Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

[Advances on cyclopeptides of plants].

Cyclopeptides isolated from a variety of plants are a class of cyclic nitrogen-containing compounds, and they are primarily formed by peptide bonds between amino acids, generally containing 2 to 37 L-configuration encoded or non-encoded amino acid residues. Cyclopeptides have significant values in scientific research as natural small-molecule metabolites produced by plants. The available studies have revealed that such natural products are ubiquitous in plants, which mainly include cyclic dipeptides, cyclic tetrapeptides, cyclic pentapeptides, cyclic hexapeptides, cyclic heptapeptides, cyclic octapeptides, cyclic nonapeptides, and cyclic decapeptides. Among them, cyclic dipeptides, cyclic hexapeptides, and cyclic octapeptides are the major active compounds. It has been reported that plant cyclopeptides have novel and unique chemical structures. They possess diverse pharmacological activities, such as antineoplastic, antimicrobial, antimalarial, anti-inflammatory, and antiviral activities. This paper summarizes the research achievements of plant cyclopeptides since 2006, aiming to provide theoretical reference for the research and application of plant cyclopeptides in medicine, health, and agriculture fields.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Gold and Silver Nanoparticles as Biosensors: Characterization of Surface and Changes in the Adsorption of Leucine Dipeptide under the Influence of Substituent Changes.

Early detection of diseases can increase the chances of successful treatment and survival. Therefore, it is necessary to develop a method for detecting or sensing biomolecules that cause trouble in living organisms. Disease sensors should possess specific properties, such as selectivity, reproducibility, stability, sensitivity, and morphology, for their routine application in medical diagnosis and treatment. This work focuses on biosensors in the form of surface-functionalized gold (AuNPs) and silver nanoparticles (AgNPs) prepared using a less-time-consuming, inexpensive, and efficient synthesis route. This allows for the production of highly pure and stable (non-aggregating without stabilizers) nanoparticles with a well-defined spherical shape, a desired diameter, and a monodisperse distribution in an aqueous environment, as confirmed by transmission electron microscopy with energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray diffraction (XRD), photoelectron spectroscopy (XPS), ultraviolet-visible (UV-VIS) spectroscopy, and dynamic light scattering (DLS). Thus, these nanoparticles can be used routinely as biomarker sensors and drug-delivery platforms for precision medicine treatment. The NPs' surface was coated with phosphonate dipeptides of L-leucine (Leu; l-Leu-C(R1)(R2)PO3H2), and their adsorption was monitored using SERS. Reproducible spectra were analyzed to determine the orientation of the dipeptides (coating layers) on the nanoparticles' surface. The appropriate R2 side chain of the dipeptide can be selected to control the arrangement of these dipeptides. This allows for the proper formation of a layer covering the nanoparticles while also simultaneously interacting with the surrounding biological environment, such as cells, tissues, and biological fluids.

A Novel Tetrapeptide Ala-Phe-Phe-Pro (AFFP) Derived from Antarctic Krill Prevents Scopolamine-Induced Memory Disorder by Balancing Lipid Metabolism of Mice Hippocampus.

Memory impairment is a serious problem with organismal aging and increased social pressure. The tetrapeptide Ala-Phe-Phe-Pro (AFFP) is a synthetic analogue of Antarctic krill derived from the memory-improving Antarctic krill peptide Ser-Ser-Asp-Ala-Phe-Phe-Pro-Phe-Arg (SSDAFFPFR) after digestion and absorption. The objective of this research was to assess the neuroprotective effects of AFFP by reducing oxidative stress and controlling lipid metabolism in the brains of mice with memory impairment caused by scopolamine. The 1H Nuclear magnetic resonance spectroscopy results showed that AFFP had three active hydrogen sites that could contribute to its antioxidant properties. The findings from in vivo tests demonstrated that AFFP greatly enhanced the mice's behavioral performance in the passive avoidance, novel object recognition, and eight-arm maze experiments. AFFP reduced oxidative stress by enhancing superoxide dismutase activity and malondialdehyde levels in mice serum, thereby decreasing reactive oxygen species level in the mice hippocampus. In addition, AFFP increased the unsaturated lipid content to balance the unsaturated lipid level against the neurotoxicity of the mice hippocampus. Our findings suggest that AFFP emerges as a potential dietary intervention for the prevention of memory impairment disorders.

Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

Exploring the role of combined external beam radiotherapy and targeted radioligand therapy with [177Lu]Lu-PSMA-617 for prostate cancer - from bench to bedside.

Management of prostate cancer (PC) might be improved by combining external beam radiotherapy (EBRT) and prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) with lutetium-177 (177Lu)-labeled PSMA inhibitors. We hypothesized a higher efficacy of the combination due to augmentation of the radiation dose to the tumor and interactions of EBRT with PSMA expression potentially increasing radiopharmaceutical uptake. Therefore, this study analyzed the influence of radiation on PSMA expression levels in vitro. The results were translated to evaluate the efficacy of the combination of photon EBRT and [177Lu]Lu-PSMA-617 in a murine PC xenograft model. Finally, a clinical case report on a combined elective field EBRT with RLT dose escalation illustrates a proof-of-concept. Methods: PSMA gene and protein expression were assessed in human PSMA-overexpressing LNCaP cells after irradiation using reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry and On-Cell Western assays. In the in vivo therapy study, LNCaP tumor-bearing BALB/c nu/nu mice were irradiated once with 2 Gy X-ray EBRT and injected with 40 MBq [177Lu]Lu-PSMA-617 after 4 h or received single or no treatment (n = 10 each). Tumor-absorbed doses by [177Lu]Lu-PSMA-617 were calculated according to the Medical Internal Radiation Dosimetry (MIRD) formalism after deriving time-activity curves using a gamma probe. An exemplified patient case is demonstrated where fractionated EBRT (54 Gy to prostate; 45 Gy to pelvic lymphatics) and three cycles of [177Lu]Lu-PSMA-617 (3.4-6.0 GBq per cycle) were sequentially combined under concurrent androgen deprivation for treating locally advanced PC. Results: At 4 h following irradiation with 2-8 Gy, LNCaP cells displayed a PSMA protein upregulation by around 18% relative to non-irradiated cells, and a stronger upregulation on mRNA level (up to 2.6-fold). This effect was reversed by 24 h when PSMA protein levels were downregulated by up to 22%. Mice treated with the combination therapy showed significantly improved outcomes regarding tumor control and median survival (p < 0.0001) as compared to single or no treatment. Relative to monotherapy with PSMA-RLT or EBRT, the tumor doubling time was prolonged 1.7- or 2.7-fold and the median survival was extended by 24% or 60% with the combination, respectively. Additionally, tumors treated with EBRT exhibited a 14% higher uptake of the radiopharmaceutical as evident from the calculated tumor-absorbed dose, albeit with high variability in the data. Concerning the patient case, the tri-modality treatment was well tolerated and the patient responded with a long-lasting complete biochemical remission for five years following end of PSMA-RLT. The patient then developed a biochemical relapse with oligo-recurrent disease on follow-up imaging. Conclusion: The present preclinical and clinical data demonstrate that the combination of EBRT with dose escalation by PSMA-RLT improves tumor control and potentially prolongs survival. This may pave the way for further clinical investigations of this approach to explore the curative potential of the combination therapy.

Methionyl-Methionine Dipeptide Enhances Mammogenesis and Lactogenesis by Suppressing the Expression of a Novel Long Noncoding RNA MGPNCR to Inhibit eIF4B Dephosphorylation.

