Accumulation Of Phenylalanine Research Articles

New findings about neuropathological outcomes in the PKU mouse throughout lifespan

Phenylketonuria (PKU, OMIM 261600) is a genetic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If left untreated, PKU leads to systemic phenylalanine (Phe) accumulation, which can result in irreversible brain damage and intellectual disabilities. In the last 60 years, early and strict dietary restriction of phenylalanine (Phe) intake proved to prevent the severe clinical phenotype of untreated PKU. While the specific mechanisms through which phenylalanine causes brain damage are still poorly understood, preclinical models have been deeply explored to characterize the neurotoxic effect of Phe on neurodevelopmental processes. At the same time, that on the aging brain still needs to be explored. In the brain of untreated PAHEnu2(−/−) mouse, we previously reported a reduction of myelin basic protein (MBP) during postnatal development up to 60 PND. Later in the diseased mouse's life, a spontaneous and persistent restoration of MBP was detected. In this present longitudinal study, ranging from 14 to 540 post-natal days (PND) of untreated PAHEnu2(−/−) mice, we further investigated: a) the long-life consistency of two Phe-related brain metabolic alterations, such as large neutral amino acids (LNAA) and biogenic amine neurotransmitters' depletion; b) the outcome of locomotor functions during the same life span; c) the integrity of myelin as assessed ex vivo by central (hippocampus) and peripheral (extensor digitorum longus-sciatic nerve) action potential conduction velocities. In contrast with the results of other studies, brain Leu, Ile, and Val concentrations were not significantly altered in the brain PAHEnu2(−/−) mouse. On the other hand, 3-O-Methyldopa (3-OMD, a biomarker of L-DOPA), serotonin, and its associated metabolites were reduced throughout most of the considered time points, with consistent reductions observed prevalently from 14 to 60 PND. Normal saltatory conduction was restored after 60 PND and remained normal at the last examination at 360 PND, resulting nonetheless in a persistent locomotor impairment throughout a lifetime. These new findings contribute to laying the foundations for the preclinical characterization of aging in PKU, confirming neurotransmitter defects as consistent metabolic traits. LNAAs have a minor role, if any, in brain damage pathogenesis. Transient myelin synthesis failure may impact brain connectivity during postnatal development but not nervous signal conduction.

Transcriptomic and metabolomic analyses revealCmMYB308 as a key regulator in the pink flower color variation of 'Dante Purple' chrysanthemum.

CmMYB308 was identified as a key regulator in chrysanthemum flower color variation from purple to pink by conducting transcriptome and metabolome analysis. CmMYB308 can inhibit anthocyanin biosynthesis by suppressing the expression of CmPAL, CmC4H, and Cm4CL. Flower color variation is a widespread natural occurrence that plays a significant role in floral breeding. We discovered a variation in the flower of the chrysanthemum cultivar 'Dante Purple' (abbreviated as 'DP'), where the flower color shifted from purple to pink. We successfully propagated these pink flowers through tissue culture and designated them as DPM. By conducting transcriptome and metabolome analysis, we identified a reduction in the expression of critical genes involved in anthocyanin biosynthesis-CmPAL, CmC4H, and Cm4CL-in the DPM. This downregulation led to an accumulation of phenylalanine and cinnamic acid within the general phenylpropanoid pathway (GPP), which prevented their conversion into cyanidin and cyanidin 3-glucoside. As a result, the flowers turned pink. Additional transformation and biochemical experiments confirmed that the upregulation of CmMYB308 gene expression in the DPM directly suppressed CmPAL-1 and CmC4H genes, which indirectly affected Cm4CL-3 expression and ultimately inhibited anthocyanin biosynthesis in the DPM. This study offers a preliminary insight into the molecular mechanism underlying chrysanthemum flower color mutation, paving the way for genetic improvements in chrysanthemum flower color breeding.

