Helicity Control of Polymeric Backbones with Alternating cis-trans Double Bonds in Cyclopolymerized Dipropargyl Amides.

We report the synthesis of new helical polymeric structures having alternating cis and trans double bonds and chiral amino acid side chains by metathesis cyclopolymerization. The polymer helicity, which is generated by the interaction between fluorenylmethyloxycarbonyl (Fmoc) groups in the side chains, is dramatically affected by solvents. A thorough experimental and theoretical analysis including nuclear magnetic resonance, atomic force microscopy, and density functional theory and molecular mechanics calculations suggests that the helicity of both backbone and side chains are determined by anti-syn rotation of the carbamate groups and by the different interactions of the Fmoc groups with solvents.

Ring Opening Metathesis Polymerization of Bicyclic α,ß-Unsaturated Anhydrides for Ready-to-be-grafted Polymers Having Tailored pH-Responsive Degradability.

Polymers having α,ß-unsaturated anhydrides as repeating units were synthesized by ring opening metathesis polymerization (ROMP). The anhydride moieties were ready-to-be-grafted with amines to form acid-labile cis-α,ß-unsaturated acid amide linkages. The pH-responsive reversible de-grafting can be controlled by changing the intramolecular accessibility between acid and amide groups. The alendronate-grafted ROMP polymers showed distinct pH-dependent cytotoxicity according to the anhydride structures.

Amine-selective affinity resins based on pH-sensitive reversible formation of covalent bonds.

A new class of affinity resins using reversible covalent bonds is introduced for the separation of amine-containing molecules. pH-sensitive reversible formation of amic acid bonds between amines and carboxylate dimethyl maleic anhydride-decorated wrinkled silica nanoparticle resins was used to selectively retain and release amine-containing molecules, by controlling the pH.

De novo biosynthesis of pterostilbene in an Escherichia coli strain using a new resveratrol O-methyltransferase from Arabidopsis.

BACKGROUND: Pterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems. RESULTS: We identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular L-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of L-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using L-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-L-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis. CONCLUSION: As a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a L-methionine containing medium in combination with a strain that had an engineered metabolic pathway for L-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme.

Production of phenylacetyl-homoserine lactone analogs by artificial biosynthetic pathway in Escherichia coli.

BACKGROUND: Quorum sensing (QS) networks are more commonly known as acyl homoserine lactone (HSL) networks. Recently, p-coumaroyl-HSL has been found in a photosynthetic bacterium. p-coumaroyl-HSL is derived from a lignin monomer, p-coumaric acid, rather than a fatty acyl group. The p-coumaroyl-HSL may serve an ecological role in diverse QS pathways between p-coumaroyl-HSL producing bacteria and specific plants. Interference with QS has been regarded as a novel way to control bacterial infections. Heterologous production of the QS molecule, p-coumaroyl-HSL, could provide a sustainable and controlled means for its large-scale production, in contrast to the restricted feedback regulation and extremely low productivity of natural producers. RESULTS: We developed an artificial biosynthetic process for phenylacetyl-homoserine lactone analogs, including cinnamoyl-HSL, p-coumaroyl-HSL, caffeoyl-HSL, and feruloyl-HSL, using a bioconversion method via E. coli (CB1) in the co-expression of the codon-optimized LuxI-type synthase (RpaI) and p-coumaroyl-CoA ligase (4CL2nt). In addition to this, we show the de novo production of p-coumaroyl-HSL in heterologous host E. coli (DN1) and tyrosine overproducing E. coli (DN2), containing the rpaI gene in addition to p-coumaroyl-CoA biosynthetic genes. The yields for p-coumaroyl-HSL reached 93.4 ± 0.6 and 142.5 ± 1.0 mg/L in the S-adenosyl-L-methionine and L-methionine feeding culture in the DN2 strain, respectively. CONCLUSIONS: This is the first report of a de novo biosynthesis in a heterologous host yielding a QS molecule, p-coumaroyl-HSL from a glucose medium using a single vector system combining p-coumaroyl-CoA biosynthetic genes and the LuxI-type synthase gene.

Artificial de novo biosynthesis of hydroxystyrene derivatives in a tyrosine overproducing Escherichia coli strain.

