Feasibility, safety, efficacy and potential scaling-up of sofosbuvir-based HCV treatment in Central and West Africa: (TAC ANRS 12311 trial).

Access to Hepatis C treatment in Sub-Saharan Africa is a clinical, public health and ethical concern. The multi-country open-label trial TAC ANRS 12311 allowed assessing the feasibility, safety, efficacy of a specific care model of HCV treatment and retreatment in patients with hepatitis C in Sub Saharan Africa. Between November 2015 and March 2017, with follow-up until mid 2019, treatment-naïve patients with HCV without decompensated cirrhosis or liver cancer were recruited to receive 12 week-treatment with either sofosbuvir + ribavirin (HCV genotype 2) or sofosbuvir + ledipasvir (genotype 1 or 4) and retreatment with sofosbuvir + velpatasvir + voxilaprevir in case of virological failure. The primary outcome was sustained virological response at 12 weeks after end of treatment (SVR12). Secondary outcomes included treatment adherence, safety and SVR12 in patients who were retreated due to non-response to first-line treatment. The model of care relied on both viral load assessment and educational sessions to increase patient awareness, adherence and health literacy. The study recruited 120 participants, 36 HIV-co-infected, and 14 cirrhotic. Only one patient discontinued treatment because of return to home country. Neither death nor severe adverse event occurred. SVR12 was reached in 107 patients (89%): (90%) in genotype 1 or 2, and 88% in GT-4. All retreated patients (n = 13) reached SVR12. HCV treatment is highly acceptable, safe and effective under this model of care. Implementation research is now needed to scale up point-of-care HCV testing and SVR assessment, along with community involvement in patient education, to achieve HCV elimination in Sub-Saharan Africa.

Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling.

Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

A high-fat diet promotes cancer progression by inducing gut microbiota-mediated leucine production and PMN-MDSC differentiation.

A high-fat diet (HFD) is a high-risk factor for the malignant progression of cancers through the disruption of the intestinal microbiota. However, the role of the HFD-related gut microbiota in cancer development remains unclear. This study found that obesity and obesity-related gut microbiota were associated with poor prognosis and advanced clinicopathological status in female patients with breast cancer. To investigate the impact of HFD-associated gut microbiota on cancer progression, we established various models, including HFD feeding, fecal microbiota transplantation, antibiotic feeding, and bacterial gavage, in tumor-bearing mice. HFD-related microbiota promotes cancer progression by generating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Mechanistically, the HFD microbiota released abundant leucine, which activated the mTORC1 signaling pathway in myeloid progenitors for PMN-MDSC differentiation. Clinically, the elevated leucine level in the peripheral blood induced by the HFD microbiota was correlated with abundant tumoral PMN-MDSC infiltration and poor clinical outcomes in female patients with breast cancer. These findings revealed that the "gut-bone marrow-tumor" axis is involved in HFD-mediated cancer progression and opens a broad avenue for anticancer therapeutic strategies by targeting the aberrant metabolism of the gut microbiota.

DNA damage induces p53-independent apoptosis through ribosome stalling.

In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Leucine improves thiram-induced tibial dyschondroplasia and gut microbiota dysbiosis in broilers.

Thiram, a commonly used agricultural insecticide and fungicide, has been found to cause tibial dyschondroplasia (TD) in broilers, leading to substantial economic losses in the poultry industry. In this study, we aimed to investigate the mechanism of action of leucine in mitigating thiram-induced TD and leucine effects on gut microbial diversity. Broiler chickens were randomly divided into five equal groups: control group (standard diet), thiram-induced group (thiram 80 mg/kg from day 3 to day 7), and different concentrations of leucine groups (0.3%, 0.6%, 0.9% leucine from day 8 to day 18). Performance indicator analysis and tibial parameter analysis showed that leucine positively affected thiram-induced TD broilers. Additionally, mRNA expressions and protein levels of HIF-1α/VEGFA and Ihh/PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. The results showed that leucine recovered lameness disorder by downregulating the expression of HIF-1α, VEGFA, and PTHrP while upregulating the expression of Ihh. Moreover, the 16 S rRNA sequencing revealed that the leucine group demonstrated a decrease in the abundance of harmful bacteria compared to the TD group, with an enrichment of beneficial bacteria responsible for producing short-chain fatty acids, including Alistipes, Paludicola, CHKCI002, Lactobacillus, and Erysipelatoclostridium. In summary, the current study suggests that leucine could improve the symptoms of thiram-induced TD and maintain gut microbiota homeostasis.