Laboratory parameters related to disease severity and physical performance after reconvalescence of acute COVID-19 infection

Research into the molecular basis of disease trajectory and Long-COVID is important to get insights toward underlying pathophysiological processes. The objective of this study was to investigate inflammation-mediated changes of metabolism in patients with acute COVID-19 infection and throughout a one-year follow up period. The study enrolled 34 patients with moderate to severe COVID-19 infection admitted to the University Clinic of Innsbruck in early 2020. The dynamics of multiple laboratory parameters (including inflammatory markers [C-reactive protein (CRP), interleukin-6 (IL-6), neopterin] as well as amino acids [tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr)], and parameters of iron and vitamin B metabolism) was related to disease severity and patients’ physical performance. Also, symptom load during acute illness and at approximately 60 days (FU1), and one year after symptom onset (FU2) were monitored and related with changes of the investigated laboratory parameters: During acute infection many investigated laboratory parameters were elevated (e.g., inflammatory markers, ferritin, kynurenine, phenylalanine) and enhanced tryptophan catabolism and phenylalanine accumulation were found. At FU2 nearly all laboratory markers had declined back to reference ranges. However, kynurenine/tryptophan ratio (Kyn/Trp) and the phenylalanine/tyrosine ratio (Phe/Tyr) were still exceeding the 95th percentile of healthy controls in about two thirds of our cohort at FU2. Lower tryptophan concentrations were associated with B vitamin availability (during acute infection and at FU1), patients with lower vitamin B12 levels at FU1 had a prolonged and more severe impairment of their physical functioning ability. Patients who had fully recovered (ECOG 0) presented with higher concentrations of iron parameters (ferritin, hepcidin, transferrin) and amino acids (phenylalanine, tyrosine) at FU2 compared to patients with restricted ability to work. Persistent symptoms at FU2 were tendentially associated with IFN-γ related parameters. Women were affected by long-term symptoms more frequently. Conclusively, inflammation-mediated biochemical changes appear to be related to symptoms of patients with acute and Long Covid.

Pegvaliase for the treatment of phenylketonuria: Final results of a long-term phase 3 clinical trial program

Phenylketonuria (PKU) is a genetic disorder caused by deficiency of the enzyme phenylalanine hydroxylase (PAH), which results in phenylalanine (Phe) accumulation in the blood and brain, and requires lifelong treatment to keep blood Phe in a safe range. Pegvaliase is an enzyme-substitution therapy approved for individuals with PKU and uncontrolled blood Phe concentrations (>600 μmol/L) despite prior management. Aggregated results from the PRISM clinical trials demonstrated substantial and sustained reductions in blood Phe with a manageable safety profile, but also noted individual variation in time to and dose needed for a first response. This analysis reports longer-term aggregate findings and characterizes individual participant responses to pegvaliase using final data from the randomized trials PRISM-1 (NCT01819727) and PRISM-2 (NCT01889862), and the open-label extension study 165–304 (NCT03694353). In 261 adult participants with a mean of 36.6 months of pegvaliase treatment, 71.3%, 65.1%, and 59.4% achieved clinically significant blood Phe levels of ≤600, ≤360, and ≤ 120 μmol/L, respectively. Some participants achieved blood Phe reductions with <20 mg/day pegvaliase, although most required higher doses. Based on Kaplan-Meier analysis, median (minimum, maximum) time to first achievement of a blood Phe threshold of ≤600, ≤360, or ≤ 120 μmol/L was 4.4 (0.0, 54.0), 8.0 (0.0, 57.0), and 11.6 (0.0, 66.0) months, respectively. Once achieved, blood Phe levels remained below clinical threshold in most participants. Sustained Phe response (SPR), a new method described within for measuring durability of blood Phe response, was achieved by 85.5%, 84.7%, and 78.1% of blood Phe responders at blood Phe thresholds of ≤600, ≤360, or ≤ 120 μmol/L, respectively. Longer-term safety data were consistent with previous reports, with the most common adverse events (AEs) being arthralgia, injection site reactions, headache, and injection site erythema. The incidence of most AEs, including hypersensitivity AEs, was higher during the early treatment phase (≤6 months) than later during treatment. In conclusion, using data from three key pegvaliase clinical trials, participants treated with pegvaliase were able to reach clinically significant blood Phe reductions to clinical thresholds of ≤600, ≤360, or ≤ 120 μmol/L during early treatment, with safety profiles improving from early to sustained treatment. This study also supports the use of participant-level data and new ways of looking at durable blood Phe responses to better characterize patients' individual PKU treatment journeys.

Roles of CcDFR and CcOMT9 in the cyanidin biosynthesis and development of Cordyceps cicadae.

