Metabolic profiling of idiopathic pulmonary fibrosis in a mouse model: implications for pathogenesis and biomarker discovery.

Background: Alterations in metabolites and metabolic pathways are thought to be important triggers of idiopathic pulmonary fibrosis (IPF), but our lack of a comprehensive understanding of this process has hampered the development of IPF-targeted drugs. Methods: To fully understand the metabolic profile of IPF, C57BL/6 J male mice were injected intratracheally with bleomycin so that it could be used to construct a mouse model of IPF, and lung tissues from 28-day and control IPF mice were analyzed by pathology and immunohistochemistry. In addition, serum metabolites from IPF mice were examined using LC-ESI-MS/MS, and the differential metabolites were analyzed for KEGG metabolic pathways and screened for biomarkers using machine learning algorithms. Results: In total, the levels of 1465 metabolites were detected, of which 104 metabolites were significantly altered after IPF formation. In IPF mouse serum, 52% of metabolite expression was downregulated, with lipids (e.g., GP, FA) and organic acids and their derivatives together accounting for more than 70% of the downregulated differentially expressed metabolites. In contrast, FA and oxidised lipids together accounted for 60% of the up-regulated differentially expressed metabolites. KEGG pathway enrichment analyses of differential metabolites were mainly enriched in the biosynthesis of unsaturated fatty acids, pentose phosphate pathway, and alanine, aspartate, and glutamate metabolism. Seven metabolites were screened by machine learning LASSO models and evaluated as ideal diagnostic tools by receiver operating characteristic curves (ROCs). Discussion: In conclusion, the serum metabolic disorders found to be associated with pulmonary fibrosis formation will help to deepen our understanding of the pathogenesis of pulmonary fibrosis.

The Complexity of Volatile Terpene Biosynthesis in Roses: Particular Insights into ß-Citronellol Production.

The fascinating scent of rose (Rosa genus) flowers has captivated human senses for centuries, making them one of the most popular and widely used floral fragrances. Despite much progress over the last decade, many biochemical pathways responsible for rose scents remain unclear. We analyzed the floral scent compositions from various rose varieties and selected the modern cultivar Rosa hybrida 'Double Delight' as a model system to unravel the formation of rose dominant volatile terpenes, which contribute substantially to the rose fragrance. Key genes involved in rose terpene biosynthesis were functionally characterized. Cytosolic geranyl diphosphate (GPP) generated by geranyl/farnesyl diphosphate synthase (G/FPPS1) catalysis, played a pivotal role in rose scent production, and terpene synthases (TPSs) in roses play an important role in the formation of most volatile terpenes, but not for geraniol, citral or ß-citronellol. Subsequently, a series of enzymes, including geraniol dehydrogenase (GeDH), geranial reductase (GER), 12-oxophytodienoate reductase (OPR) and citronellal reductase (CAR), were characterized as involved in the transformation of geraniol to ß-citronellol in roses through three successive steps. Interestingly, the ß-citronellol biosynthesis pathway appears to be conserved in other horticultural plants like Lagerstroemia caudata and Paeonia lactiflora. Our findings provide valuable insights into the biosynthesis of rose volatile terpenoid compounds and offer essential gene resources for future breeding and molecular modification efforts.

Corrigendum: The central inflammatory regulator IκBζ: induction regulation and physiological functions.

[This corrects the article DOI: 10.3389/fimmu.2023.1188253.].

Characterization of pneumococcal serotype 7F in vaccine conjugation.

Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.

Characterization and anti-aging effects of polysaccharide from Gomphus clavatus Gray.

