Elucidation of the role of XBP1 in the progression of complete hydatidiform mole to invasive mole through RNA-seq.

OBJECTIVE: A complete hydatidiform mole (CHM) is a common disease and is known to develop post-molar gestational trophoblast neoplasia (GTN). However, the molecular mechanisms underlying the progression of CHM to post-molar GTN remain largely unknown. In this study, we investigated the molecular factors associated with the progression using RNA-seq. METHODS: We included 13 patients with CHM and performed RNA-seq using freshly frozen samples. We identified differentially expressed genes between patients who developed GTN (GTN group) and those who achieved spontaneous remission after uterine evacuation (SR group), and performed pathway analysis. Then, functional analyses were performed on choriocarcinoma (JAR and JEG-3) and CHM (Hmol1-3B and Hmol1-2C) cells. Moreover, we evaluated the in vivo tumorigenicity of XBP1-overexpressed Hmol1-3B cells. RESULTS: The gene expression profiles were separated into two groups, and an upstream regulator analysis was performed using 281 differentially expressed genes. We focused on transcription factors and identified that 33 transcription factors were activated in the GTN group. Then, excluding those with low expression levels in clinical samples and cell lines, XBP1 was selected for further analysis. Additionally, XBP1 downregulation significantly decreased the migration and invasive abilities of choriocarcinoma cells, whereas XBP1 overexpression significantly increased the migration and invasive abilities of CHM cells. Furthermore, animal experiments showed that tumor weight and blood human chorionic gonadotropin (hCG) levels were significantly higher in the XBP1-overexpressing Hmol1-3B-bearing mice than those in the control mice. CONCLUSION: RNA-seq identified XBP1 as a key factor in post-molar GTN, suggesting it contributes to the development of post-molar GTN.

Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Characterization of Bisphosphonate Hydrate Crystals by Phosphorus K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy.

X-ray absorption near-edge structure (XANES) spectroscopy is a new method for the characterization of active pharmaceutical ingredients. XANES spectra show unique features depending on the electronic states of the X-ray absorbing elements and provide information about the chemical environment that affects the electronic states. In this study, six bisphosphonate hydrate crystals were used to investigate, for the first time, how the phosphorus K-edge XANES spectra are affected by the interatomic interactions and charged states of phosphonate moieties. Phosphorus K-edge XANES spectra showed several differences among the bisphosphonates. In particular, the chlorine atoms covalently bonded near the phosphonate and the number of electric charges of the phosphonate moieties seemed to have large effects on peak shape in XANES spectra. Unique shapes of the XANES spectra demonstrated that differences in interactions at the oxygen atoms of the phosphonate moieties could change the shapes of the XANES spectrum peaks to the extent that each material was distinguished based on the spectra. Since slight differences in interatomic interactions and charged states lead to variations in the spectra, XANES spectroscopy could be widely applied as the fingerprint method to evaluate active pharmaceutical ingredients.

Heart Rate Variability Measurement Can Be a Point-of-Care Sensing Tool for Screening Postpartum Depression: Differentiation from Adjustment Disorder.

Postpartum depression (PPD) is a serious mental health issue among women after childbirth, and screening systems that incorporate questionnaires have been utilized to screen for PPD. These questionnaires are sensitive but less specific, and the additional use of objective measures could be helpful. The present study aimed to verify the usefulness of a measure of autonomic function, heart rate variability (HRV), which has been reported to be dysregulated in people with depression. Among 935 women who had experienced childbirth and completed the Edinburgh Postnatal Depression Scale (EPDS), HRV was measured in EPDS-positive women (n = 45) 1 to 4 weeks after childbirth using a wearable device. The measurement was based on a three-behavioral-state paradigm with a 5 min duration, consisting of rest (Rest), task load (Task), and rest-after-task (After) states, and the low-frequency power (LF), the high-frequency power (HF), and their ratio (LF/HF) were calculated. Among the women included in this study, 12 were diagnosed with PPD and 33 were diagnosed with adjustment disorder (AJD). Women with PPD showed a lack of adequate HRV regulation in response to the task load, accompanying a high LF/HF score in the Rest state. On the other hand, women with AJD exhibited high HF and reduced LF/HF during the After state. A linear discriminant analysis using HRV indices and heart rate (HR) revealed that both the differentiation of PPD and AJD patients from the controls and that of PPD patients from AJD patients were possible. The sensitivity and specificity for PPD vs. AJD were 75.0% and 90.9%, respectively. Using this paradigm, an HRV measurement revealed the characteristic autonomic profiles of PPD and AJD, suggesting that it may serve as a point-of-care sensing tool in PPD screening systems.

