Lenalidomide promotes the development of TP53-mutated therapy-related myeloid neoplasms.

There is a growing body of evidence that therapy-related myeloid neoplasms (t-MNs) with driver gene mutations arise in the background of clonal hematopoiesis (CH) under the positive selective pressure of chemo- and radiation therapies. Uncovering the exposure relationships that provide selective advantage to specific CH mutations is critical to understanding the pathogenesis and etiology of t-MNs. In a systematic analysis of 416 patients with t-MN and detailed prior exposure history, we found that TP53 mutations were significantly associated with prior treatment with thalidomide analogs, specifically lenalidomide. We demonstrated experimentally that lenalidomide treatment provides a selective advantage to Trp53-mutant hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, the effect of which was specific to Trp53-mutant HSPCs and was not observed in HSPCs with other CH mutations. Because of the differences in CK1α degradation, pomalidomide treatment did not provide an equivalent level of selective advantage to Trp53-mutant HSPCs, providing a biological rationale for its use in patients at high risk for t-MN. These findings highlight the role of lenalidomide treatment in promoting TP53-mutated t-MNs and offer a potential alternative strategy to mitigate the risk of t-MN development.

Modeling and targeting of erythroleukemia by hematopoietic genome editing.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

A 17-gene stemness score for rapid determination of risk in acute leukaemia.

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC+ and 89 LSC- cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

Association of frailty with clinical outcomes in myelofibrosis: a retrospective cohort study.

There is limited understanding of the impact of frailty on clinical outcomes in patients with myelofibrosis (MF). In this retrospective cohort study on 439 chronic phase MF patients [mean age: 68·7 ± 12 years; median follow-up: 3·4 years (IQR 0·4-8·6)] from 2004 till 2018, we used a 35-variable frailty index (FI) to categorise patient's frailty status as fit (FI < 0·2, reference), prefrail (FI 0·2-0·29) or frail (FI ≥ 0·3). The association of frailty with overall survival (OS) and cumulative JAK inhibitor (JAKi) therapy failure was measured using hazard ratio (HR, 95% CI). In multivariable analysis, prefrail (HR 1·7, 1·1-2·5) and frail patients (HR 2·9, 1·6-5·5), those with higher DIPSS score (HR 2·5, 1·6-3·9) and transfusion dependency (HR 1·9, 1·3-2·9) had shorter OS. In a subset analysis of patients on JAKi treatment (n = 222), frail patients (HR 2·5, 1·1-5·7), patients with higher DIPSS score (HR 1·7, 1·0-3·1) and transfusion dependence (HR 1·7, 1·1-2·7) had higher cumulative incidence of JAKi failure. Age, comorbidities, ECOG performance status, and MPN driver mutations did not impact outcomes. Thus, higher frailty scores are associated with worse OS and increased JAKi failure in MF, and is a superior indicator of fitness in comparison to age, comorbidities, and performance status.

Cementless unicompartmental knee replacement achieves better ten-year clinical outcomes than cemented: a systematic review.

PURPOSE: The aim of this study was to report and compare the long-term revision rate, revision indications and patient reported outcome measures of cemented and cementless unicompartmental knee replacements (UKR). METHODS: Databases Medline, Embase and Cochrane Central of Controlled Trials were searched to identify all UKR studies reporting the ≥ 10 year clinical outcomes. Revision rates per 100 component years [% per annum (% pa)] were calculated by fixation type and then, subgroup analyses for fixed and mobile bearing UKRs were performed. Mechanisms of failure and patient reported outcome measures are reported. RESULTS: 25 studies were eligible for inclusion with a total of 10,736 UKRs, in which there were 8790 cemented and 1946 cementless knee replacements. The revision rate was 0.73% pa (CI 0.66-0.80) and 0.45% pa (CI 0.34-0.58) per 100 component years, respectively, with the cementless having a significantly (p < 0.001) lower overall revision rate. Therefore, based on these studies, the expected 10-year survival of cementless UKR would be 95.5% and cemented 92.7%. Subgroup analysis revealed this difference remained significant for the Oxford UKR (0.37% pa vs 0.77% pa, p < 0.001), but for non-Oxford UKRs there were no significant differences in revision rates of cemented and cementless UKRs (0.57% pa vs 0.69% pa, p = 0.41). Mobile bearing UKRs had significantly lower revision rates than fixed bearing UKRs in cementless (p = 0.001), but not cemented groups (p = 0.13). Overall the revision rates for aseptic loosening and disease progression were significantly lower (p = 0.02 and p = 0.009 respectively) in the cementless group compared to the cemented group (0.06 vs 0.13% pa and 0.10 vs 0.21% pa respectively). CONCLUSIONS: Cementless fixation had reduced long-term revision rates compared to cemented for the Oxford UKR. For the non-Oxford UKRs, the revision rates of cementless and cemented fixation types were equivalent. Therefore, cementless UKRs offer at least equivalent if not lower revision rates compared to cemented UKRs. LEVEL OF EVIDENCE: III.

Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice.

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the Crbn I391V model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of Trp53 causes lenalidomide resistance. We further demonstrate that Crbn I391V is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the Crbn I391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Threshold for Computer- and Robot-Assisted Knee and Hip Replacements in the English National Health Service.

OBJECTIVES: To estimate threshold prices for computer- and robot-assisted knee and hip replacement. METHODS: A lifetime cohort Markov model provided the framework for analysis. Linked primary care and inpatient hospital records informed estimates of outcomes under current practice. Outcomes were estimated under a range of hypothetical relative improvements in quality of life if unrevised and in revision risk after computer or robot-assisted surgery. Threshold prices, a price at which the net health benefit from funding the intervention would be zero, for these improvements were estimated for a cost-effectiveness threshold of £20 000 per additional quality-adjusted life-year (QALY) gained. RESULTS: For average patient profiles under current knee and hip replacement practice, lifetime QALYs were 10.3 (9.9 to 10.7) and 11.0 (10.6 to 11.4), with costs of £6060 (£5947 to £6203) and £6506 (£6335 to £6710) for knee and hip replacement, respectively. A combined 50% relative reduction in risk of revision and 5% improvement in postoperative quality of life if unrevised would, for example, result in QALYs increasing to 10.9 (10.4 to 11.3) and 11.6 (11.2 to 12.0), and costs falling to £5880 (£5816 to £5956) and £6258 (£6149 to £6376) after knee and hip replacement, respectively. These particular improvements would have an associated threshold price of £11 182 (£10 691 to £11 721) for knee replacement and £12 134 (£11 616 to £12 701) for hip replacement. The 50% reduction in revision rate alone would have associated threshold prices of £1094 (£788 to £1488) and £1347 (£961 to £1842), and the 5% improvement in quality of life alone would have associated threshold prices of £9911 (£9476 to £10 296) and £10 578 (£10 171 to £10 982). CONCLUSIONS: At current prices, computer- and robot-assisted knee and hip replacement will likely need to lead to improvements in patient-reported outcomes in addition to any reduction in the risk revision.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Most unicompartmental knee replacement revisions could be avoided: a radiographic evaluation of revised Oxford knees in the National Joint Registry.

PURPOSE: The purpose of this study was to understand why the revision rate of unicompartmental knee replacement (UKR) in the National Joint Registry (NJR) is so high. Using radiographs, the appropriateness of patient selection for primary surgery, surgical technique, and indications for revision were determined. In addition, the alignment of the radiographs was assessed. METHODS: Oxford UKR registered with the NJR between 2006 and 2010 and subsequently revised were identified by the NJR. A blinded review was undertaken of pre-primary, post-primary, and pre-revision anteroposterior and lateral radiographs of a sample of 107 cases from multiple centres. RESULTS: The recommended indications were satisfied in 70%, with 29% not demonstrating bone-on-bone arthritis. Major technical errors, likely leading to revision, were seen in 6%. Pre-revision radiographs were malaligned and, therefore, difficult to interpret in 53%. No reason for revision was seen in 67%. Reasons for revision included lateral compartment arthritis (10%), tibial loosening (7%), bearing dislocation (7%), infection (6%), femoral loosening (3%), and peri-prosthetic fracture (2%, one femoral, one tibial). CONCLUSIONS: Only 20% of the revised UKR were implanted for the recommended indications, using appropriate surgical technique and had a mechanical problem necessitating revision. One-third of primary surgeries were undertaken in patients with early arthritis, which is contraindicated. Two-thirds were presumably revised for unexplained pain, which is not advised as it tends not to help the pain. This study suggests that variable and inappropriate indications for primary and revision surgery are responsible for the high rates of revision seen in registries. LEVEL OF EVIDENCE: III, Therapeutic study.

