Region Sequences Research Articles

Mutations on the A and C genomes of Brassica napus confer herbicide resistance to acetohydroxyacid synthase inhibitors

ABSTRACT The point mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, are reported for two Brassica napus lines (19c and 30a) developed by seed mutagenesis using ethyl methanesulfonate. Crosses with the herbicide-susceptible wild-type line generated F2 and backcrossed progeny and confirmed that chlorsulfuron resistance was inherited as a dominant mutation at a single locus in both lines. F2 progeny from crossing 19c and 30a segregated in a 15 resistant: 1 susceptible ratio, establishing that the two herbicide-resistant loci are independently inherited and that the mutations for resistance occur at different loci. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene revealed two separate point mutations at different nucleotides in the same codon. This results in the substitution of proline-197 for leucine in 19c and proline-197 for serine in 30a. The different amino acid substitutions in the acetohydroxyacid synthase enzyme explain the differing responses of 19c and 30a to sulfonylurea and imidazolinone herbicides. Based on attempts at introgression of chlorsulfuron resistance to a wide range of forage brassicas, we conclude that the 19c mutation resides in the Brassica C genome, whereas the 30a mutation is in the Brassica A genome.

Obstacle avoidance and target attainment of planar quadrotors via control barrier function

In this article, a safety-critical controller and a novel algorithm based on a quadratic program for a class of planar quadrotors are proposed to ensure obstacle avoidance and target attainment. First, we define a circular safe region and design a safety-critical controller based on the control barrier function. Also, the designed controller can guarantee that the planar quadrotor remains within the safe region and eventually reaches the center of the safe region. Second, we design an algorithm to generate a sequence of safe regions that progressively approach the target point. With the designed controller, the planar quadrotor gradually reaches the center of each safe region and eventually reaches the target point. Finally, the effectiveness of the proposed controller and algorithm is verified through two simulation experiments.

Beyond Poliomyelitis: A 21-Year Study of Non-Polio Enterovirus Genotyping and Its Relevance in Acute Flaccid Paralysis in São Paulo, Brazil

In the context of the near-global eradication of wild poliovirus, the significance of non-polio enteroviruses (NPEVs) in causing acute flaccid paralysis (AFP) and their impact on public health has gained increased attention. This research, conducted from 2001 to 2021, examined stool samples from 1597 children under 15 years in São Paulo, Brazil, through the AFP/Poliomyelitis Surveillance Program, detecting NPEVs in 6.9% of cases. Among the 100 NPEV-positive strains analyzed, 90 were genotyped through genomic sequencing of the partial VP1 region, revealing a predominance of EV-B species (58.9%), followed by EV-A (27.8%) and EV-C (13.3%). This study identified 31 unique NPEV types, including EV-A71, CVB2, and E11, as the most prevalent, along with the first documented occurrence of CVA19 in Brazil. These findings emphasize the importance of NPEV genotyping in distinguishing AFP from poliomyelitis, enhancing understanding of these viruses’ epidemiology. Moreover, it ensures that AFP cases are correctly classified, contributing to the effective surveillance and eradication efforts for poliomyelitis.

Influence of hormonal factors, number of sexual partners, surgical intervention on gastrointestinal and urogenital microbiota of patients endometriosis