In this study, a highly branched polysaccharide (GPF, 112.0 kDa) was isolated and purified from Gomphus clavatus Gray fruiting bodies. GPF was primarily composed of mannose, galactose, arabinose, xylose, and glucose at a molar ratio of 3.2:1.9:1.6:1.2:1.0. GPF was a highly branched heteropolysaccharide composed of 13 glucosidic bonds, with a degree of branching (DB) of 48.85 %. GPF exhibited anti-aging activity in vivo, significantly increased antioxidant enzymes activities (SOD, CAT and GSH-Px), improved total antioxidant capability (T-AOC) and decreased MDA level in the serum and brain of d-Gal induced aging mice. Behavioral experiments showed that GPF effectively improved learning and memory deficits in d-Gal induced aging mice. Mechanistic studies indicated that GPF could activate AMPK by increasing AMPK phosphorylation and upregulating SIRT1 and PGC-1α expression. These findings suggest that GPF has significant potential as a natural candidate to slow down aging and prevent aging-related diseases.

The central inflammatory regulator IκBζ: induction, regulation and physiological functions.

IκBζ (encoded by NFKBIZ) is the most recently identified IkappaB family protein. As an atypical member of the IkappaB protein family, NFKBIZ has been the focus of recent studies because of its role in inflammation. Specifically, it is a key gene in the regulation of a variety of inflammatory factors in the NF-KB pathway, thereby affecting the progression of related diseases. In recent years, investigations into NFKBIZ have led to greater understanding of this gene. In this review, we summarize the induction of NFKBIZ and then elucidate its transcription, translation, molecular mechanism and physiological function. Finally, the roles played by NFKBIZ in psoriasis, cancer, kidney injury, autoimmune diseases and other diseases are described. NFKBIZ functions are universal and bidirectional, and therefore, this gene may exert a great influence on the regulation of inflammation and inflammation-related diseases.

Persistent Facial Flushing in a Patient with Telangiectasia Macularis Eruptiva Perstans: An Unusual but Should Emphasized Clinical Finding.

Facial flushing is one of the common conditions in dermatology, which affects the aesthetic of patients to a great extent, and even leads to psychological and economic burdens. The most common causes of facial flushing are often inflammatory skin diseases such as rosacea, contact dermatitis, and others, but the facial flushing as a sign can also be the cutaneous manifestation of systemic disease. Telangiectasia macularis eruptiva perstans (TMEP) is a rare disease associated with mast cells. Here, we describe an unusual clinical finding with persistent facial flushing in a patient with TMEP.

Serum Level of Brain-Derived Neurotrophic Factor (BDNF) Associated with Depression in Patients with Rosacea: A Candidate Predictive Biomarker.

Background: The biomarker to predict the depression in patients with rosacea was absent. Objective: We aimed to explore the potential association between BDNF and depression in patients with rosacea, and also to determine whether serum BDNF level is a potential biomarker for identifying depression in patients with rosacea. Methods: The patients with rosacea, rosacea with depression and healthy control were included, clinical evaluation (DLQI, RSSs, BDI-II) and serum BDNF levels detection were performed on subjects, the comparisons and correlation analysis of the obtained data were performed. Results: In clinical evaluation, whether DLQI or RSSs, rosacea with depression group was significantly higher compared to rosacea group. Besides, we found the serum BDNF levels were lower in patients with rosacea and rosacea with depression compared to healthy controls, also in the rosacea with depression group, serum BDNF levels were lower than in rosacea patients. Whatever in rosacea or rosacea with depression group, the statistical significance of serum BDNF levels between the different subtypes like the ETR and PPR was not found. In further correlation analysis, we found no correlation between serum BDNF and RSSs in patients with rosacea whatever the subtype of ETR or PPR. Interestingly, we found a negative correlation between serum BDNF levels and BDI-II in rosacea with depression group, the decreased serum BDNF levels were associated with the increased BDI-II, also the ROC confirmed it can evaluate the depression in patients with rosacea. Conclusion: Serum BDNF level is a potential biomarker for identifying depression in patients with rosacea.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Discovery of Insulin Receptor Partial Agonists MK-5160 and MK-1092 as Novel Basal Insulins with Potential to Improve Therapeutic Index.

We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of ∼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.

Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations.

The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.