Ten-year clinical and radiographic results of 1000 cementless Oxford unicompartmental knee replacements.

PURPOSE: Unicompartmental knee replacement (UKR) has substantial benefits over total knee replacement (TKR) but has higher revision rates. The cementless Oxford UKR was introduced to address this but there are concerns about fixation and tibial plateau fractures. The first long-term study of the device with clinical and radiographic outcomes is reported. METHODS: The first 1000 medial cementless Oxford UKR were prospectively identified and followed up by independent physiotherapists. Survival was calculated using the endpoints reoperation, revision, revision to TKR, major revision requiring revision TKR components and patient mortality. The Oxford Knee Score (OKS), Tegner Activity Score and American Knee Society Score (AKSS) were recorded and radiographs analysed. RESULTS: The ten year survival was 96.6% (CI 94.8-97.8), 97.5% (CI 95.7-98.5), 98.9% (CI 97.7-99.4) and 99.6% (CI 98.8-99.9) using reoperation, revision, revision to TKR and major revision as the endpoint, respectively. Commonest causes for revision were bearing dislocation (n = 7, 0.7%), disease progression (n = 4, 0.4%) and pain (n = 2, 0.2%). There was one lateral tibial plateau fracture and one femoral component loosening. At 10 years, the mean OKS was 41.2 (SD 9.8), Tegner 2.8 (SD 1.3), AKSS-O 89.1 (SD 13.0) and AKSS-F 80.4 (SD 14.6). There were no pathological radiolucencies or complete radiolucent lines. There were no implant-related deaths. CONCLUSIONS: The cementless Oxford UKR is a safe procedure with excellent long-term clinical results. Our results suggest that reliable fixation was achieved with only one (0.1%) revision for loosening (femoral), no radiographic evidence of loosening in the remaining cases and no fractures related to implantation. LEVEL OF EVIDENCE: III.

Age and Outcomes of Medial Meniscal-Bearing Unicompartmental Knee Arthroplasty.

BACKGROUND: National joint registries report increasing revision rates with decreasing patient age for all types of joint arthroplasty. This study aimed to explore the effect of age on function and revision risk in patients undergoing medial meniscal-bearing UKA. METHODS: A prospectively followed cohort of 1000 consecutive medial meniscal-bearing UKAs at a designer center was analyzed. All knees were implanted for recommended indications and had mean 10-year follow-up. Patients were grouped by age at surgery (<55, 55 to <65, 65 to <75, 75+). Oxford Knee Scores (OKS) were assessed at 5 and 10 years. Component-time revision incidence rates and Kaplan-Meier implant survival were calculated. RESULTS: Mean patient age at surgery was 66.6 years (range, 33-88). All age-groups had significant (P < .001) improvement in OKS over time, and at 5 years achieved a median OKS of 44. At 10 years, median OKS, from youngest group to eldest, were 44, 45, 42, and 39, with the eldest group having a significantly lower OKS (P < .01). Ten-year implant survival rates were 97%, 94%, 94%, and 93%, respectively, and was not significantly associated with age at UKA. CONCLUSION: Medial meniscal-bearing UKA provides good functional outcomes in all age-groups; however, in older patients (75+), the functional outcome deteriorated at 10 years presumably due to deteriorating health. Contrary to registry observations, the revision rate was not higher in younger patients. These results suggest that, with correct indications, patient age should not be considered a contraindication to medial meniscal-bearing UKA.

Treatment outcomes following leukemic transformation in Philadelphia-negative myeloproliferative neoplasms.

