UHPLC-HRMS/MS Chemical Fingerprinting of the Bioactive Partition from Cultivated Piper aduncum L.

Piper aduncum L. is widely distributed in tropical regions and the ethnobotanical uses of this species encompass medicinal applications for the treatment of respiratory, antimicrobial, and gynecological diseases. Chemical studies reveal a diverse array of secondary metabolites, including terpenes, flavonoids, and prenylated compounds. Extracts from P. aduncum have shown antibacterial, antifungal, and larvicidal activities. Our study explores the activity of extracts and partitions against Mycobacterium tuberculosis H37Rv, as well as the chemical diversity of the bioactive partition. This marks the first investigation of the bioactive partition of P. aduncum from agroecological cultivation. The ethyl acetate partition from the ethanolic leaf extract (PAEPL) was found to be the most active. PAEPL was subjected to column chromatography using Sephadex LH-20 and the obtained fractions were analyzed using UHPLC-HRMS/MS. The MS/MS data from the fractions were submitted to the online GNPS platform for the generation of the molecular network, which displayed 1714 nodes and 167 clusters. Compounds were identified via manual inspection and different libraries, allowing the annotation of 83 compounds, including flavonoids, benzoic acid derivatives, glycosides, free fatty acids, and glycerol-esterified fatty acids. This study provides the first chemical fingerprint of an antimycobacterial sample from P. aduncum cultivated in an agroecological system.

Centromere diversity: How different repeat-based holocentromeres may have evolved.

In addition to monocentric eukaryotes, which have a single localized centromere on each chromosome, there are holocentric species, with extended repeat-based or repeat-less centromeres distributed over the entire chromosome length. At least two types of repeat-based holocentromeres exist, one composed of many small repeat-based centromere units (small unit-type), and another one characterized by a few large centromere units (large unit-type). We hypothesize that the transposable element-mediated dispersal of hundreds of short satellite arrays formed the small centromere unit-type holocentromere in Rhynchospora pubera. The large centromere unit-type of the plant Chionographis japonica is likely a product of simultaneous DNA double-strand breaks (DSBs), which initiated the de novo formation of repeat-based holocentromeres via insertion of satellite DNA, derived from extra-chromosomal circular DNAs (eccDNAs). The number of initial DSBs along the chromosomes must be higher than the number of centromere units since only a portion of the breaks will have incorporated eccDNA at an appropriate position to serve as future centromere unit sites. Subsequently, preferential incorporation of the centromeric histone H3 variant at these positions is assumed. The identification of repeat-based holocentromeres across lineages will unveil the centromere plasticity and elucidate the mechanisms underlying the diverse formation of holocentromeres.

How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus (Juncaceae).

Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole-genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1-155 or JefSAT2-180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono- and holocentricity.

Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

Meiotic recombination dynamics in plants with repeat-based holocentromeres shed light on the primary drivers of crossover patterning.

Centromeres strongly affect (epi)genomic architecture and meiotic recombination dynamics, influencing the overall distribution and frequency of crossovers. Here we show how recombination is regulated and distributed in the holocentric plant Rhynchospora breviuscula, a species with diffused centromeres. Combining immunocytochemistry, chromatin analysis and high-throughput single-pollen sequencing, we discovered that crossover frequency is distally biased, in sharp contrast to the diffused distribution of hundreds of centromeric units and (epi)genomic features. Remarkably, we found that crossovers were abolished inside centromeric units but not in their proximity, indicating the absence of a canonical centromere effect. We further propose that telomere-led synapsis of homologues is the feature that best explains the observed recombination landscape. Our results hint at the primary influence of mechanistic features of meiotic pairing and synapsis rather than (epi)genomic features and centromere organization in determining the distally biased crossover distribution in R. breviuscula, whereas centromeres and (epi)genetic properties only affect crossover positioning locally.

Mitochondrial genome of Hancornia speciosa gomes: intergenic regions containing retrotransposons and predicted genes.

