Synthesis and biological evaluation of orally active anti-Trypanosoma agents.

In previous studies, we developed anti-trypanosome tubulin inhibitors with promising in vitro selectivity and activity against Human African Trypanosomiasis (HAT). However, for such agents, oral activity is crucial. This study focused on further optimizing these compounds to enhance their ligand efficiency, aiming to reduce bulkiness and hydrophobicity, which should improve solubility and, consequently, oral bioavailability. Using Trypanosoma brucei brucei cells as the parasite model and human normal kidney cells and mouse macrophage cells as the host model, we evaluated 30 new analogs synthesized through combinatorial chemistry. These analogs have fewer aromatic moieties and lower molecular weights than their predecessors. Several new analogs demonstrated IC50s in the low micromolar range, effectively inhibiting trypanosome cell growth without harming mammalian cells at the same concentration. We conducted a detailed structure-activity relationship (SAR) analysis and a docking study to assess the compounds' binding affinity to trypanosome tubulin homolog. The results revealed a correlation between binding energy and anti-Trypanosoma activity. Importantly, compound 7 displayed significant oral activity, effectively inhibiting trypanosome cell proliferation in mice.

Folic Acid and Taurine Alleviate the Impairment of Redox Status, Immunity, Rumen Microbial Composition and Fermentation of Lambs under Heat Stress.

The aim of this study was to investigate if the supplementation of folic acid and taurine can relieve the adverse effects of different levels of heat stress (HS) on growth performance, physiological indices, antioxidative capacity, immunity, rumen fermentation and microbiota. A total of 24 Dorper × Hu crossbred lambs (27.51 ± 0.96 kg) were divided into four groups: control group (C, 25 °C), moderate HS group (MHS, 35 °C), severe HS group (SHS, 40 °C), and the treatment group, under severe HS (RHS, 40 °C, 4 and 40 mg/kg BW/d coated folic acid and taurine, respectively). Results showed that, compared with Group C, HS significantly decreased the ADG of lambs (p < 0.05), and the ADG in the RHS group was markedly higher than in the MHS and SHS group (p < 0.05). HS had significant detrimental effects on physiological indices, antioxidative indices and immune status on the 4th day (p < 0.05). The physiological indices, such as RR and ST, increased significantly (p < 0.05) with the HS level and were significantly decreased in the RHS group, compared to the SHS group (p < 0.05). HS induced the significant increase of MDA, TNF-α, and IL-ß, and the decrease of T-AOC, SOD, GPx, IL-10, IL-13, IgA, IgG, and IgM (p < 0.05). However, there was a significant improvement in these indices after the supplementation of folic acid and taurine under HS. Moreover, there were a significant increase in Quinella and Succinivibrio, and an evident decrease of the genera Rikenellaceae_RC9_gut_group and Asteroleplasma under HS (p < 0.05). The LEfSe analysis showed that the genera Butyrivibrio, Eubacterium_ventriosum_group, and f_Bifidobacteriaceae were enriched in the MHS, SHS and RHS groups, respectively. Correlated analysis indicated that the genus Rikenellaceae_RC9_gut_group was positively associated with MDA, while it was negatively involved in IL-10, IgA, IgM, and SOD (p < 0.05); The genus Anaeroplasma was positively associated with the propionate and valerate, while the genus Succinivibrio was negatively involved in TNF-α (p < 0.05). In conclusion, folic acid and taurine may alleviate the adverse effects of HS on antioxidant capacity, immunomodulation, and rumen fermentation of lambs by inducing changes in the microbiome that improve animal growth performance.

Uptake and detection rate of colorectal cancer screening with colonoscopy in China: A population-based, prospective cohort study.