Small Extracellular Vesicles from adipose-derived stem cells suppress cell proliferation by delivering the let-7 family of microRNAs in ovarian cancer.

INTRODUCTION: Ovarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines. MATERIALS AND METHODS: ADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis. RESULTS: ADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients. CONCLUSION: ADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs.

Characterization of Ambroxol and Its Hydrochloride Salt Crystals by Bromine K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and X-Ray Crystal Structure Analysis.

Polymorphic crystals of ambroxol, forms I and II, and form A ambroxol hydrochloride crystals were characterized with bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy and single-crystal X-ray structure analysis. The XANES spectra had unique shapes depending on the crystal forms. Refined single-crystal structures revealed different interatomic interactions around bromine atoms, such as C-H…Br and N-H…Br hydrogen bonds, Br…O halogen bonds, and N-H…π interactions. Differences in these weak interactions could affect the electronic states of the bromines, resulting in differences in the XANES spectra. The results demonstrated that weak non-conventional interatomic interactions could alter the shape of XANES spectra. Hence, the spectra could be used for evaluating polymorphs of active pharmaceutical ingredients.

Novel Pharmaceutical Cocrystals and Solvate Crystals of Nobiletin, a Citrus Flavonoid with Potent Pharmacological Activity.

Nobiletin (NOB) is a flavonoid with attractive pharmaceutical characteristics, including anti-Alzheimer's, anti-inflammation, and anti-cancer properties, but it has low solubility in water, resulting in reduced bioavailability. Its solubility must be improved to develop NOB as a drug. Cocrystal engineering can change the physicochemical properties of an active pharmaceutical ingredient and generate remarkable drug candidates that are superior in drug formulation. In this report, extensive co-crystal screening of NOBs with 31 cocrystal formers (coformers) with various functional groups was carried out by the liquid-assisted grinding method. As a result, four cocrystals (NOB with urea (URE), oxalic acid, gallic acid and salicylic acid) and one solvate crystal (NOB with formic acid (FOR)) were found. Powder X-ray diffraction and thermal analysis revealed the unique crystal morphology of all the obtained samples. In addition, the crystal structures of two of them (NOB-URE and NOB-FOR) were determined by single crystal X-ray diffraction. The results revealed that NOB-URE and NOB-FOR are new cocrystals or solvate crystals consisting of molar ratios of 1 : 2 and 1 : 0.73, respectively. In NOB-URE, we could observe a transient increase in solubility due to supersaturation, suggesting that URE is one of the better coformers of NOB.

The prognostic impact of limited-staging surgery in patients with stage IA epithelial ovarian cancer: a multi-center study with a propensity score-adjusted analysis.

OBJECTIVE: Complete-staging surgery is recommended for stage IA ovarian cancer, but may be omitted for various reasons, including the preservation of fertility and an advanced age. We herein investigated the prognostic impact of limited-staging surgery in patients with stage IA epithelial ovarian cancer. METHODS: We retrospectively collected data on 4730 patients with malignant ovarian tumors from the databases of multiple institutions and ultimately included 293 with stage IA epithelial ovarian cancer. Limited-staging surgery was defined as one that did not involve hysterectomy, systematic retroperitoneal lymphadenectomy or the collection of ascites cytology. We used an inverse probability of treatment weighting analysis with propensity scores and estimated the hazard ratios of recurrence and death with limited-staging surgery. RESULTS: In total, 176 out of 293 patients (39.9%) were assigned to the limited-staging surgery group. After propensity score adjustments, no significant differences were observed in recurrence-free survival or overall survival between the limited- and complete-staging surgery groups. Even in the subgroup analysis with age stratification, recurrence-free survival and overall survival were similar in the limited- and complete-staging surgery groups. CONCLUSIONS: The present results indicate the limited prognostic impact of limited-staging surgery for stage IA epithelial ovarian cancer.

