Neurotoxic non-protein amino acids in commercially harvested Lobsters (Homarus americanus H. Milne-Edwards).

Cyanobacteria produce neurotoxic non-protein amino acids (NPAAs) that accumulate in ecosystems and food webs. American lobsters (Homarus americanus H. Milne-Edwards) are one of the most valuable seafood industries in Canada with exports valued at > $2 billion. Two previous studies have assessed the occurrence of ß-N-methylamino-L-alanine (BMAA) in a small number of lobster tissues but a complete study has not previously been undertaken. We measured NPAAs in eyeballs, brain, legs, claws, tails, and eggs of 4 lobsters per year for the 2021 and 2022 harvests. Our study included 4 male and 4 female lobsters. We detected BMAA and its isomers, N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB) and ß-aminomethyl-L-alanine (BAMA) by a fully validated reverse phase chromatography-tandem mass spectrometry method. We quantified BMAA, DAB, AEG and BAMA in all of the lobster tissues. Our quantification data varied by individual lobster, sex and collection year. Significantly more BMAA was quantified in lobsters harvested in 2021 than 2022. Interestingly, more BAMA was quantified in lobsters harvested in 2022 than 2021. Monitoring of lobster harvests for cyanobacterial neurotoxins when harmful algal bloom events occur could mitigate risks to human health.

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: role of the "sleeping beauty" amino acid exporters.

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or degradation of proteins and peptides. The accumulation of several amino acids inhibits the growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, ß-alanine, and 2,3-diaminopropionic acid, as well as the two-carbon amino acid glycine. Resistance to the non-proteinogenic amino acid ß-alanine, which is a precursor for coenzyme A biosynthesis, is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations affecting the transcription factor AerA (previously YdeC) can result in strong constitutive aexA expression. AexA is the first characterized member of a group of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this group "sleeping beauty amino acid exporters." 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance toward 2,3-diaminopropionic acid. Moreover, our work shows how low-specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis.IMPORTANCEEven though Bacillus subtilis is one of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a group of very poorly expressed amino acid exporters in B. subtilis that we call "sleeping beauty amino acid exporters." The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience selective pressure by toxic compounds, and they protect the cells from rare but potentially dangerous encounters with such compounds.

Maternal separation regulates sensitivity of stress-induced depression in mice by affecting hippocampal metabolism.

Depression is a serious mental illness. Previous studies found that early life stress (ELS) plays a vital role in the onset and progression of depression. However, relevant studies have not yet been able to explain the specific effects of early stress on stress-induced depression sensitivity and individual behavior during growth. Therefore, we constructed a maternal separation (MS) model and administered chronic social frustration stress at different stages of their growth while conducting metabolomics analysis on the hippocampus of mice. Our results showed that the immobility time of mice in the forced swimming test was significantly reduced at the end of MS. Meanwhile, mice with MS experience significantly decreased total movement distance in the open field test and sucrose preference ratio in the sucrose preference test when subjected to chronic social defeat stress (CSDS) during adolescence. In adulthood, the results were the opposite. In addition, we found that level changes in metabolites such as Beta-alanine, l-aspartic acid, 2-aminoadipic acid, and Glycine are closely related to behavioral changes. These metabolites are mainly enriched in Pantothenate, CoA biosynthesis, and Beta Alanine metabolism pathways. Our experiment revealed that the effects of ELS vary across different age groups. It will increase an individual's sensitivity to depression when facing CSDS in adolescence, but it will reduce their sensitivity to depression when facing CSDS in adulthood. This may be achieved by regulating the hippocampus's Pantothenate and CoA biosynthesis and Beta Alanine metabolism pathways represented by Beta-alanine, l-Aspartic acid, 2-aminoadipic acid, and Glycine metabolites.

Topical netarsudil for the treatment of primary corneal endothelial degeneration in dogs.