BACKGROUND: Colorectal cancer is the leading cause of cancer-related death worldwide. Colonoscopy is widely used as a screening test for detecting colorectal cancer in many countries. However, there is little evidence regarding the uptake and diagnostic yields of colonoscopy in population-based screening programs in countries with limited medical resources. OBJECTIVE: We reported the uptake of colonoscopy and the detection of colorectal lesions and explored related factors based on a colorectal cancer screening program in China. DESIGN: Individuals aged 45-74 years who were asymptomatic for colorectal cancer and had no history of colorectal cancer were recruited. An established risk score system was used to identify individuals at high risk for colorectal cancer, and they were subsequently recommended for colonoscopy. SETTING: A population-based, prospective cohort study was implemented in 169 communities, 14 districts of Chongqing, Southwest China. PARTICIPANTS: A total of 288,150 eligible participants were recruited from November 2013 to June 2021, and 41,315 participants were identified to be at high risk of colorectal cancer. METHODS: Generalized linear mixed model was used to explore the individual and community structural characteristics associated with uptake of colonoscopy. Additionally, the detection rate of colorectal lesions under colonoscopy screening was also reported, and their associated factors were explored. RESULTS: 7859 subjects underwent colonoscopy, with an uptake rate of 19.02 % (95 % CI 18.64 %-19.40 %). Lower uptake rates were associated with older age, lower education, more physical activity, and structural characteristics, including residing in developing areas (OR 0.73, 95 % CI 0.69-0.78), residing more than 5 km from screening hospital (5-10 km: OR 0.85, 95 % CI 0.79-0.91; >10 km: OR 0.85, 95 % CI 0.80-0.91), and not being exposed to social media publicity (OR 0.63, 95 % CI 0.53-0.75). Overall, 8 colorectal cancers (0.10 %), 423 advanced adenomas (5.38 %), 820 nonadvanced adenomas (10.43 %), and 684 hyperplastic polyps (8.70 %) were detected, with an adenoma detection rate of 15.92 %. Several factors, including older age, male, current smoking and a family history of colorectal cancer, were positively related to colorectal neoplasms. CONCLUSIONS: The uptake of colonoscopy for colorectal cancer screening was not optimal among a socioeconomically diverse high-risk population. The screening strategy should attempt to ensure equitable access to screening according to regional characteristics, and enhance the uptake of colonoscopy by recommended multifaceted interventions, which focus on individuals with poor compliance, select a closer screening hospital, and strengthen social media publicity at the structural level.

Two Energetic Framework Materials Based on DNM-TNBI as Host Molecule: Effectively Coordinated by Different Cations.

With the demand of develop outstanding-performance energetic materials, 1-(dinitromethyl)-4,4',5,5'-tetranitro-1H,1'H-2,2'-biimidazole (DNM-TNBI) emerged as a great contender (D: 9102 m ⋅ s-1; P: 37.6 GPa). However, the relatively poor thermal stability (Td: 142 °C) limits its practical application. In this study, DNM-TNBI as a host molecule to synthesize two new energetic open-framework materials by effectively coordinated with different cations. Their supramolecular structures were investigated and indicated that [DNM-TNBI2 -][2NH4 +] and [DNM-TNBI2 -][2K+] can be classified as a new energetic hydrogen-bonded ammonium framework (EHAF) and an energetic metal organic framework (EMOF). Meanwhile, their thermal stabilities are higher than that of DNM-TNBI and have satisfactory detonation performance ([DNM-TNBI2 -][2NH4 +], D: 8050 m ⋅ s-1, P: 26.4 GPa; [DNM-TNBI2 -][2K+], D: 8301 m ⋅ s-1, P: 30.8 GPa).

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1's functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

Telomere maintenance in African trypanosomes.

Telomere maintenance is essential for genome integrity and chromosome stability in eukaryotic cells harboring linear chromosomes, as telomere forms a specialized structure to mask the natural chromosome ends from DNA damage repair machineries and to prevent nucleolytic degradation of the telomeric DNA. In Trypanosoma brucei and several other microbial pathogens, virulence genes involved in antigenic variation, a key pathogenesis mechanism essential for host immune evasion and long-term infections, are located at subtelomeres, and expression and switching of these major surface antigens are regulated by telomere proteins and the telomere structure. Therefore, understanding telomere maintenance mechanisms and how these pathogens achieve a balance between stability and plasticity at telomere/subtelomere will help develop better means to eradicate human diseases caused by these pathogens. Telomere replication faces several challenges, and the "end replication problem" is a key obstacle that can cause progressive telomere shortening in proliferating cells. To overcome this challenge, most eukaryotes use telomerase to extend the G-rich telomere strand. In addition, a number of telomere proteins use sophisticated mechanisms to coordinate the telomerase-mediated de novo telomere G-strand synthesis and the telomere C-strand fill-in, which has been extensively studied in mammalian cells. However, we recently discovered that trypanosomes lack many telomere proteins identified in its mammalian host that are critical for telomere end processing. Rather, T. brucei uses a unique DNA polymerase, PolIE that belongs to the DNA polymerase A family (E. coli DNA PolI family), to coordinate the telomere G- and C-strand syntheses. In this review, I will first briefly summarize current understanding of telomere end processing in mammals. Subsequently, I will describe PolIE-mediated coordination of telomere G- and C-strand synthesis in T. brucei and implication of this recent discovery.