BACKGROUND: Plant mitochondrial genomes are characterized by high homologous recombination, extensive intergenic spacers, conservation in DNA sequences, and gene content. The Hancornia genus belongs to the Apocynaceae family, with H. speciosa Gomes being the sole species in the genus. It is an siganificant commercial fruit crop; however, only a number of studies have been conducted. In this study, we present the mitochondrial genome of H. speciosa and compare it with other mitochondrial genomes within the Apocynaceae family. METHODS AND RESULTS: A total of 2.8 Gb of Illumina paired-end reads were used to obtain the mitogenome, resulting in 22 contigs that were merged using 6.1 Gb of Illumina mate-pair reads to obtain a circular chromosome. The mitochondrial genome of H. speciosa is circular, containing 63 predicted functional genes, spanning a length of 741,811 bp, with a CG content of 44%. Within the mitogenome, 50 chloroplast DNA sequences, equivalent to 1.72% of the genome, were detected. However, intergenic spaces accounted for 703,139 bp (94.79% of the genome), and 287 genes were predicted, totaling 173,721 bp. CONCLUSION: This suggests the incorporation of nuclear DNA into the mitogenome of H. speciosa and self duplication. Comparative analysis among the mitogenomes in the Apocynaceae family revealed a diversity in the structure mediated by recombination, with similar gene content and large intergenic spaces.

Toll-like receptors in inflammatory bowel disease: A review of the role of phytochemicals.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammation within the gastrointestinal tract with a remarkable impact on patients' quality of life. Toll-like receptors (TLR), as a key contributor of immune system in inflammation, has a critical role in the pathogenesis of IBD and thus, can be a suitable target of therapeutic agents. Medicinal plants have long been considered as a source of bioactive agents for different diseases, including IBD. PURPOSE: This review discusses current state of the art on the role of plant-derived compounds for the management of IBD with a focus on TLRs. METHODS: Electronic database including PubMed, Web of Science, and Scopus were searched up to January 2023 and all studies in which anticolitis effects of a phytochemical was assessed via modulation of TLRs were considered. RESULTS: Different categories of phytochemicals, including flavonoids, lignans, alkaloids, terpenes, saccharides, and saponins have demonstrated modulatory effects on TLR in different animal and cell models of bowel inflammation. Flavonoids were the most studied phytochemicals amongst others. Also, TLR4 was the most important type of TLRs which were modulated by phytochemicals. Other mechanisms such as inhibition of pro-inflammatory cytokines, nuclear factor-κB pathway, nitric oxide synthesis pathway, cyclooxygenase-2, lipid peroxidation, as well as induction of endogenous antioxidant defense mechanisms were also reported for phytochemicals in various IBD models. CONCLUSION: Taken together, a growing body of pre-clinical evidence support the efficacy of herbal compounds for the treatment of IBD via modulation of TLRs. Future clinical studies are recommended to assess the safety and efficacy of these compounds in human.

Cholesteryl Hemiazelate Present in Cardiovascular Disease Patients Causes Lysosome Dysfunction in Murine Fibroblasts.

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.

Subgenome dominance shapes novel gene evolution in the decaploid pitcher plant Nepenthes gracilis.

Subgenome dominance after whole-genome duplication generates distinction in gene number and expression at the level of chromosome sets, but it remains unclear how this process may be involved in evolutionary novelty. Here we generated a chromosome-scale genome assembly of the Asian pitcher plant Nepenthes gracilis to analyse how its novel traits (dioecy and carnivorous pitcher leaves) are linked to genomic evolution. We found a decaploid karyotype and a clear indication of subgenome dominance. A male-linked and pericentromerically located region on the putative sex chromosome was identified in a recessive subgenome and was found to harbour three transcription factors involved in flower and pollen development, including a likely neofunctionalized LEAFY duplicate. Transcriptomic and syntenic analyses of carnivory-related genes suggested that the paleopolyploidization events seeded genes that subsequently formed tandem clusters in recessive subgenomes with specific expression in the digestive zone of the pitcher, where specialized cells digest prey and absorb derived nutrients. A genome-scale analysis suggested that subgenome dominance likely contributed to evolutionary innovation by permitting recessive subgenomes to diversify functions of novel tissue-specific duplicates. Our results provide insight into how polyploidy can give rise to novel traits in divergent and successful high-ploidy lineages.