This study evaluated the tolerability and efficacy of the topical rho-kinase inhibitor netarsudil for canine primary corneal endothelial degeneration (PCED). Twenty-six eyes of 21 client-owned dogs with PCED were enrolled in a prospective, randomized, vehicle control clinical trial and received topical netarsudil 0.02% (Rhopressa®) or vehicle control twice daily (BID) for the first 4 months. Then, all patients received netarsudil for the next 4 or 8 months. Complete ophthalmic examination, ultrasonic pachymetry, Fourier-domain optical coherence tomography, and in vivo confocal microscopy were performed at baseline and 1, 2, 4, 6, 8 and 12 months. Effect of netarsudil on central corneal thickness (CCT), percentage of cornea with edema, and endothelial cell density (ECD) were evaluated by repeated measures ANOVA. Kaplan-Meier curves and log-rank test were used to compare corneal edema and clinical progression of eyes in netarsudil versus vehicle control groups. All dogs developed conjunctival hyperemia in at least one eye while receiving netarsudil. Unilateral transient reticulated intraepithelial bullae and stromal hemorrhage were observed respectively in 2 dogs in the netarsudil group. Two dogs showed persistently decreased tear production while receiving netarsudil, requiring topical immunomodulatory treatment. No significant differences in CCT, ECD, corneal edema or clinical progression were observed between netarsudil or vehicle treated eyes. When comparing efficacy of topical netarsudil BID and topical ripasudil 0.4% administered four times daily from our previous study, dogs receiving ripasudil had significantly less progression than those receiving netarsudil.

Integrative proteomics and metabolomics data analysis exploring the mechanism of brain injury after cardiac surgery in chronic stress rats.

OBJECTIVE: Preoperative chronic stress (CS) is associated with postoperative brain injury in patients undergoing open heart cardiac surgery. This research is to explore the potential molecular biological mechanisms of brain damage following cardiac surgery in preoperative CS rats by the analyses combining proteomics and metabolomics. METHODS: We constructed the chronic unpredictable stress (CUS) and cardiac surgery models in adult rats. We proved the brain injury in CUS cardiac surgery rats by Hematoxylin-Eosin (H&E) staining, followed by separating the hippocampal tissue and investigating the potential mechanisms of brain injury by the methods of data-independent acquisition proteomics and untargeted metabolomics. RESULTS: The signaling pathways of glycoproteins and metabolism of amino acids were the main possible mechanisms of brain injury in CUS rats following cardiac surgery according to the proteomics and metabolomics. In addition, the pathways of animo acids metabolism such as the pathways of lysine degradation and ß-alanine metabolism may be the main mechanism of cardiac surgery related brain injury in preoperative CUS rats. CONCLUSIONS: The pathways of animo acids metabolism such as lysine degradation and ß-alanine metabolism may be the potential mechanisms of brain injury in CUS rats following cardiac surgery. We should focus on the varieties of bioproteins and metabolites in these pathways, and related changes in other signaling pathways induced by the two pathways.

Molecular properties of linear amino acids in water.

Four linear amino acids of increased separation of the carboxyl and amino groups, namely glycine (aminoacetic acid), ß-alanine (3-aminopropanoic acid), GABA (4-aminobutanoic acid) and DAVA (5-aminopentanoic acid), have been studied by quantum chemical ab initio and DFT methods including the solvent effect in order to get electronic structure and molecular descriptors, such as ionisation energy, electron affinity, molecular electronegativity, chemical hardness, electrophilicity index, dipole moment, quadrupole moment and dipole polarizability. Thermodynamic functions (zero-point energy, inner energy, enthalpy, entropy, and the Gibbs energy) were evaluated after the complete vibrational analysis at the true energy minimum provided by the full geometry optimization. Reaction Gibbs energy allows evaluating the absolute redox potentials on reduction and/or oxidation. The non-local non-additive molecular descriptors were compared along the series showing which of them behave as extensive, varying in match with the molar mass and/or separation of the carboxyl and amino groups. Amino acidic forms and zwitterionic forms of the substances were studied in parallel in order to compare their relative stability and redox properties. In total, 24 species were investigated by B3LYP/def2-TZVPD method (M1) including neutral molecules, molecular cations and molecular anions. For comparison, MP2/def2-TZVPD method (M2) with full geometry optimization and vibrational analysis in water has been applied for 12 species; analogously, for 24 substances, DLPNO-CCSD(T)/aug-cc-pVTZ method (M3) has been applied in the geometry obtained by MP2 and/or B3LYP. It was found that the absolute oxidation potential correlates with the adiabatic ionisation energy; the absolute reduction potential correlates with the adiabatic electron affinity and the electrophilicity index. In order to validate the used methodology with experimental vertical ionisation energies and vibrational spectrum obtained in gas phase, calculations were done also in vacuo.

