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Background: Growth hormone deficiency(GHD) and idiopathic short stature(ISS) are the primary causes of short stature in children. Animal experiments have revealed a link between growth hormone(GH), gut microbiota and metabolism, however, limited information is available from human trials., Methods: Fecal samples collected from GHD (n = 36), ISS (n = 32) and healthy control (HC) children(n = 16) were subjected to microbiome (16 S rRNA gene sequencing) and metabolome (nuclear magnetic resonance,NMR) analyses., Results: GHD, ISS and HC exhibit distinct differences in beta diversity of gut microbiota.In addition, short stature (GHD and ISS) exhibit higher relative abundance of Prevotellaceae_NK3B31_group at genus level compared to HC, whereas Rodentibacter, Rothia, and Pelomonas showed lower abundance. Additionally,Fusobacterium_mortiferum was identified as the characteristic species of GHD. Moreover, glucose metabolism, pyruvate metabolism and pyrimidine metabolism might play significant roles for distinguishing between GHD and normal GH groups (ISS and HC). Furthermore, a disease prediction model based on differential bacteria and metabolites between GHD and ISS exhibited high diagnostic value., Conclusion: These findings highlight the characteristics of different GH levels on the gut microbiota and metabolism in children, providing novel perspectives for early diagnosis and prognostic treatment of short stature with abnormal GH levels., Impact: The key message of our study is to identify human-relevant gut microbiota and host metabolic patterns that are interfered with growth hormone levels, and to develop biomarker models to identify short stature associated with growth hormone deficiency. We used idiopathic short stature as a control group for growth hormone deficiency, complementing the absence of height as a factor in the existing literature. Our study ultimately hopes to shed new light on the diagnosis and treatment of short stature children associated with growth hormone deficiency., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Objectives: The study aimed to explore the underlying mechanisms of OSA-related cognitive impairment by investigating the altered topology of brain white matter networks in children with OSA., Methods: Graph theory was used to examine white matter networks' network topological properties in 46 OSA and 31 non-OSA children. All participants underwent MRI, polysomnography, and cognitive testing. The effects of the obstructive apnea-hypopnea index (OAHI) on topological properties of white matter networks and network properties on cognition were studied using hierarchical linear regression. Mediation analyses were used to explore whether white matter network properties mediated the effects of OAHI on cognition., Results: Children with OSA had significantly higher assortativity than non-OSA children. Furthermore, OAHI was associated with the nodal properties of several brain regions, primarily in the frontal and temporal lobes. The relationship between OAHI and verbal comprehension index was mediated through clustering coefficients in the right temporal pole of the superior temporal gyrus., Conclusions: OSA affects the development of white matter networks in children's brains. Besides, the mediating role of white matter network properties between the OAHI and the verbal comprehension index provided neuroimaging evidence of impaired cognitive function in children with OSA., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biofouling and bacterial infections are significant challenges in biomedical devices. In this study, a biocompatible dual-functional coating with antimicrobial and antifouling properties is developed by co-depositing the zwitterionic copolymer and silver nanoparticles via a dopamine-assisted strategy. Inspired by mussel adhesion, the coating exhibits substrate-independent adhesion as a result of the formation of irreversible covalent bonds. The zwitterionic copolymer in the dual coating plays a crucial role in improving surface wettability and reducing protein adsorption and platelet and bacterial adhesion, thereby improving its antifouling property significantly. The silver nanoparticles reduced by self-polymerized polydopamine without the addition of any chemical reductants can effectively improve the antimicrobial activity. Furthermore, as the zwitterion content in the zwitterion polymer increases, the antibacterial and antifouling properties of the coating can be further advanced. The simple and effective approach presented here provides a promising pathway for constructing potent antibacterial and antifouling surfaces, demonstrating great potential for clinical applications in implanted materials.