Endometriosis is associated with gastrointestinal (GI) and urogenital (UG) microbial dysbiosis in patients with endometriosis (P-EOSIS). Sexual partner exposure may contribute to microbial dysbiosis but has not been studied in P-EOSIS. We hypothesized that sexual partner number, hormonal and surgical therapy would affect GI/UG microbial dysbiosis in P-EOSIS. Urine, fecal and vaginal swabs from control (n = 15) and P-EOSIS (n = 33) were collected on the day of surgery (DOS) and ∼1-3 weeks post-surgical intervention (PSI). Control and P-EOSIS were grouped based on hormonal therapy (HT) to determine the effect of HT on microbial profiles, Control (HT n = 8; no HT n = 7) and P-EOSIS (HT n = 18; no HT n = 15). Samples underwent DNA extraction and sequencing of the V4 region of 16S rRNA gene. Sequences were processed using QIIME2 and amplicon sequence variants (ASV) were analyzed for microbial differences. Pearson's and Spearman correlation analyses determined associations among microbial features and sexual partner exposure. P-EOSIS had microbial dysbiosis characterized by unique GI/UG bacteria and altered microbial richness and diversity. Hormonal and surgical intervention in P-EOSIS restored GI microbial diversity. Increased sexual partner exposure decreased GI/UG microbial diversity. P-EOSIS who had 10 or more sexual partners had greater microbial dysbiosis compared to 4-6 partners. Surgical intervention negatively correlated with sexual partner numbers and GI/UG microbial abundance. Increased sexual partner exposure may enhance microbial dysbiosis in P-EOSIS and diminish the effectiveness of HT and surgical interventions.

Unique genital microbiota in male lichen sclerosus urethral stricture associated with urine exposure

BackgroundLichen sclerosus (LS) is a chronic inflammatory cicatricial skin disease that can lead to urethral stricture or even malignant transformation and the etiology is still unknown. This study comparatively analyzed the balanopreputial swab and urine microbiota simultaneously between male patients with LS urethral stricture (LSUS) and non-LS urethral stricture (non-LSUS).MethodsWe prospectively included 31 male patients with LSUS and 30 with non-LSUS in this case–control study. Midstream urine samples and balanopreputial swabs were collected from each patient for the 16S V3-V4 hypervariable region sequencing. Operational taxonomic units were defined using a > 97% sequence similarity threshold. We compared the differences in alpha diversity, beta diversity, and microbial structure between the two groups.ResultsWhether in swab or urine samples, there was no significant difference in alpha diversity between the two groups. Swab samples showed a significant difference in beta diversity (p = 0.001). For all individuals, composition analyses showed that the most abundant phyla were Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes in both samples. Additionally, the microbial communities of swab samples were significantly more similar to the communities of urine samples in the LSUS group (p = 0.047).ConclusionsMicrobiota showed significant variation between LSUS and non-LSUS groups, suggesting that microecological imbalance may be closely related to the occurrence of LS. Urinary irritation may be related to the unique microbiota on the genital skin of patients with LSUS.

First report of Phyllosticta capitalensis causing leaf blotch on Chinese olive (Canarium album) in Guangdong province of China.

Chinese olive (Canarium album) is a well-known plant used both as food and medicine, extensively cultivated in southern China (Kuo et al., 2016). Its production is challenged by various fungal diseases. From July to October 2023, Chinese olive plants in an approximately 400 hectare conventional hillside orchard in Chaozhou, Guangdong province, China (23°39'45.36″N, 116°37'45.48″E), where sustainable Chinese olive farming has been practiced for over a decade, exhibited leaf blotch symptoms with a disease incidence of 40% (n = 100 plants investigated). Symptoms include yellow or brown leaf blotches, followed by drying, curling, and eventual leaf drop. To identify the causal agent, seven symptomatic leaves from seven trees were collected. The symptomatic leaves were cut into pieces of about 0.3 mm2 at the necrotic lesion edges and surface disinfected. The pieces were placed on potato dextrose agar (PDA), incubated in the dark at 28°C for 5 days, then subcultured for 7 days. Four fungal isolates were obtained with an isolation frequency of 57%. Genomic DNA was extracted from the isolates. Morphological analysis and internal transcribed spacer (ITS) region sequencing revealed no observable differences among isolates. The representative isolate (LB-1) cultured on PDA was initially white, turning light grey with dense aerial mycelium after 7 days. The transparent hyphae displayed intricate branching patterns and multiple cellular structures. The conidia were single-celled, hyaline, fusiform structures measuring 3.6-7.8 × 1.17-2.83 μm (n = 50). Morphologically, the fungal isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). Primers ITS1/ITS5, ACT-512F/ACT-783R, and EF1/EF2 were used to amplify the ITS region, the actin (ACT) gene, and the transcription elongation factor (TEF), respectively (Wang et al. 2023). The sequences obtained from the LB-1 isolate have been deposited in NCBI GenBank (accession nos. PP922193; PP960207 and PP960206). A phylogenetic tree was constructed based on aligned sequences (Wang et al. 2023). BLAST analysis showed LB-1 isolate shares 99.4%-100% similarity with sequences KY855645, KY855590, and KY855919 from P. capitalensis CPC27084. Based on morphological and molecular analysis, isolate LB-1 was identified as P. capitalensis. Pathogenicity assays of the LB-1 isolate were conducted on 2-year-old Canarium album seedlings. A 10⁶ spores/mL LB-1 suspension was applied to 3 mm spots on the leaves of four plants for inoculation. Controls leaves was treated with sterile water. The inoculated plants were placed in a greenhouse at 28°C with 80% humidity. The experiment was repeated 3 times. Leaves inoculated with LB-1 isolates showed blotch symptoms similar to those in the field, the controls remained healthy. Reisolation of P. capitalensis was performed confirmed by morphology and DNA sequencing, fulfilling Koch's postulates. Previous studies have shown that P. capitalensis causes fungal diseases on fruit trees such as Citrus lalifolia and Mangifera indica (Wikee et al. 2013). To our knowledge, this is the first report of Phyllosticta capitalensis causing leaf blotch on Chinese olive in China. Preliminary observations suggest it may be present in other olive-producing regions of Guangdong. It causes significant leaf wilting and shedding, potentially reduces fruit quality, and lowers overall yield. This discovery will offer valuable insights for creating effective control measures against this recently emerged severe disease.