Simultaneous Determination of 25 Ginsenosides by UPLC-HRMS via Quantitative Analysis of Multicomponents by Single Marker.

A method using UPLC-HRMS has been developed for a rapid, simultaneous qualitative and quantitative analysis of twenty-five ginsenosides. Chromatographic separation was achieved on a C18 analytical column with an elution gradient comprising 0.1% aqueous formate/acetonitrile as the mobile phase. HRMS detection acquired full mass data for quantification and fullms-ddms2 (i.e., data-dependent scan mode) yielded product ion spectra for identification. Furthermore, quantitative analysis of multiginsenosides by single marker (QAMS) was developed and validated using a relative correction factor. Under optimal conditions, we could simultaneously separate eight groups of isomers of the 25 ginsenosides. Good linearity was observed over the validated concentration range for each analyte (r 2 > 0.9924), showing excellent sensitivity (LODs, 0.003-0.349 ng/mL) and lower limit quantification (LOQs, 0.015-1.163 ng/mL). The LC-MS external standard method (ESM) and QAMS were compared and successfully applied to analyze the ginsenoside content from Panax ginseng roots. Overall, our UPLC-HRMS/QAMS approach provides high precision, stability, and reproducibility and can be used for high-throughput analysis of complex ginsenosides and quantitative analysis of multiple components and quality control of traditional Chinese medicines (TCM).

A Co-Processed API Approach for a Shear Sensitive Compound Affording Improved Chemical Stability and Streamlined Drug Product Processing.

The physical properties of active pharmaceutical ingredients (API) are critical to both drug substance (DS) isolation and drying operations, as well as streamlined drug product (DP) processing and the quality of final dosage units. High aspect ratio, low bulk density, API 'needles' in particular are a hindrance to efficient processing, with a low probability that conventional crystallization routes can modify the challenging morphology. The compound evaluated in this manuscript demonstrated this non-ideal morphology, with the added complexity of shear sensitivity. Modest shear exposure resulted in conversion of the thermodynamically stable crystalline phase to the amorphous phase, with the amorphous phase then undergoing accelerated chemical degradation. Slow filtration during DS isolation resulted in uncontrolled and elevated amorphous levels, while subsequent DP operations including blending, densification and compression increased amorphous content still further. A chemically stable final dosage unit would ideally involve a high bulk density, free flowing API that did not require densification in order to be commercialized as an oral dosage form with direct encapsulation of a single dosage unit. Despite every effort to modify the crystallization process, the physical properties of the API could not be improved. Here, an innovative isolation strategy using a thin film evaporation (TFE) process in the presence of a water soluble polymer alleviated filtration and drying risks and consistently achieved a high bulk density, free flowing co-processed API amenable to direct encapsulation. Characterization of the engineered materials suggested the lower amorphous levels and reduced shear sensitivity were achieved by coating surfaces of the API at relatively low polymer loads. This particle engineering route blurred conventional DS/DP boundaries that not only achieved improved chemical stability but also resulted in a optimized material, with simplified and more robust processing operations for both drug substance and drug product.

Chromosome karyotype and stability of new synthetic hexaploid wheat.

Synthetic hexaploid wheat offers breeders ready access to potentially novel genetic variation in wild ancestral species. In this study, we crossed MY3478 (2n = 4x = 28, AABB) as the maternal parent with the stripe rust-resistant SY41 (2n = 2x = 14, DD) as the paternal parent to construct the new hexaploid wheat line NA0928 through natural allopolyploidization. Agronomic traits and the cytology of the S8-S9 generations of NA0928 were analyzed. Abundant variation in agronomic traits was observed among each strain of NA0928 in the S8 generation. Agronomic traits were superior in strains resistant to stripe rust compared with those of highly susceptible strains. The rank order of the coefficients of variation were tiller number (55.3%) > spike length (15.3%) > number of spikelets (13.9%) > plant height (8.7). Number of tillers and spike length are important traits in wheat breeding to improve yield. Cytological observation and fluorescence in situ hybridization showed that the chromosome number and configuration showed rich variation among NA0928 strains in the S9 generation. Chromosome number ranged from 36 to 44. Variation in chromosome karyotype was detected in the A and B subgenomes. Meiotic chromosome behavior in pollen mother cells and multicolor genomic in situ hybridization revealed that two new synthetic hexaploid wheat strains showed genetic stability; one strain was resistant to stripe rust and developed multiple tillers, and the other strain was susceptible to stripe rust, but both showed improved thousand-kernel weight (TKW) weight and produced multiple tillers. The two strains will be valuable germplasm resources for use in wheat breeding.