Prulifloxacin, a broad-spectrum quinolone antibiotic, exhibits three distinct crystal forms, each with different bioavailability and therapeutic properties. It is imperative to assess and control the proportion of each crystal form during the production of raw materials and preparations. Therefore, it is necessary to establish an analytical method that can determine the content of each crystal form in the ternary polycrystalline mixtures. In this study, prulifloxacin crystal forms were analyzed and quantitatively measured using Raman spectroscopy. First, three pure crystal forms of prulifloxacin were prepared under different crystallization conditions and mixed into ternary mixtures at the designed proportions. Subsequently, the ternary mixed crystal samples were analyzed using a Raman microscope.Then run a partial least squares regression analysis to establish a PLS quantitative model using the average spectra data, and a non-negative least squares analysis to establish an area percentage quantitative model using Raman imaging data.The method validation results showed that the two models successfully predicted the proportion of each crystal form within the prulifloxacin polycrystalline mixtures, with a prediction accuracy of less than ± 10 %. Raman spectroscopy was thus established as an effective method for crystal form analysis and quantitative measurement of prulifloxacin., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liwen ZHANG reports financial support was provided by Science and Technology Program of Guangzhou,Science and Technology Program of Guangzhou Municipal Public Security Bureau., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Anthraquinone is a redox mediator that can effectively catalyze the degradation of azo dyes by promoting the electron transfer. In this study, a mediator membrane with poly (vinylidene fluoride) (PVDF) as the membrane support and 1,8-dichloroanthraquinone (1,8-AQ) and graphene oxide (GO) as the additives was prepared and characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP), atomic force microscopy (AFM) and water contact angle. The introduction of GO increases the pure water flux of the membrane to 258.56 ±12.93 L/(m 2 ·h). Its catalytic performances for the biodegradation of azo dyes were evaluated. Under the optimized conditions, the 1,8-AQ/GO/PVDF composite membrane is able to improve the dye degradation efficiency 2.2 times for reactive red X-3B and 1.1 times for acid red B, as compared with PVDF membrane. In addition, the mediator membrane maintains stable and high catalytic efficiency in the cyclic test and over 90 % dye degradation efficiency is still obtained after 5 cycles of decolorization. These results suggest the great application potentials of the 1,8-AQ/GO/PVDF membrane in the dye wastewater treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Highly nutritious traditional plants which are rich in bioactive substances are attracting increasing attention. In this study, the nutritional value, chemical composition, biological activities, and feed indices of different parts of Millettia speciosa were comprehensively evaluated. In terms of its nutritional value, this study demonstrated that the leaves, flowers and seeds of M. speciosa were rich in elements and amino acids; the biological values (BVs) of these ingredients ranged from 85% to 100%, showing the extremely high nutritional value of this plant. GC-MS analysis suggested that the main chemical components of the flower volatile oil were n -hexadecanoic acid (21.73%), tetracosane (19.96%), and pentacosane (5.86%). The antibacterial activities of the flower and seed extracts were significantly stronger than those of the leaves and branches. The leaf extract displayed the strongest antifungal activities (EC 50 values: 18.28 ± 0.54 μg/mL for Pseudocryphonectria elaeocarpicola and 568.21 ± 33.60 μg/mL for Colletotrichum gloeosporioides ) and were the least toxic to mouse fibroblasts (L929) (IC 50 value: 0.71 ± 0.04 mg/mL), while flowers were the most toxic (IC 50 value: 0.27 ± 0.03 mg/mL). In addition, the abundance of fiber, protein, mineral elements, and functional metabolite contents indicated the potential applicability of M. speciosa as an animal feed. In conclusion, as a traditional herbal plant used for medicinal and food purposes, M. speciosa shows potential for safe and multifunctional development.