Trimethylamine N-oxide, ß-alanine, tryptophan index, and vitamin B6-related dietary patterns in association with stroke risk.

BACKGROUND AND AIMS: The aim of this study was to examine the associations of dietary patterns derived by reduced-rank regression (RRR) model reflecting variation in novel biomarkers (trimethylamine N-oxide, ß-alanine, tryptophan index, and vitamin B6) with stroke risk. METHODS AND RESULTS: We performed analyses based on a community-based cohort study "the Prospective Follow-up Study on Cardiovascular Morbidity and Mortality in China (PFS-CMMC)". Factor loadings were calculated by RRR using 11 food groups collected via a validated food frequency questionnaire and the four response variables based on its nested case-control data (393 cases of stroke vs. 393 matched controls). Dietary pattern scores were derived by applying the factor loadings to the food groups in the entire cohort (n = 15,518). The associations of dietary pattern with the stroke risk were assessed using Cox proportional hazards models. The dietary pattern characterized with higher intakes of red meat and poultry but lower intakes of fresh vegetables, fresh fruits, and fish/seafoods were identified for further analyses. The hazard ratios (HR) for the highest vs. lowest quartile was 1.55 [95 % confidence interval (CI): 1.18-2.03, P trend = 0.001] for total stroke, 2.96 [95 % CI: 1.53-5.71, P trend <0.001] for non-ischemic stroke, after adjustment for sex, age, educational attainment, current smoking, current drinking, body mass index, total energy intake, family history of stroke, hypertension, diabetes, hyperlipidemia, and estimated glomerular filtration rate. CONCLUSION: Our findings highlight the importance of limited meat intake and increased intakes of fresh vegetables, fruits, and fish/seafoods in the prevention of stroke among Chinese adults.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

The Role of Rho Kinase Inhibitors in Corneal Diseases.

The cornea, as the outermost layer of the eye, plays a crucial role in vision by focusing light onto the retina. Various diseases and injuries can compromise its clarity, leading to impaired vision. This review aims to provide a thorough overview of the pharmacological properties, therapeutic potential and associated risks of Rho-associated protein kinase (ROCK) inhibitors in the management of corneal diseases. The article focuses on four key ROCK inhibitors: Y-27632, fasudil, ripasudil, and netarsudil, providing a comparative examination. Studies supporting the use of ROCK inhibitors highlight their efficacy across diverse corneal conditions. In Fuchs' endothelial corneal dystrophy, studies on the application of Y-27632, ripasudil, and netarsudil demonstrated noteworthy enhancements in corneal clarity, endothelial cell density, and visual acuity. In pseudophakic bullous keratopathy, the injection of Y-27632 together with cultured corneal endothelial cells into the anterior chamber lead to enhanced corneal endothelial cell density and improved visual acuity. Animal models simulating chemical injury to the cornea showed a reduction of neovascularization and epithelial defects after application of fasudil and in a case of iridocorneal endothelial syndrome netarsudil improved corneal edema. Addressing safety considerations, netarsudil and ripasudil, both clinically approved, exhibit adverse events such as conjunctival hyperemia, conjunctival hemorrhage, cornea verticillata, conjunctivitis, and blepharitis. Monitoring patients during treatment becomes crucial to balancing the potential therapeutic benefits with these associated risks. In conclusion, ROCK inhibitors, particularly netarsudil and ripasudil, offer promise in managing corneal diseases. The comparative analysis of their pharmacological properties and studies supporting their efficacy underscore their potential therapeutic significance. However, ongoing research is paramount to comprehensively understand their safety profiles and long-term outcomes in diverse corneal conditions, guiding their optimal application in clinical practice.

Integrative Placental Multi-Omics Analysis Reveals Perturbed Pathways and Potential Prognostic Biomarkers in Gestational Hypertension.