Cordyceps cicadae is a traditional Chinese medicinal fungus known for its rich production of bioactive substances, particularly cyanidin, an anthocyanin commonly found in plants with notable anti-inflammatory, anti-tumor, antiviral, and antibacterial properties. This study revealed two key genes, CcDFR and CcOMT9, affecting cyanidin biosynthesis in C. cicadae. The roles of these genes in cyanidin production, growth, and development were elucidated through the gene knockout method, phenotypic analysis, transcriptomics, and metabolomics. CcDFR deletion led to reduced cyanidin-3-O-glucoside (C3G), suppressed expression of cyanidin biosynthesis genes, impaired synnemata formation, decreased polysaccharide and adenosine content, and diminished chitinase activity. Meanwhile, the ΔCcOMT9 mutant exhibited an increase in C3G production, promoted expression of cyanidin biosynthesis genes and rising bioactive compounds, suppressed RNA methylation, and led to phenylalanine accumulation with no effect on fruiting body formation. We revealed a distinct anthocyanin biosynthesis pathway in C. cicadae and identified two genes with opposite functions, laying the foundation for future genetic modification of cyanidin-producing strains using modern biological techniques. This will shorten the production period of this valuable compound, facilitating the industrial-scale production of cyanidin.

Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Program freezing of diapausing embryos in the mouse

Embryonal diapause is a characteristic feature of about 130 mammalian species. However, very few studies have addressed cryopreservation of diapausing embryos. This work is aimed to apply program freezing to blastocysts obtained from CD1 mice, which were at diapause state after ovariectomy and the subsequent hormonal therapy. Blastocysts collected from non-operated mice of the same strain served as controls. Some diapausing as well as non-diapausing frozen-thawed blastocysts demonstrated blastocoel re-expansion after 24 h of in vitro culture (IVC) indicating their viability after cryopreservation. Raman spectroscopy assessment of phenylalanine accumulation revealed that the fraction of new synthesized proteins was lower for non-frozen as well as for frozen-thawed diapausing blastocysts compared to non-diapausing ones. Although protein metabolism was reduced in diapausing embryos, most of the protein synthesis remained active. Cell number increased after 24 h of IVC in non-frozen as well as in the frozen-thawed blastocysts of the control but not of the diapause group. However, cell numbers were increased in frozen-thawed diapausing blastocysts after 47 h of IVC in a medium supplemented with putrescine. This indicates viability of frozen-thawed diapausing embryos after cryopreservation. Besides, protein metabolism was not affected by cryopreservation in diapausing and non-diapausing murine embryos indicating their viability. Our results demonstrated the possibility of successful cryopreservation of diapausing murine embryos.

Evaluating change in diet with pegvaliase treatment in adults with phenylketonuria: Analysis of phase 3 clinical trial data

Phenylketonuria (PKU), a genetic disorder characterized by phenylalanine hydroxylase (PAH) deficiency and phenylalanine (Phe) accumulation, is primarily managed with a protein-restricted diet and PKU-specific medical foods. Pegvaliase is an enzyme substitution therapy approved for individuals with PKU and uncontrolled blood Phe concentrations (>600 μmol/L) despite prior management. This analysis assessed the effect of pegvaliase on dietary intake using data from the Phase 3 PRISM-1 (NCT01819727), PRISM-2 (NCT01889862), and 165–304 (NCT03694353) clinical trials. Participants (N = 250) had a baseline diet assessment, blood Phe ≥600 μmol/L, and had discontinued sapropterin; they were not required to follow a Phe-restricted diet. Outcomes were analyzed by baseline dietary group, categorized as >75%, some (>0% but ≤75%), or no protein intake from medical food. At baseline, mean age was 29.1 years, 49.2% were female, mean body mass index was 28.4 kg/m2, and mean blood Phe was 1237.0 μmol/L. Total protein intake was stable up to 48 months for all 3 baseline dietary groups. Over this time, intact protein intake increased in all groups, and medical protein intake decreased in those who consumed any medical protein at baseline. Of participants consuming some or >75% medical protein at baseline, 49.1% and 34.1% were consuming no medical protein at last assessment, respectively. Following a first hypophenylalaninemia (HypoPhe; 2 consecutive blood Phe measurements <30 μmol/L) event, consumption of medical protein decreased and consumption of intact protein increased. Substantial and sustained Phe reductions were achieved in all 3 baseline dietary groups. The probability of achieving sustained Phe response (SPR) at ≤600 μmol/L was significantly greater for participants consuming medical protein versus no medical protein in an unadjusted analysis, but no statistically significant difference between groups was observed for probability of achieving SPR ≤360 or SPR ≤120 μmol/L. Participants with alopecia (n = 49) had longer pegvaliase treatment durations, reached HypoPhe sooner, and spent longer in HypoPhe than those who did not have alopecia. Most (87.8%) had an identifiable blood Phe drop before their first alopecia episode, and 51.0% (n = 21/41) of first alopecia episodes with known duration resolved before the end of the HypoPhe episode. In conclusion, pegvaliase treatment allowed adults with PKU to lower their blood Phe, reduce their reliance on medical protein, and increase their intact and total protein intake. Results also suggest that HypoPhe does not increase the risk of protein malnutrition in adults with PKU receiving pegvaliase.

