Individual dynamic prediction and prognostic analysis for long-term allograft survival after kidney transplantation.

BACKGROUND: Predicting allograft survival is vital for efficient transplant success. With dynamic changes in patient conditions, clinical indicators may change longitudinally, and doctors' judgments may be highly variable. It is necessary to establish a dynamic model to precisely predict the individual risk/survival of new allografts. METHODS: The follow-up data of 407 patients were obtained from a renal allograft failure study. We introduced a landmarking-based dynamic Cox model that incorporated baseline values (age at transplantation, sex, weight) and longitudinal changes (glomerular filtration rate, proteinuria, hematocrit). Model performance was evaluated using Harrell's C-index and the Brier score. RESULTS: Six predictors were included in our analysis. The Kaplan-Meier estimates of survival at baseline showed an overall 5-year survival rate of 87.2%. The dynamic Cox model showed the individual survival prediction with more accuracy at different time points (for the 5-year survival prediction, the C-index = 0.789 and Brier score = 0.065 for the average of all time points) than the static Cox model at baseline (C-index = 0.558, Brier score = 0.095). Longitudinal covariate prognostic analysis (with time-varying effects) was performed. CONCLUSIONS: The dynamic Cox model can utilize clinical follow-up data, including longitudinal patient information. Dynamic prediction and prognostic analysis can be used to provide evidence and a reference to better guide clinical decision-making for applying early treatment to patients at high risk.

Characterization of Multifunctional and Non-stereoselective Oxidoreductase RubE7/IstO, Expanding the Functional Diversity of the Flavoenzyme Superfamily.

Flavin-dependent enzymes enable a broad range of redox transformations and generally act as monofunctional and stereoselective catalysts. Herein, we report the investigation of a multifunctional and non-stereoselective FMN-dependent oxidoreductase RubE7 from the rubrolone biosynthetic pathway. Our study outlines a single RubE7-catalysed sequential reduction of three spatially distinct bonds in a tropolone ring and a reversible double-bond reduction and dehydrogenation. The crystal structure of IstO (a RubE7 homologue) with 2.0 Šresolution reveals the location of the active site at the interface of two monomers, and the size of active site is large enough to permit both flipping and free rotation of the substrate, resulting in multiple nonselective reduction reactions. Molecular docking and site mutation studies demonstrate that His106 is oriented towards the substrate and is important for the reverse dehydrogenation reaction.

Bisaspochalasins D and E: Two Heterocycle-Fused Cytochalasan Homodimers from an Endophytic Aspergillus flavipes.

Two heterocycle-fused cytochalasan homodimers, bisaspochalasins D (1) and E (2), were isolated from an endophytic Aspergillus flavipes. Their chemical structures were elucidated using a combination of HRESIMS, NMR, theoretical calculations, and crystallographic techniques. Bisaspochalasin D (1) is dimerized by the first reported naturally occurring triple heterobridged 3,8-dioxa-6-azabicyclo[3.2.1]octane framework, while bisaspochalasin E (2) employs a pyrrole ring as the linking moiety. Possible dimerization mechanisms of bisaspochalasins D and E were proposed. The bioassay screening revealed that bisaspochalasin D showed cytotoxic activities against five cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) with IC50 values ranging from 4.45 to 22.99 µM. Additionally, bisaspochalasin D exhibited neurotrophic activities in a PC12 cell-based assay. At a concentration of 10 µM, bisaspochalasin D can promote neurite growth by inducing a differentiation rate of 12.52% for PC12 cells.

Microbispora fusca sp. nov., a novel actinomycete isolated from the ear of wheat (Triticum aestivum L.).