Previous research has demonstrated that in pregnant mice deficient in l-methionine (Met), the mixture of the dipeptide l-methionyl-l-methionine (Met-Met) with Met was more effective than Met alone in promoting mammogenesis and lactogenesis. This study aimed to investigate the role of a novel long noncoding RNA (lncRNA), named mammary gland proliferation-associated lncRNA (MGPNCR), in these processes. Transcriptomic analysis of mammary tissues from Met-deficient mice, supplemented either with a Met-Met/Met mixture or with Met alone, revealed significantly higher MGPNCR expression in the Met group compared to the mixture group, a finding recapitulated in a mammary epithelial cell model. Our findings suggested that MGPNCR hindered mammogenesis and milk protein synthesis by binding to eukaryotic initiation factor 4B (eIF4B). This interaction promoted the dephosphorylation of eIF4B at serine-422 by enhancing its association with protein phosphatase 2A (PP2A). Our study sheds light on the regulatory mechanisms of lncRNA-mediated dipeptide effects on mammary cell proliferation and milk protein synthesis. These insights underscore the potential benefits of utilizing dipeptides to improve milk protein in animals and potentially in humans.

Tutorial on how to build non-Markovian dynamic models from molecular dynamics simulations for studying protein conformational changes.

Protein conformational changes play crucial roles in their biological functions. In recent years, the Markov State Model (MSM) constructed from extensive Molecular Dynamics (MD) simulations has emerged as a powerful tool for modeling complex protein conformational changes. In MSMs, dynamics are modeled as a sequence of Markovian transitions among metastable conformational states at discrete time intervals (called lag time). A major challenge for MSMs is that the lag time must be long enough to allow transitions among states to become memoryless (or Markovian). However, this lag time is constrained by the length of individual MD simulations available to track these transitions. To address this challenge, we have recently developed Generalized Master Equation (GME)-based approaches, encoding non-Markovian dynamics using a time-dependent memory kernel. In this Tutorial, we introduce the theory behind two recently developed GME-based non-Markovian dynamic models: the quasi-Markov State Model (qMSM) and the Integrative Generalized Master Equation (IGME). We subsequently outline the procedures for constructing these models and provide a step-by-step tutorial on applying qMSM and IGME to study two peptide systems: alanine dipeptide and villin headpiece. This Tutorial is available at https://github.com/xuhuihuang/GME_tutorials. The protocols detailed in this Tutorial aim to be accessible for non-experts interested in studying the biomolecular dynamics using these non-Markovian dynamic models.

[161Tb]Tb-PSMA-617 radioligand therapy in patients with mCRPC: preliminary dosimetry results and intra-individual head-to-head comparison to [177Lu]Lu-PSMA-617.

Rationale: Evaluation of alternative radionuclides for use in prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) is currently focusing on 161Tb, which may provide advantages by emitting additional Auger and conversion electrons. In this pilot study, we present preliminary dosimetry data for [161Tb]Tb-PSMA-617 RLT in a direct comparison with [177Lu]Lu-PSMA-617. Method: Six patients with metastatic castration-resistant prostate cancer (mCRPC) underwent treatment with [177Lu]Lu-PSMA-617 and subsequently - after inadequate response - with [161Tb]Tb-PSMA-617. Whole-body planar and SPECT imaging-based dosimetry of organs at risk (kidneys and salivary glands) and tumor lesions were calculated using IDAC for 177Lu and OLINDA/EXM for 161Tb. The therapeutic index (TI) of mean tumor-absorbed doses over relevant organs at risk was calculated. Results: Mean absorbed doses to organs at risk of PSMA-RLT were slightly higher for [161Tb]Tb-PSMA-617 compared to [177Lu]Lu-PSMA-617 (kidneys: 0.643 ± 0.247 vs. 0.545 ± 0.231 Gy/GBq, factor 1.18; parotid gland: 0.367 ± 0.198 vs. 0.329 ± 0.180 Gy/GBq, factor 1.10), but markedly higher regarding tumor lesions (6.10 ± 6.59 vs 2.59 ± 3.30 Gy/GBq, factor 2.40, p < 0.001). Consequently, the mean TI was higher for [161Tb]Tb-PSMA-617 compared to [177Lu]Lu-PSMA-617 for both, the kidneys (11.54 ± 9.74 vs. 5.28 ± 5.13, p = 0.002) and the parotid gland (16.77 ± 13.10 vs. 12.51 ± 18.09, p = 0.008). Conclusion: In this intra-individual head-to-head pilot study, [161Tb]Tb-PSMA-617 delivered higher tumor-absorbed doses and resulted in superior TI compared to [177Lu]Lu-PSMA-617. This preliminary data support 161Tb as a promising radionuclide for PSMA-RLT in mCRPC.