Long-term comparative effectiveness of pegvaliase versus medical nutrition therapy with and without sapropterin in adults with phenylketonuria

Phenylketonuria is characterized by intellectual disability and behavioral, psychiatric, and movement disorders resulting from phenylalanine (Phe) accumulation. Standard-of-care treatment involves a Phe-restricted diet plus medical nutrition therapy (MNT), with or without sapropterin dihydrochloride, to reduce blood Phe levels. Pegvaliase is an injectable enzyme substitution treatment approved for adult patients with blood Phe >600 μmol/L despite ongoing management. A previous comparative effectiveness analysis using data from the Phase 3 PRISM trials of pegvaliase (NCT01819727 and NCT01889862) and the Phenylketonuria Demographics, Outcomes and Safety Registry (PKUDOS; NCT00778206) suggested that pegvaliase was more effective at lowering mean blood Phe levels than sapropterin + MNT or MNT alone at 1 and 2 years of treatment. The current work augments and complements the previous analysis by including additional follow-up from the completed studies, robust methods reflecting careful consideration of issues with the distribution of Phe, and alternative methods for adjustment that are important for control of potential confounding in comparative effectiveness. Median blood Phe levels were lower, and median intact protein intakes were higher, in the pegvaliase group (n = 183) than in the sapropterin + MNT (n = 82) and MNT (n = 67) groups at Years 1, 2, and 3. In the pegvaliase group, median blood Phe levels decreased from baseline (1244 μmol/L) to Year 1 (535 μmol/L), Year 2 (142 μmol/L), and Year 3 (167 μmol/L). In the sapropterin + MNT group, median blood Phe levels decreased from baseline (900 μmol/L) to Year 1 (588 μmol/L) and Year 2 (592 μmol/L), and increased at Year 3 (660 μmol/L). In the MNT group, median blood Phe levels decreased slightly from baseline (984 μmol/L) to Year 1 (939 μmol/L) and Year 2 (941 μmol/L), and exceeded baseline levels at Year 3 (1157 μmol/L). The model-estimated proportions of participants achieving blood Phe ≤600 μmol/L were 41%, 100%, and 100% in the pegvaliase group at Years 1, 2, and 3, respectively, compared with 55%, 58%, and 38% in the sapropterin + MNT group and 5%, 16%, and 0% in the MNT group. The estimated proportions of participants achieving more stringent blood Phe targets of ≤360 μmol/L and ≤120 μmol/L were also higher in the pegvaliase group than in the other groups at Years 2 and 3. Overall, our results indicate that, compared with standard therapy, pegvaliase induces a substantial, progressive, and sustained decrease in blood Phe levels – to a much greater extent than sapropterin + MNT or MNT alone – which is expected to improve long-term outcomes in patients with phenylketonuria.

Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need

The c.1222C>T (p.Arg408Trp) variant in the phenylalanine hydroxylase gene (PAH) is the most frequent cause of phenylketonuria (PKU), the most common inborn error of metabolism. This autosomal-recessive disorder is characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Using real-world data, we observed that despite dietary and medical interventions, most PKU individuals harboring at least one c.1222C>T variant experience chronic, severe Phe elevations and do not comply with Phe monitoring guidelines. Motivated by these findings, we generated an edited c.1222C>T hepatocyte cell line and humanized c.1222C>T mouse models, with which we demonstrated efficient invitro and invivo correction of the variant with prime editing. Delivery via adeno-associated viral (AAV) vectors reproducibly achieved complete normalization of blood Phe levels in PKU mice, with up to 52% whole-liver corrective PAH editing. These studies validate a strategy involving prime editing as a potential treatment for a large proportion of individuals with PKU.