A novel actinomycete, designated strain NEAU-HEGS1-5T, was isolated from the ear of wheat (Triticum aestivum L.) and characterized using a polyphasic approach. The morphological and chemotaxonomic characteristics of the strain coincided with those of members of the genus Microbispora. The results of 16S rRNA gene sequence analysis showed that the isolate was most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.3 %), Microbispora camponoti 2C-HV3T (99.2 %), Microbispora amethystogenes JCM 3021T (99.1 %) and Microbispora rosea subsp. rosea JCM 3006T (98.5 %). However, two tree-making algorithms supported the position that strain NEAU-HEGS1-5T formed a distinct clade with M. bryophytorum NEAU-TX2-2T, M. camponoti 2C-HV3T and M. rosea subsp. rosea JCM 3006T. Furthermore, multilocus sequence analysis based on the 16S-gyr B-rpo B genes and a combination of DNA-DNA hybridization results and some physiological and biochemical properties demonstrated that the strain could be distinguished from its closest relatives. Therefore, it is proposed that strain NEAU-HEGS1-5T should be classified as representative of a novel species of the genus Microbispora, for which the name Microbispora fusca sp. nov. is proposed. The type strain is NEAU-HEGS1-5T (=CCTCC AA 2019030T=DSM 104648T).

Streptomyces triticagri sp. nov. and Streptomyces triticirhizae sp. nov., two novel Actinobacteria isolated from the rhizosphere soil of wheat (Triticum aestivum L.).

Two novel Actinobacteria, designated strains NEAU-YY421T and NEAU-YY642T, were isolated from the rhizosphere soil of wheat (Triticum aestivum L.) collected from Zhumadian, Henan Province, PR China and characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains NEAU-YY421T and NEAU-YY642T belonged to the genus Streptomyces and strain NEAU-YY421T was most closely related to Streptomyces fumanus CGMCC 4.1732T (97.9 % sequence similarity) and Streptomyces naganishii DSM 40282T (97.8 %), and that of strain NEAU-YY642T to Streptomyces zhaozhouensis LZS-5T (98.0 %) and Streptomyces sedi YIM 65188T (97.5 %). The cell walls of the two strains contained ll-diaminopimelic acid as the diagnostic diamino acid and the whole-cell hydrolysates were glucose and ribose. Multilocus sequence analysis using the concatenated sequences of the atp D, gyr B, rec A, rpo B and trp B genes showed that the two strains formed separate branches in the genus Streptomyces. Moreover, a combination of DNA-DNA hybridization results and cultural and physiological properties indicated that the two strains can be distinguished from their closest phylogenetic relatives. Therefore, strains NEAU-YY421T and NEAU-YY642T belong to two novel species in the genus Streptomyces, for which the names Streptomyces triticagri sp. nov. (NEAU-YY421T=CGMCC 4.7476T=DSM 106775T) and Streptomyces triticirhizae sp. nov. (NEAU-YY642T=CCTCC AA 2018092T=DSM 107172T) are proposed, respectively.

Benwamycins A-G, Trialkyl-Substituted Benzene Derivatives from a Soil-Derived Streptomyces.

Seven new trialkyl-substituted benzene derivatives named benwamycins A-G (1-7), together with three known congeners, 8-10, were isolated from culture broth of the soil-derived Streptomyces sp. KIB-H1471. Their structures were elucidated by using 1D and 2D NMR analyses in combination with HRESIMS data. The absolute configurations of 1-9 were determined by chemical conversion and comparison of circular dichroism spectra and confirmed for 1 by single-crystal X-ray crystallography. Compounds 6 and 7 have a unique γ-pyrone-like ring on one side chain. Compounds 2 and 6 inhibited human T cell proliferation with IC50 values of 14.3 and 12.5 µM, respectively, without obvious cytotoxicity for naïve human T cells. Compounds 3 and 6 could weakly enhance insulin-stimulated glucose uptake.

Microbispora triticiradicis sp. nov., a novel actinomycete isolated from the root of wheat (Triticum aestivum L.).