Exploring the digesta- and mucosa-associated microbial community dynamics in the rumen and hindgut of goats from birth to adult.

Recently, the relationship between the goat host and its gastrointestinal microbiome has emerged as a hallmark of host-microbiota symbiosis, which was indispensable for the proper physiological function that convert the plant biomass to livestock products. However, little integrative information about the establishment of gastrointestinal microflora in goats exists. Herein, we characterized the colonizing process of the bacterial community in the digesta and mucosa of the rumen, cecum, and colon of the cashmere goat from birth to adulthood to compare its spatiotemporal difference via 16S rRNA gene sequencing. A total of 1,003 genera belonging to 43 phyla were identified. Principal coordinate analysis unveiled the similarity of microbial community between or within each age group increased and gradually developed toward mature whatever in digesta or mucosa. In the rumen, the composition of the bacterial community in digesta differed significantly from in mucosa across age groups; whereas in the hindgut, there was a high similarity of bacterial composition between the in digesta and mucosa in each age group before weaning, while the bacterial community structure differed markedly between these two types of samples after weaning. Taxonomic analysis indicated that 25 and 21 core genera coexisted in digesta and mucosa of the rumen and hindgut, respectively; but their abundances differed considerably by GIT region and/or age. In digesta, as goats aged, a lower abundance of Bacillus was observed with higher abundances of Prevotella 1 and Rikenellaceae RC9 in the rumen; while in the hindgut, the genera Escherichia-Shigella, Variovorax, and Stenotrophomonas decreased and Ruminococcaceae UCG-005, Ruminococcaceae UCG-010, and Alistipes increased with age increased. In mucosa, the rumen showed microbial dynamics with increases of Butyrivibrio 2 and Prevotellaceae UCG-001 and decreases of unclassified_f_Pasteurellaceae; while the genera Treponema 2 and Ruminococcaceae UCG-010 increased and Escherichia-Shigella decreased in the hindgut as goats aged. These results shed light on the colonization process of microbiota in the rumen and hindgut, which mainly include the initial, transit, and mature phases. Furthermore, there is a significant difference in the microbial composition between in digesta and mucosa, and both these exhibit a considerable spatiotemporal specificity.

Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei.

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression.

Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level expression.

Dietary Nutritional Level Affects Intestinal Microbiota and Health of Goats.

The intestine is a complex micro-ecosystem, and its stability determines the health of animals. Different dietary nutritional levels affect the intestinal microbiota and health. In this study, the nutritional levels of energy and protein in the diet of goats were changed, and the body weight was measured every 15 days. In the late feeding period, 16 S rRNA sequencing technology was used to detect the content of microorganisms. A meteorological chromatograph was used to detect volatile fatty acids in the cecum and colon of goats. In the feeding stage, reducing the nutritional level of the diet significantly reduced the weight of the lamb (p < 0.05). In the cecum, the abundance of potentially harmful bacteria, such as Sphingomonas, Marvinbryantia, and Eisenbergiella, were significantly enriched in goats fed with the standard nutritional level diets (p < 0.05). Additionally, the contents of acetate (p = 0.037) and total VFAs (p = 0.041) increased. In the colon, the abundance of SCFAs-producing bacteria, such as Ruminococcaceae, Christensenellaceae, and Papillibacter, decreased as the nutritional level in the diet increased (p < 0.05). In conclusion, the increase in nutritional level could affect the growth performance and composition of intestinal microbiota.