BACKGROUND: Styrene and its derivatives as monomers and petroleum-based feedstocks are valuable as raw materials in industrial processes. The chemical reaction for styrene production uses harsh reaction conditions such as high temperatures or pressures, or requires base catalysis with microwave heating. On the other hand, production of styrene and its derivatives in Escherichia coli is an environmental friendly process to produce conventional petroleum-based feedstocks. RESULTS: An artificial biosynthetic pathway was developed in E. coli that yields 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene from simple carbon sources. This artificial biosynthetic pathway has a codon-optimized phenolic acid decarboxylase (pad) gene from Bacillus and some of the phenolic acid biosynthetic genes. E. coli strains with the tal and pad genes, the tal, sam5, and pad genes, and the tal, sam5, com, and pad genes produced 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. Furthermore, these pathways were expressed in a tyrosine overproducing E. coli. The yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation. CONCLUSIONS: Our system is the first to use E. coli with artificial biosynthetic pathways for the de novo synthesis of 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in a simple glucose medium. Similar approaches using microbial synthesis from simple sugar could be useful in the synthesis of plant-based aromatic chemicals.

Intra- and inter-examiner reliability of goniometer and inclinometer use in Craig's test.

[Purpose] The purpose of this study was to determine the inter- and intra-examiner reliability of measurement methods for femoral anterversion during Craig's test. [Subjects and Methods] The study included 37 healthy participants (20 males and 17 females). Two novice examiners (Department of Physical Therapy students at Silla University) used three different methods to measure the femoral anterversion during Craig's test: a goniometer, a goniometer with a laser beam, and an inclinometer. [Results] The intra-examiner reliability was high for both examiners with all three measurement methods, with scores of 0.82, 0.86, and 0.73 for examiner 1 and 0.74, 0.78, and 0.72 for examiner 2 for the goniometer, goniometer with the laser beam, and inclinometer, respectively. The inter-examiner reliability during Craig's test was below moderate for both the goniometer (0.25) and inclinometer (0.27) and moderate for the goniometer with the laser beam (0.62). [Conclusion] This study found that Craig's test using a goniometer with a laser beam had high intra-examiner reliability and moderate inter-examiner reliability. Clinically, these findings may supplement existing measurement skills and reduce the difficulty of locating the goniometer axis during Craig's test.

Biosynthesis of methylated resveratrol analogs through the construction of an artificial biosynthetic pathway in E. coli.

BACKGROUND: Methylated resveratrol analogs show similar biological activities that are comparable with those of the resveratrol. However, the methylated resveratrol analogs exhibit better bioavailability as they are more easily transported into the cell and more resistant to degradation. Although these compounds are widely used in human health care and in industrial materials, at present they are mainly obtained by extraction from raw plant sources. Accordingly their production can suffer from a variety of economic problems, including low levels of productivity and/or heterogeneous quality. On this backdrop, large-scale production of plant metabolites via microbial approaches is a promising alternative to chemical synthesis and extraction from plant sources. RESULTS: An Escherichia coli system containing an artificial biosynthetic pathway that produces methylated resveratrol analogues, such as pinostilbene (3,4'-dihydroxy-5-methoxystilbene), 3,5-dihydroxy-4'-methoxystilbene, 3,4'-dimethoxy-5-hydroxystilbene, and 3,5,4'-trimethoxystilbene, from simple carbon sources is developed. These artificial biosynthetic pathways contain a series of codon-optimized O-methyltransferase genes from sorghum in addition to the resveratrol biosynthetic genes. The E. coli cells that harbor pET-opTLO1S or pET-opTLO3S produce the one-methyl resveratrol analogues of 3,5-dihydroxy-4'-methoxystilbene and pinostilbene, respectively. Furthermore, the E. coli cells that harbor pET-opTLO13S produce 3,5-dihydroxy-4'-methoxystilbene, bis-methyl resveratrol (3,4'-dimethoxy-5-hydroxystilbene), and tri-methyl resveratrol (3,5,4'-trimethoxystilbene). CONCLUSIONS: Our strategy demonstrates the first harness microorganisms for de novo synthesis of methylated resveratrol analogs used a single vector system joined with resveratrol biosynthetic genes and sorghum two resveratrol O-methyltransferase genes. Thus, this is also the first report on the production of the methylated resveratrol compounds bis-methyl and tri-methyl resveratrol (3,4'-dimethoxy-5-hydroxystilbene and 3,5,4'-trimethoxystilbene) in the E. coli culture. Thus, the production of the methylated resveratrol compounds was performed on the simple E. coli medium without precursor feeding in the culture.