A novel actinomycete, designated strain NEAU-HRDPA2-9T, was isolated from the roots of wheat (Triticumaestivum L.) and characterized using a polyphasic approach. The morphological and chemotaxonomic characteristics of the strain coincided with those of members of the genus Microbispora. The 16S rRNA gene sequence analysis showed that the isolate was most closely related to Microbispora bryophytorum NEAU-TX2-2T (98.6 %), Microbispora hainanensis DSM 45428T (98.5 %), Microbispora camponoti 2C-HV3T (98.5 %), Microbispora amethystogenes JCM 3021T (98.2 %), Microbispora siamensis NBRC 104113T (98.1 %), Microbispora corallina JCM 10267T (98.0 %) and Microbispora roseasubsp.rosea JCM 3006T (97.9 %). However, two tree-making algorithms supported the position that strain NEAU-HRDPA2-9T formed a distinct clade with M. siamensis NBRC 104113T and M. hainanensis DSM 45428T. Furthermore, a combination of DNA-DNA hybridization results and some physiological and biochemical properties demonstrated that the strain could be distinguished from its closest relatives. Therefore, it is proposed that strain NEAU-HRDPA2-9T should be classified as representative of a novel species of the genus Microbispora, for which the name Microbisporatriticiradicis sp. nov. is proposed. The type strain is NEAU-HRDPA2-9T (=CGMCC 4.7399T=DSM 104649T).

Mutations in X-linked PORCN, a putative regulator of Wnt signaling, cause focal dermal hypoplasia.

Focal dermal hypoplasia is an X-linked dominant disorder characterized by patchy hypoplastic skin and digital, ocular and dental malformations. We used array comparative genomic hybridization to identify a 219-kb deletion in Xp11.23 in two affected females. We sequenced genes in this region and found heterozygous and mosaic mutations in PORCN in other affected females and males, respectively. PORCN encodes the human homolog of Drosophila melanogaster porcupine, an endoplasmic reticulum protein involved in secretion of Wnt proteins.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ~86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1(eyes2) mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1(eyes2) embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

Mouse model reveals the role of SOX7 in the development of congenital diaphragmatic hernia associated with recurrent deletions of 8p23.1.

Recurrent microdeletions of 8p23.1 that include GATA4 and SOX7 confer a high risk of both congenital diaphragmatic hernia (CDH) and cardiac defects. Although GATA4-deficient mice have both CDH and cardiac defects, no humans with cardiac defects attributed to GATA4 mutations have been reported to have CDH. We were also unable to identify deleterious GATA4 sequence changes in a CDH cohort. This suggested that haploinsufficiency of another 8p23.1 gene may contribute, along with GATA4, to the development of CDH. To determine if haploinsufficiency of SOX7-another transcription factor encoding gene-contributes to the development of CDH, we generated mice with a deletion of the second exon of Sox7. A portion of these Sox7(Δex2/+) mice developed retrosternal diaphragmatic hernias located in the anterior muscular portion of the diaphragm. Anterior CDH is also seen in Gata4(+/-) mice and has been described in association with 8p23.1 deletions in humans. Immunohistochemistry revealed that SOX7 is expressed in the vascular endothelial cells of the developing diaphragm and may be weakly expressed in some diaphragmatic muscle cells. Sox7(Δex2/Δex2) embryos die prior to diaphragm development with dilated pericardial sacs and failure of yolk sac remodeling suggestive of cardiovascular failure. Similar to our experience screening GATA4, no clearly deleterious SOX7 sequence changes were identified in our CDH cohort. We conclude that haploinsufficiency of Sox7 or Gata4 is sufficient to produce anterior CDH in mice and that haploinsufficiency of SOX7 and GATA4 may each contribute to the development of CDH in individuals with 8p23.1 deletions.

Four unreported aporphine alkaloids with antifungal activities from Artabotrys hexapetalus.

In this study, the extract from Artabotrys hexapetalus showed strong antifungal activity against phytopathogenic fungi in vitro. Four unreported aporphine alkaloids, hexapetalusine A-D (1-4), were isolated from stems and roots of Artabotrys hexapetalus (L.f.) Bhandari, along with six known aporphine alkaloids (5-10). Their chemical structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1-3 were determined using single-crystal X-ray diffractions and ECD calculations. Hexapetalusine A-C (1-3) were special amidic isomers. Additionally, all isolated compounds were evaluated for their antifungal activity against four phytopathogenic fungi in vitro. Hexapetalusine D (4) exhibited weak antifungal activity against Curvularia lunata. Liriodenine (5) displayed significant antifungal activity against Fusarium proliferatum and Fusarium oxysporum f. sp. vasinfectum, which is obviously better than positive control nystatin, suggesting that it had great potential to be developed into an effective and eco-friendly fungicide.

