Genome-wide functional analysis of pathogenicity genes in the rice blast fungus.

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.

Arabidopsis ecotype Ct-1, with its altered nitrate sensing ability, exhibits enhanced growth under low nitrate conditions in comparison to Col-0.

To address the urgent need for sustainable solutions to the increased use of nitrogen fertilizers in agriculture, it is imperative to acquire an in-depth comprehension of the intricate interplay between plants and nitrogen. In this context, our research aimed to elucidate the molecular mechanism behind NO3- sensing/signaling in plants, which can enhance nitrogen utilization efficiency. Previous reports have revealed that the density and quantity of root hairs exhibit responsive behavior to varying levels of NO3-, while the precise molecular mechanisms governing these changes remain elusive. To further investigate this phenomenon, we specifically selected the Ct-1 ecotype, which manifested a greater abundance of root hairs compared to the Col-0 ecotype under conditions of low NO3-. Our investigations unveiled that the dissimilarities in the amino acid sequence of NRT1.1, a transceptor responsible for regulating nitrate signaling and transport, accounted for the observed variation in root hair numbers. These results suggest that NRT1.1 represents a promising target for gene editing technology, offering potential applications in enhancing the efficiency of nitrogen utilization in agricultural crops.

Enhanced growth performance of abi5 plants under high salt and nitrate is associated with reduced nitric oxide levels.

Numerous environmental stresses have a significant impact on plant growth and development. By 2050, it is anticipated that high salinity will destroy more than fifty percent of the world's agricultural land. Understanding how plants react to the excessive use of nitrogen fertilizers and salt stress is crucial for enhancing crop yield. However, the effect of excessive nitrate treatment on plant development is disputed and poorly understood; so, we evaluated the effect of excessive nitrate supply and high salinity on abi5 plant growth performance. We demonstrated that abi5 plants are tolerant to the harmful environmental conditions of excessive nitrate and salt. abi5 plants have lower amounts of endogenous nitric oxide than Arabidopsis thaliana Columbia-0 plants due to their decreased nitrate reductase activity, caused by a decrease in the transcript level of NIA2, a gene encoding nitrate reductase. Nitric oxide appeared to have a critical role in reducing the salt stress tolerance of plants, which was diminished by an excess of nitrate. Discovering regulators such as ABI5 that can modulate nitrate reductase activity and comprehending the molecular activities of these regulators are crucial for the application of gene-editing techniques. This would result in the appropriate buildup of nitric oxide to increase the production of crops subjected to a variety of environmental stresses.

Comparison of 3-month cytogenetic and molecular assays for early assessment of long-term clinical impact after BCR-ABL1 tyrosine kinase inhibitor treatment in chronic myeloid leukemia.

To compare the clinical significance of 3-month cytogenetic and molecular monitoring, we analyzed 1,410 paired cytogenetic and molecular data from 705 chronic-phase chronic myeloid leukemia patients. Based on early cytogenetic response (ECyR, Ph+≤35 %) and molecular response (EMR, BCR-ABL1IS≤10 %) at 3 months, the patients were divided into four groups (group 1: ECyR + EMR, n = 560; group 2: no ECyR + EMR, n = 27; group 3: ECyR + no EMR, n = 55; group 4: no ECyR + no EMR, n = 63). By 10 years, major molecular response (MMR), deep molecular response (MR4.5), overall survival (OS), and progression-free survival (PFS) rates were significantly high in group 1 (P < 0.001). Comparing groups 2 and 3, the MMR (P = 0.096), MR4.5 (P = 0.945), OS (P = 0.832), and PFS (P = 0.627) rates tended to be higher in group 2, although not significantly. Thus, the cytogenetic assay can not only be useful but its addition may also provide a more precise prediction of MR4.5.

A Phase 3, Multicenter, Randomized, Controlled Trial to Evaluate Immune Equivalence and Safety of Multidose and Single-dose Formulations of Vi-DT Typhoid Conjugate Vaccine in Healthy Filipino Individuals 6 Months to 45 Years of Age.

Trial Design: Phase 3, randomized, controlled, multicenter, equivalence trial. Methods: Recruitment of participants occurred between 04Februray2020 and 15July2020 at four centers in the Philippines: University of the East - Ramon Magsaysay Memorial Medical Center Inc., Quezon City; University of Philippines Manila - National Institute of Health, Ermita Manila; Asian Hospital and Medical Center, Metro Manila, Philippines Study; and Medical Research Unit, Tropical Disease Foundation, Makati City, Metro Manila, Philippines. Participants: 1800 adults and children 6-months to 45-years of age. Interventions: Participants received a single injection of multidose (MD) or single dose (SD) Vi-DT as test vaccines or meningococcal conjugate vaccine as a comparator. Objective: To evaluate immune equivalence of SD and MD formulations of Vi-DT, and to assess the safety of both formulations compared with comparator vaccine. Outcome Measurement: Blood draw for immunogenicity was performed at baseline prior to vaccine receipt and at four weeks after vaccination for a subset of participants to determine anti-Vi IgG geometric mean titers (GMT) and seroconversion rates. The primary outcome was comparison of anti Vi-IgG seroconversion and GMT between the two formulations of Vi-DT at 4 weeks following vaccine administration. Immune equivalence of MD and SD formulations was confirmed when the two-tailed 95% confidence interval (CI) of the GMT ratio is within [0.67, 1.5] at a two-sided significance level of 0.05. All participants were followed for safety events for six months after vaccine administration. Randomization: Participants were randomized to receive SD Vi-DT, MD Vi-DT, or meningococcal conjugate vaccines in 2.5:2.5:1 allocation ratio. Blinding: Study participants and observers were blinded to treatment assignment. Findings: Immune equivalence of SD (n=252) and MD (n=247) formulations was confirmed by anti-Vi IgG GMT ratio of 1.14 (95%CI: 0.91, 1.43) with respective GMTs in the MD and SD groups of 640.62 IU/mL (95%CI: 546.39, 751.11) and 562.57 IU/mL (95%CI: 478.80, 661.00) (p=0.259). Similarly, anti-Vi IgG seroconversion rate difference between the two formulations of ‒0.43% (95%CI: -4.42, 3.56) confirmed immune equivalence with corresponding seroconversion rates of 98.38% (95%CI: 95.91, 99.37) and 98.81% (95%CI: 96.56, 99.59) in MD and SD Vi-DT formulations, respectively (p=0.722). Both formulations of Vi-DT had a satisfactory safety profile - all five serious adverse events reported during the study were unrelated to the investigational product. Interpretation: The MD and SD formulations of Vi-DT elicited robust and equivalent immune responses following one dose vaccination, and both formulations demonstrated a favorable safety profile. Trial Registration: ClinicalTrials.gov: NCT04204096. Funding: This study was funded by the Bill & Melinda Gates Foundation (OPP 1115556).

Immune persistence and response to booster dose of Vi-DT vaccine at 27.5 months post-first dose.

Vaccination with typhoid conjugate vaccines (TCV) is a major part of typhoid prevention. However, little is known about long-term immune persistence following vaccination with TCVs. In this phase-2, randomized double-blind trial (NCT03527355), 285 children aged 6-23 months were randomized to one of three groups: (1) the group that received a first dose of Vi polysaccharide conjugated to diphtheria-toxoid (Vi-DT) vaccine followed by an "early booster" at 24 weeks, (2) the group that which received a first dose of Vi-DT followed by a "late booster" at 96 or 110 weeks, and (3) comparator group. Safety and immunogenicity of anti-Vi IgG GMTs were assessed at weeks 0, 4, 24, 28, 60, 96, 110, and 114 since the first dose. Here, we describe persistence of immune responses at weeks 60, 96, 110, and 114 post first dose. The anti-Vi IgG seroconversion rate after 27.5 months of follow-up was 88.16% (95% CI: 79.00, 93.64) in late-booster and 94.76% (95% CI: 86.91, 97.88) in early booster Vi-DT groups (p = 0.081). Whereas anti-Vi IgG GMTs were significantly higher in the early booster group (11.95 [95% CI: 9.65, 14.81]) than prebooster GMTs in the late booster group (5.50 [95% CI: 4.44, 6.80], p < 0.0001). GMT in the late booster group significantly increased to 351.76 (95% CI: 265.01, 466.93) (p < 0.0001) when measured 4 weeks after they received their "late-booster" shot. In conclusion, late booster dosing with Vi-DT at 27.5 months post first dose was safe and elicited robust anti-Vi IgG immune responses. Anti-Vi IgG seroconversion rates were persistently comparable in early and late-booster Vi-DT groups.

Comprehensive analyses of safety and efficacy toward individualizing imatinib dosage in patients with chronic myeloid leukemia.

Safety and efficacy outcomes of imatinib treatment were evaluated using extensive clinical data collected from a total of 1003 patients with newly diagnosed chronic myeloid leukemia in chronic phase between 2001 and 2018. By 12 months of imatinib treatment at a fixed dose of 400 mg/day, 45.4% of patients experienced at least one type of dose-limiting toxicities (DLTs). The DLTs that frequently occurred first were thrombocytopenia (40.0%), neutropenia/leukopenia (14.3%) and dermatological reactions (12.1%). Patients with lighter body weight (≤ 64 kg) and older age (> 43 years) experienced a markedly higher occurrence of first DLTs by 12 months than heavier and younger patients (57.9% vs. 30.1%, p < 0.001). On the other hand, 38.9% of patients achieved major molecular response (MMR) at 12 months at the fixed dose. Female patients achieved a greater rate of MMR than male patients (45.6% vs. 35.5%, p = 0.028). In conclusion, patients with light weight and old age are more vulnerable to DLTs, whereas female patients gain more efficacy benefit at the fixed dose. The authors suggest that the initial dose of imatinib should be reduced to 300 mg/day or lower for patients vulnerable to DLTs to diminish the risk of DLTs without compromising the achievement of MMR.

Immunogenicity, safety and reactogenicity of a Phase II trial of Vi-DT typhoid conjugate vaccine in healthy Filipino infants and toddlers: A preliminary report.

BACKGROUND: Typhoid fever remains an important public health problem in developing countries and is endemic in many parts of Asia and Africa where the incidence of disease typically peaks in school-aged children. Age restrictions and other limitations of existing oral live-attenuated typhoid and parenteral Vi polysaccharide vaccines have triggered the development of Vi conjugate vaccines with improved immunological properties, use in younger age range, and longer durability of protection. We present the safety, reactogenicity, and immunogenicity data from a Phase II study after a single dose of Vi polysaccharide conjugated to diphtheria toxoid (Vi-DT) conducted in 6-23-month old Filipino children. METHODS: This is a randomized, observer-blinded Phase II study to assess the immunogenicity, safety and reactogenicity of Vi-DT compared to placebo, conducted in Muntinlupa City, The Philippines. Participants aged 6-23 months were enrolled and randomized to Vi-DT (25 µg) or placebo (0.9% sodium chloride) and evaluated for immunogenicity and overall safety 28 days post vaccination. RESULTS: A total of 285 participants were enrolled and age-stratified: 6 to < 9 months, 9-12 months, and 13-23 months. Seventy-six (76) participants received Vi-DT and 19 received placebo per each strata. All participants seroconverted after a single dose of Vi-DT versus 7% of placebo recipients. Anti-Vi IgG GMT was 444.38 [95% CI (400.28; 493.34)] after a single dose of Vi-DT; there was no change in GMT after placebo administration, 0.41 [95% CI (0.33; 0.51), p < 0.0001]. A similar pattern of immunogenicity was reported across all age strata. The vaccine reported to be safe and well tolerated. CONCLUSIONS: Vi-DT vaccine was immunogenic, safe, and well tolerated in children aged 6-23 months. ClinicalTrials.gov registration number: NCT03527355.

Safety and immunogenicity of Vi-DT conjugate vaccine among 6-23-month-old children: Phase II, randomized, dose-scheduling, observer-blind Study.

BACKGROUND: Typhoid causes significant mortality among young children in resource-limited settings. Conjugate typhoid vaccines could significantly reduce typhoid-related child deaths, but only one WHO-prequalified typhoid conjugate vaccine exists for young children. To address this gap, we investigated the safety, immunogenicity and dose-scheduling of Vi-DT typhoid conjugate vaccine among children aged 6-23 months. METHODS: In this single center, observer blind, phase II trial, participants were randomly assigned (2:2:1) to receive one or two doses of Vi-DT or comparator vaccine. Anti-Vi IgG titer and geometric mean titers (GMT) were determined at 0, 4, 24 and 28 weeks. Data were analyzed using per-protocol and immunogenicity (a subset of intention-to-treat analysis) sets. The trial is registered with ClinicalTrials.gov (NCT03527355). FINDINGS: Between April and July 2018, 285 children were randomized; 114 received one or two doses of Vi-DT while 57 received comparator. 277 completed the study follow-up per protocol; 112 and 110 from single- and two-dose Vi-DT schedules, respectively and 55 from the placebo group were included in the per protocol analysis. Safety profile is satisfactory. Thirteen serious adverse events were reported during the 28-week follow-up, none of which were related to Vi-DT. The seroconversion rate four weeks after the first dose was 100% (95% CI 98·3-100) in Vi-DT recipients and 7·0% (95% CI 2·8-16·7) in comparator recipients (p<0·0001). Similarly, the seroconversion rate 4 weeks after the second dose was 98·2% (95% CI 93· 6-99·5) and 21·8% (95% CI 13·0-34·4) among Vi-DT and comparator groups, respectively (p<0·0001). Anti-Vi IgG GMT was significantly higher in Vi-DT than in control group at all post-vaccination visits (p<0·0001). INTERPRETATION: Both single and two doses of Vi-DT vaccine are safe, well tolerated, and immunogenic for infants and toddlers in a moderately endemic setting.

Compound mutations involving T315I and P-loop mutations are the major components of multiple mutations detected in tyrosine kinase inhibitor resistant chronic myeloid leukemia.

To analyze the pattern of multiple mutations detected by Sanger sequencing (SS), we performed subcloning sequencing using 218 samples from 45 patients with tyrosine kinase inhibitor resistant chronic myeloid leukemia. At the first time of multiple mutation detection by SS (baseline), a total of 19 major mutations from 45 samples were detected; these mutations were found in the following order: T315I (68.9%), E255 K (33.3%), Y253H (13.3%), G250E (13.3%), and F317 L (11.1%). Subcloning sequencing of 900 baseline colonies identified 556 different mutant types, and 791 among the 900 were colonies with major mutations (87.9%). The mutations were found in the following order: T315I (36.4%), E255 K (16.2%), Y253H (7.0%), G250E (6.7%), M351 T (6.6%), and E255 V (5.3%). In subcloning sequencing with 4357 colonies of 218 serial samples, 2506 colonies (57.5%) had compound mutations, among which 2238 colonies (89.3%) had at least one major mutation. The median number of mutations in compound mutant colonies was 2 (range, 2-7), and most were double (52.9%) or triple (28.7%) mutations. Additionally, some mutations in allosteric binding sites were detected as low level mutation in 13 patients. With the available retrospective samples before baseline, subcloning sequencing identified low-level mutations of various frequencies (median, 10%) to be major mutations in 20 patients. Thus, compound mutations involving T315I and P-loop mutations were the major components of multiple mutations, and some low-level mutations with potential clinical significance were detected by subcloning sequencing. Hence, more sensitive sequencing assays are needed in patients with multiple mutations.

BCR-ABL1 transcript levels at 4 weeks have prognostic significance for time-specific responses and for predicting survival in chronic-phase chronic myeloid leukemia patients treated with various tyrosine kinase inhibitors.

The present study aimed to assess the clinical impact of BCR-ABL1 transcript levels determined at an earlier time point than the 3-month early molecular response (EMR) in chronic-phase chronic myeloid leukemia (CML-CP) patients. BCR-ABL1 transcript levels of CML-CP patients (n = 258; median age, 43 [range, 18-81] years) treated with various tyrosine kinase inhibitors (TKIs) were determined at 4 weeks (28 ± 3 days) and at every 3 months of treatment initiation. At 4 weeks, receiver operating characteristic curves revealed that cutoff values of BCR-ABL1 transcripts for achieving major molecular responses (MMRs) by 12 and 60 months were 40.89% and 39.16%, respectively (95% CI, 0.658-0.772 and 95% CI, 0.643-0.758; P < 0.0001). With 40% of BCR-ABL1 transcripts at 4 weeks (very early MR; VEMR), patients with VEMR achieved higher 3-month EMR and 4-week VEMR significantly associated with higher cumulative incidences of 5-year MMR (89.1% vs 72.3%; P < 0.001) and 5-year deep molecular response (DMR) (56.5% vs 29.4%; P = 0.001). Furthermore, event-free survival (EFS)-a (93.0% vs 84.8%; P = 0.068) and EFS-b (71.1% vs 57.9%; P = 0.061) by 5 years were also marginally significant. VEMR and 3-month EMR were achieved in 89 patients, with significantly superior outcomes. In multivariate analyses, lower leukocyte count (P = 0.008) and frontline second-generation TKI therapy size (P < 0.001) were significantly associated with VEMR achievement, but not baseline BCR-ABL1 level and CML duration. In conclusion, the 4-week BCR-ABL1 transcript levels including VEMR could be important to predict long-term outcomes and may provide additional information about innate intrinsic sensitivity to CML among individuals.

Safety and immunogenicity of a Vi-DT typhoid conjugate vaccine: Phase I trial in Healthy Filipino adults and children.

BACKGROUND: Typhoid fever remains a major public health problem in low- and middle-income countries where children aged 2-14 years bear the greatest burden. Vi polysaccharide is poorly immunogenic in children <2 years of age, and protection in adults is modest. The limitations of Vi polysaccharide vaccines can be overcome by conjugation of the Vi to a carrier protein. A typhoid conjugate vaccine composed of Vi polysaccharide conjugated to diphtheria toxoid (Vi-DT) has been developed. The Phase I study results are presented here. METHODS: This was a randomized, observer-blinded Phase I study to assess the safety and immunogenicity of Vi-DT compared to Vi polysaccharide vaccine, conducted in Manila, Philippines. Participants enrolled in an age de-escalation manner (18-45, 6-17 and 2-5 years) were randomized between Test (Vi-DT, 25 µg) administered at 0 and 4 weeks and Comparator (Vi polysaccharide, Typhim Vi® and Vaxigrip®, Sanofi Pasteur) vaccines. RESULTS: A total of 144 participants were enrolled (48 by age strata, 24 in Test and Comparator groups each). No serious adverse event was reported in either group. Solicited and unsolicited adverse events were mild or moderate in both groups with the exception of a 4-year old girl in Test group with grade 3 fever which resolved without sequelae. All participants in Test group seroconverted after first and second doses of Vi-DT while the proportions in the Comparator group were 97.1% and 97.2%, after first dose of Typhim Vi® and second dose of Vaxigrip®, respectively. Vi-DT showed 4-fold higher Geometric Mean Titers (GMT) compared to Typhim Vi® (adjusted for age strata, p < 0.001). No further increase of GMT was detected after the second dose of Vi-DT. Anti-DT IgG seroresponse rates were 81.2% and 84.5% post first and second Vi-DT doses, respectively. CONCLUSIONS: Vi-DT vaccine was safe, well-tolerated and immunogenic in participants aged 2-45 years. ClinicalTrials.gov registration number: NCT02645032.