Engineering Enhanced Antimicrobial Properties in α-Conotoxin RgIA through D-Type Amino Acid Substitution and Incorporation of Lysine and Leucine Residues.

Antimicrobial peptides (AMPs), acknowledged as host defense peptides, constitute a category of predominant cationic peptides prevalent in diverse life forms. This study explored the antibacterial activity of α-conotoxin RgIA, and to enhance its stability and efficacy, D-amino acid substitution was employed, resulting in the synthesis of nine RgIA mutant analogs. Results revealed that several modified RgIA mutants displayed inhibitory efficacy against various pathogenic bacteria and fungi, including Candida tropicalis and Escherichia coli. Mechanistic investigations elucidated that these polypeptides achieved antibacterial effects through the disruption of bacterial cell membranes. The study further assessed the designed peptides' hemolytic activity, cytotoxicity, and safety. Mutants with antibacterial activity exhibited lower hemolytic activity and cytotoxicity, with Pep 8 demonstrating favorable safety in mice. RgIA mutants incorporating D-amino acids exhibited notable stability and adaptability, sustaining antibacterial properties across diverse environmental conditions. This research underscores the potential of the peptide to advance innovative oral antibiotics, offering a novel approach to address bacterial infections.

Leucine alleviates cytokine storm syndrome by regulating macrophage polarization via the mTORC1/LXRα signaling pathway.

Cytokine storms are associated with severe pathological damage and death in some diseases. Excessive activation of M1 macrophages and the subsequent secretion of pro-inflammatory cytokines are a major cause of cytokine storms. Therefore, promoting the polarization of M2 macrophages to restore immune balance is a promising therapeutic strategy for treating cytokine storm syndrome (CSS). This study was aimed at investigating the potential protective effects of leucine on lipopolysaccharide (LPS)-induced CSS in mice and exploring the underlying mechanisms. CSS was induced by LPS administration in mice, which were concurrently administered leucine orally. In vitro, bone marrow derived macrophages (BMDMs) were polarized to M1 and M2 phenotypes with LPS and interleukin-4 (IL-4), respectively, and treated with leucine. Leucine decreased mortality in mice treated with lethal doses of LPS. Specifically, leucine decreased M1 polarization and promoted M2 polarization, thus diminishing pro-inflammatory cytokine levels and ameliorating CSS in mice. Further studies revealed that leucine-induced macrophage polarization through the mechanistic target of rapamycin complex 1 (mTORC1)/liver X receptor α (LXRα) pathway, which synergistically enhanced the expression of the IL-4-induced M2 marker Arg1 and subsequent M2 polarization. In summary, this study revealed that leucine ameliorates CSS in LPS mice by promoting M2 polarization through the mTORC1/LXRα/Arg1 signaling pathway. Our findings indicate that a fundamental link between metabolism and immunity contributes to the resolution of inflammation and the repair of damaged tissues.

Real-Life Comparison of Antivirals for SARS-CoV-2 Omicron Infection in Patients With Hematologic Malignancies.