BACKGROUND: Gestational hypertension (GH) is a severe complication that occurs after 20 weeks of pregnancy; however, its molecular mechanisms are not yet fully understood. OBJECTIVE: Through this case-control discovery phase study, we aimed to find disease-specific candidate placental microRNAs (miRNAs) and metabolite markers for differentiating GH by integrating next-generation sequencing and metabolomics multi-omics analysis of placenta. Using small RNA sequencing and metabolomics of placental tissues of healthy pregnant (HP, n = 24) and GH subjects (n = 20), the transcriptome and metabolome were characterized in both groups. RESULTS: The study identified a total of 44 downregulated placental miRNAs which includes three novel, three mature and 38 precursor miRNAs. Six miRNAs including three mature (hsa-miR-181a-5p, hsa-miR-498-5p, and hsa-miR-26b-5p) and three novel (NC_000016.10_1061, NC_000005.10_475, and NC_000001.11_53) were considered for final target prediction and functional annotation. Integrative analysis of differentially expressed miRNAs and metabolites yielded five pathways such as purine, glutathione, glycerophospholipid, inositol phosphate and ß-alanine to be significantly perturbed in GH. We present fourteen genes (LPCAT1, LPCAT2, DGKH, PISD, GPAT2, PTEN, SACM1L, PGM2, AMPD3, AK7, AK3, CNDP1, IDH2, and ODC1) and eight metabolites (xanthosine, xanthine, spermine, glycine, CDP-Choline, glyceraldehyde 3-phosphate, ß-alanine, and histidine) with potential to distinguish GH and HP. CONCLUSION: The differential expression of miRNAs, their target genes, altered metabolites and metabolic pathways in GH patients were identified for the first time in our study. Further, the altered miRNAs and metabolites were integrated to build their inter-connectivity network. The findings obtained from our study may be used as a valuable source to further unravel the molecular pathways associated with GH and also for the evaluation of prognostic markers.

Assessment of breast cancer chemotherapy dose reduction in an integrated healthcare delivery system.

PURPOSE: Most cytotoxic drugs are dosed using body surface area (BSA), yet not all cancer patients receive the full BSA-determined dose. Prior work suggests that breast cancer patients who are obese are more likely to experience dose reduction than normal weight patients. However, the factors driving dose reduction remain unclear. METHODS: In 452 women diagnosed with stage I-IIIA primary breast cancer at Kaiser Permanente Northern California, we evaluated the association between obesity and dose reduction, and further explored other factors in relation to dose reduction, including various sociodemographic characteristics, tumor characteristics, and comorbidities. Study participants were a part of the Pathways Study, diagnosed between 2006 and 2013 and treated with cyclophosphamide + doxorubicin, followed by paclitaxel (ACT). Dose reduction was assessed using first cycle dose proportion (FCDP) and average relative dose intensity (ARDI), a metric of dose intensity over the course of chemotherapy. RESULTS: Overall, 8% of participants received a FCDP < 90% and 21.2% had an ARDI < 90%, with dose reduction increasing with body mass index. In adjusted logistic regression models, obese women had 4.1-fold higher odds of receiving an ARDI < 90% than normal weight women (95% CI: 1.9-8.9; p-trend = 0.0006). Increasing age was positively associated with an ADRI < 90%, as was the presence of comorbidity. Dose reduction was less common in later calendar years. CONCLUSION: Results offer insight on factors associated with chemotherapy dosing for a common breast cancer regimen. Larger studies are required to evaluate relevance to other regimens, and further work will be needed to determine whether dose reductions impact outcomes in obese women.

MERCURY-3: a randomized comparison of netarsudil/latanoprost and bimatoprost/timolol in open-angle glaucoma and ocular hypertension.