Dynamic effects of prognostic factors and individual survival prediction for amyotrophic lateral sclerosis disease.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons, with broad heterogeneity in disease progression and survival in different patients. Therefore, an accurate prediction model will be crucial to implement timely interventions and prolong patient survival time. METHODS: A total of 1260 ALS patients from the PRO-ACT database were included in the analysis. Their demographics, clinical variables, and death reports were included. We constructed an ALS dynamic Cox model through the landmarking approach. The predictive performance of the model at different landmark time points was evaluated by calculating the area under the curve (AUC) and Brier score. RESULTS: Three baseline covariates and seven time-dependent covariates were selected to construct the ALS dynamic Cox model. For better prognostic analysis, this model identified dynamic effects of treatment, albumin, creatinine, calcium, hematocrit, and hemoglobin. Its prediction performance (at all landmark time points, AUC ≥ 0.70 and Brier score ≤ 0.12) was better than that of the traditional Cox model, and it predicted the dynamic 6-month survival probability according to the longitudinal information of individual patients. INTERPRETATION: We developed an ALS dynamic Cox model with ALS longitudinal clinical trial datasets as the inputs. This model can not only capture the dynamic prognostic effect of both baseline and longitudinal covariates but also make individual survival predictions in real time, which are valuable for improving the prognosis of ALS patients and providing a reference for clinicians to make clinical decisions.

Discovery and biosynthesis of karnamicins as angiotensin converting enzyme inhibitors.

Angiotensin-converting enzyme inhibitors are widely used for treatment of hypertension and related diseases. Here, six karnamicins E1-E6 (1-6), which bear fully substituted hydroxypyridine and thiazole moieties are characterized from the rare actinobacterium Lechevalieria rhizosphaerae NEAU-A2. Through a combination of isotopic labeling, genome mining, and enzymatic characterization studies, the programmed assembly of the fully substituted hydroxypyridine moiety in karnamicin is proposed to be due to sequential operation of a hybrid polyketide synthase-nonribosomal peptide synthetase, two regioselective pyridine ring flavoprotein hydroxylases, and a methyltransferase. Based on AlphaFold protein structures predictions, molecular docking, and site-directed mutagenesis, we find that two pyridine hydroxylases deploy active site residues distinct from other flavoprotein monooxygenases to direct the chemo- and regioselective hydroxylation of the pyridine nucleus. Pleasingly, karnamicins show significant angiotensin-converting enzyme inhibitory activity with IC50 values ranging from 0.24 to 5.81 µM, suggesting their potential use for the treatment of hypertension and related diseases.

Characterization of two putative norlaudanosoline methyltransferases from Aristolochia debilis.

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

Corrigendum: Dynamic risk prediction of BK polyomavirus reactivation after renal transplantation.

[This corrects the article DOI: 10.3389/fimmu.2022.971531.].

Genomic alterations that contribute to the development of isolated and non-isolated congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life threatening birth defect. Most of the genetic factors that contribute to the development of CDH remain unidentified. OBJECTIVE: To identify genomic alterations that contribute to the development of diaphragmatic defects. METHODS: A cohort of 45 unrelated patients with CDH or diaphragmatic eventrations was screened for genomic alterations by array comparative genomic hybridisation or single nucleotide polymorphism based copy number analysis. RESULTS: Genomic alterations that were likely to have contributed to the development of CDH were identified in 8 patients. Inherited deletions of ZFPM2 were identified in 2 patients with isolated diaphragmatic defects and a large de novo 8q deletion overlapping the same gene was found in a patient with non-isolated CDH. A de novo microdeletion of chromosome 1q41q42 and two de novo microdeletions on chromosome 16p11.2 were identified in patients with non-isolated CDH. Duplications of distal 11q and proximal 13q were found in a patient with non-isolated CDH and a de novo single gene deletion of FZD2 was identified in a patient with a partial pentalogy of Cantrell phenotype. CONCLUSIONS: Haploinsufficiency of ZFPM2 can cause dominantly inherited isolated diaphragmatic defects with incomplete penetrance. These data define a new minimal deleted region for CDH on 1q41q42, provide evidence for the existence of CDH related genes on chromosomes 16p11.2, 11q23-24 and 13q12, and suggest a possible role for FZD2 and Wnt signalling in pentalogy of Cantrell phenotypes. These results demonstrate the clinical utility of screening for genomic alterations in individuals with both isolated and non-isolated diaphragmatic defects.

miRNA-34c Suppresses Osteosarcoma Progression In Vivo by Targeting Notch and E2F.