Unlocking the pH-responsive degradability of fumaramic acid derivatives using photoisomerization.

Fumaramic acid derivatives can be converted into their cis isomer maleamic acid derivatives under UV illumination, and these maleamic acid derivatives show pH-responsive degradability at acidic pH only after the preceding photoisomerization. The rate of the tandem photoisomerization-degradation of fumaramic acid derivatives can be finely controlled by changing the substituents on the double bond. Photoisomerization-based unlocking of the pH-responsive degradability of fumaramic acid derivatives has strong potential for the development of multisignal-responsive smart materials in biomedical applications.

Comparison of pH-sensitive degradability of maleic acid amide derivatives.

We synthesized five maleic acid amide derivatives (maleic, citraconic, cis-aconitic, 2-(2'-carboxyethyl) maleic, 1-methyl-2-(2'-carboxyethyl) maleic acid amide), and compared their degradability for the future development of pH-sensitive biomaterials with tailored kinetics of the release of drugs, the change of charge density, and the degradation of scaffolds. The degradation kinetics was highly dependent upon the substituents on the cis-double bond. Among the maleic acid amide derivatives, 2-(2'-carboxyethyl) maleic acid amide with one carboxyethyl and one hydrogen substituent showed appropriate degradability at weakly acidic pH, and the additional carboxyl group can be used as a pH-sensitive linker.

Lower critical solution temperature (LCST) phase separation of glycol ethers for forward osmotic control.

Lower critical solution temperature (LCST) phase transition of glycol ether (GE)-water mixtures induces an abrupt change in osmotic pressure driven by a mild temperature change. The temperature-controlled osmotic change was applied for the forward osmosis (FO) desalination. Among three GEs evaluated, di(ethylene glycol) n-hexyl ether (DEH) was selected as a potential FO draw solute. A DEH-water mixture with a high osmotic pressure could draw fresh water from a high-salt feed solution such as seawater through a semipermeable membrane at around 10 °C. The water-drawn DEH-water mixture was phase-separated into a water-rich phase and a DEH-rich phase at around 30 °C. The water-rich phase with a much reduced osmotic pressure released water into a low-salt solution, and the DEH-rich phase was recovered into the initial DEH-water mixture. The phase separation behaviour, the residual GE concentration in the water-rich phase, the osmotic pressure of the DEH-water mixture, and the osmotic flux between the DEH-water mixture and salt solutions were carefully analysed for FO desalination. The liquid-liquid phase separation of the GE-water mixture driven by the mild temperature change between 10 °C and 30 °C is very attractive for the development of an ideal draw solute for future practical FO desalination.

Diagnostic Accuracy of Preoperative Radiologic Findings in Papillary Thyroid Microcarcinoma: Discrepancies with the Postoperative Pathologic Diagnosis and Implications for Clinical Outcomes.

BACKGRUOUND: The diagnostic accuracy of preoperative radiologic findings in predicting the tumor characteristics and clinical outcomes of papillary thyroid microcarcinoma (PTMC) was evaluated across all risk groups. METHODS: In total, 939 PTMC patients, comprising both low-risk and non-low-risk groups, who underwent surgery were enrolled. The preoperative tumor size and lymph node metastasis (LNM) were evaluated by ultrasonography within 6 months before surgery and compared with the postoperative pathologic findings. Discrepancies between the preoperative and postoperative tumor sizes were analyzed, and clinical outcomes were assessed. RESULTS: The agreement rate between radiological and pathological tumor size was approximately 60%. Significant discrepancies were noted, including an increase in tumor size in 24.3% of cases. Notably, in 10.8% of patients, the postoperative tumor size exceeded 1 cm, despite being initially classified as 0.5 to 1.0 cm based on preoperative imaging. A postoperative tumor size >1 cm was associated with aggressive pathologic factors such as multiplicity, microscopic extrathyroidal extension, and LNM, as well as a higher risk of distant metastasis. In 30.1% of patients, LNM was diagnosed after surgery despite not being suspected before the procedure. This group was characterized by smaller metastatic foci and lower risks of distant metastasis or recurrence than patients with LNM detected both before and after surgery. CONCLUSION: Among all risk groups of PTMCs, a subset showed an increase in tumor size, reaching 1 cm after surgery. These cases require special consideration due to their association with adverse clinical outcomes, including an elevated risk of distant metastasis.

Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain.

BACKGROUND: The phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms. RESULTS: We develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation. CONCLUSIONS: We achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

Ethanolamine bacterial microcompartments: from structure, function studies to bioengineering applications.

Two decades of structural and functional studies have revealed functions, structures and diversity of bacterial microcompartments. The protein-based organelles encapsulate diverse metabolic pathways in semipermeable, icosahedral or pseudo-icosahedral shells. One of the first discovered and characterized microcompartments are those involved in ethanolamine degradation. This review will summarize their function and assembly along with shared and unique characteristics with other microcompartment types. The modularity and self-assembling properties of their shell proteins make them valuable targets for bioengineering. Advances and prospects for shell protein engineering in vivo and in vitro for synthetic biology and biotechnology applications will be discussed.

Comparison of Korean vs. American Thyroid Imaging Reporting and Data System in Malignancy Risk Assessment of Indeterminate Thyroid Nodules.

BACKGROUND: The management of cytologically indeterminate thyroid nodules is challenging for clinicians. This study aimed to compare the diagnostic performance of the Korean Thyroid Imaging Reporting and Data Systems (K-TIRADS) with that of the American College of Radiology (ACR)-TIRADS for predicting the malignancy risk of indeterminate thyroid nodules. METHODS: Thyroid nodules diagnosed by fine-needle aspiration (FNA) followed by surgery or core needle biopsy at a single referral hospital were enrolled. RESULTS: Among 200 thyroid nodules, 78 (39.0%) nodules were classified as indeterminate by FNA (Bethesda category III, IV, and V), and 114 (57.0%) nodules were finally diagnosed as malignancy by surgery or core needle biopsy. The area under the curve (AUC) was higher for FNA than for either TIRADS system in all nodules, while all three methods showed similar AUCs for indeterminate nodules. However, for Bethesda category III nodules, applying K-TIRADS 5 significantly increased the risk of malignancy compared to a cytological examination alone (50.0% vs. 26.5%, P=0.028), whereas applying ACR-TIRADS did not lead to a change. CONCLUSION: K-TIRADS and ACR-TIRADS showed similar diagnostic performance in assessing indeterminate thyroid nodules, and K-TIRADS had beneficial effects for malignancy prediction in Bethesda category III nodules.

Engineering Bacillus subtilis for the formation of a durable living biocomposite material.

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.

A dimeric α-helical cell penetrating peptide mounted with an HER2-selective affibody.

We have developed a cell penetrating peptide (CPP) system with high selectivity and penetrability at nanomolar concentrations with a combination of an HER2-selective affibody, ZHER2:342 (ZHER2), and a dimeric α-helical leucine- and lysine-rich peptide, LK-2. ZHER2 and LK-2 are linearly fused together and expressed in a prokaryotic system to create the LK-2-ZHER2 protein, which can successfully distinguish and penetrate HER2-overexpressing cancer cells at nanomolar concentrations. LK-2-ZHER2 has the ability to intracellularly deliver doxorubicin as a conjugate form to enhance its anti-cancer effect on HER2-overexpressing breast cancer cells with a great selectivity. The selective penetrability was confirmed in vitro, in 3D spheroids, and in in vivo models. LK-2-ZHER2 has the capability to overcome the weak points of current CPPs, such as poor penetrability at low concentrations and a lack of selectivity, by combining powerful CPP and affibody sequences.

Use of serotonin reuptake inhibitors and risk of subsequent bone loss in a nationwide population-based cohort study.