New consensus indicates type 2 diabetes mellitus (T2DM) and periodontitis as comorbidity and may share common pathways of disease progression. Sulfonylureas have been reported to improve the periodontal status in periodontitis patients. Glipizide, a sulfonylurea widely used in the treatment of T2DM, has also been reported to inhibit inflammation and angiogenesis. The effect of glipizide on the pathogenicity of periodontitis, however, has not been studied. We developed ligature-induced periodontitis in mice and treated them with different concentrations of glipizide and then analyzed the level of periodontal tissue inflammation, alveolar bone resorption, and osteoclast differentiation. Inflammatory cell infiltration and angiogenesis were analyzed using immunohistochemistry, RT-qPCR, and ELISA. Transwell assay and Western bolt analyzed macrophage migration and polarization. 16S rRNA sequencing analyzed the effect of glipizide on the oral microbial flora. mRNA sequencing of bone marrow-derived macrophages (BMMs) stimulated by P. gingivalis lipopolysaccharide (Pg-LPS) after treatment with glipizide was analyzed. Glipizide decreases alveolar bone resorption, periodontal tissue degradation, and the number of osteoclasts in periodontal tissue affected by periodontitis (PAPT). Glipizide-treated periodontitis mice showed reduced micro-vessel density and leukocyte/macrophage infiltration in PAPT. Glipizide significantly inhibited osteoclast differentiation in vitro experiments. Glipizide treatment did not affect the oral microbiome of periodontitis mice. mRNA sequencing and KEGG analysis showed that glipizide activated PI3K/AKT signaling in LPS-stimulated BMMs. Glipizide inhibited the LPS-induced migration of BMMs but promoted M2/M1 macrophage ratio in LPS-induced BMMs via activation of PI3K/AKT signaling. In conclusion, glipizide inhibits angiogenesis, macrophage inflammatory phenotype, and osteoclastogenesis to alleviate periodontitis pathogenicity suggesting its' possible application in the treatment of periodontitis and diabetes comorbidity., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Serum amyloid P component (SAP) regulates the innate immune system and microbial diseases. Periodontitis is an inflammatory oral disease developed by the host immune system's interaction with the dysbiotic oral microbiome, thereby SAP could play a role in periodontitis pathogenicity., Objectives: To investigate the role of SAP in oral microbiome modulation and peridontitis pathogenicity., Methods: In this study, wildtype and SAP-knockout (KO) mice were used. Ligature-based periodontitis was developed in mice. Oral microbiome diversity was analyzed by 16 s rRNA sequencing. Macrophages and Porphyromonas gingivalis (P. gingivalis) co-culture system analyzed the effect of SAP in macrophage phagocytosis of P. gingivalis., Results: The level of SAP was upregulated in the periodontitis-affected periodontium of humans and mice but not in the liver and blood circulation. Periodontal macrophages were the key source of upregulated SAP in periodontitis. SAP-KO aggravated periodontal inflammation, periodontitis, and a higher number of M1-type inflammatory macrophage infiltration in the periodontium. The oral microbiome of SAP-KO periodontitis mice was altered with a higher abundance of Porphyromonas at the genus level. SAP-KO macrophages showed compromised phagocytosis of P. gingivalis in the co-culture system. Co-culture of SAP-KO macrophages and P. gingivalis induced the C5a expression and exogenous SAP treatment nullified this effect. Exogenous recombinant SAP treatment did not affect P. gingivalis growth and opsonization. PMX205, an antagonist of C5a, treatment robustly enhanced P. gingivalis phagocytosis by SAP-KO macrophages, indicating the involvement of the C5a-C5aR signaling in the compromised P. gingivalis phagocytosis by SAP-KO macrophages., Conclusion: SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of P. gingivalis. A higher abundance of P. gingivalis during SAP deficiency could promote M1 macrophage polarization and periodontitis. This finding suggests the possible protecting role of elevated levels of periodontal SAP against periodontitis progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Objective: Obstructive sleep apnea (OSA) seriously affects the children's cognitive functions, but the neuroimaging mechanism of cognitive impairment is still unclear. The purpose of our study was to explore the difference in brain local gray matter volume (GMV) between children with OSA and non-OSA, and the correlation between the difference regions of brain gray matter volume and cognitive, the severity of OSA., Method: Eighty-three children aged 8-13 years were recruited in our study, 52 children were diagnosed as OSA by polysomnography, and 31 as the non-OSA. All the subjects were underwent high-resolution 3-dimensional T1-weighted magnetic resonance images. The voxel-based morphometry (VBM) was be used to analyse the local GMV. The Das-Naglieri cognitive assessment system (DN: CAS) was used to assess the subjects' cognitive. The difference of local GMV between the two groups was analyzed by two-sample T -test. The PSG variables and the scores of DN: CAS between the OSA group and non-OSA group were compared by independent samples t -tests. Pearson correlation was used to calculate the association between the difference areas of gray matter volumes in brain and DN: CAS scores, obstructive apnea/hypopnea index (OAHI, an index of the severity of OSA)., Results: The gray matter volume of the right Middle Frontal Gyrus (MFG_R) in OSA children were larger than the non-OSA children, and the OSA children had lower scores of the Word Series in DN: CAS. There was negative correlation between the scores of Expressive Attention in DN: CAS and the gray matter volume of the right middle frontal gyrus, and it was no significantly correlation between OAHI and the gray matter volume of the right middle frontal gyrus., Conclusion: Our results suggest that the development of gray matter volume in frontal cortex, which associated with attention, were sensitive to the effects of OSA, provides neuroimaging evidence for cognitive impairment in children with OSA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yu, Fu, Lu, Huang, Chen, Wei, Li, Ampadu, Wang, Zheng, Jiang, Li, Lui and Cai.)

