NLR Outperforms Low Hemoglobin and High Platelet Count as Predictive and Prognostic Biomarker in Metastatic Renal Cell Carcinoma Treated with Immune Checkpoint Inhibitors.

BACKGROUND: Reliable biomarkers in renal cell carcinoma (RCC) remain elusive. While several markers have been shown to be associated with prognosis, and may aid in risk assessment, predictive biomarkers of response to immune checkpoint inhibitors (ICIs) have not been established. Previous studies have shown that a high pretreatment neutrophil-lymphocyte ratio (NLR) is a negative prognostic factor in RCC. However, a clinically useful cut-off for the predictive and prognostic value of NLR has not been well defined. METHODS: We conducted a retrospective analysis of 132 patients with previously untreated metastatic clear cell RCC (ccRCC) who received first line ICI-based therapy. ICI-based therapy included anti-PD-1/PD-L1 alone or in combination with anti-CTLA-4 or VEGF-TKI. Platelet, haemoglobin, neutrophil and lymphocyte counts were collected prior to treatment and at 12-weeks after treatment initiation. Radiologic response at 12-weeks and overall survival (OS) data was also collected. RESULTS: Low haemoglobin, high platelet count, and NLR ≥3 were statistically significant negative predictive biomarkers when assessed at 12-weeks, but not at baseline. Median OS was shorter in patients with low haemoglobin (20.3 months vs. 51.6 months, P = .009), high platelet count (14.3 months vs. 43.8 months, P = .003), and NLR ≥ 3 (17.5 months vs. 40.3 months, P < .001) at 12-weeks. In an IMDC-risk adjusted analysis, only NLR ≥3 at 12-weeks remained statistically significant (OR of 2.11, P = .003) A dynamic change towards lower absolute NLR overtime was associated with longer OS. In patients who had baseline NLR ≥ 3, those who achieved NLR < 3 at 12-weeks demonstrated significant longer median OS compared to those whose NLR remained persistently ≥ 3 (40.3 months vs. 14.7 months, P = .004). CONCLUSION: NLR ≥3, low haemoglobin and elevated platelet count after 12-weeks of ICI-based first line therapy were negatively prognostic and predictive in patients with metastatic RCC. Normalization of NLR in patients with baseline elevation was associated with longer median OS and response to therapy. These results suggest that monitoring of routine haematologic biomarkers during therapy may provide important predictive and prognostic information, beyond what is available with baseline risk assessment scoring systems.

Treatment Patterns and Survival Outcomes Before and After Access to Immune Checkpoint Inhibitors for Patients With Metastatic Urothelial Carcinoma: A Single-Center Retrospective Study From 2004 to 2021.

INTRODUCTION: Metastatic urothelial carcinoma (mUC) is a lethal disease with limited treatment options. We aimed to compare the treatment patterns and outcomes of patients with mUC who were treated before and after the introduction of immune checkpoint inhibitors (ICIs) at a tertiary hospital in Barcelona. METHODS: Single-center retrospective study from 2004 to 2021. Access to ICIs began in December 2014. We analyzed differences in clinical characteristics and survival outcomes, such as overall survival (OS), progression-free survival (PFS), and restricted mean survival time (RMST). RESULTS: A total of 206 patients were included. The median follow-up was 48.6 months. Ninety and 116 patients were treated during the pre-ICIs and the post-ICIs eras, respectively. We found high treatment attrition rates, with no differences in the number of patients who received second-line (48%) and third-line (26%) therapies between the two eras. In the second-line, ICIs became the predominant therapy (58%), leading to a 30% reduction in the utilisation of platinum-based ChT and non-platinum ChT. Innovative approaches including ICIs in the first-line treatment (18%) and targeted therapies in the third-line setting (34%) were observed. We found no differences in the median OS, 2-year OS, or 24-month RMST between the two periods. CONCLUSION: ICIs have emerged as a transformative treatment option, reshaping the treatment landscape. Nevertheless, substantial attrition rates from first-line to subsequent lines of systemic therapies might impede the potential impact of ICIs on long-term survival outcomes across the entire population.

Cumulative incidences of hypogonadism, hypertension, and dyslipidaemia in patients with stage I seminoma treated with a risk-adapted strategy: a Spanish single-centre retrospective analysis.