This study examined whether the use of SRIs is associated with an increased risk of bone loss using a nested case-control design with a nationwide population-based cohort in Korea. Using the Korean National Health Screening Cohort, subjects newly diagnosed with osteoporosis or osteopenia (n = 55,799) were matched with controls (n = 278,995) at a ratio of 1:5. We stratified the participants by their time-dependent use of SRIs and sex and controlled for various confounders, including lifestyle habits, laboratory data, and comorbidities. Conditional logistic regression showed that both recent and former users of SRIs had an increased risk of subsequent bone loss compared with non-users: men [recent users: odds ratio (OR) 1.35, 95% confidential interval (CI) 1.20, 1.53; former-users: OR 1.10, 95% CI 1.01, 1.20]; women (recent users: OR 1.38, 95% CI 1.28-1.48; former-users: OR 1.07, 95% CI 1.02, 1.21). The use of SRIs was associated with an increased risk of bone loss in both men and women. In particular, the association was stronger in recent users. These findings provide population-level evidence for the risk of bone loss associated with SRI exposure and highlight the importance of monitoring the bone health of SRI users.

Sex differences in sarcopenia and frailty among community-dwelling Korean older adults with diabetes: The Korean Frailty and Aging Cohort Study.

AIMS/INTRODUCTION: We aimed to examine the prevalence of sarcopenia and frailty in Korean older adults with diabetes compared with individuals without diabetes. MATERIALS AND METHODS: We analyzed the data of 2,403 participants aged 70-84 years enrolled in the Korean Frailty and Aging Cohort Study. Sarcopenia was defined using the Asian Working Group for Sarcopenia and the Foundation for the National Institutes of Health. Frailty was assessed by the Cardiovascular Health Study frailty phenotype criteria. RESULTS: The mean age of the participants was 76.0 ± 3.9 years, and 47.2% were men. The prevalence of diabetes was 30.2% in men and 25.8% in women. Adults with diabetes showed a lower muscle mass index (appendicular skeletal muscle mass/body mass index) and handgrip strength in both sexes, but only the women showed decreased physical performance. Women with diabetes presented a higher prevalence of sarcopenia diagnosed by the Foundation for the National Institutes of Health criteria, and frailty compared with participants without diabetes (sarcopenia 14.7% vs 8.5%, P = 0.001; frailty 9.5% vs 4.9%, P = 0.003). Men in the high and middle tertiles for homeostatic model assessment of insulin resistance presented a significantly higher prevalence of sarcopenia, compared with men in the low tertile homeostatic model assessment of insulin resistance (high tertile 16.6%, middle tertile 13.3%, low tertile 8.6%). CONCLUSIONS: In older adults with diabetes, muscle mass index and muscle strength were lower than in those without diabetes. However, the prevalence of sarcopenia and frailty was higher and physical performance was lower only in women with diabetes.

Dynamics and Entropy of Cyclohexane Rings Control pH-Responsive Reactivity.

Activation entropy (ΔS ‡) is not normally considered the main factor in determining the reactivity of unimolecular reactions. Here, we report that the intramolecular degradation of six-membered ring compounds is mainly determined by the ΔS ‡, which is strongly influenced by the ring-flipping motion and substituent geometry. Starting from the unique difference between the pH-dependent degradation kinetics of geometric isomers of 1,2-cyclohexanecarboxylic acid amide (1,2-CHCAA), where only the cis isomer can readily degrade under weakly acidic conditions (pH < 5.5), we found that the difference originated from the large difference in ΔS ‡ of 16.02 cal·mol-1·K-1. While cis-1,2-CHCAA maintains a preference for the classical chair cyclohexane conformation, trans-1,2-CHCAA shows dynamic interconversion between the chair and twisted boat conformations, which was supported by both MD simulations and VT-NMR analysis. Steric repulsion between the bulky 1,2-substituents of the trans isomer is one of the main reasons for the reduced energy barrier between ring conformations that facilitates dynamic ring inversion motions. Consequently, the more dynamic trans isomer exhibits much a larger loss in entropy during the activation process due to the prepositioning of the reactant than the cis isomer, and the pH-dependent degradation of the trans isomer is effectively suppressed. When the ring inversion motion is inhibited by an additional methyl substituent on the cyclohexane ring, the pH degradability can be dramatically enhanced for even the trans isomer. This study shows a unique example in which spatial arrangement and dynamic properties can strongly influence molecular reactivity in unimolecular reactions, and it will be helpful for the future design of a reactive structure depending on dynamic conformational changes.