Mitochondrial control region sequences show high genetic connectivity in the brownstripe snapper, Lutjanus vitta (Quoy and Gaimard, 1824) from the east coast of Peninsular Malaysia

Mitochondrial control region sequences show high genetic connectivity in the brownstripe snapper, Lutjanus vitta (Quoy and Gaimard, 1824) from the east coast of Peninsular Malaysia

Monoclonal antibodies against jellyfish collagen.

Collagens are abundant structural proteins found in both mammalian and marine species, and attractive biomaterials used in various fields. Jellyfish collagen-based products have become increasingly popular because of their clinically proven health benefits such as the effects of skin wound healing and immune stimulation. To develop detection tools for jellyfish collagen, we generated four monoclonal antibodies, MCOL1, 2, 3, and 4, by immunizing mice with moon jellyfish collagen. The nucleotide and amino acid sequences of the variable regions of the monoclonal antibodies were determined. The antibody-binding kinetics toward collagens from moon jellyfish were evaluated using a surface plasmon resonance (SPR) biosensor, and the binding specificity was evaluated in comparison with binding to collagens from edible jellyfish, fish scales, and pig and cow skins. MCOL1, 3, and 4 specifically bound to moon jellyfish collagen, whereas MCOL2 bound to both moon and edible jellyfish collagens. Considering the results showing that the SPR responses of MCOL2 binding were greater than those seen with the other antibodies, MCOL2 could recognize the common and repetitive sequences of the two jellyfish collagens. Therefore, this monoclonal antibody will be most applicable for detecting jellyfish collagen.

The saNeuroGut Initiative: Investigating the gut microbiome and symptoms of anxiety, depression and posttraumatic stress.

Common mental disorders, such as anxiety disorders, depression, and posttraumatic stress disorder (PTSD), present a substantial health and economic burden. The gut microbiome has been associated with these psychiatric disorders via the microbiome-gut-brain axis. However, previous studies have focused on the associations between the gut microbiome and common mental disorders in European, North American and Asian populations. As part of the saNeuroGut Initiative, we assessed associations between gut microbial composition and self-reported symptoms of anxiety, depression and posttraumatic stress among South African adults. Participants completed validated, online self-report questionnaires to evaluate symptoms of state anxiety, trait anxiety, depression, and PTSD. Eighty-six stool-derived microbial DNA samples underwent sequencing of the V4 region of the 16S rRNA gene to characterise gut bacterial taxa in the sample. No significant associations were observed between symptom severity scores and alpha (Shannon and Simpson indices) and beta (Aitchison distances) diversity metrics. Linear regression models revealed that the abundances of Catenibacterium, Collinsella and Holdemanella were significantly positively associated with the severity of posttraumatic stress (PTS) symptoms. Catenibacterium, Collinsella and Holdemanella have each previously been associated with various psychiatric disorders, with Catenibacterium having been positively associated with symptoms of PTSD in another South African cohort. This study sheds light on the relationship between the human gut microbiome and symptoms of anxiety, depression, and PTS in a South African adult sample.