Transmembrane protein 16A/anoctamin 1 inhibitor T16Ainh-A01 reversed monocrotaline-induced rat pulmonary arterial hypertension.

Transmembrane protein 16A was involved in the development of the monocrotaline-induced pulmonary arterial hypertension model through ERK1/2 activation, and it was considered as potential target for pulmonary arterial hypertension treatment. A pulmonary arterial hypertension rat model was established by intraperitoneal administration of monocrotaline. Noninvasive pulsed-wave Doppler and histological analysis was performed, and it revealed proliferation and remodeling of pulmonary arterioles and right ventricle hypertrophy. In addition, transmembrane protein 16A, proliferating cell nuclear antigen-a proliferate marker, P-ERK1/2 increased following monocrotaline treatment. Expression of transmembrane protein 16A in the pulmonary arteries was co-localized with a specific marker of vascular smooth muscle α-actin. Then, a specific inhibitor of transmembrane protein 16A-T16Ainh-A01 was administered to pulmonary arterial hypertension rats. It was found to alleviate the remodeling of pulmonary arterioles and right ventricle hypertrophy significantly, and decrease the upregulation of proliferating cell nuclear antigen in monocrotaline-induced pulmonary arteries. In addition, T16Ainh-A01 could inhibit the activation of ERK1/2 in pulmonary arterial hypertension model. Transmembrane protein 16A mediated the proliferation and remodeling of pulmonary arterioles in the monocrotaline-induced pulmonary arterial hypertension model. ERK1/2 pathway is one of downstream factors. Long-term use of T16Ainh-A01 in vivo could alleviate remodeling and pressure in pulmonary arterial hypertension.

Ultrahigh performance liquid chromatography methods facilitate the development of glucose-responsive insulin therapeutics.

Insulin oligosaccharide conjugates hold promise as potential glucose-responsive insulins (GRIs), which can improve the therapeutic index of insulins and mitigate the risk of hypoglycemia. A key challenge for the analytical development of such molecules is finding an efficient method to characterize the purity and impurities of conjugated insulins. Using the S-Matrix Fusion QbD-ultrahigh performance liquid chromatography (UHPLC) integrated system, we were able to quickly screen and develop two short UHPLC methods. These methods were used to support process development, clinical batch drug substance (DS) release, and stability studies of MK-2640, an insulin oligosaccharide conjugate. Both methods used a Waters CSH C18 column, with a shallow gradient of acetonitrile to aqueous mobile phase containing 25 mM sodium perchlorate and 0.05% perchloric acid. The 10-min run time method was well suited for process development and monitoring as it was able to separate the main product, MK-2640, six oligosaccharide-substituted recombinant human insulin (RHI) impurities, A21 deamidated MK-2640, and the starting material RHI. The 13-min run time method provided improved separation of the major impurities and demonstrated good chromatographic reproducibility on different instruments or using columns from different lots of stationary phase, which made it ideal for the final DS release. Validation of the 13-min method demonstrated great linearity for both the MK-2640 main peak and its related impurities, low limit of detection (0.02%), and limit of quantitation (0.05%). The high specificity of the method allowed the separation of the degradation products from main peak, thus makes it suitable for stability monitoring. The major impurities in the DS were characterized by two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS).