What Is the Appropriate Antibiotic Administration During Tooth Extractions in Patients Receiving High-Dose Denosumab?

PURPOSE: Medication-related osteonecrosis of the jaw (MRONJ) occasionally occurs following tooth extractions in cancer patients receiving denosumab (Dmab). However, there are currently no established guidelines for perioperative antibiotic administration during tooth extraction in these patients. The primary objective was to develop guidelines for the dose and frequency of antibiotics during tooth extraction by investigating the correlation between the current status of antibiotic administration and the development of MRONJ. METHODS: This study included 68 cancer patients receiving high-dose Dmab who had tooth extractions between 2012 and 2022 at 10 hospitals. The relationship between the way of perioperative antibiotic administration and the development of MRONJ was analyzed. A P-value < .05 was considered significant. RESULTS: There was considerable variability across hospitals and surgeons regarding the type, dosage, and duration of antibiotic administration. Amoxicillin (AMPC) was the most commonly used antibiotic. Focusing exclusively on teeth extracted under AMPC administration, MRONJ developed in 21 out of 123 teeth (17.0%). No significant relationship was found between the development of MRONJ and the dosage or duration of perioperative AMPC administration. CONCLUSION: Perioperative antibiotic administration alone may not be sufficient to prevent MRONJ. Therefore, a single preoperative dose is likely adequate for effective and appropriate AMPC administration.　.

An observational study on the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma.

BACKGROUND: Pulmonary sarcomatoid carcinoma is a rare tumor that is resistant to cytotoxic agents. This observational study aimed to evaluate the detection rate of driver gene alteration and the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma. METHODS: We established a database of patients with pulmonary sarcomatoid carcinoma and their clinical information, including EGFR mutation, ALK fusion gene, ROS1 fusion gene, BRAF mutation, and MET exon 14 skipping mutation. The present study retrieved and analyzed the data of patients with pulmonary sarcomatoid carcinoma in whom driver gene alterations were evaluated, and the survival duration after the initiation of treatment with targeted therapy was examined. RESULTS: A total of 44 patients were included in the present study. The EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in 2/43 patients (4.7%), 2/34 patients (5.9%), and 2/16 patients (12.5%), respectively. The ROS1 fusion gene (0/18 patients) and BRAF mutation (0/15 patients) were not detected. Female patients (P = 0.063, Fisher's exact test) and patients without smoking history (P = 0.025, Fisher's exact test) were the dominant groups in which any driver mutation was detected. Five patients with driver gene alterations were treated with targeted therapy. Progression-free survival (PFS) was 1.3 months and 1.6 months in 2 of the patients treated with gefitinib. Two patients with the ALK fusion gene showed 2.1 and 14.0 months of PFS from the initiation of treatment with crizotinib, and a patient with the MET exon 14 skipping mutation showed 9.7 months of PFS from the initiation of treatment with tepotinib. CONCLUSION: The EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in patients with pulmonary sarcomatoid carcinoma in clinical practice, and some patients achieved long survival times after receiving targeted therapy. Further investigation is necessary to evaluate the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma.

Comparison of the efficacy of first­/second­generation EGFR­tyrosine kinase inhibitors and osimertinib for EGFR­mutant lung cancer with negative or low PD­L1 expression.

In epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) with negative or low programmed death ligand-1 (PD-L1) expression, the acquisition rate of the T790M mutation is higher after treatment with first-/second-generation EGFR-tyrosine kinase inhibitors (TKIs) and the progression-free survival (PFS) is longer in patients treated with osimertinib. The present study compared the clinical course after the initiation of each EGFR-TKI monotherapy in patients with EGFR-mutant NSCLC with negative or low PD-L1 expression. Data of patients with EGFR-mutant NSCLC with negative or low PD-L1 expression who were treated with EGFR-TKI monotherapy were retrieved and retrospectively analyzed. Between June 2013 and November 2023, 26 and 29 patients were treated with first-/second-generation EGFR-TKIs and osimertinib, respectively. The PFS time was longer in patients treated with osimertinib (median, 22.5 months) than in those treated with first-/second-generation EGFR-TKIs (median, 12.9 months). However, the EGFR-TKI treatment duration, defined as the PFS for osimertinib, or the sum of the PFS for first-/second-generation EGFR-TKIs and sequential osimertinib therapy after the acquisition of the T790M mutation, was similar between patients treated with first-/second-generation EGFR-TKIs (median, 23.0 months) and osimertinib (median, 22.5 months). The Cox proportional hazard model suggested that there was no significant difference in the EGFR-TKI treatment duration between patients treated with first-/second-generation EGFR-TKIs and patients treated with osimertinib (hazard ratio, 1.31, 95% CI, 0.55-3.13). In conclusion, first-/second-generation EGFR-TKIs and osimertinib were associated with a similar EGFR-TKI treatment duration in patients with EGFR-mutant NSCLC with negative or low PD-L1 expression. The findings suggested that both treatments are promising for this population.

Pure Red Cell Aplasia and Chromosomal Abnormality in a Patient With Lung Adenocarcinoma Receiving Immune Checkpoint Inhibitors: A Case Report.

BACKGROUND/AIM: Immune checkpoint inhibitors can induce immune-related adverse events in various organs, thus careful observation is required. CASE REPORT: A 69-year-old man was diagnosed with advanced lung adenocarcinoma and treated with combined therapy of carboplatin plus pemetrexed plus pembrolizumab. After two cycles of treatment, anemia was noted. Myelosuppression due to cytotoxic anticancer agents was suspected and the cytotoxic agents were discontinued, followed by three courses of pembrolizumab monotherapy. However, the anemia persisted, requiring red blood cell transfusions. A bone marrow biopsy revealed erythroblast hypoplasia and chromosomal abnormalities, resulting in a diagnosis of pure red cell aplasia. These adverse events were considered immune-related because of the treatment history with an immune checkpoint inhibitor, and 60 mg/day (1 mg/kg/day) of prednisolone was initiated. Anemia improved, and it did not recur during the tapering of prednisolone. CONCLUSION: Immune-related pure red cell aplasia should be considered for patients presenting anemia during treatment with immune checkpoint inhibitors.

Immune Checkpoint Inhibitor Therapy for Patients With Non-small Cell Lung Cancer Ineligible for Platinum Doublet Chemotherapy.

BACKGROUND/AIM: Combined therapy with immune checkpoint inhibitors plus platinum doublet chemotherapy has a survival advantage over platinum doublet chemotherapy in patients with non-small cell lung cancer. However, a variety of factors make it difficult to administer treatment with platinum doublet chemotherapy in many patients in clinical practice and there are few reports on the efficacy and safety of first-line treatments with immune checkpoint inhibitors for patients who are ineligible for platinum doublet chemotherapy. This observational study aimed to evaluate the efficacy and safety of first-line immune checkpoint inhibitor therapy for this population. PATIENTS AND METHODS: We retrospectively assessed the survival and adverse events from the initiation of first-line immune checkpoint inhibitor therapy, including pembrolizumab or nivolumab plus ipilimumab in patients with non-small cell lung cancer who were ineligible for platinum doublet chemotherapy. RESULTS: Data from 48 patients were analyzed. Seventeen patients showed a performance status (PS) of ≥2 while 16 and 15 patients were considered ineligible for platinum doublet chemotherapy because of age and comorbidities, respectively. The median (95% confidential interval, CI) progression-free survival (PFS) and overall survival (OS) of the 48 patients were 7.1 (1.7-13.7) and 31.7 (8.8-not estimated) months, respectively. The two-year PFS and OS rates (95% CI) were 30.8% (18.2%-47.2%) and 50.7% (33.7%-67.7%), respectively. In patients with a PS of ≥2, the median (95% CI) PFS and OS were 1.6 (1.2-not estimated) and 5.5 (2.3-not estimated) months, respectively. The two-year PFS and OS rates (95% CI) were 34.3% (15.8%-59.2%) and 45.3% (22.2%-70.7%), respectively. CONCLUSION: Patients with non-small cell lung cancer and a PS of 0-1 who were ineligible for platinum doublet chemotherapy had favorable outcome after the initiation of ICI therapy, and even in patients with a PS of ≥2, they achieved high two-year PFS and OS rates.

Efficient selection of a biallelic and nonchimeric gene-edited tree using Oxford Nanopore Technologies sequencing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e., indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation.

Formation of xylem tissues and secondary cell walls is diminished by severe and consecutive insect defoliation.

PREMISE: Insect defoliation of trees causes unusual changes to wood anatomy and slows radial growth that decreases tree value; however, the characteristics of these anatomical changes in hardwoods remain unclear. The aim of this study was to characterize the anatomy and histochemistry of the wood in trunks of Betula maximowicziana trees after severe insect defoliation. METHODS: Secondary xylem tissues were sampled from trunks that had been defoliated by Caligula japonica at Naie and Furano in central Hokkaido during 2006-2012, then cross-dated and examined microscopically and stained histochemically to characterize anatomical and chemical changes in the cells. RESULTS: White rings with thin-walled wood fibers and greatly reduced annual ring width in the subsequent year were observed in samples from both sites. From these results, the year that the white rings formed was determined, and severe defoliation was confirmed to trigger white ring formation. The characteristics may prove useful to detect the formation year of white rings. Scanning electron microscopy and histochemical analyses of the white rings indicated that the thickness of the S2 layer in the wall of wood fiber cells decreased, but xylan and lignin were still deposited in the cell walls of wood fibers. However, the walls of the fibers rethickened after the defoliation. CONCLUSIONS: Our results suggest that B. maximowicziana responds to a temporary lack of carbon inputs due to insect defoliation by regulating the thickness of the S2 layer of the cell wall of wood fibers. For B. maximowicziana, insect defoliation late in the growing season has serious deleterious effects on wood formation and radial growth.

Barriers to undergoing cervical cancer screening among health sciences university students in Japan: A cross-sectional study.

In most developed countries, cervical cancer screening and human papillomavirus vaccination have reduced cervical cancer incidence. However, the incidence has been increasing in Japan, possibly because of the low screening rate. Although cervical cancer incidence has increased in people in their 20s, the screening rate among 20-24-year-olds in Japan is only 10.2%, meaning that cervical cancer screening rates should be increased among young Japanese women. We conducted a questionnaire survey among students at health sciences universities to determine their knowledge of cervical cancer, screening rates, and barriers to screening. Students taking specialized medical courses were highly knowledgeable; recognition of the facts that "cervical cancer can be prevented through screening" and that "the risk of cervical cancer increases in one's 20s" was significantly high among those who underwent screening. On the other hand, only 7.5% of students used the free coupons provided for screening. Knowledge of cervical cancer improves screening rates. Therefore, educational programs to raise awareness of the importance of cervical cancer screening among non-medical and health sciences university students and young women in general are required.

Peripheral CD4 memory T cells predict the efficacy of immune checkpoint inhibitor therapy in patients with non-small cell lung cancer.

Immune checkpoint inhibitors have significantly improved the prognosis in patients with non-small cell lung cancer, compared with cytotoxic agents. However, the prediction of treatment response is often difficult, even after assessing the tumor programmed death-ligand 1 expression. We conducted this observational study to analyze the association between the differentiation of peripheral CD4 + T cells and the efficacy of immune checkpoint inhibitor therapy. We enrolled patients who were diagnosed with non-small cell lung cancer and received immune checkpoint inhibitor therapy between 2020 and 2022. Blood samples were collected at the start of immune checkpoint inhibitor therapy, and the expressions of PD-1, CCR7, and CD45RA in peripheral CD4 + T cells were analyzed by flow cytometry. The association between the findings of flow cytometry and survival after the initiation of the immune checkpoint inhibitor therapy was evaluated. Forty patients with non-small cell lung cancer were enrolled. The Cox proportional hazards model showed that an increased proportion of CD45RA-CD4 + T cells was associated with a reduced risk of progression after adjustment for performance status, tumor programmed death-ligand 1 expression level, mutation status of the epidermal growth factor receptor gene, and combined therapy with cytotoxic agents. The present study showed that the proportion of peripheral CD45RA- CD4 + T cells was associated with progression-free survival after the initiation of immune checkpoint inhibitor therapy, independent of several clinical factors.

Is the LRINEC score useful for predicting necrotizing fasciitis as a complication of MRONJ?

INTRODUCTION: Necrotizing fasciitis as a complication of medication-related osteonecrosis of the jaw (MRONJ), which we named "ONJ-NF", has been sometimes reported. This study aimed to investigate the usefulness of the Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score for predicting ONJ-NF. MATERIALS AND METHODS: We included patients with acute MRONJ who required hospitalization at a single institution from April 2013 to June 2022. They were divided into two groups: patients with ONJ-NF and those with severe cellulitis as a complication of MRONJ, which we named "ONJ-SC." LRINEC scores were compared between the groups and the cut-off value of the score was set by creating a receiver operating characteristic curve. RESULTS: Eight patients with ONJ-NF and 22 patients with ONJ-SC were included. The LRINEC score was significantly higher in patients with ONJ-NF (median: 8.0 points, range 6-10 points) than in those with ONJ-SC (median: 2.5 points, range 0-6 points). A LRINEC score of ≥ 6 points had a sensitivity of 100.0%, a specificity of 77.3%, and an area under the curve of 0.97. Among 6 parameters of LRINEC score, only C-reactive protein (CRP) and white blood cell count (WBC) had significant differences between two groups. Most of the patients with ONJ-NF were rescued by antibiotic therapy and surgical drainage including debridement of necrotic tissues, but unfortunately, one patient did not survive. CONCLUSION: Our results suggested that the LRINEC score may be a useful diagnostic tool to predict ONJ-NF but valuating only CRP and WBC may be sufficient particularly in patients with osteoporosis.

Efficacy of immune checkpoint inhibitor therapy in patients with pulmonary sarcomatoid carcinoma in clinical practice.

OBJECTIVE: Studies have suggested the potential efficacy of immune checkpoint inhibitors (ICIs) for pulmonary sarcomatoid carcinoma. This multicenter observational study was conducted to evaluate the efficacy of systemic ICI therapy and chemoradiation followed by durvalumab therapy for pulmonary sarcomatoid carcinoma. METHODS: We analyzed the data of patients with pulmonary sarcomatoid carcinoma who received systemic ICI therapy or chemoradiation followed by durvalumab therapy between 2016 and 2022. RESULTS: In this study, data of a total of 22 patients who received systemic ICI therapy and four patients who received chemoradiation followed by durvalumab therapy were analyzed. In the patients who received systemic ICI therapy, the median progression-free survival after initiation of therapy was 9.6 months, and the overall survival did not reach the median. The 1-year progression-free survival rate and overall survival rate were estimated to be 45.5% and 50.1%, respectively. Although the log-rank test revealed no significant association between the tumor expression level of programmed death ligand-1 (tumor proportion score evaluated using 22C3 antibody: ≥50% vs. <50%) and the survival duration, the majority of patients showing long-term survival showed a tumor proportion score of ≥50%. Of four patients treated with chemoradiation followed by durvalumab therapy, two patients showed an overall survival of ≥30 months, whereas the remaining two patients died within 12 months. CONCLUSION: The progression-free survival of patients who received systemic ICI therapy was 9.6 months, suggesting that ICI therapy might be effective in patients with pulmonary sarcomatoid carcinoma.

Hematologic and inflammatory parameters for determining severity of odontogenic infections at admission: a retrospective study.

BACKGROUND: Severe odontogenic infections in the head and neck region, especially necrotizing soft tissue infection (NSTI) and deep neck abscess, are potentially fatal due to their delayed diagnosis and treatment. Clinically, it is often difficult to distinguish NSTI and deep neck abscess in its early stage from cellulitis, and the decision to perform contrast-enhanced computed tomography imaging for detection is often a challenge. This retrospective case-control study aimed to examine the utility of routine blood tests as an adjunctive diagnostic tool for NSTI in the head and neck region and deep neck abscesses. METHODS: Patients with severe odontogenic infections in the head and neck region that required hospitalization were classified into four groups. At admission, hematologic and inflammatory parameters were calculated according to the blood test results. In addition, a decision tree analysis was performed to detect NSTI and deep neck abscesses. RESULTS: There were 271 patients, 45.4% in Group I (cellulitis), 22.5% in Group II (cellulitis with shallow abscess formation), 27.3% in Group III (deep neck abscess), and 4.8% in Group IV (NSTI). All hematologic and inflammatory parameters were higher in Groups III and IV. The Laboratory Risk Indicator for Necrotizing Fasciitis score, with a cut-off value of 6 and C-reactive protein (CRP) + the neutrophil-to-lymphocyte ratio (NLR), with a cut-off of 27, were remarkably useful for the exclusion diagnosis for Group IV. The decision tree analysis showed that the systemic immune-inflammation index (SII) of ≥ 282 or < 282 but with a CRP + NLR of ≥ 25 suggests Group III + IV and the classification accuracy was 89.3%. CONCLUSIONS: Hematologic and inflammatory parameters calculated using routine blood tests can be helpful as an adjunctive diagnostic tool in the early diagnosis of potentially fatal odontogenic infections. An SII of ≥ 282 or < 282 but with a CRP + NLR of ≥ 25 can be useful in the decision-making for performing contrast-enhanced computed tomography imaging.

Microstructural Variations in Laser Powder Bed Fused Al-15%Fe Alloy at Intermediate Temperatures.

The samples of the Al-15Fe (mass%) binary alloy that were additively manufactured by laser powder bed fusion (L-PBF) were exposed to intermediate temperatures (300 and 500 °C), and the thermally induced variations in their microstructural characteristics were investigated. The L-PBF-manufactured sample was found to have a microstructure comprising a stable θ-Al13Fe4 phase localized around melt-pool boundaries and several spherical metastable Al6Fe-phase particles surrounded by a nanoscale α-Al/Al6Fe cellular structure in the melt pools. The morphology of the θ phase remained almost unchanged even after 1000 h of exposure at 300 °C. Moreover, the nanoscale α-Al/Al6Fe cellular structure dissolved in the α-Al matrix; this was followed by the growth (and nucleation) of the spherical Al6Fe-phase particles and the precipitation of the θ phase. Numerous equiaxed grains were formed in the α-Al matrix during the thermal exposure, which led to the formation of a relatively homogenous microstructure. The variations in these microstructural characteristics were more pronounced at the higher investigated temperature of 500 °C.

Tracheobronchitis in patients with diffuse wall thickening: Three case reports.

We herein report the cases of three patients with chest symptoms or fever and diffuse wall thickening of the trachea and main bronchi on chest CT. They were diagnosed with various causes of inflammations of the trachea and main bronchi using bronchial or tracheal biopsy specimens and flexible bronchoscopy.

Rare MYH9-ROS1 Fusion Gene-Positive Lung Adenocarcinoma Showing Response to Entrectinib Treatment: A Case Study.

The c-ros oncogene 1 (ROS1) fusion gene is a rare genomic alteration detected in nearly 1-2% of lung adenocarcinomas. The major partner genes of ROS1 include CD74, SDC4, and EZR. Here, we report a case of MYH9-ROS1 fusion gene-positive lung adenocarcinoma, a rare ROS1 fusion gene. The patient was a woman in her 40s who was diagnosed with advanced primary lung adenocarcinoma after a thorough examination. Initial genetic testing conducted using mediastinal lymph node biopsy specimens collected by endobronchial ultrasound-guided transbronchial needle aspiration revealed no driver gene mutations, including the ROS1 fusion gene. The patient was treated with four courses of immunochemotherapy. As the disease worsened, another genetic test was conducted using FoundationOne® CDx, and the MYH9-ROS1 fusion gene was detected. Multiple lung metastases disappeared after the administration of entrectinib; the response persisted up to a year. Adverse events of rash, dysgeusia, and peripheral edema were observed, and the patient required temporary drug interruption; however, we were able to continue entrectinib following a short-term drug interruption. This is the first report on the effectiveness of entrectinib against lung adenocarcinoma with the rare MYH9-ROS1 fusion gene.

ZEITLUPE Promotes ABA-Induced Stomatal Closure in Arabidopsis and Populus.

Plants balance water availability with gas exchange and photosynthesis by controlling stomatal aperture. This control is regulated in part by the circadian clock, but it remains unclear how signalling pathways of daily rhythms are integrated into stress responses. The serine/threonine protein kinase OPEN STOMATA 1 (OST1) contributes to the regulation of stomatal closure via activation of S-type anion channels. OST1 also mediates gene regulation in response to ABA/drought stress. We show that ZEITLUPE (ZTL), a blue light photoreceptor and clock component, also regulates ABA-induced stomatal closure in Arabidopsis thaliana, establishing a link between clock and ABA-signalling pathways. ZTL sustains expression of OST1 and ABA-signalling genes. Stomatal closure in response to ABA is reduced in ztl mutants, which maintain wider stomatal apertures and show higher rates of gas exchange and water loss than wild-type plants. Detached rosette leaf assays revealed a stronger water loss phenotype in ztl-3, ost1-3 double mutants, indicating that ZTL and OST1 contributed synergistically to the control of stomatal aperture. Experimental studies of Populus sp., revealed that ZTL regulated the circadian clock and stomata, indicating ZTL function was similar in these trees and Arabidopsis. PSEUDO-RESPONSE REGULATOR 5 (PRR5), a known target of ZTL, affects ABA-induced responses, including stomatal regulation. Like ZTL, PRR5 interacted physically with OST1 and contributed to the integration of ABA responses with circadian clock signalling. This suggests a novel mechanism whereby the PRR proteins-which are expressed from dawn to dusk-interact with OST1 to mediate ABA-dependent plant responses to reduce water loss in time of stress.

Risk factors associated with post-extraction bleeding in patients on warfarin or direct-acting oral anticoagulants: a retrospective cohort study.

PURPOSE: The purpose of this study was to investigate the risk factors associated with post-extraction persistent bleeding in patients on warfarin or direct-acting oral anticoagulants (DOACs) and the ability of risk scores to predict post-extraction bleeding. METHODS: Three hundred ninety-one patients taking warfarin or DOACs underwent tooth extractions. Various risk factors for post-extraction bleeding, including number of tooth extraction, with antiplatelet therapy, and risk scores, were investigated by univariate and multivariate analyses. A post-extraction bleeding was classified into grades 1-3. RESULTS: The incidence of post-extraction bleeding was 26.8% (77 out of 287 patients; grade 1: 63, grade 2:14) in patients taking warfarin, and 26.0% (27 out of 104 patients; grade 1: 20, grade 2:7) in patients taking warfarin DOACs. Multivariate analyses showed that multiple teeth extractions and HAS-BLED scores (above 3 points) in patients taking warfarin, and only multiple teeth extractions in patients taking DOAC, were significantly associated with post-extraction bleeding, respectively. CONCLUSION: Most of the post-extraction bleedings were grade 1, which can be stopped by eligibly pressing gauze by surgeons. If patients taking anticoagulants are scheduled to undergo multiple teeth extractions or their HAS-BLED score are above 3 points (if warfarin), we recommend informing patients risk of post-extraction bleeding before operation, taking carefully hemostasis, and instructing patients to bite down accurately on the gauze for longer than usual.

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Monitoring Seasonal Bud Set, Bud Burst, and Cold Hardiness in Populus.

Using a perennial model plant allows the study of reoccurring seasonal events in a way that is not possible using a fast-growing annual such as A. thaliana (Arabidopsis). In this study, we present a hybrid aspen (Populus tremula × P. tremuloides) as our perennial model plant. These plants can be grown in growth chambers to shorten growth periods and manipulate day length and temperature in ways that would be impossible under natural conditions. In addition, the use of growth chambers allows easy monitoring of height and diameter expansion, accelerating the collection of data from new strategies that allow evaluation of promoters or inhibitors of growth. Here, we describe how to study and quantify responses to seasonal changes (mainly using P. tremula × P. tremuloides) by measuring growth rate and key events under different photoperiodic cycles.