A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant

The c.1222C>T (p.Arg408Trp) phenylalanine hydroxylase (PAH) variant is the most frequent cause of phenylketonuria (PKU), an autosomal recessive disorder characterized by accumulation of blood phenylalanine (Phe) to neurotoxic levels. Here we devised a therapeutic base editing strategy to correct the variant, using prime-edited hepatocyte cell lines engineered with the c.1222C>T variant to screen a variety of adenine base editors and guide RNAs invitro, followed by assessment in c.1222C>T humanized mice invivo. We found that upon delivery of a selected adenine base editor mRNA/guide RNA combination into mice via lipid nanoparticles (LNPs), there was sufficient PAH editing in the liver to fully normalize blood Phe levels within 48 h. This work establishes the viability of a base editing strategy to correct the most common pathogenic variant found in individuals with the most common inborn error of metabolism, albeit with potential limitations compared with other genome editing approaches.

The effects of phenylalanine and tyrosine levels on dopamine production in rat PC12 cells. Implications for treatment of phenylketonuria, tyrosinemia type 1 and comorbid neurodevelopmental disorders

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in phenylalanine accumulation and impaired tyrosine production. In Tyrosinemia type 1 (TYRSN1) mutations affect fumarylacetoacetate hydrolase, leading to accumulation of toxic intermediates of tyrosine catabolism. Treatment of TYRSN1 with nitisinone results in extreme tissue levels of tyrosine. Although PKU and TYRSN1 have opposite effects on tyrosine levels, both conditions have been associated with neuro-psychiatric symptoms typically present in ADHD, possibly indicating an impaired dopamine (DA) synthesis. However, concrete in vivo data on the possible molecular basis for disrupted DA production under disease mimicking conditions have been lacking. In pursuit to uncover associated molecular mechanisms, we exposed an established, DA producing cell line (PC12) to different concentrations of phenylalanine and tyrosine in culture media. We measured the effects on viability, proteomic composition, tyrosine, DA and tyrosine hydroxylase (TH) levels and TH phosphorylation. TH catalyzes the rate-limiting step in DA synthesis. High extracellular levels of phenylalanine depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 μM), either low (35 μM) or high concentrations of tyrosine (275 or 835 μM) decreased cellular DA, TH protein, and its phosphorylation levels. Using deep proteomic analysis, we identified multiple proteins, biological processes and pathways that were altered, including enzymes and transporters involved in amino acid metabolism. Using this information and published data, we developed a mathematical model to predict how extracellular levels of aromatic amino acids can affect the cellular synthesis of DA via different mechanisms. Together, these data provide new information about the normal regulation of neurotransmitter synthesis and how this may be altered in neurometabolic disorders, such as PKU and TYRSN1, with implications for the treatment of cognitive symptoms resulting from comorbid neurodevelopmental disorders.

Low bone mineralization in phenylketonuria may be due to undiagnosed metabolic acidosis

BackgroundDietary intervention is to date the mainstay treatment to prevent toxic phenylalanine (Phe) accumulation in PKU patients. Despite success preventing central nervous system damage, there is increasing evidence of possible other unfavorable outcomes affecting other systems, e.g. kidney and bone; underlying mechanisms are yet to be fully elucidated. MethodsThis observational, cross-sectional and descriptive study investigated 20 adult with PKU evaluating biochemical parameters, BMD measurements and extrapolating data from 3-days food records and protein substitutes (PS) and special low protein foods (SLPF) composition. ResultsBlood gas venous analysis (VBG) indices were indicative of metabolic acidosis in 60% of PKU patients and VBG pH significantly correlated with BMD's Z-score (p-value = 0.022) even if its overall mean was in range (−1.29). Low bone mineral density for chronological age (Z-score < − 2.0) was found in 4 patients (20%). Indices of kidney function were not impaired. All used PS had a moderate excess of acidity, while SLPF were alkalizing and type/variety of consumed vegetables did not determine significant changes in acid-base equilibrium. Total intakes of potassium and magnesium were lower than expected. DiscussionPKU patients seem to be at risk of metabolic acidosis, directly linked to possible low bone mineralization. This may be related to the acidic composition of PS, potentially capable of acidifying the entire diet. Reported low intakes of potassium and magnesium may be relevant to these observations. Further studies are needed to better address these topics.