Environmental factors and potential probiotic lineages shape the active prokaryotic communities associated with healthy Penaeus stylirostris larvae and their rearing water.

Microbial dysbiosis is hypothesized to cause larval mass mortalities in New-Caledonian shrimp hatcheries. In order to confirm this hypothesis and allow further microbial comparisons, we studied the active prokaryotic communities of healthy Penaeus stylirostris larvae and their surrounding environment during the first 10 days of larval rearing. Using daily nutrient concentration quantitative analyses and spectrophotometric organic matter analyses, we highlighted a global eutrophication of the rearing environment. We also evidenced drastic bacterial community modifications in the water and the larvae samples using Illumina HiSeq sequencing of the V4 region of the 16S rRNA gene. We confirmed that Alteromonadales, Rhodobacterales, Flavobacteriales, Oceanospirillales and Vibrionales members formed the core bacteriota of shrimp larvae. We also identified, in the water and the larvae samples, several potential probiotic bacterial strains which could lead to rethink probiotic use in aquaculture (AEGEAN 169 marine group, OM27 clade, Ruegeria, Leisingera, Pseudoalteromonas and Roseobacter). Finally, investigating the existing correlations between the environmental factors and the major bacterial taxa of the water and the larvae samples, we suggested that deterministic and stochastic processes were involved in the assembly of prokaryotic communities during the larval rearing of P. stylirostris. Overall, our results showed that drastic changes mostly occurred during the zoea stages suggesting that this larval phase is crucial during shrimp larval development.

Role of gut microbiome in developing necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most devastating intestinal diseases observed in preterm in the first days of life. Researchers have recently focused on potential predictive biomarkers for early and concomitant diagnoses. Thus, we inquired about the linkage of intestinal dysbiosis, one of the most important factors in NEC development to the gut microbiota. In this study, the systematic differences in the bacterial composition between neonates affected by NEC and healthy newborns were highlighted by metagenomic analysis. The next-generation sequencing of the V3-V4 variable region of the 16S rRNA gene and gene-specific qPCR analyzed the untargeted gut microbiota. Total bacteria, total and fecal coliform loads in stool samples with NEC were higher than control. OTU-level relative abundances of NEC infant was characterized by Firmicutes and Bacteroidetes at phylum levels. At the genus level, NEC stool was identified by the lack of Klebsiella and the presence of Roseburia, Blautia, and Parasutterella. Finally, Clostridium fessum was the predominant species of Clostridium genus in disease and healthy specimens at the species level, whereas Clostridium jeddahitimonense was at NEC diagnosis. Despite a strong relationship between pathophysiology and characterization of gut microbiota at a clinical diagnosis of NEC, our results emphasize the broad difficulty in identifying potential biomarkers.

Phylogeny and host specificity of Fusarium solani species complex isolated from chickpea, lentil and common bean

Using a combination of morphological, molecular, and cross-pathogenicity assessments, populations of Fusarium solani recovered from symptomatic chickpeas, lentils, and common beans were examined. From the numerous isolates within the Fusarium solani species complex (FSSC), four isolates from common beans, three from lentils, and two from chickpeas were selected for host specificity and phylogenetic analysis. The selected isolates were cross-inoculated on chickpeas, common beans, and lentils. Although the average disease intensity varied among hosts, no significant differential response was observed to indicate host specificity. Sequencing of the partial regions of TEF-1α and ITS-rDNA was conducted. The phylogenetic tree reconstruction revealed that two isolates clustered within the phylogenetic species of F. falciforme, while the others grouped within Fusarium solani sensu stricto. This study demonstrated that FSSC isolates from the tested pulse crops, regardless of their phylogenetic classification, do not exhibit host specificity.