Unlocking phenotypic plasticity provides novel insights for immunity and personalized therapy in lung adenocarcinoma.

Background: Unlocking phenotype plasticity (UPP) has been shown to have an essential role in the mechanism of tumor development and therapeutic response. However, the clinical significance of unlocking phenotypic plasticity in patients with lung adenocarcinoma is unclear. This study aimed to explore the roles of unlocking phenotypic plasticity in immune status, prognosis, and treatment in patients with lung adenocarcinoma (LUAD). Methods: Differentially expressed genes (DEGs) and clinical information of UPP were selected from the cancer genome atlas (TCGA) database, and the GO, KEGG enrichment analyses were performed. The independent prognostic genes were determined by univariate and multivariate Cox regression, and the UPP signature score was constructed. Patients with LUAD were divided into high- and low-risk groups according to the median of score, and the immunocytes and immune function, the gene mutation, and drug sensitivities between the two groups were analyzed. Finally, the results were validated in the GEO database. Results: Thirty-nine significantly DEGs were determined. Enrichment analysis showed that UPP-related genes were related to protein polysaccharides and drug resistance. The prognostic results showed that the survival of patients in the high-risk group was poorer than that in the low-risk group (p < 0.001). In the high- and low-risk groups, single nucleotide polymorphism (SNP) and C > T are the most common dissent mutations. The contents of immune cells were significantly different between high- and low-risk groups. And the immune functions were also significantly different, indicating that UPP affects the immunity in LUAD. The results from TCGA were validated in the GEO. Conclusion: Our research has proposed a new and reliable prognosis indicator to predict the overall survival. Evaluation of the UPP could help the clinician to predict therapeutic responses and make individualized treatment plans in patients with LUAD.

Identification of Prazosin as a Potential Flagellum Attachment Zone 1(FAZ1) Inhibitor for the Treatment of Human African Trypanosomiasis.

Human African trypanosomiasis (HAT) remains a health threat to sub-Saharan Africa. The current treatments suffer from drug resistance and life-threatening side effects, making drug discovery for HAT still important. A high-throughput screening of the library of pharmaceutically active compounds identified prazosin, an α-adrenoceptor antagonist, that showed selective activity toward Trypanosoma brucei brucei. Furthermore, a series of prazosin analogues were examined, and overall, the new analogues had improved activity and selectivity. To elucidate the binding partner, a biotin-conjugated probe was synthesized, and a protein pulldown assay combined with a proteomic analysis identified the flagellum attachment zone 1 (FAZ1) filament as an interacting partner. Additionally, prazosin treatment resulted in dysfunction of the flagellum of trypanosome cells, which is indicative of a FAZ1 irregularity. We also examined the drug distribution by utilizing immunofluorescence with a designed fluorescent analogue that showed partial colocalization with FAZ1. With the activity of the prazosin analogues, a structure-activity relationship (SAR) was summarized for future lead optimization. Our findings provide a new group of FAZ1 inhibitors as novel antitrypanosomal agents.

pH-Responsive hyaluronic acid-enveloped ZIF-8 nanoparticles for anti-atherosclerosis therapy.

Nanomedicines represent new promising strategies for treating atherosclerosis (AS), because they enhance drug bioavailability and have lower side effects. Nevertheless, nanomedicines have several challenges with these advantages, including a limited circulation life, lack of precise targeting, and insufficient control of drug release. Accordingly, the development of drug delivery systems (DDSs) with abilities to enhance the payload delivery to the AS plaque lesion and to control drug release can boost the therapeutic efficacy and safety for AS treatment. Herein, we employed a one-step self-assembly approach for effectively encapsulating the anti-AS drug simvastatin (SIM) in zeolitic imidazolate framework-8 (ZIF-8) (SIM/ZIF-8), and then coated it with hyaluronic acid (HA) to fabricate the SIM/ZIF-8@HA nanoplatform. The resulting nanoplatform could efficiently accumulate in plaque regions through the specific recognition between HA and CD44. Meanwhile, the acid environment breaks down ZIF-8 to release SIM. The in vitro and in vivo experiments demonstrated that SIM/ZIF-8@HA could inhibit the proliferation of smooth muscle cells and have good biocompatibility. Moreover, SIM/ZIF-8@HA can effectively suppress the development of AS plaques without any considerable side effects in mice treatments. The findings revealed that SIM/ZIF-8@HA may be a promising nanomedicine for safe and efficient anti-AS applications.