Comparison of the physical properties of disodium etidronate amorphous forms prepared by different manufacturing methods.

Amorphous forms of disodium etidronate were prepared by three manufacturing methods, heat drying, freeze drying, and anti-solvent precipitation, and the effects of these methods on the physical properties of disodium etidronate amorphous forms were evaluated for the first time. Variable temperature X-ray powder diffraction and thermal analyses revealed that these amorphous forms had different physical properties such as glass transition point, water desorption, and crystallization temperatures. These differences can be explained by the molecular mobility and water content in amorphous forms. The differences in the structural characteristics related to the differences in these physical properties could not be detected clearly by the spectroscopic methods like Raman spectroscopy and X-ray absorption near-edge spectroscopy. Dynamic vapor sorption analyses demonstrated that all amorphous forms were hydrated to form I, a tetrahydrated form, at above 50% relative humidity, and the transition to form I was irreversible. These amorphous forms require strict humidity control to avoid crystallization. Among the three amorphous forms of disodium etidronate, the amorphous form prepared by heat drying was the most suitable for manufacturing the solid formulation, considering the low water content and low molecular mobility.

Analysis of Cimetidine Crystal Polymorphs by X-ray Absorption Near-Edge Spectroscopy.

Sulfur K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of the active pharmaceutical ingredients (APIs) containing sulfur atoms. Cimetidine (CIM) was used as a model API. Each crystal form of CIM has its own XANES spectrum, so we can discriminate the crystal form by its spectrum. The analysis of the crystal structure of CIM revealed that the difference in the shape of XANES spectra was ascribable to the difference in the C-S-C bond angle of CIM molecules and the intermolecular hydrogen bonds, such as C-H···S and N-H···S, and S-S interaction. It was found that the peak shape of the XANES spectrum is gentle when the C-S-C bond angle is large, while the peak shape can be steep when the C-S-C bond angle is small. Furthermore, it was found that the peak energy values varied depending on the hydrogen bonds and S-S interaction. By linear combination fitting using XANES spectra, it was possible to quantify the ratio of CIM form A crystal in mixed powders of form A and monohydrate crystals. These results indicate that XANES measurements can be a useful technique to evaluate the crystal polymorphism of APIs containing S atom in pharmaceutical formulation.

C-H▪▪▪S Hydrogen Bonds in Ampicillin and Amoxicillin Crystals Investigated by Sulfur K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and Single-Crystal X-Ray Structure Analysis.

Sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was evaluated for its ability to detect non-conventional C-H▪▪▪S hydrogen bonds in crystals of the sulfur-containing penam antibiotics ampicillin and amoxicillin. The XANES spectra of the nearly isomorphous crystals of ampicillin trihydrate and amoxicillin trihydrate were very similar, whereas that of ampicillin anhydrate displayed unique features. Single-crystal X-ray structure analyses revealed that the C-H▪▪▪S hydrogen bond geometries and the chemical types of the hydrogen donors differed between the isomorphous trihydrate crystals and ampicillin anhydrate crystal. These observations demonstrate that the shapes of the sulfur K-edge XANES spectra are dependent on the nature of the C-H▪▪▪S hydrogen bonds. Sulfur K-edge XANES spectroscopy shows promise for use in the detection and analysis of non-covalent interactions, including hydrogen bonds to sulfur atoms, within active pharmaceutical ingredients.

Direct detection of the crystal form of an active pharmaceutical ingredient in tablets by X-ray absorption fine structure spectroscopy.

Different crystal forms of active pharmaceutical ingredients (APIs) may display variations in physicochemical properties. During the drug development process, the definitive purpose is to maintain homogeneous quality in a single crystalline form. Hence, it is important to evaluate and understand the properties of each crystal form of APIs in pharmaceutics. In this study, forms 0, Ⅰ, Ⅱ, III of bromhexine hydrochloride, and form S of bromhexine were characterized by the commonly used methods X-ray powder diffraction, thermogravimetry-differential thermal analysis, and single crystal structure X-ray diffraction. Additionally, X-ray absorption fine structure spectroscopy (XAFS), a seldom used method in the pharmaceutics discipline was also applied to explore the chemical environment of bromine atoms in forms 0, Ⅰ, Ⅱ and S as well as chloride ions in forms 0 to Ⅱ. The XAFS spectra of each form were different from each of the other forms which indicated the chemical environment around target elements in the crystal polymorphs were distinct. Then, we measured the commercial bromhexine hydrochloride tablets with XAFS measurement and found that XAFS could distinguish the crystal form in the tablets. Hence, we demonstrated that XAFS measurements would be applicable as one of the methods for the direct detection of APIs in the tablets.