The QS regulator AphBvp promotes expression of the AHPND PirAvp and PirBvp toxins and may enhance virulence under acidic conditions

Shrimp acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases to impact the global shrimp farming industry, with a mortality rate of 70 %–100 %. The key virulence factors are a pair of Photorhabdus insect-related (Pir)-like toxins, PirAvp and PirBvp. In this study, by using an in vitro transcription and translation assay, we first confirmed that the quorum sensing transcriptional regulator AphBvp could trigger the expression of its downstream genes after binding to the AphBvp binding sequence in the promoter region of the pirAvp/pirBvp operon. Next, we showed that AphBvp was essential for the expression of these toxins by using an aphBvp-deletion mutant (ΔaphBvp) derived from the AHPND-causing Vibrio parahaemolyticus. Lastly, we discovered that the expression levels of PirAvp and PirBvp were up-regulated under acidic conditions (pH 4.5), and further showed that an acidic environment promoted the binding of AphBvp to the pirABvp promoter. We speculate that this was because the acidic environment favored the formation of AphBvp tetramers, which is important for binding to DNA. Taken together, these findings improve our understanding of the gene regulatory mechanisms of pirAvp and pirBvp, and suggest that the pH value of the environment might affect the virulence of AHPND-causing V. parahaemolyticus.

Participation of retroelements in chromoanagenesis in cancer development

Purpose of the study: to determine the role of retroelements in chromoanagenesis mechanisms in cancer etiopathogenesis.Material and Methods. The search for relevant sources was carried out in the Scopus, Web of Science, PubMed, Elibrary systems, including publications from February 2002 to December 2023. Of the 864 scientifc articles found, 60 were used to write a systematic review.Results. According to original works and meta-analyses results, the cause of complex chromosomal rearrangements during cancer development may be retroelement pathological activation. Chromoanagenesis involves LINE1, SVA, Alu, HERV, which cause double-stranded DNA breaks, insertions in tumor suppressor genes region, the formation of chimeric oncogenes due to retroelement use as new promoters, and function as molecular “band-aids” in non-homologous end junctions and form bridges of distal DNA fragments. Global structural rearrangements of chromosomes observed during chromoanagenesis may be consequences of retroelements activation, which participate in non-allelic homologous recombination and in microhomology-mediated joining of ends characteristic. Certain types of neoplasms, such as colon cancer, are characterized by both high levels of chromothripsis and retroelement activity. In head and neck squamous cell carcinoma, chromoplexy is specifc, the sources of sequences at the breakpoints of which are retroelements. During chromoanagenesis, activation of proto-oncogenes and inactivation of tumor suppressor genes are observed, which is also a consequence of retroelement activation. This is due to the presence of retroelement sequences in proto-oncogenes promoter regions and introns (which become the basis for chimeric oncogene formation) and hot spots of insertional mutagenesis in tumor suppressor genes (transpositions into these regions inactivate these genes).Conclusion. The results obtained on the driver effect of retroelements in chromothripsis, chromoplexy and chromoanasynthesis mechanisms, which are the basis for the formation of clonal evolution of tumors, indicate promise of targeted therapy aimed at silencing the activity of retroelements in cancer patients treatment. For this purpose, it is possible to use microRNAs complementary to retroelements, which are also involved in tumor development, as tools.