Effects of Comparative Metabolism on Tomato Fruit Quality under Different Levels of Root Restriction

In a soilless culture (perlite substrate), root restriction cannot only reduce production costs but also improve fruit quality. Therefore, this study used different levels of root restriction [T1: 0.5 L, T2: 4 L, nonrestriction treatment (CK): 35 L] on tomatoes to explore their impact on quality. Results showed that total soluble solids (TSS), glucose, fructose, and sucrose contents were increased, whereas L-tryptophan, L-tyrosine, and titratable acidity were decreased under two restriction treatments. Meanwhile, root restriction also promoted the accumulation of phenylalanine and proline. For lycopene and flavonoid biosynthesis (prunin, naringin, naringenin), the restriction groups were significantly higher than those in the control group. Overall, T1 and T2 treatment had a better effect than CK treatment. This study provided an idea for improving substrate use efficiency and tomato quality.

Rapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing

Phenylketonuria (PKU), an autosomal recessive disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, results in the accumulation of blood phenylalanine (Phe) to neurotoxic levels. Current dietary and medical treatments are chronic and reduce, rather than normalize, blood Phe levels. Among the most frequently occurring PAH variants in PKU patients is the P281L (c.842C>T) variant. Using a CRISPR prime-edited hepatocyte cell line and a humanized PKU mouse model, we demonstrate efficient in vitro and in vivo correction of the P281L variant with adenine base editing. With the delivery of ABE8.8 mRNA and either of two guide RNAs in vivo using lipid nanoparticles (LNPs) in humanized PKU mice, we observe complete and durable normalization of blood Phe levels within 48 h of treatment, resulting from corrective PAH editing in the liver. These studies nominate a drug candidate for further development as a definitive treatment for a subset of PKU patients.

Comparative estimation of the lysine requirements in two generations of improved strain of Nile tilapia (Oreochromis niloticus) at the grow-out stage

A 3 × 2 factorial experiment was conducted to investigate the effects of dietary lysine on growth performance, body indices, feed intake, feed efficiency, whole body nutrient composition and amino acid deposition in two successive generations (16th and 17th) of GIFT (Oreochromis niloticus). Three diets containing different levels of lysine at 1.16%, 1.56% and 2.41% were prepared for the feeding trial. Triplicate groups of fish with an initial body weight of 155 g were fed to apparent satiation for 10 weeks in a recirculating aquaculture system. Apparent digestibility coefficients (ADC) of dry matter, crude protein, crude lipids, and total carbohydrates were measured in the experimental diets. At the end of the experiment, no interactions between dietary lysine levels in diet and fish generation were observed on all parameters except for the condition factor (CF) and ADC of crude protein. However, dietary lysine level significantly affected the final weight, weight gain, thermal unit growth coefficient (TGC), protein efficiency ratio (PER) and ADC of dry matter regardless of the fish generation. Final weight, weight gain and TGC of fish were the highest in fish fed 2.41% dietary lysine in diet or 6.52% lysine in the protein. PER was the lowest in fish fed 1.16% dietary lysine. The final weight and the body's accumulation of isoleucine, phenylalanine, and alanine were significantly affected by the fish generation, with the 17th generation having the best performance. Increase growth and higher lysine requirement observed in the improved generation (17th) compared to the (16th) generation at grow out phase indicating that genetic improvement may have changed the dietary lysine requirement.

Targeted metabolomics unravels altered phenylalanine levels in piglets receiving total parenteral nutrition.