Synthesis and biological evaluation of imidamide analogs as selective anti-trypanosomal agents.

Human African trypanosomiasis is caused by a protozoan parasite Trypanosoma brucei majorly infecting people living in sub-Saharan Africa. Current limited available treatments suffer from drug resistance, severe adverse effects, low efficacy, and costly administrative procedures in African countries with limited medical resources. Therefore, there is always a perpetual demand for advanced drug development and invention of new strategies to combat the disease. Previous work in our lab generated a library of sulfonamide analogs as selective tubulin inhibitors, based on the structural difference between mammalian and trypanosome tubulin proteins. Further lead derivatization was performed in the current study and generated 25 potential drug candidates to improve the drug efficacy and uptake by selectively targeting the parasite's P2 membrane transporter protein with imidamide moiety. One of the newly synthesized analogs, compound 25 with a di-imidamide moiety, has shown greater potency with an IC50 of 1 nM to selectively inhibit the growth of trypanosome cells without affecting the viability of mammalian cells. Western blot analyses reveal that the compound suppressed tubulin polymerization in T. brucei cells. A detailed structure-activity relationship (SAR) was summarized that will be used to guide future lead optimization.

Assessment of Sexual Outcomes in Patients Undergoing Thulium Laser Prostate Surgery for Management of Benign Prostate Hyperplasia: A Systematic Review and Meta-analysis.

BACKGROUND: Thulium laser (Tm:YAG) prostate surgery is a safe and effective procedure with low morbidity and comparable clinical outcomes to those of transurethral resection of the prostate (TURP). However, the sexual function outcomes (erectile and ejaculatory function) have been scarcely studied. AIM: We aimed to assess the impact of Tm:YAG prostate surgery on sexual outcomes (erectile and ejaculatory function) and compare them with those patients undergoing TURP. MATERIAL AND METHODS: We searched digital databases like PUBMED, SCOPUS, CENTRAL and EMBASE using relevant keywords to identify comparative studies on TURP and non-comparative studies on Tm:YAG prostate surgery that assessed sexual outcomes. We performed qualitative and quantitative analyses with the extracted data. We carried out a meta-analysis to compare postoperative International Index of Erectile Function (IIEF-5) scores and incidences of retrograde ejaculation (RE) in patients undergoing either Tm:YAG or TURP. The pre-operative and post-operative IIEF-5 scores were pooled to estimate overall scores. RESULTS: We included 5 comparative and 8 non-comparative studies in this review. We found the postoperative IIEF-5 score improvements to be significantly higher in the Tm:YAG prostate surgery group than in the TURP group with a significant mean difference (MD) of 0.45 (95% CI, 0.18 to 0.72; P = .001). We found no significant associations between the procedures. The pooled OR for the association of RE was estimated at 0.90 (95% CI, 0.50 to 1.60; P = .71; I2 = 0%). CONCLUSION: Tm:YAG prostate surgery improves erectile function more than TURP, according to our findings. Tm:YAG prostate aided surgery also outperforms TURP in terms of preserving sexual function following surgery.However, We found similar or no difference in incidence of RE between Tm:YAG prostate surgery and TURP. Bibo L, Hao L, Pang K, et al. Assessment of Sexual Outcomes in Patients Undergoing Thulium Laser Prostate Surgery for Management of Benign Prostate Hyperplasia: A Systematic Review and Meta-analysis. Sex Med 2022;10:100483.

POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.