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders., Competing Interests: D.J.Z. and X.C. are inventors on an intellectual property disclosure related to the technology described in the manuscript that has been filed with Johns Hopkins University, which seeks to license the technology., (© 2023 The Author(s).)

MicroRNA-155 (miR155) is overexpressed in various inflammatory diseases and cancer, in which bone resorption and osteolysis are frequently observed. However, the role of miR155 on osteogenesis and bone mass phenotype is still unknown. Here, we report a low bone mass phenotype in the long bone of Mir155 -Tg mice compared with wild-type mice. In contrast, Mir155 -KO mice showed a high bone mass phenotype and protective effect against inflammation-induced bone loss. Mir155 -KO mice showed robust bone regeneration in the ectopic and orthotopic model, but Mir155 -Tg mice showed compromised bone regeneration compared with the wild-type mice. Similarly, the osteogenic differentiation potential of bone marrow stromal stem cells (BMSCs) from Mir155 -KO mice was robust and Mir155 -Tg was compromised compared with that of wild-type mice. Moreover, Mir155 knockdown in BMSCs from wild-type mice showed higher osteogenic differentiation potential, supporting the results from Mir155 -KO mice. TargetScan analysis predicted sphingosine 1-phosphate receptor-1 ( S1pr1 ) as a target gene of Mir155 , which was further confirmed by luciferase assay and Mir155 knockdown. S1pr1 overexpression in BMSCs robustly promoted osteogenic differentiation without affecting cell viability and proliferation. Furthermore, osteoclastogenic differentiation of Mir155 -Tg bone marrow-derived macrophages was inhibited compared with that of wild-type mice. Thus, Mir155 showed a catabolic effect on osteogenesis and bone mass phenotype via interaction with the S1pr1 gene, suggesting inhibition of Mir155 as a potential strategy for bone regeneration and bone defect healing., Competing Interests: ZZ, LW, ZL, RT, HL, YH, TW, SX, HC, ZY, DX, XL, GW, RJ, JP No competing interests declared, (© 2023, Zheng, Wu, Li et al.)

Background: Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood., Results: In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects., Conclusions: This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