PURPOSE   : To compare the efficacy and safety of the fixed-dose combination (FDC) of netarsudil 0.02%/latanoprost 0.005% ophthalmic solution (NET/LAT; Roclanda®) with bimatoprost 0.03%/timolol maleate 0.5% (BIM/TIM; Ganfort®) ophthalmic solution in the treatment of open-angle glaucoma (OAG) and ocular hypertension (OHT). METHODS: MERCURY-3 was a 6-month prospective, double-masked, randomized, multicenter, active-controlled, parallel-group, non-inferiority study. Patients (≥ 18 years) with a diagnosis of OAG or OHT in both eyes that was insufficiently controlled with topical medication (IOP ≥ 17 mmHg in ≥ 1 eye and < 28 mmHg in both eyes) were included. Following washout, patients were randomized to once-daily NET/LAT or BIM/TIM for up to 6 months; efficacy was assessed at Week 2, Week 4, and Month 3; safety was evaluated for 6 months. Comparison of NET/LAT relative to BIM/TIM for mean IOP at 08:00, 10:00, and 16:00 h was assessed at Week 2, Week 6, and Month 3. Non-inferiority of NET/LAT to BIM/TIM was defined as a difference of ≤ 1.5 mmHg at all nine time points through Month 3 and ≤ 1.0 mmHg at five or more of nine time points through Month 3. RESULTS: Overall, 430 patients were randomized (NET/LAT, n = 218; BIM/TIM, n = 212), and all received at least one dose of study medication. Efficacy analyses were performed at Month 3 on 388 patients (NET/LAT, n = 184; BIM/TIM, n = 204). NET/LAT demonstrated non-inferiority to BIM/TIM, with a between-treatment difference in IOP of ≤ 1.5 mmHg achieved at all time points and ≤ 1.0 mmHg at the majority of time points (six of nine) through Month 3. Mean diurnal IOP during the study ranged from 15.4 to 15.6 mmHg and 15.2 to 15.6 mmHg in the NET/LAT and BIM/TIM groups respectively, with no between-group statistically significant difference. No significant differences were observed in key secondary endpoints. No serious, treatment-related adverse events (AEs) were observed, and AEs were typically mild/moderate in severity. The most common treatment-related AEs were conjunctival hyperemia (NET/LAT, 30.7%; BIM/TIM, 9.0%) and cornea verticillata (NET/LAT, 11.0%; BIM/TIM, 0%). CONCLUSIONS: Once-daily NET/LAT was non-inferior to BIM/TIM in IOP reduction in OAG and OHT, with AEs consistent with previous findings. NET/LAT offers a compelling alternative FDC treatment option for OAG and OHT.

Comparison of safety and efficacy of Netarsudil 0.02% and Bimatoprost 0.01% monotherapy and combination therapy in primary open-angle glaucoma and ocular hypertension.

PURPOSE: To study and compare the efficacy and safety profile of Rho-kinase inhibitor (netarsudil 0.02%) and prostaglandin analog (bimatoprost 0.01%) both as monotherapy and in combination. DESIGN: Prospective, randomized, monocentric, open-label clinical trial. METHODS: Patients ≥20 years of age with primary open-angle glaucoma or ocular hypertension (IOP >21 mmHg) were recruited and randomized to receive either netarsudil 0.02%, netarsudil 0.02% + bimatoprost 0.01%, or bimatoprost 0.01% once daily for a period of 12 weeks. IOP and side effects were documented at 4, 8, and 12 weeks. RESULTS: The mean treated IOP ranged 17.51-18.57 mmHg for netarsudil, 15.80-16.46 mmHg for bimatoprost, and 14.00-14.87 mmHg for the combination therapy group. The mean IOP reduction from baseline at 4, 8, and 12 weeks was found to be statistically significant ( P < 0.001) in all three groups. The safety profile of netarsudil/bimatoprost combination was consistent with each constituent individually. The only frequently observed ocular adverse event was conjunctival hyperemia, which was seen mostly in netarsudil and netarsudil + bimatoprost groups ( P < 0.001). CONCLUSION: The IOP-lowering effect of netarsudil 0.02% once daily is non-inferior to bimatoprost 0.01% in patients with POAG and ocular hypertension with acceptable ocular safety, and the combination therapy achieved a higher IOP-lowering effect. This group of medications can be a useful adjunct in patients on maximal therapy.

Content of amino acids and biogenic amines in stored meat as a result of a broiler diet supplemented with ß-alanine and garlic extract.