BACKGROUND: We aimed to describe a cohort of hematologic patients with COVID-19 treated with antivirals early. METHODS: Non-interventional chart review study. Comparison of baseline characteristics and outcomes in high-risk hematologic patients treated with remdesivir between December 2021 and April 2022 versus those treated with nirmatrelvir/ritonavir between May and August 2022. RESULTS: Eighty-three patients were analyzed. Forty-two received remdesivir, and 41 nirmatrelvir/ritonavir. Patients with remdesivir were younger, vaccinated with lower number of doses, and received prior corticosteroids less frequently and sotrovimab, hyperimmune plasma and corticosteroids more often. Viral shedding median (IQR) duration was 18 (13-23) and 11 (8-21) days in the remdesivir and nirmatrelvir/ritonavir groups, respectively (p = 0.004). Median (IQR) Ct values before treatment were similar in both groups. Within 5 days of treatment, median (IQR) Ct values were 26 (23-29) and 33 (30-37) in the remdesivir and nirmatrelvir/ritonavir groups, respectively (p < 0.0001). All patients were hospitalized for remdesivir administration and only four (9.8%) in the nirmatrelvir/ritonavir group. The overall outcomes in this cohort of COVID-19 patients with Omicron variant was good, as no patient needed oxygen or ICU admission. One patient in remdesivir group died from septic shock. No severe adverse event was recorded in both treatment groups. CONCLUSIONS: Patients with hematologic malignancies and non-severe COVID-19 who received nirmatrelvir/ritonavir experienced faster decrease in viral load and shorter viral shedding. Furthermore, besides the advantage of oral administration, nirmatrelvir/ritonavir administration reduced the need of hospital admission.

Leucine-Rich Glioma-Inactivated 1 (LGI1) Protein Stimulates Proliferation and IL-10 Production in Peripheral Blood Mononuclear Cells of Patients with LGI1 Antibody-Mediated Autoimmune Encephalitis In Vitro.

Limbic encephalitis (LE) due to anti-leucine-rich glioma-inactivated 1 (LGI1) antibodies is an autoimmune disease characterized by distinct clinical features unique to LGI1 LE, such as faciobrachial dystonic seizures. However, it is unclear whether an additional disease-related LGI1 antigen-specific T cell response is involved in the pathogenesis of this disease. To address this question, we studied the effect of recombinant LGI1 on the proliferation and effector-specific cytokine production (IFN-γ, IL-5, IL-10, and IL-17) of peripheral blood mononuclear cells (PBMCs) from patients with LGI1 LE and healthy controls. We observed that recombinant LGI1 stimulated the proliferation of PBMCs from patients with LGI1 LE, but not from healthy controls. Cytokine measurement of cell culture supernatants from PBMCs incubated with recombinant LGI1 revealed a highly significant increase in IL-10 release in PBMCs from patients with LGI1 LE in comparison with healthy controls. These results suggest that LGI1-mediated stimulation of PBMCs from patients with LGI1 LE leads to the establishment of an IL-10-dominated immunosuppressive cytokine milieu, which may inhibit Th1 differentiation and support B cell proliferation, IgG production, and IgG subclass switching.

The Upregulation of Leucine-Rich Repeat Containing 1 Expression Activates Hepatic Stellate Cells and Promotes Liver Fibrosis by Stabilizing Phosphorylated Smad2/3.

Liver fibrosis poses a significant global health risk due to its association with hepatocellular carcinoma (HCC) and the lack of effective treatments. Thus, the need to discover additional novel therapeutic targets to attenuate liver diseases is urgent. Leucine-rich repeat containing 1 (LRRC1) reportedly promotes HCC development. Previously, we found that LRRC1 was significantly upregulated in rat fibrotic liver according to the transcriptome sequencing data. Herein, in the current work, we aimed to explore the role of LRRC1 in liver fibrosis and the underlying mechanisms involved. LRRC1 expression was positively correlated with liver fibrosis severity and significantly elevated in both human and murine fibrotic liver tissues. LRRC1 knockdown or overexpression inhibited or enhanced the proliferation, migration, and expression of fibrogenic genes in the human hepatic stellate cell line LX-2. More importantly, LRRC1 inhibition in vivo significantly alleviated CCl4-induced liver fibrosis by reducing collagen accumulation and hepatic stellate cells' (HSCs) activation in mice. Mechanistically, LRRC1 promoted HSC activation and liver fibrogenesis by preventing the ubiquitin-mediated degradation of phosphorylated mothers against decapentaplegic homolog (Smad) 2/3 (p-Smad2/3), thereby activating the TGF-ß1/Smad pathway. Collectively, these results clarify a novel role for LRRC1 as a regulator of liver fibrosis and indicate that LRRC1 is a promising target for antifibrotic therapies.