Parenteral nutrition, received by many patients with intestinal failure, can induce hepatobiliary complications, which is termed as parenteral nutrition-associated liver disease (PNALD). The spectrum of PNALD ranges from cholestasis and steatosis to fibrosis and cirrhosis. Although many factors contribute to the pathogenesis of PNALD, the underlying mechanisms remain unclear. In this study, we performed targeted metabolomics to characterize the metabolomic profile in neonatal piglets receiving total parenteral nutrition (TPN) or enteral nutrition (EN) for 1 or 2 weeks. Overall, the metabolomic signature of TPN groups differed from EN groups at both time points. Among the 20 acylcarnitines identified, a majority of them were significantly reduced in TPN groups. KEGG pathway analysis showed that phenylalanine metabolism-associated pathways were dysregulated accompanied by more progressive liver steatosis associated with TPN. Next, we evaluated phenylalanine catabolism and its association with fatty acid oxidation in piglets and rats with PNALD. We showed that the hepatic expression of phenylalanine-degrading enzyme phenylalanine hydroxylase (PAH) was reduced and systemic phenylalanine levels were increased in both animal models of PNALD. Moreover, carnitine palmitoyltransferase 1A, a central regulator of fatty acid oxidation, was downregulated and its expression was negatively correlated with phenylalanine levels in TPN-fed animals. To explore the effects of phenylalanine accumulation on lipid metabolism, we treated HepG2 cells with phenylalanine co-cultured with sodium palmitate or soybean oil emulsion to induce lipid accumulation. We found that phenylalanine treatment exacerbated lipid accumulation by inhibiting fatty acid oxidation without affecting fatty acid synthesis. In summary, our findings establish a pathogenic role of increased phenylalanine levels in driving liver steatosis, linking dysregulation of phenylalanine catabolism with lipid accumulation in the context of PNALD.

Integrated Transcriptome and Metabolome Analyses Reveal Details of the Molecular Regulation of Resistance to Stem Nematode in Sweet Potato.

Stem nematode disease can seriously reduce the yield of sweet potato (Ipomoea batatas (L.) Lam). To explore resistance mechanism to stem nematode in sweet potato, transcriptomes and metabolomes were sequenced and compared between two sweet potato cultivars, the resistant Zhenghong 22 and susceptible Longshu 9, at different times after stem nematode infection. In the transcriptional regulatory pathway, mitogen-activated protein kinase signaling was initiated in Zhenghong 22 at the early stage of infection to activate genes related to ethylene production. Stem nematode infection in Zhenghong 22 also triggered fatty acid metabolism and the activity of respiratory burst oxidase in the metabolic pathway, which further stimulated the glycolytic and shikimic pathways to provide raw materials for secondary metabolite biosynthesis. An integrated analysis of the secondary metabolic regulation pathway in the resistant cultivar Zhenghong 22 revealed the accumulation of tryptophan, phenylalanine, and tyrosine, leading to increased biosynthesis of phenylpropanoids and salicylic acid and enhanced activity of the alkaloid pathway. Stem nematode infection also activated the biosynthesis of terpenoids, abscisic acid, zeatin, indole, and brassinosteroid, resulting in improved resistance to stem nematode. Finally, analyses of the resistance regulation pathway and a weighted gene co-expression network analysis highlighted the importance of the genes itf14g17940 and itf12g18840, encoding a leucine-rich receptor-like protein and 1-aminocyclopropane-1-carboxylate synthase, respectively. These are candidate target genes for increasing the strength of the defense response. These results provide new ideas and a theoretical basis for understanding the mechanism of resistance to stem nematode in sweet potato.

Phenylketonuria and the brain

Classic phenylketonuria (PKU) is caused by defective activity of phenylalanine hydroxylase (PAH), the enzyme that coverts phenylalanine (Phe) to tyrosine. Toxic accumulation of phenylalanine and its metabolites, left untreated, affects brain development and function depending on the timing of exposure to elevated levels. The specific mechanisms of Phe-induced brain damage are not completely understood, but they correlate to phenylalanine levels and on the stage of brain growth. During fetal life, high levels of phenylalanine such as those seen in maternal PKU can result in microcephaly, neuronal loss and corpus callosum hypoplasia. Elevated phenylalanine levels during the first few years of life can cause acquired microcephaly, severe cognitive impairment and epilepsy, likely due to the impairment of synaptogenesis. During late childhood, elevated phenylalanine can cause alterations in neurological functioning, leading to ADHD, speech delay and mild IQ reduction. In adolescents and adults, executive function and mood are affected, with some of the abnormalities reversed by better control of phenylalanine levels. Altered brain myelination can be present at this stage. In this article, we review the current knowledge about the consequences of high phenylalanine levels in PKU patients and animal models through different stages of brain development and its effect on cognitive, behavioural and neuropsychological function.