Poultry meat is a highly esteemed product among consumers. However, the emphasis on increasing body weight has led to a rise in the proportion of rapidly shrinking fibers, adversely affecting the quality and shelf life of poultry meat. With a growing awareness of dietetics among consumers, there is an increasing challenge to produce chicken meat that is not only free of antibiotics but also beneficial for dietary and health reasons. Biogenic amines (BA) can serve as indicators of meat freshness and quality. While they play vital roles in the body, excessive consumption of BA can have toxic and carcinogenic effects. The objective of this study was to examine the impact of supplementing feed with garlic extract and ß-alanine (ß-Ala) on the formation of BA and amino acid (AA) levels in the breast and leg muscles of chickens stored under aerobic chilling conditions. The muscles were obtained from chickens fed with garlic extract and ß-Ala in quantities of 0.5 and 2% for each additive, as well as 0.5 and 2% of their combination. Analyses were conducted on d 1, 3, 5, 7, and 10 of storage. ß-Ala supplementation increased the proportion of this AA in breast (P < 0.01) and leg muscles (P < 0.01), along with a rise in the proportion of nonessential AA (NEAA; sum of aspartic, aspartic acid, glutamic, glutamic acid, serine, ß-Ala, and proline) (P < 0.01). The levels of BA changed during storage in breast and leg muscles (P < 0.001). The applied diet significantly influenced the formation of putrescine (P = 0.030), phenylethylamine (P = 0.003), agmatine (P = 0.025), and total BA (P < 0.001) in breast muscles. On the 10 d of storage, the breast muscles exhibited the lowest BA index (BAI) in the group, with a diet supplemented with 0.5% garlic extract and 0.5% ß-Ala (P < 0.05). The leg muscles showed a similar BA trend as the breast muscles. These supplements may be utilized in production to augment the protein content of chicken muscles and potentially decrease the BAI index during meat storage.

Peripheral signaling pathways contributing to non-histaminergic itch in humans.

BACKGROUND: Chronic itch (chronic pruritus) is a major therapeutic challenge that remains poorly understood despite the extensive recent analysis of human pruriceptors. It is unclear how the peripheral nervous system differentiates the signaling of non-histaminergic itch and pain. METHODS: Here we used psychophysical analysis and microneurography (single nerve fiber recordings) in healthy human volunteers to explore the distinct signaling mechanisms of itch, using the pruritogens ß-alanine, BAM 8-22 and cowhage extract. RESULTS: The mode of application (injection or focal application using inactivated cowhage spicules) influenced the itch/pain ratio in sensations induced by BAM 8-22 and cowhage but not ß-alanine. We found that sensitizing pre-injections of prostaglandin E2 increased the pain component of BAM 8-22 but not the other pruritogens. A-fibers contributed only to itch induced by ß-alanine. TRPV1 and TRPA1 were necessary for itch signaling induced by all three pruritogens. In single-fiber recordings, we found that BAM 8-22 and ß-alanine injection activated nearly all CM-fibers (to different extents) but not CMi-fibers, whereas cowhage extract injection activated only 56% of CM-fibers but also 25% of CMi-fibers. A "slow bursting discharge pattern" was evoked in 25% of CM-fibers by ß-alanine, in 35% by BAM 8-22, but in only 10% by cowhage extract. CONCLUSION: Our results indicate that no labeled line exists for these pruritogens in humans. A combination of different mechanisms, specific for each pruritogen, leads to itching sensations rather than pain. Notably, non-receptor-based mechanisms such as spatial contrast or discharge pattern coding seem to be important processes. These findings will facilitate the discovery of therapeutic targets for chronic pruritus, which are unlikely to be treated effectively by single receptor blockade.

Symptomatic Netarsudil-Induced Verticillata.

This case report discusses a diagnosis of bilateral corneal verticillata in a woman aged 80 years who was using netarsudil eye drops for the treatment of primary open-angle glaucoma.

Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

BACKGROUND: Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. RESULTS: In this study, production of 3-HP via the synthetic ß-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCß21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCß21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCß21. CONCLUSIONS: Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the ß-alanine pathway.

Surface Modification of Silica with ß-Alanine Derivatives for Unique Applications in Liquid Chromatography.