177Lu-PSMA-617 in Metastatic Castration-Resistant Prostate Cancer: A Review of the Evidence and Implications for Canadian Clinical Practice.

Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer and a therapeutic target. Lutetium-177 (177Lu)-PSMA-617 is the first radioligand therapy to be approved in Canada for use in patients with metastatic castration-resistant prostate cancer (mCRPC). As this treatment represents a new therapeutic class, guidance regarding how to integrate it into clinical practice is needed. This article aims to review the evidence from prospective phase 2 and 3 clinical trials and meta-analyses of observational studies on the use of 177Lu-PSMA-617 in prostate cancer and discuss how Canadian clinicians might best apply these data in practice. The selection of appropriate patients, the practicalities of treatment administration, including necessary facilities for treatment procedures, the assessment of treatment response, and the management of adverse events are considered. Survival benefits were observed in clinical trials of 177Lu-PSMA-617 in patients with progressive, PSMA-positive mCRPC who were pretreated with androgen receptor pathway inhibitors and taxanes, as well as in taxane-naïve patients. However, the results of ongoing trials are awaited to clarify questions regarding the optimal sequencing of 177Lu-PSMA-617 with other therapies, as well as the implications of predictive biomarkers, personalized dosimetry, and combinations with other therapies.

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

Exploring the potential of Mustard (Brassica spp.) seeds through 'Kolhu' traditional method of extraction and novel identification of an anti-cancer dipeptide, Aurantiamide acetate (Asperglaucide) on ultra-performance liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (UPLC/MS-QToF) analytical platform.

Mustard (Brassica spp.) is one of the world's oldest condiments in the food basket, which holds a significant place in the global culinary landscape due to historical prominence and perceived health benefits. This study explores the extraction of oils from Mustard seeds by employing traditional 'Kolhu' method, modern supercritical fluid, and solvent extraction techniques. This study, for the first-time, identified Aurantiamide acetate, a potent anti-cancer dipeptide in Mustard seeds using ultra-performance liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (UPLC/MS-QToF) analytical platform. The analytical methodology was meticulously validated encompassing optimal parameters such as limit of detection, limit of quantification, precision, accuracy, linearity and robustness, within the range. Interestingly, 'Kolhu' method of oil extraction exhibited better yield of Aurantiamide acetate, suggesting superior efficiency of traditional methods. This study accentuates the importance of classical extraction methods, used traditionally, and emphasizes that naturally occurring substances indeed could be harnessed for better health.

Supramolecular Hydrogels and Water Channels of Differing Diameters from Dipeptide Isomers.

Dipeptides stereoisomers and regioisomers composed of norleucine (Nle) and phenylalanine (Phe) self-assemble into hydrogels under physiological conditions that are suitable for cell culture. The supramolecular behavior, however, differs as the packing modes comprise amphipathic layers or water channels, whose diameter is defined by either four or six dipeptide molecules. A variety of spectroscopy, microscopy, and synchrotron-radiation-based techniques unveil fine details of intermolecular interactions that pinpoint the relationship between the chemical structure and ability to form supramolecular architectures that define soft biomaterials.

Prognostic Performance of RECIP 1.0 Based on [18F]PSMA-1007 PET in Prostate Cancer Patients Treated with [177Lu]Lu-PSMA I&T.