PURPOSE: To describe the incidences of hypogonadism, hypertension, and dyslipidaemia in patients with stage 1 seminoma (S1S) testicular cancer (TC) treated with a risk-adapted strategy. METHODS: A retrospective analysis from 2000 to 2020 was conducted. Active surveillance (AS), carboplatin one cycle, and carboplatin two cycles were offered according to risk factors. Cumulative incidences and relapse-free survival (RFS) were estimated. RESULTS: Of the 145 patients, 8 (5.4%) were excluded due to bilateral TC or hypogonadism at diagnosis. Median follow-up time was 8.2 years. Eighty-four, 30, and 33 patients were treated with AS, carboplatin one cycle, and carboplatin two cycles, respectively. In the overall population, the 5-year and 10-year cumulative incidences were 1.6% and 5.3% for hypogonadism; 2.0% and 8.6% for hypertension; and 12.4% and 25.1% for dyslipidaemia. No statistically significant differences were found in the incidences among the three adjuvant strategies. Five-year and 10-year RFS were 85.9% and 83.3% for AS; 92.4% and 84.0% for carboplatin one cycle; and 96.7% at both times for carboplatin two cycles. CONCLUSION: There were no statistically differences in cumulative incidences of hypogonadism, hypertension, and dyslipidaemia in S1S patients treated with a risk-adapted strategy.

Genome sequencing and de novo assembly of Trichoderma longibrachiatum isolate collected from Florida agricultural soils.

We report a draft genome assembly of Trichoderma longibrachiatum isolate GEV 3550, obtained from Florida, United States of America.

Comparative genomics of the niche-specific plant pathogen Streptomyces ipomoeae reveal novel genome content and organization.

IMPORTANCE: While most plant-pathogenic Streptomyces species cause scab disease on a variety of plant hosts, Streptomyces ipomoeae is the sole causative agent of soil rot disease of sweet potato and closely related plant species. Here, genome sequencing of virulent and avirulent S. ipomoeae strains coupled with comparative genomic analyses has identified genome content and organization features unique to this streptomycete plant pathogen. The results here will enable future research into the mechanisms used by S. ipomoeae to cause disease and to persist in its niche environment.

Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

A snapshot of the transcriptome of Medicago truncatula (Fabales: Fabaceae) shoots and roots in response to an arbuscular mycorrhizal fungus and the pea aphid (Acyrthosiphon pisum) (Hemiptera: Aphididae).

Plants simultaneously interact with belowground symbionts such as arbuscular mycorrhizal (AM) fungi and aboveground antagonists such as aphids. Generally, plants gain access to valuable resources including nutrients and water through the AM symbiosis and are more resistant to pests. Nevertheless, aphids' performance improves on mycorrhizal plants, and it remains unclear whether a more nutritious food source and/or attenuated defenses are the contributing factors. This study examined the shoot and root transcriptome of barrel medic (Medicago truncatula Gaertn.) plants highly colonized by the AM fungus Rhizophagus irregularis (Blaszk., Wubet, Renker, and Buscot) C. Walker and A. Schüßler (Glomerales: Glomeraceae) and exposed to 7 days of mixed age pea aphid (Acyrthosiphon pisum (Harris)) herbivory. The RNA-seq samples chosen for this study showed that aphids were heavier when fed mycorrhizal plants compared to nonmycorrhizal plants. We hypothesized that (i) insect-related plant defense pathways will be downregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; (ii) pathways involved in nutrient acquisition, carbohydrate-related and amino acid transport will be upregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; and (iii) roots of mycorrhizal plants with aphids will exhibit mycorrhiza-induced resistance. The transcriptome data revealed that the gene repertoire related to defenses, nutrient transport, and carbohydrates differs between nonmycorrhizal and mycorrhizal plants with aphids, which could explain the weight gain in aphids. We also identified novel candidate genes that are differentially expressed in nonmycorrhizal plants with aphids, thus setting the stage for future functional studies.

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus.

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees.

A synthetic 'essentialome' for axenic culturing of 'Candidatus Liberibacter asiaticus'.

OBJECTIVE: 'Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus 'greening' disease. All attempts to achieve axenic growth and complete Koch's postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic 'Ca. Liberibacter' spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. RESULTS: Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of 'essential' Lcr genes to CLas.

Shared in planta population and transcriptomic features of nonpathogenic members of endophytic phyllosphere microbiota.

SignificancePlants evolved in an environment colonized by a vast number of microbes, which collectively constitute the plant microbiota. The majority of microbiota taxa are nonpathogenic and may be beneficial to plants under certain ecological or environmental conditions. We conducted experiments to understand the features of long-term interactions of nonpathogenic microbiota members with plants. We found that a multiplication-death equilibrium explained the shared long-term static populations of nonpathogenic bacteria and that in planta bacterial transcriptomic signatures were characteristic of the stationary phase, a physiological state in which stress protection responses are induced. These results may have significant implications in understanding the bulk of "nonpathogenic" plant-microbiota interactions that occur in agricultural and natural ecosystems.

Comparative Genomics of Fusarium circinatum Isolates Used to Screen Southern Pines for Pitch Canker Resistance.

Pitch canker, caused by the fungal pathogen Fusarium circinatum, is a global disease affecting many Pinus spp. Often fatal, this disease causes significant mortality in both commercially grown and natural pine forests and is an issue of current and growing concern. F. circinatum isolates collected from three locations in the U.S. state of Florida were shown to be virulent on both slash and loblolly pine, with two of the isolates causing equivalent and significantly larger lesions than those caused by the third isolate during pathogenicity trials. In addition, significant genetic variation in lesion length in the pedigreed slash pine population was evident and rankings of parents for lesion length were similar across isolates. Experimental data demonstrate that both host and pathogen genetics contribute to disease severity. High-quality genomic assemblies of all three isolates were created and compared for structural differences and gene content. No major structural differences were observed among the isolates; however, missing or altered genes do contribute to genomic variation in the pathogen population. This work evaluates in planta virulence among three isolates of F. circinatum, provides genomic resources to facilitate study of this organism, and details comparative genomic methods that may be used to explore the pathogen's contribution to disease development.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Draft Genome Sequence and De Novo Assembly of a Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia.

We report a draft genome assembly of the causal agent of tomato vascular wilt, Fusarium oxysporum f. sp. lycopersici isolate 59, obtained from the Andean region in Colombia.

The Xa7 resistance gene guards the rice susceptibility gene SWEET14 against exploitation by the bacterial blight pathogen.

Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.

Fungal Epithiodiketopiperazines Carrying α,ß-Polysulfide Bridges from Penicillium steckii YE, and Their Chemical Interconversion.

Some fungal epithiodiketopiperazine alkaloids display α,ß-polysulfide bridges alongside diverse structural variations. However, the logic of their chemical diversity has rarely been explored. Here, we report the identification of three new (2, 3, 8) and five known (1, 4-7) epithiodiketopiperazines of this subtype from a marine-derived Penicillium sp. The structure elucidation was supported by multiple spectroscopic analyses. Importantly, we observed multiple nonenzymatic interconversions of these analogues in aqueous solutions and organic solvents. Furthermore, the same biosynthetic origin of these compounds was supported by one mined gene cluster. The dominant analogue (1) demonstrated selective cytotoxicity to androgen-sensitive prostate cancer cells and HIF-depleted colorectal cells and mild antiaging activities, linking the bioactivity to oxidative stress. These results provide crucial insight into the formation of fungal epithiodiketopiperazines through chemical interconversions.

Draft Genome Sequences of Three Fusarium circinatum Isolates Used To Inoculate a Pedigreed Population of Pinus elliottii Seedlings.

Here, we announce the draft genome sequences of three Fusarium circinatum isolates that were used to inoculate slash pines (Pinus elliottii) at the U.S. Forest Service Resistance Screening Center in Asheville, North Carolina. The genomes of these isolates were similar to other publicly available genomes, with average nucleotide identity values of >0.98.

A draft genome of Steinernema diaprepesi.

Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.

Waikikiamides A-C: Complex Diketopiperazine Dimer and Diketopiperazine-Polyketide Hybrids from a Hawaiian Marine Fungal Strain Aspergillus sp. FM242.

Waikikiamides A-C (1-3), structurally complex diketopiperazine derivatives, and putative biogenic precursors, (+)-semivioxanthin (4), notoamide F (5), and (-)-notoamide A (6), were isolated from Aspergillus sp. FM242. 1 and 2, bearing a hendecacyclic ring system, represent a novel skeleton. 3 features the first unique heterodimer of two notoamide analogs with an N-O-C bridge. Compounds 1 and 3 exhibit antiproliferative activity with IC50 values in the range of 0.56 to 1.86 µM. The gene clusters mined from the sequenced genome support their putative biosynthetic pathways.

Broad-spectrum resistance to bacterial blight in rice using genome editing.

Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.

Diagnostic kit for rice blight resistance.

Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.

Xanthomonas translucens commandeers the host rate-limiting step in ABA biosynthesis for disease susceptibility.

Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1 The results represent an appropriation of host physiology by a bacterial virulence effector.