Column purchasing cost is an important issue for an analyst to analyze complex sample matrices. Here, we report the development of an amino acid (ß-alanine)-derived stationary phase (Sil-Ala-C12) with strategic and effective interaction sites (amide and urea as embedded polar groups with C12 alkyl chain) able to separate various kinds of analytes. Owing to the balanced hydrophobicity and hydrophilicity of the phase, it showed exceptional separation abilities in both reversed-phase high-performance liquid chromatography (RP-HPLC) as a hydrophobic phase and hydrophilic interaction chromatography (HILIC) as a hydrophilic phase. Remarkably, the baseline separation was achieved for the challenging ß- and γ-isomers of tocopherol. Usually, three columns such as pentafluorophenyl or C30, C18, and sulfobetaine HILIC are required for the analysis of vitamin E, capsaicinoids, and vitamin C in chili peppers (Capsicum spp.), respectively. However, only Sil-Ala-C12 was able to separate these analytes. A single column can serve 3-4 purposes, which suggests that Sil-Ala-C12 had the potential to reduce column purchasing costs.

Muscle growth affects the metabolome of the pectoralis major muscle in red-winged tinamou (Rhynchotus rufescens).

The aim of the present study was to identify and quantify the metabolites (metabolome analysis) of the pectoralis major muscle in male red-winged tinamou (Rhynchotus rufescens) selected for growth traits. A selection index was developed for females [body weight (BW), chest circumference (CC), and thigh circumference (TC)] and males [BW, CC, TC, semen volume, and sperm concentration] in order to divide the animals into 2 experimental groups: selection group with a higher index (TinamouS) and commercial group with a lower index (TinamouC). Twenty male offspring of the 2 groups (TinamouS, n = 10; TinamouC, n = 10) were confined for 350 d. The birds were slaughtered and pectoralis major muscle samples were collected, subjected to polar and apolar metabolites extractions and analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. Analysis of the polar metabolomic profile identified 65 metabolites; 29 of them were differentially expressed between the experimental groups (P < 0.05). The TinamouS groups exhibited significantly higher concentrations (P < 0.05) of 25 metabolites, including anserine, aspartate, betaine, carnosine, creatine, glutamate, threonine, 3-methylhistidine, NAD+, pyruvate, and taurine. Significantly higher concentrations of cysteine, beta-alanine, lactose, and choline were observed in the TinamouC group (P < 0.05). The metabolites identified in the muscle provided information about the main metabolic pathways (higher impact value and P < 0.05), for example, phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; D-glutamine and D-glutamate metabolism; ß-alanine metabolism; glycine, serine and threonine metabolism; taurine and hypotaurine metabolism; histidine metabolism; phenylalanine metabolism. The NMR spectra of apolar fraction showed 8 classes of chemical compounds. The metabolome analysis shows that the selection index resulted in the upregulation of polyunsaturated fatty acids, unsaturated fatty acids, phosphocholines, phosphoethanolamines, triacylglycerols, and glycerophospholipids. The present study suggests that, despite few generations, the selection based on muscle growth traits promoted changes in metabolite concentrations in red-winged tinamou.

Use of Buffers in Specific Contexts: Highly Trained Female Athletes, Extreme Environments and Combined Buffering Agents-A Narrative Review.

This narrative review evaluated the evidence for buffering agents (sodium bicarbonate, sodium citrate and beta-alanine), with specific consideration of three discrete scenarios: female athletes, extreme environments and combined buffering agents. Studies were screened according to exclusion and inclusion criteria and were analysed on three levels: (1) moderating variables (supplement dose and timing, and exercise test duration and intensity), (2) design factors (e.g., use of crossover or matched group study design, familiarisation trials) and (3) athlete-specific factors (recruitment of highly trained participants, buffering capacity and reported performance improvements). Only 19% of the included studies for the three buffering agents reported a performance benefit, and only 10% recruited highly trained athletes. This low transferability of research findings to athletes' real-world practices may be due to factors including the small number of sodium citrate studies in females (n = 2), no studies controlling for the menstrual cycle (MC) or menstrual status using methods described in recently established frameworks, and the limited number of beta-alanine studies using performance tests replicating real-world performance efforts (n = 3). We recommend further research into buffering agents in highly trained female athletes that control or account for the MC, studies that replicate the demands of athletes' heat and altitude camps, and investigations of highly trained athletes' use of combined buffering agents. In a practical context, we recommend developing evidence-based buffering protocols for individual athletes which feature co-supplementation with other evidence-based products, reduce the likelihood of side-effects, and optimise key moderating factors: supplement dose and timing, and exercise duration and intensity.