In metastatic castration-resistant prostate cancer (mCRPC) patients treated with prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT), the recently proposed criteria for evaluating response to PSMA PET (RECIP 1.0) based on 68Ga- and 18F-labeled PET agents provided prognostic information in addition to changes in prostate-specific antigen (PSA) levels. Our aim was to evaluate the prognostic performance of this framework for overall survival (OS) in patients undergoing RLT and imaged with [18F]PSMA-1007 PET/CT and compare the prognostic performance with the PSA-based response assessment. Methods: In total, 73 patients with mCRPC who were scanned with [18F]PSMA-1007 PET/CT before and after 2 cycles of RLT were retrospectively analyzed. We calculated the changes in serum PSA levels (ΔPSA) and quantitative PET parameters for the whole-body tumor burden (SUVmean, SUVmax, PSMA tumor volume, and total lesion PSMA). Men were also classified following the Prostate Cancer Working Group 3 (PCWG3) criteria for ΔPSA and RECIP 1.0 for PET imaging response. We performed univariable Cox regression analysis, followed by multivariable and Kaplan-Meier analyses. Results: Median OS was 15 mo with a median follow-up time of 14 mo. Univariable Cox regression analysis provided significant associations with OS for ΔPSA (per percentage, hazard ratio [HR], 1.004; 95% CI, 1.002-1.007; P < 0.001) and PSMA tumor volume (per unit, HR, 1.003; 95% CI, 1.000-1.005; P = 0.03). Multivariable Cox regression analysis confirmed ΔPSA (per percentage, HR, 1.004; 95% CI, 1.001-1.006; P = 0.006) as an independent prognosticator for OS. Kaplan-Meier analyses provided significant segregation between individuals with versus those without any PSA response (19 mo vs. 14 mo; HR, 2.00; 95% CI, 0.95-4.18; P = 0.04). Differentiation between patients with or without progressive disease (PD) was also feasible when applying PSA-based PCWG3 (19 mo vs. 9 mo for non-PD and PD, respectively; HR, 2.29; 95% CI, 1.03-5.09; P = 0.01) but slightly failed when applying RECIP 1.0 (P = 0.08). A combination of both response systems (PCWG3 and RECIP 1.0), however, yielded the best discrimination between individuals without versus those with PD (19 mo vs. 8 mo; HR, 2.78; 95% CI, 1.32-5.86; P = 0.002). Conclusion: In patients with mCRPC treated with RLT and imaged with [18F]PSMA-1007, frameworks integrating both the biochemical (PCWG3) and PET-based response (RECIP 1.0) may best assist in identifying subjects prone to disease progression.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

AIM: Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. METHODS: A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. RESULTS: CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CONCLUSION: CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve in vivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Adiponectin-Receptor Agonistic Dipeptide Tyr-Pro Stimulates the Acetylcholine Nervous System in NE-4C Cells.

The dipeptide Tyr-Pro has physiological potential for intact transportability into the brain parenchyma, prevention of cognitive impairment, and an adiponectin receptor 1 (AdipoR1) agonistic effect. The present study aimed to understand the effect of Tyr-Pro on the acetylcholine (ACh) nervous system and its underlying mechanism in NE-4C nerve cells. Concentration-dependent ACh production was induced by stimulation with Tyr-Pro and AdipoRon (an AdipoR1 agonist), along with the expression of AdipoR1 and choline acetyltransferase (ChAT) in NE-4C cells. By knocking down AdipoR1 in the cells, Tyr-Pro promoted ChAT expression, along with the activations of AMPK and ERK 1/2. Tyr-Pro did not alter acetylcholinesterase or ACh receptors, indicating that the dipeptide might operate as an ACh accelerator in nerve cells. This study provides the first evidence that the AdipoR1 agonistic Tyr-Pro is a promising dipeptide responsible for the stimulation of the ACh nervous system by AdipoR1-induced ChAT activation.

Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Due to the lack of trunk neuromuscular organoids (NMOs) from ALS patients' induced pluripotent stem cells (iPSCs), an organoid system was missing to model the trunk spinal neuromuscular neurodegeneration. With the C9orf72 ALS patient-derived iPSCs and isogenic controls, we used an NMO system containing trunk spinal cord neural and peripheral muscular tissues to show that the ALS NMOs could model peripheral defects in ALS, including contraction weakness, neural denervation, and loss of Schwann cells. The neurons and astrocytes in ALS NMOs manifested the RNA foci and dipeptide repeat proteins. Acute treatment with the unfolded protein response inhibitor GSK2606414 increased the glutamatergic muscular contraction 2-fold and reduced the dipeptide repeat protein aggregation and autophagy. This study provides an organoid system for spinal neuromuscular pathologies in ALS and its application for drug testing.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.