Bromine K-Edge X-Ray Absorption Near-Edge Structure Analysis on Hydrobromide-Salt Crystals and the Solid Dispersion of Active Pharmaceutical Ingredients.

Bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy analyses were used to evaluate the crystals of the active pharmaceutical ingredients, eletriptan hydrobromide, dextromethorphan hydrobromide and scopolamine hydrobromide salts and the solid dispersion of eletriptan hydrobromide. The crystals and the solid dispersion of the active pharmaceutical ingredient (API) salts could be discriminated based on the shape of the XANES spectra. The differences in the shape of XANES spectra was ascribable to the differences in the interatomic interactions of the bromine ions based on the crystal structures. Ratio of the eletriptan hydrobromide α-form crystal in mixed powders of α-form and monohydrate crystals could be quantified by the linear-combination fitting using their XANES spectra. These results indicated that the XANES spectroscopy are a potent method for evaluating the APIs of pharmaceutical formulations even at the higher energy region around the bromine K-edge of 13470 eV.

Investigation of Physical Properties of Disodium Etidronate Tetrahydrate and Application of Phosphorus K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy.

PURPOSE: Disodium etidronate is a bisphosphonate, compounds that are widely used in the treatment of bone disorders such as osteoporosis and Paget's disease. We investigated the physical properties of disodium etidronate tetrahydrate crystal, form I. METHODS: We used X-ray powder diffraction (XRPD), thermal analysis, dynamic vapor sorption (DVS), X-ray single crystal structure analysis, and phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy for the first time. RESULTS: XRPD and thermal analyses demonstrated that form I was dehydrated and transformed to an amorphous form, to a crystalline form II, and finally to a form III by heating. DVS measurements revealed that the amorphous form, form II, and form III were rehydrated to form I by humidification, and form I was stable even at 0% relative humidity. These results indicate that form I is the most stable solid-state under ambient conditions and is suitable as an API for manufacture in solid formulations. The phosphorus K-edge XANES spectra differed among form I, the amorphous form, and form II, which may be ascribed to the difference in the coordinate bond schemes between the phosphate moieties and sodium ions. The results demonstrated that the phosphorus K-edge XANES spectroscopy could be applied to the identification or the discrimination of crystal forms of the APIs containing phosphate moieties. CONCLUSIONS: Acquired information about physical properties are crucial for manufacturing of solid formulations of disodium etidronate. XANES spectroscopy is a promising alternative method for evaluating the solid-state forms of APIs.

A Fabry-Pérot cavity coupled surface plasmon photodiode for electrical biomolecular sensing.

Surface plasmon resonance is a well-established technology for real-time highly sensitive label-free detection and measurement of binding kinetics between biological samples. A common drawback, however, of surface plasmon resonance detection is the necessity for far field angular resolved measurement of specular reflection, which increases the size as well as requiring precise calibration of the optical apparatus. Here we present an alternative optoelectronic approach in which the plasmonic sensor is integrated within a photovoltaic cell. Incident light generates an electronic signal that is sensitive to the refractive index of a solution via interaction with the plasmon. The photogenerated current is enhanced due to the coupling of the plasmon mode with Fabry-Pérot modes in the absorbing layer of the photovoltaic cell. The near field electrical detection of surface plasmon resonance we demonstrate will enable a next generation of cheap, compact and high throughput biosensors.

Reduced deliquescency of isosorbide by cocrystallization and mechanisms for hygroscopicity.