Repeat-based phylogenomics shed light on unclear relationships in the monocentric genus Juncus L. (Juncaceae).

The repetitive fraction (repeatome) of eukaryotic genomes is diverse and usually fast evolving, being an important tool for clarify plant systematics. The genus Juncus L. comprises 332 species, karyotypically recognized by having holocentric chromosomes. However, four species were recently described as monocentric, yet our understanding of their genome evolution is largely masked by unclear phylogenetic relationships. Here, we reassess the current Juncus systematics using low-coverage genome skimming data of 33 taxa to construct repeats, nuclear rDNA and plastome-based phylogenetic hypothesis. Furthermore, we characterize the repeatome and chromosomal distribution of Juncus-specific centromeric repeats/CENH3 protein to test the monocentricity reach in the genus. Repeat-base phylogenies revealed topologies congruent with the rDNA tree, but not with the plastome tree. The incongruence between nuclear and plastome chloroplast dataset suggest an ancient hybridization in the divergence of Juncotypus and Tenageia sections 40 Myr ago. The phylogenetic resolution at section level was better fitted with the rDNA/repeat-based approaches, with the recognition of two monophyletic sections (Stygiopsis and Tenageia). We found specific repeatome trends for the main lineages, such as the higher abundances of TEs in the Caespitosi and Iridifolii + Ozophyllum clades. CENH3 immunostaining confirmed the monocentricity of Juncus, which can be a generic synapomorphy for the genus. The heterogeneity of the repeatomes, with high phylogenetic informativeness, identified here may be correlated with their ancient origin (56 Mya) and reveals the potential of comparative genomic analyses for understanding plant systematics and evolution.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation.

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1ß and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.

Genomic hotspots of chromosome rearrangements explain conserved synteny despite high rates of chromosome evolution in a holocentric lineage.

Holocentric organisms, unlike typical monocentric organisms, have kinetochore activity distributed along almost the whole length of the chromosome. Because of this, chromosome rearrangements through fission and fusion are more likely to become fixed in holocentric species, which may account for the extraordinary rates of chromosome evolution that many holocentric lineages exhibit. Long blocks of genome synteny have been reported in animals with holocentric chromosomes despite high rates of chromosome rearrangements. Nothing is known from plants, however, despite the fact that holocentricity appears to have played a key role in the diversification of one of the largest angiosperm genera, Carex (Cyperaceae). In the current study, we compared genomes of Carex species and a distantly related Cyperaceae species to characterize conserved and rearranged genome regions. Our analyses span divergence times ranging between 2 and 50 million years. We also compared a C. scoparia chromosome-level genome assembly with a linkage map of the same species to study rearrangements at a population level and suppression of recombination patterns. We found longer genome synteny blocks than expected under a null model of random rearrangement breakpoints, even between very distantly related species. We also found repetitive DNA to be non-randomly associated with holocentromeres and rearranged regions of the genome. The evidence of conserved synteny in sedges despite high rates of chromosome fission and fusion suggests that conserved genomic hotspots of chromosome evolution related to repetitive DNA shape the evolution of recombination, gene order and crossability in sedges. This finding may help explain why sedges are able to maintain species cohesion even in the face of high interspecific chromosome rearrangements.

New Insights on the Mechanisms of Myocardial Injury in Hypertensive Patients With COVID-19.