Neutrophil profiling illuminates anti-tumor antigen-presenting potency.

Neutrophils, the most abundant and efficient defenders against pathogens, exert opposing functions across cancer types. However, given their short half-life, it remains challenging to explore how neutrophils adopt specific fates in cancer. Here, we generated and integrated single-cell neutrophil transcriptomes from 17 cancer types (225 samples from 143 patients). Neutrophils exhibited extraordinary complexity, with 10 distinct states including inflammation, angiogenesis, and antigen presentation. Notably, the antigen-presenting program was associated with favorable survival in most cancers and could be evoked by leucine metabolism and subsequent histone H3K27ac modification. These neutrophils could further invoke both (neo)antigen-specific and antigen-independent T cell responses. Neutrophil delivery or a leucine diet fine-tuned the immune balance to enhance anti-PD-1 therapy in various murine cancer models. In summary, these data not only indicate the neutrophil divergence across cancers but also suggest therapeutic opportunities such as antigen-presenting neutrophil delivery.

Role of leucine-rich repeat kinase 2 in severe acute pancreatitis.

Introduction: Intrapancreatic activation of trypsinogen caused by alcohol or high-fat intake and the subsequent autodigestion of the pancreas tissues by trypsin are indispensable events in the development of acute pancreatitis. In addition to this trypsin-centered paradigm, recent studies provide evidence that innate immune responses triggered by translocation of intestinal bacteria to the pancreas due to intestinal barrier dysfunction underlie the immunopathogenesis of acute pancreatitis. Although severe acute pancreatitis is often associated with pancreatic colonization by fungi, the molecular mechanisms linking fungus-induced immune responses to the development of severe acute pancreatitis are poorly understood. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that mediates innate immune responses to fungi and bacteria. Mutations in Lrrk2 is a risk factor for Parkinson's disease and Crohn's disease, both of which are driven by innate immune responses to gut organisms. Discussion: In this Minireview article, we discuss how activation of LRRK2 by the recognition of fungi induces severe acute pancreatitis.

Comparison of Glecaprevir/Pibrentasvir and Sofosbuvir/Ledipasvir in Patients with Hepatitis C Virus Genotype 1 and 2 in South Korea.

Background/Aims: This study compared the effectiveness and safety of glecaprevir/pibrentasvir (GLE/PIB) and sofosbuvir/ledipasvir (SOF/LDV) in real-life clinical practice. Methods: The data from genotype 1 or 2 chronic hepatitis C patients treated with GLE/PIB or sofosbuvir + ribavirin or SOF/LDV in South Korea were collected retrospectively. The analysis included the treatment completion rate, sustained virologic response at 12 weeks (SVR12) test rate, treatment effectiveness, and adverse events. Results: Seven hundred and eighty-two patients with genotype 1 or 2 chronic hepatitis C who were treated with GLE/PIB (n=575) or SOF/LDV (n=207) were included in this retrospective study. The baseline demographic and clinical characteristics revealed significant statistical differences in age, genotype, ascites, liver cirrhosis, and hepatocellular carcinoma between the GLE/PIB and SOF/LDV groups. Twenty-two patients did not complete the treatment protocol. The treatment completion rate was high for both regimens without statistical significance (97.7% vs. 95.7%, p=0.08). The overall SVR12 of intention-to-treat analysis was 81.2% vs. 80.7% without statistical significance (p=0.87). The overall SVR12 of per protocol analysis was 98.7% vs. 100% without statistical significance (p=0.14). Six patients treated with GLE/PIB experienced treatment failure. They were all male, genotype 2, and showed a negative hepatitis C virus RNA level at the end of treatment. Two patients treated with GLE/PIB stopped medication because of fever and abdominal discomfort. Conclusions: Both regimens had similar treatment completion rates, effectiveness, and safety profiles. Therefore, the SOF/LDV regimen can also be considered a viable DAA for the treatment of patients with genotype 1 or 2 chronic hepatitis C.