Trypanosoma brucei causes human African trypanosomiasis and sequentially expresses distinct VSGs, its major surface antigen, to achieve host immune evasion. VSGs are monoallelically expressed from subtelomeric loci, and telomere proteins regulate VSG monoallelic expression and VSG switching. T. brucei telomerase is essential for telomere maintenance, but no regulators of telomerase have been identified. T. brucei appears to lack OB fold-containing telomere-specific ssDNA binding factors that are critical for coordinating telomere G- and C-strand syntheses in higher eukaryotes. We identify POLIE as a telomere protein essential for telomere integrity. POLIE-depleted cells have more frequent VSG gene conversion-mediated VSG switching and an increased amount of telomeric circles (T-circles), indicating that POLIE suppresses DNA recombination at the telomere/subtelomere. POLIE-depletion elongates telomere 3' overhangs dramatically, indicating that POLIE is essential for coordinating DNA syntheses of the two telomere strands. POLIE depletion increases the level of telomerase-dependent telomere G-strand extension, identifying POLIE as the first T. brucei telomere protein that suppresses telomerase. Furthermore, depletion of POLIE results in an elevated telomeric C-circle level, suggesting that the telomere C-strand experiences replication stress and that POLIE may promote telomere C-strand synthesis. Therefore, T. brucei uses a novel mechanism to coordinate the telomere G- and C-strand DNA syntheses.

In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei.

Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.

Regulation of Antigenic Variation by Trypanosoma brucei Telomere Proteins Depends on Their Unique DNA Binding Activities.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoprotein (VSG), to evade the host immune response. Such antigenic variation is a key pathogenesis mechanism that enables T. brucei to establish long-term infections. VSG is expressed exclusively from subtelomere loci in a strictly monoallelic manner, and DNA recombination is an important VSG switching pathway. The integrity of telomere and subtelomere structure, maintained by multiple telomere proteins, is essential for T. brucei viability and for regulating the monoallelic VSG expression and VSG switching. Here we will focus on T. brucei TRF and RAP1, two telomere proteins with unique nucleic acid binding activities, and summarize their functions in telomere integrity and stability, VSG switching, and monoallelic VSG expression. Targeting the unique features of TbTRF and TbRAP1's nucleic acid binding activities to perturb the integrity of telomere structure and disrupt VSG monoallelic expression may serve as potential therapeutic strategy against T. brucei.

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion.

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial-mesenchymal transition (EMT) inducer, by directly binding to TBXT 3'-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.

Role of NRP1 in Bladder Cancer Pathogenesis and Progression.

Bladder urothelial carcinoma (BC) is a fatal invasive malignancy and the most common malignancy of the urinary system. In the current study, we investigated the function and mechanisms of Neuropilin-1 (NRP1), the co-receptor for vascular endothelial growth factor, in BC pathogenesis and progression. The expression of NRP1 was evaluated using data extracted from GEO and HPA databases and examined in BC cell lines. The effect on proliferation, apoptosis, angiogenesis, migration, and invasion of BC cells were validated after NRP1 knockdown. After identifying differentially expressed genes (DEGs) induced by NRP1 silencing, GO/KEGG and IPA® bioinformatics analyses were performed and specific predicted pathways and targets were confirmed in vitro. Additionally, the co-expressed genes and ceRNA network were predicted using data downloaded from CCLE and TCGA databases, respectively. High expression of NRP1 was observed in BC tissues and cells. NRP1 knockdown promoted apoptosis and suppressed proliferation, angiogenesis, migration, and invasion of BC cells. Additionally, after NRP1 silencing the activity of MAPK signaling and molecular mechanisms of cancer pathways were predicted by KEGG and IPA® pathway analysis and validated using western blot in BC cells. NRP1 knockdown also affected various biological functions, including antiviral response, immune response, cell cycle, proliferation and migration of cells, and neovascularisation. Furthermore, the main upstream molecule of the DEGs induced by NRP1 knockdown may be NUPR1, and NRP1 was also the downstream target of NUPR1 and essential for regulation of FOXP3 expression to activate neovascularisation. DCBLD2 was positively regulated by NRP1, and PPAR signaling was significantly associated with low NRP1 expression. We also found that NRP1 was a predicted target of miR-204, miR-143, miR-145, and miR-195 in BC development. Our data provide evidence for the biological function and molecular aetiology of NRP1 in BC and for the first time demonstrated an association between NRP1 and NUPR1, FOXP3, and DCBLD2. Specifically, downregulation of NRP1 contributes to BC progression, which is associated with activation of MAPK signaling and molecular mechanisms involved in cancer pathways. Therefore, NRP1 may serve as a target for new therapeutic strategies to treat BC and other cancers.