PURPOSE: Myocardial injury is common in hypertensive patients with 2019 coronavirus disease (COVID-19). Immune dysregulation could be associated to cardiac injury in these patients, but the underlying mechanism has not been fully elucidated. METHODS: All patients were selected prospectively from a multicenter registry of adults hospitalized with confirmed COVID-19. Cases had hypertension and myocardial injury, defined by troponin levels above the 99th percentile upper reference limit, and controls were hypertensive patients with no myocardial injury. Biomarkers and immune cell subsets were quantified and compared between the two groups. A multiple logistic regression model was used to analyze the associations of clinical and immune variables with myocardial injury. RESULTS: The sample comprised 193 patients divided into two groups: 47 cases and 146 controls. Relative to controls, cases had lower total lymphocyte count, percentage of T lymphocytes, CD8+CD38+ mean fluorescence intensity (MFI), and percentage of CD8+ human leukocyte antigen DR isotope (HLA-DR)+ CD38-cells and higher percentage of natural killer lymphocytes, natural killer group 2A (NKG2A)+ MFI, percentage of CD8+CD38+cells, CD8+HLA-DR+MFI, CD8+NKG2A+MFI, and percentage of CD8+HLA-DR-CD38+cells. On multivariate regression, the CD8+HLA-DR+MFI, CD8+CD38+MFI, and total lymphocyte count were associated significantly with myocardial injury. CONCLUSION: Our findings suggest that lymphopenia, CD8+CD38+MFI, and CD8+HLA-DR+MFI are immune biomarkers of myocardial injury in hypertensive patients with COVID-19. The immune signature described here may aid in understanding the mechanisms underlying myocardial injury in these patients. The study data might open a new window for improvement in the treatment of hypertensive patients with COVID-19 and myocardial injury.

Holocentromeres can consist of merely a few megabase-sized satellite arrays.

The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.

Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Bioactive limonoids from Carapa guianensis seeds oil and the sustainable use of its by-products.

Carapa guianensis (Andiroba, Meliaceae) is considered a multipurpose tree. In Brazil, Indigenous people have used it as insect repellent and in the treatment of various diseases. Most biological activities and popular uses are attributed to limonoids, which are highly oxygenated tetranortriterpenoids. More than 300 limonoids have been described in Meliaceae family. Limonoids from Andiroba oil have shown high anti-inflammatory and anti-allergic activities in vivo, by inhibiting platelet activating factors and many inflammatory mediators such as IL-5, IL-1ß and TNF-α. It also reduced T lymphocytes, eosinophils and mast cells. In corroboration with the wide popular use of Andiroba oil, no significant cytotoxicity or genotoxicity in vivo was reported. This oil promotes apoptosis in a gastric cancer cell line (ACP02) at high concentrations, without showing mutagenic effects, and is suggested to increase the body's nonspecific resistance and adaptive capacity to stressors, exhibit some antioxidant activity, and protect against oxidative DNA damages. Recently, new methodologies of toxicological assays have been applied. They include in chemico, in vitro, in silico and ex vivo procedures, and take place to substitute the use of laboratory animals. Andiroba by-products have been used in sustainable oil production processes and as fertilizers and soil conditioners, raw material for soap production, biodegradable surfactants and an alternative natural source of biodegradable polymer in order to reduce environmental impacts. This review reinforces the relevance of Andiroba and highlights its ability to add value to its by-products and to minimize possible risks to the health of the Amazonian population.

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

Aurelianolides from Aureliana fasciculata var. fasciculata Trigger Apoptosis With Caspase Activation in Human Leukemia Cells.

BACKGROUND/AIM: Aurelianolide A and B were identified and isolated from Aureliana fasciculata var. fasciculata leaves. Withanolides are naturally occurring C-28 steroidal lactone triterpenoids with cytotoxic and anticancer properties, among other relevant pharmacological activities. Herein we have described, for the first time, the cytotoxic effects of aurelianolides on human cancer cells. MATERIALS AND METHODS: Aurelianolide A and B were tested on human leukemia cell lines: THP-1, MOLT-4, Jurkat, K562 and K562-Lucena 1. RESULTS: For aurelianolide A, MOLT-4 had the lower IC50 (1.17 µM) and for aurelianolide B, Jurkat was the most susceptible cell line (IC50 2.25 µM). On the other hand, the multidrug resistant (MDR) cell line K562-Lucena 1 showed higher IC50 for both aurelianolides. Using 293T, a non-tumor embryonic kidney cell line, we observed an excellent selectivity index for both aurelianolides, from 2.24 (aurelianolide B in K562-Lucena 1) to 45.5 (aurelianolide A in MOLT-4). Aurelianolide A and B activated caspase 3/7 with consequent induction of apoptosis on Jurkat and K562-Lucena 1 cell lines. We have not observed induction of necrosis. CONCLUSION: Aurelianolides A and B have important cytotoxic effects on human leukemia cell lines by the activation of the caspase pathway.