Piriformospora indica, a root endophytic fungus, has been shown to enhance biomass production and confer tolerance to various abiotic and biotic stresses in many plant hosts. A growth chamber experiment of soybean (Glycine max) colonized by P. indica compared to uninoculated control plants showed that the fungus significantly increased shoot dry weight, nutrient content, and rhizobial biomass. RNA-Seq analyses of root tissue showed upregulation of 61 genes and downregulation of 238 genes in colonized plants. Gene Ontology (GO) enrichment analyses demonstrated that upregulated genes were most significantly enriched in GO categories related to lignin biosynthesis and regulation of iron transport and metabolism but also mapped to categories of nutrient acquisition, hormone signaling, and response to drought stress. Metabolic pathway analysis revealed upregulation of genes within the phenylpropanoid and derivative pathways such as biosynthesis of monolignol subunits, flavonoids and flavonols (luteolin and quercetin), and iron scavenging siderophores. Highly enriched downregulated GO categories included heat shock proteins involved in response to heat, high-light intensity, hydrogen peroxide, and several related to plant defense. Overall, these results suggest that soybean maintains an association with this root endosymbiotic fungus that improves plant growth and nutrient acquisition, modulates abiotic stress, and promotes synergistic interactions with rhizobia.

Schistosomiasis japonica, caused by Schistosoma japonicum , is an endemic, zoonotic parasitic disease. Domestic animals, particularly bovines, are thought to play an important role in transmission of the disease. Historically, China was the country mostly severely impacted by schistosomiasis japonica, but now prevalence and morbidity have been greatly reduced. Since the mid-1950s when China launched the National Schistosomiasis Control Program, the control of schistosomiasis in domestic animals has been carried out almost synchronously with that of human schistosomiasis, and this concept has been proven to be effective. Generally, the campaign of schistosomiasis japonica control in domestic animals in China went through four phases over the past six decades, namely, the large-scale epidemiological investigation phase, the case screening and small-scale chemotherapy phase, the mass chemotherapy phase, and the infection source control phase. These distinct phases were responsive to changing disease epidemiology, socioeconomic development, and technological advances, resulting in successful attainment of disease control goals.

Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by (60)Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

A 4.6-fold increase in interstitial glutamate was observed following the reverse microdialysis of 5 mM glutamine into the rat hippocampus. Two possible mechanisms of glutamine hydrolysis were examined: (a) an enzymatic glutaminase activity and (b) a non-enzymatic mechanism. Injection of 14C-glutamine at the site of microdialysis followed by microdialysis with artificial cerebral spinal fluid allowed isolation of 14C-glutamine (63%), 14C-glutamate (14%), and a compound tentatively identified as pyroglutamate (22%). In this study, we determined if non-enzymatic pyroglutamate formation from glutamine contributed to the synthesis of glutamate. Pyroglutamate is in chemical equilibrium with glutamate, although under physiological conditions, the chemical equilibrium is strongly in the direction of pyroglutamate. In vitro stability studies indicated that 14C-glutamine and 14C-pyroglutamate are not subject to significant non-enzymatic breakdown at pH 6.5-7.5 at 37 degrees C for up to 8 h. Reverse microdialysis with 1 mM pyroglutamate did not increase interstitial glutamate levels. Following injection of 14C-pyroglutamate and microdialysis, radioactivity was recovered in 14C-pyroglutamate (88%) and 14C-glutamine (11%). Less than 1% of the radioactivity was recovered as glutamate. Our data do not support a role of pyroglutamate as an intermediate in the formation of extracellular glutamate following the infusion of glutamine. However, it confirms that pyroglutamate, a known constituent in brain, is actively metabolized in brain cells and contributes to glutamine in the interstitial space.

Identification of antibody targets associated with malignant disease is indispensable to developing passive and active antibody-based therapeutics or diagnostic agents. We have developed a novel technique combining Western blotting, genetic profiling, and mass spectroscopy that allows for the rapid and unambiguous identification of such antigens in a high-throughput manner. Herein, we demonstrate this technique, designated Ab SCAN, by deducing the known target of a monoclonal antibody and by identifying a new antigen that was observed to be the frequent target of humoral immune responses in prostate cancer patients. In both instances, a specific antigen emerged as the sole protein candidate. The newly identified antigen, mannose-6-phosphate/IGF II receptor, may be an important naturally immunogenic antigen involved in prostate cancer. The Ab SCAN technique is uniquely suited to the analysis of longitudinal serum samples from clinical studies and could be a powerful tool to correlate humoral immune responses directed against discreet antigens with clinical events.