Leukemic transformation (LT) is a rare but fatal complication of Philadelphia-negative myeloproliferative neoplasms (MPNs) for which optimal treatment strategies are not known. At our center, we have adopted a treatment approach for LT where patients within the transplant age group who have a reasonable fitness level are treated with curative intent and offered induction chemotherapy. Subsequently, those who respond and have a suitable donor are considered for allogeneic hematopoietic cell transplantation (HCT). In this study, we evaluated the clinical outcomes of this treatment approach in 75 patients with LT. The 2-year overall survival (OS) from the time of LT was 15%. A total of 39 patients (52%) were treated with curative intent (induction ± HCT) and had a 2-y OS of 26% compared with 3% in those noncuratively treated (P < .0001). In the curative intent group, 18 individuals (46%) achieved complete remission (CR) or CR with incomplete recovery and 12 (31%) reverted to a chronic MPN phase, with 17 patients undergoing HCT. Survival of patients posttransplant was significantly improved compared with those who responded to induction but were not transplanted (2-y OS of 47% vs 15%; P = .03). Thus, induction chemotherapy followed by HCT has the potential for long-term disease control in select patients with LT preceded by a MPN.

Napa Valley Cabernet Sauvignon Proanthocyanidin Changes During Fruit Ripening: A Multi-Appellation Survey.

In 2015, an experiment was designed to investigate the distribution and variance of in winegrape flavonoids across the ripening phase in the Napa Valley. This Cabernet Sauvignon experiment was intended to evaluate the polyphenol differences across Napa Valley in order to understand parameters controlling "proanthocyanidin activity." This method has shown promise in understanding proanthocyanidin (PA) astringency based on size distribution, pigmentation, conformation, and composition. Results from whole berry partial extractions showed that seed PA material was driving PA activity early in the ripening phase, while the formation of the pigmented polymer led to a decrease later in the growing season. Multivariate analysis showed that the main drivers of changes across the ripening phase were the molecular masses of PAs and the amount of pigmentation. Given the high amount of variability seen in the experiment between sites in such a small geographical area, the results suggest that manipulation of PA activity may be possible in the vineyard, perhaps explaining variations in wine mouthfeel attributes between locations. These results can be used to develop furthermore controlled experiments targeting the variables responsible for PA activity changes.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Outcomes of intensive and nonintensive blast-reduction strategies in accelerated and blast-phase MPN.

ABSTRACT: Transformation of BCR::ABL1-negative myeloproliferative neoplasms (MPN) to an accelerated or blast phase is associated with poor outcomes. The efficacy of acute myeloid leukemia (AML)-type intensive and nonintensive hypomethylating agent-based regimens is not well studied. We therefore performed a retrospective analysis of patients with MPN-AP/BP (N = 138) treated with intensive (N = 81) and nonintensive (N = 57) blast-reduction strategies. We used clinically relatable response criteria developed at the Princess Margaret Cancer Centre. The overall best response, comprising complete remission (CR), complete remission with incomplete hematologic recovery (CRi), and reversion to chronic phase MPN (cMPN), in the intensive and nonintensive groups was 77% (62 of 81) and 39% (21 of 54), respectively. Similar overall best response rates were observed in patients receiving induction with daunorubicin combined with cytarabine arabinoside (daunorubicin + ara-C) (74% [23 of 31]) or FLAG-IDA/NOVE-HiDAC (78% [39 of 50], P = .78). However, patients receiving daunorubicin + ara-C more often required second inductions (29% [9 of 31] vs 4% [2 of 50], P = .002). Most responses in the entire cohort were reversions to cMPN (55 of 83 [66%]). CR and CRi comprised 30% (25 of 83) and 4% (3 of 83) of responses, respectively. Mutations in TP53 (overall response [OR] 8.2 [95% confidence interval [CI] 2.01, 37.1], P = .004) and RAS pathway (OR 5.1 [95%CI 1.2, 23.7], P = .03) were associated with inferior treatment response for intensively treated patients, and poorer performance status (Eastern Cooperative Oncology Group) was associated with inferior treatment response in both intensively (OR 10.4 [95% CI 2.0, 78.5], P = .009) and nonintensively treated groups (OR 12 [95% CI 2.04, 230.3], P = .02). In patients with paired samples before and after therapy (N = 26), there was a significant residual mutation burden remaining irrespective of response to blast-reduction therapy.

Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.