A case report of drug interaction between co-packaged nirmatrelvir-ritonavir and tacrolimus causing hyponatremia in a lung transplant recipient.

BACKGROUND: Coronavirus disease 2019 (COVID-19) infection in lung transplant recipients can be lethal owing to the use of immunosuppressants. Antiviral agents may be administered to these patients. Co-packaged nirmatrelvir-ritonavir is a new agent currently being used in combination. CASE PRESENTATION: In this report, we present a case of a 64-year-old woman, a lung transplant recipient, who experienced hyponatremia and showed a high serum tacrolimus concentration following the administration of the co-packaged nirmatrelvir-ritonavir combination. CONCLUSION: Although the nirmatrelvir-ritonavir and tacrolimus combination is not contraindicated, other treatment strategies should be considered first, if available, and the dose of tacrolimus should be reduced when using the nirmatrelvir-ritonavir combination. In cases where combination therapy is necessary, serum tacrolimus levels should be closely monitored in lung transplant recipients. Documentation of more such reports is important to identify drug interactions between nirmatrelvir-ritonavir and other agents, with the aim of preventing severe adverse effects.

The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

Repurposed Drugs Celecoxib and Fmoc-L-Leucine Alone and in Combination as Temozolomide-Resistant Antiglioma Agents-Comparative Studies on Normal and Immortalized Cell Lines, and on C. elegans.

Glioblastoma multiforme therapy remains a significant challenge since there is a lack of effective treatment for this cancer. As most of the examined gliomas express or overexpress cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptors γ (PPARγ), we decided to use these proteins as therapeutic targets. Toxicity, antiproliferative, proapoptotic, and antimigratory activity of COX-2 inhibitor (celecoxib-CXB) and/or PPARγ agonist (Fmoc-L-Leucine-FL) was examined in vitro on temozolomide resistant U-118 MG glioma cell line and comparatively on BJ normal fibroblasts and immortalized HaCaT keratinocytes. The in vivo activity of both agents was studied on C. elegans nematode. Both drugs effectively destroyed U-118 MG glioma cells via antiproliferative, pro-apoptotic, and anti-migratory effects in a concentration range 50-100 µM. The mechanism of action of CXB and FL against glioma was COX-2 and PPARγ dependent and resulted in up-regulation of these factors. Unlike reports by other authors, we did not observe the expected synergistic or additive effect of both drugs. Comparative studies on normal BJ fibroblast cells and immortalized HaCaT keratinocytes showed that the tested drugs did not have a selective effect on glioma cells and their mechanism of action differs significantly from that observed in the case of glioma. HaCaTs did not react with concomitant changes in the expression of COX-2 and PPARγ and were resistant to FL. Safety tests of repurposing drugs used in cancer therapy tested on C. elegans nematode indicated that CXB, FL, or their mixture at a concentration of up to 100 µM had no significant effect on the entire nematode organism up to 4th day of incubation. After a 7-day treatment, CXB significantly shortened the lifespan of C. elegans at 25-400 µM concentration and body length at 50-400 µM concentration.

Leucine-Rich Repeat in Polycystin-1 Suppresses Cystogenesis in a Zebrafish (Danio rerio) Model of Autosomal-Dominant Polycystic Kidney Disease.

Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5ß1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.