Genetic Characterization of Sumatran Mirah Chicken Based on Mitochondrial D-loop Region Sequence

Mirah chickens are Indonesian indigenous chickens that originate from Simalungun Regency, North Sumatera Province of Indonesia. The study aimed to determine the genetic characterization of Mirah chicken based on the mitochondrial D-loop region (838 bp). Twenty Mirah cocks from Simalungun Regency of Indonesia were used in the present study. The results showed that fourteen haplotypes were found in the studies of birds based on sixteen mutation sites. Therefore, these haplotype and nucleotide diversities in the partial D-loop region of the Mirah chicken were classified as high. The Median-joining tree revealed that the Mirah chickens were classified in a similar cluster with the Red jungle fowl (Gallus gallus). Ten haplotypes of birds were close to G. g. bankiva, and four haplotypes of birds were close to G. g. gallus. In conclusion, Mirah chickens had the genetic introgression from two sub-species of Red junglefowl, i.e., G.g. gallus and G.g. bankiva. Hence, the pure breeding program for Mirah chickens is important to conserve their genetic resources from extinction.

"Elucidating the immunomodulatory role of endocannabinoids in intervertebral disc degeneration".

The endocannabinoid system (ECS) has been well-established to play a crucial role in the regulation of several physiological processes as well as many inflammatory disease conditions. However, its role in intervertebral disc degeneration has been least explored. We aim to investigate the immunomodulatory role of endocannabinoids in regulating IVD health. The study population included 20 healthy volunteers (controls) and 40 patients with disc degeneration (disease group) (20 Modic and 20 Non Modic). 16S metagenome sequencing of the V3-V4 region was performed for the DNA extracted from NP tissue samples of both control and disease groups. Sequencing was carried out using the Novaseq 6000 platform using 250bp paired-end chemistry. A global metabolic profile was obtained using the uHPLC system coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Our study revealed a higher prevalence of gram-negative bacteria, particularly opportunistic pathogens like Pseudomonas, in diseased discs (71-81%) compared to healthy controls (54%). Further investigation using metabolomics identified significant changes in the lipid profiles of diseased discs. We found that the signalling molecules of the ECS, 2-arachidonylglycerol (2-AG) and N-arachidonoylethanolamine (AEA), were significantly lower in diseased discs compared to controls (Log2FC -2.62 for 2-AG and -3.15 for AEA). Conversely, pro-inflammatory metabolites like LTA4, HPETE, HETE, and Prostaglandin G2 were elevated in diseased discs, with a Log2 fold increase greater than 2.5. The study reveals that the endocannabinoid metabolites (2-AG and AEA) of the ECS could be a significant molecule influencing susceptibility to infection and inflammation within the intervertebral discs, which could be a potential target for improving disc health. Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding.

Species Identification and Genotyping of Cutaneous Leishmaniasis in Clinical Samples Based on ITS1-PCR-Sequencing in Southeast Iran.

Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases in many regions of Iran. It has a major role in deprived societies. We aimed to identify Leishmania species based on molecular as ITS1-rDNA-PCR internal transcribed spacer 1 (ITS1) region, microscopy, and culture techniques in diagnosing cutaneous leishmaniasis. From April 2018 to May 2020, we conducted a cross-sectional study involving 32 patients with suspected CL lesions in Sistan and Baluchistan Province, located in southeast Iran. Samples were subjected to microscopic examination, culture, and PCR amplification targeting the internal transcribed spacer 1 (ITS1) region. DNA sequencing was performed on PCR-positive samples for species identification and phylogenetic analysis. PCR demonstrated superior sensitivity (93.75%, 30/32) compared to culture (56.25%, 18/32) and microscopic examination (53.1%, 17/32). Molecular analysis revealed that L. major was the predominant causative agent of CL in the study area, with L. tropica occurring less frequently. Sequencing and phylogenetic analysis of the ITS1 region showed high intraspecies similarity among L. tropica isolates, while L. major isolates exhibited greater genetic diversity. This study shows the co-existence of L. major and L. tropica in Mirjaveh, southeast Iran, with L. major as the primary cause. While L. tropica isolates displayed high genetic similarity, L. major samples were more diverse, indicating different epidemiological patterns. These findings highlight the importance of molecular methods for accurately identifying Leishmania species and understanding CL epidemiology in the region.

Microbiomes of urban trees: unveiling contributions to atmospheric pollution mitigation.