The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR-34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR-34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR-34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast-specific overexpression of miR-34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR-34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1, and HEY2) and in p53-mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2, and HDAC1). More interestingly, we found that the metastatic-free survival probability was increased among a patient cohort from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS, which has lower expression of direct targets of miR-34c that was identified in our transcriptome analysis, such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR-34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR-34c in OS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dynamic risk prediction of BK polyomavirus reactivation after renal transplantation.

Purpose: To construct a dynamic prediction model for BK polyomavirus (BKV) reactivation during the early period after renal transplantation and to provide a statistical basis for the identification of and intervention for high-risk populations. Methods: A retrospective study of 312 first renal allograft recipients with strictly punctual follow-ups was conducted between January 2015 and March 2022. The covariates were screened using univariable time-dependent Cox regression, and those with P<0.1 were included in the dynamic and static analyses. We constructed a prediction model for BKV reactivation from 2.5 to 8.5 months after renal transplantation using dynamic Cox regression based on the landmarking method and evaluated its performance using the area under the curve (AUC) value and Brier score. Monte-Carlo cross-validation was done to avoid overfitting. The above evaluation and validation process were repeated in the static model (Cox regression model) to compare the performance. Two patients were presented to illustrate the application of the dynamic model. Results: We constructed a dynamic prediction model with 18 covariates that could predict the probability of BKV reactivation from 2.5 to 8.5 months after renal transplantation. Elder age, basiliximab combined with cyclophosphamide for immune induction, acute graft rejection, higher body mass index, estimated glomerular filtration rate, urinary protein level, urinary leukocyte level, and blood neutrophil count were positively correlated with BKV reactivation, whereas male sex, higher serum albumin level, and platelet count served as protective factors. The AUC value and Brier score of the static model were 0.64 and 0.14, respectively, whereas those of the dynamic model were 0.79 ± 0.05 and 0.08 ± 0.01, respectively. In the cross-validation, the AUC values of the static and dynamic models decreased to 0.63 and 0.70 ± 0.03, respectively, whereas the Brier score changed to 0.11 and 0.09 ± 0.01, respectively. Conclusion: Dynamic Cox regression based on the landmarking method is effective in the assessment of the risk of BKV reactivation in the early period after renal transplantation and serves as a guide for clinical intervention.

Mono-/Bis-Alkenoic Acid Derivatives From an Endophytic Fungus Scopulariopsis candelabrum and Their Antifungal Activity.

During a screening for antifungal secondary metabolites, six new mono-/bis-alkenoic acid derivatives (2-7) and one known alkenoic acid derivative (1) were isolated from an endophytic fungi Scopulariopsis candelabrum. Their chemical structures were identified by 1H-NMR, 13C-NMR, 2D NMR, and high-resolution mass spectrometry, as well as comparisons with previously reported literatures. Among them, fusariumesters C‒F (2-5) are bis-alkenoic acid derivatives dimerized by an ester bond, while acetylfusaridioic acid A (6) and fusaridioic acid D (7) are alkenoic acid monomers. All the isolates were submitted to an antifungal assay against Candida albicans and the corn pathogen Exserohilum turcicum using the filter paper agar diffusion method. As a result, only compound 1 decorating with ß-lactone ring turned out to be active against these two tested fungi. The broth microdilution assay against Candida albicans showed the minimum inhibitory concentration (MIC) value of 1 to be 20 µg/ml, while the minimum inhibitory concentration value of the positive control (naystatin) was 10 µg/ml. And the half maximal inhibitory concentration (IC50) value (21.23 µg/ml) of 1 against Exserohilum turcicum was determined by analyzing its inhibition effect on the mycelial growth, using cycloheximide (IC50 = 46.70 µg/ml) as the positive control.