural analysis illustrated that PebHLH proteins of the specific subfamily are relatively conserved. Collinearity analysis indicated that the expansion of the PebHLH gene family mainly took place by segmental duplication, and the structural diversity of duplicated genes might contribute to their functional diversity. PebHLHs, which potentially regulate different floral organ and fruit development, were further screened out, and many of these genes were differentially expressed under various stress treatments. The co-presence of different cis-regulatory elements involved in developmental regulation, hormone and stress responses in the promoter regions of PebHLHs might be closely related to their diverse regulatory roles. Overall, this study will be helpful for further functional investigation of PebHLHs and provides clues for improvement of the passion fruit breeding.

Ponderations around Akamon: from Chinese visiting scholars in the Department of Hepatobiliary-Pancreatic Surgery of Tokyo University Hospital / 中华消化外科杂志

It is well known that Tokyo University Hospital (TUH) is one of the most famous medical centers around the world in Japan. Among several departments in TUH, the Department of Hepatobiliary-Pancreatic Surgery and Artificial Organ and Transplantation Division are representative predominant divisions, which have high-quality diagnostic and treatment service systems being at the leading position in the world. The authors visit the Department of Hepatobiliary-Pancreatic Surgery and Artificial Organ and Transplantation Division in 2022. When studying and working in TUH, the authors make a multi-angle comparisons of the development status and technical levels of hepatobiliary and pancreatic surgery among TUH, western countries and China. It aims to share and exchange with fellow surgeons at home and abroad, and bring benefits or enlightenment to the professional advancement of them.

PeHVA22 gene family in passion fruit (Passiflora edulis): initial characterization and expression profiling diversity.

Passion fruit, an economically valuable fruit crop, is highly vulnerable to adverse climate conditions. The HVA22 genes, recognized as abscisic acid (ABA) and stress-inducible, play vital roles in stress response and growth regulation in diverse eukaryotic organisms. Here, six HVA22 genes were firstly identified in passion fruit genome and all predicted to be localized within the endoplasmic reticulum. Phylogenetic analyses showed that all PeHVA22s were divided into four subgroups. The gene structural features of PeHVA22 genes clustered in the same subgroup were relatively conserved, while the gene structure characteristics of PeHVA22s from different subgroups varied significantly. PeHVA22A and PeHVA22C closely clustered with barley HVA22 in Group II, were also induced by ABA and drought stress treatment, suggesting conserved roles similar to barley HVA22. Meanwhile, most PeHVA22s exhibited induced expression post-drought treatment but were suppressed under salt, low and high-temperature conditions, indicating a unique role in drought response. Additionally, PeHVA22s displayed tissue-specific expression patterns across diverse tissues, except for PeHVA22B which maybe a pseudogene. Notably, PeHVA22C, PeHVA22E, and PeHVA22F predominantly expressed in fruit, indicating their involvement in fruit development. Almost all PeHVA22s showed variable expression at different developmental stages of stamens or ovules, implying their roles in passion fruit's sexual reproduction. The intricate roles of PeHVA22s may result from diverse regulatory factors including transcription factors and CREs related to plant growth and development, hormone and stress responsiveness. These observations highlighted that PeHVA22s might play conserved roles in ABA response and drought stress tolerance, and also be participated in the regulation of passion fruit growth and floral development.

Correlation between active component content and color of Atractylodes Lancea and A. chinensis based on color difference principle / 中国中药杂志

We explored the correlations between the color difference values [ΔL~*(lightness), Δa~*(red-green), Δb~*(yellow-blue)] and the content of four active components(including sesquiterpenoids and polyacetylenes) in the powder of Atractylodes lancea and A. chinensis, aiming to provide reference for the quality evaluation of Atractylodis Rhizoma and establish a qualitative model that can distinguish between A. lancea and A. chinensis based on the chromatic values. The tristimulus values(L~*, a~*, and b~*) of 23 batches of A. lancea and A. chinensis were measured by a color difference meter. The content of atractylenolide Ⅱ, β-eudesmol, atractylodin, and atractylone in the 23 batches of samples were measured by high performance liquid chromatography(HPLC). Principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were performed to establish the qualitative models for distinguishing between A. lancea and A. chinensis. SPSS was employed to analyze the correlations between the tristimulus values and the content of the four index components. The results showed that the established PCA and PLS-DA models can divide the A. lancea and A. chinensis samples into two regions, and the tristimulus values of A. lancea and A. chinensis were positively correlated with the content of β-eudesmol and atractylodin. Therefore, the PCA and PLS-DA models can successfully identify A. lancea and A. chinensis, and the appearance color can be used to quickly predict the internal quality of Atractylodis Rhizoma. This study provides a reference for the quality evaluation of Atractylodis Rhizoma and the modern research on the color of Chinese medicinal materials.

Part Affinity Fields and CoordConv for Detecting Landmarks of Lumbar Vertebrae and Sacrum in X-ray Images.

With the prevalence of degenerative diseases due to the increase in the aging population, we have encountered many spine-related disorders. Since the spine is a crucial part of the body, fast and accurate diagnosis is critically important. Generally, clinicians use X-ray images to diagnose the spine, but X-ray images are commonly occluded by the shadows of some bones, making it hard to identify the whole spine. Therefore, recently, various deep-learning-based spinal X-ray image analysis approaches have been proposed to help diagnose the spine. However, these approaches did not consider the characteristics of frequent occlusion in the X-ray image and the properties of the vertebra shape. Therefore, based on the X-ray image properties and vertebra shape, we present a novel landmark detection network specialized in lumbar X-ray images. The proposed network consists of two stages: The first step detects the centers of the lumbar vertebrae and the upper end plate of the first sacral vertebra (S1), and the second step detects the four corner points of each lumbar vertebra and two corner points of S1 from the image obtained in the first step. We used random spine cutout augmentation in the first step to robustify the network against the commonly obscured X-ray images. Furthermore, in the second step, we used CoordConv to make the network recognize the location distribution of landmarks and part affinity fields to understand the morphological features of the vertebrae, resulting in more accurate landmark detection. The proposed network was evaluated using 304 X-ray images, and it achieved 98.02% accuracy in center detection and 8.34% relative distance error in corner detection. This indicates that our network can detect spinal landmarks reliably enough to support radiologists in analyzing the lumbar X-ray images.