Urban trees are crucial in delivering essential ecosystem services, including air pollution mitigation. This service is influenced by plant associated microbiomes, which can degrade hydrocarbons, support tree health, and influence ecological processes. Yet, our understanding of tree microbiomes remains limited, thus affecting our ability to assess and quantify the ecosystem services provided by trees as complex systems. The main hypothesis of this work was that tree microbiomes concur to hydrocarbon biodegradation, and was tested through three case studies, which collectively investigated two tree micro-habitats (phyllosphere and tree cavity organic soil-TCOS) under various conditions representing diverse ecological scenarios, by applying different culture-based and molecular techniques and at different scales. The integration of all results provided a more comprehensive understanding of the role of microbiomes in urban trees. Firstly, bacterial strains isolated from the phyllosphere of Quercus ilex were characterized, indicating the presence of Plant-Growth Promoting bacteria and strains able to catabolize PAHs, particularly naphthalene and phenanthrene. Secondly, naphthalene biodegradation on artificially spiked Hedera helix leaves was quantified in greenhouse experiments on inoculated and untreated plants. The persistence of the inoculated strain and community structure of epiphytic bacteria were assessed by Illumina sequencing of V5-V6 hypervariable regions of 16S rRNA gene. Results showed that naphthalene degradation was initially faster on inoculated plants but later the degradation rates became similar, probably because bacterial populations with hydrocarbon-degrading abilities gradually developed also on non-inoculated plants. Finally, we explored bacterial and fungal biodiversity hosted by TCOS samples, collected from six large trees located in an urban park and belonging to different species. Microbial communities were characterized by Illumina sequencing of V5-V6 hypervariable regions of bacterial gene 16S rRNA and of fungal ITS1. Results indicated TCOS as a distinct substrate, whose microbiome is determined both by the host tree and by canopy environmental conditions and has a pronounced aerobic hydrocarbon degradation potential. Overall, a better assessment of biodiversity associated with trees and the subsequent provision of ecosystem services constitute a first step toward developing future new microbe-driven sustainable solutions, especially in terms of support for urban green planning and management policy.

CTIM-11. LONG-TERM SURVIVORS FROM A PHASE 1B STUDY OF IGV-001 IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

Abstract Imvax has developed the Goldspire™ platform to create IGV-001, an autologous biologic-device combination product for the treatment of newly diagnosed glioblastoma (ndGBM). IGV-001 consists of autologous GBM tumor cells and an antisense oligonucleotide against IGF-1R mRNA (IMV-001), irradiated and administered via biodiffusion chambers (BDCs) implanted in the abdomen. Together, these components stimulate immunogenic cell death and antigen release. IGV-001 was well tolerated and multiple efficacy signals were observed in a Phase 1b study in patients with ndGBM (Andrews et al., 2021), including significant improvements in progression-free survival (PFS), radiographic evidence of tumor response, and changes in immune response biomarkers. The Phase 1b study enrolled 33 patients in four different cohorts implanted with 10 or 20 BDCs for 24 or 48 hours. Here we report on 6 subjects with survival beyond 4 years, including 5 subjects who survived 5 years or more (15.2%). There were 4 male and 2 female subjects, with a median age of 52 years (range 32-75). At diagnosis the MGMT promoter was methylated in 4 of 5 subjects that survived 5 years or more. We also report on T cell receptor Vβ CDR3 region sequencing of peripheral blood mononuclear cells and tissue infiltrated lymphocytes that was performed in a subset of 9 patients, including 3 subjects who survived beyond 4 years. Immune correlates of IGV-001 suggest an association between peripheral T cell clonal expansion and PFS/OS outcomes. A Phase 2b randomized, multicenter, double-blind, placebo-controlled study to assess the safety and efficacy of IGV-001 in patients with ndGBM (NCT04485949) has completed enrollment and results are expected to be available in 2025.

RNA-First Approach Identifies Deep Intronic PHEX Variants in X-Linked Hypophosphatemic Rickets.

Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. To identify intronic variants causing PHEX mis-splicing in patients with XLH. The metabolic bone clinic of a paediatric orthopedic hospital. Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. RNA and DNA sequence analysis of PHEX. Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768+173A>G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.