Isosorbide (ISO) is an effective hyperosmotic agent that can be administrated orally and is used as a therapeutic agent for brain pressure drop, glaucoma, and Meniere's disease. However, the critical relative humidity (CRH) of ISO is about 48% RH at 25 °C, and it deliquesces in humid environments. In this study, we attempted to reduce the deliquescence of ISO using cocrystallization and analyze the water adsorption mechanism from the crystal structure. Four new ISO cocrystals with piperazine (PZ), hydrochlorothiazide (HCT), 3,5-dihydroxybenzoic acid (35DHBA), or gallic acid (GA) were identified. The dynamic vapor sorption analyses demonstrated that all the cocrystals showed higher CRHs than the ISO crystal. Although water adsorption below the CRH was observed for all cocrystals, the water molecules adsorbed in the ISO-PZ and ISO-GA cocrystals were lower than those in the ISO crystal. Investigation of the crystal structures suggested that the amount of water adsorbed might be related to the degree of exposure of the ISO hydroxyl groups on the crystal surface. Given the CRH, water adsorption below the CRH, thermal stability, apparent dissolution rate, and toxicity level of the coformer, the ISO-GA cocrystal is the most suitable for preparing a solid formulation of ISO.

X-ray Absorption Near-Edge Spectroscopy Analysis of Indomethacin in Crystalline Forms and in Amorphous Solid Dispersions.

Chlorine K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of identical active pharmaceutical ingredients (APIs) containing chloride atoms and their amorphous solid dispersions (ASDs). Indomethacin (IMC), of which three crystal forms (α, ß, and γ) have been reported, was used as a model API. The shape of XANES spectra was unique to each IMC crystal. The analysis of the crystal structures of IMC revealed that chlorine atoms of the IMCα form had unique intermolecular interactions and halogen bonds with oxygen atoms, while those of the IMCγ form do not have any notable interactions. This result showed that XANES measurements can detect weak interatomic interactions. The shapes of the ASD spectra were clearly different from those of the crystals, suggesting that the environment around the Cl atom of IMC was different from that of the crystals. A thermal stress test was then performed to study the transformation from the amorphous form to the crystalline form of IMC in the ASD. The powder X-ray diffraction (PXRD) patterns indicated that amorphous IMC transformed into crystals during the thermal stress test. In accordance with the PXRD results, the XANES spectra also transformed from ASD to crystalline form. These results indicate that the IMC transformation could be monitored by XANES measurement. Our findings led us to conclude that XANES measurement is a novel approach for the evaluation of crystal polymorphs of APIs and the crystalline state of APIs in ASDs.

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

Chlorine K-Edge X-Ray Absorption Near-Edge Structure Analysis of Clarithromycin Hydrochloride Metastable Crystal.

X-ray absorption fine structure (XAFS) spectra, especially X-ray absorption near-edge structure (XANES), show unique features depending on the chemical states and structures in the vicinity of the X-ray absorbing elements. As such, they can be used to identify the chemical environment of elements. XAFS spectroscopy was applied to investigate the chemical environment of chloride ions in a metastable form A crystal of the antibiotic clarithromycin hydrochloride hydrate. The XANES of the form A crystals showed low similarity to that of the active pharmaceutical ingredient (API) hydrochloride salt crystals, but showed a high similarity to that of a hydrochloride aqueous solution. This indicated that the chloride ion in the form A crystals predominantly interacted with water molecules and was fully hydrated, which was consistent with the previous presumption that form A may be high water-content crystals. This study demonstrated that this XAFS spectroscopy method would be a potent alternative technique for evaluating APIs.

Sub-Ångstrom structure of collagen model peptide (GPO)10 shows a hydrated triple helix with pitch variation and two proline ring conformations.

Collagens are large structural proteins that are prevalent in mammalian connective tissue. Peptides designed to include a glycine-proline-hydroxyproline (GPO) amino acid triad are biomimetic analogs of the collagen triple helix, a fold that is a hallmark of collagen-like sequences. To inform the rational engineering of collagen-like peptides and proteins for food systems, we report the crystal structure of the (GPO)10 peptide at 0.89-Å resolution, solved using direct methods. We determined that a single chain in the asymmetric unit forms a pseudo-hexagonal network of triple helices that have a pitch variation consistent with the model 7/2 helix (3.5 residues per turn). The proline rings occupied one of two states, while the helix was found to have a well-defined hydration shell involved in the stabilization of the inter-helix crystal network. This structure offers a new high-resolution basis for understanding the hierarchical assembly of native collagens, which will aid the food industry in engineering new sustainable food systems.