Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis.

We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent in vitro activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, FM09h, was highly active with IC50 of 0.3 µM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of FM09h remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of FM09h (10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).

Genome screening, reporting, and genetic counseling for healthy populations.

Rapid advancements of genome sequencing (GS) technologies have enhanced our understanding of the relationship between genes and human disease. To incorporate genomic information into the practice of medicine, new processes for the analysis, reporting, and communication of GS data are needed. Blood samples were collected from adults with a PCR-confirmed SARS-CoV-2 (COVID-19) diagnosis (target N = 1500). GS was performed. Data were filtered and analyzed using custom pipelines and gene panels. We developed unique patient-facing materials, including an online intake survey, group counseling presentation, and consultation letters in addition to a comprehensive GS report. The final report includes results generated from GS data: (1) monogenic disease risks; (2) carrier status; (3) pharmacogenomic variants; (4) polygenic risk scores for common conditions; (5) HLA genotype; (6) genetic ancestry; (7) blood group; and, (8) COVID-19 viral lineage. Participants complete pre-test genetic counseling and confirm preferences for secondary findings before receiving results. Counseling and referrals are initiated for clinically significant findings. We developed a genetic counseling, reporting, and return of results framework that integrates GS information across multiple areas of human health, presenting possibilities for the clinical application of comprehensive GS data in healthy individuals.

In Vivo Reversal of P-Glycoprotein-Mediated Drug Resistance in a Breast Cancer Xenograft and in Leukemia Models Using a Novel, Potent, and Nontoxic Epicatechin EC31.

The modulation of P-glycoprotein (P-gp, ABCB1) can reverse multidrug resistance (MDR) and potentiate the efficacy of anticancer drugs. Tea polyphenols, such as epigallocatechin gallate (EGCG), have low P-gp-modulating activity, with an EC50 over 10 µM. In this study, we optimized a series of tea polyphenol derivatives and demonstrated that epicatechin EC31 was a potent and nontoxic P-gp inhibitor. Its EC50 for reversing paclitaxel, doxorubicin, and vincristine resistance in three P-gp-overexpressing cell lines ranged from 37 to 249 nM. Mechanistic studies revealed that EC31 restored intracellular drug accumulation by inhibiting P-gp-mediated drug efflux. It did not downregulate the plasma membrane P-gp level nor inhibit P-gp ATPase. It was not a transport substrate of P-gp. A pharmacokinetic study revealed that the intraperitoneal administration of 30 mg/kg of EC31 could achieve a plasma concentration above its in vitro EC50 (94 nM) for more than 18 h. It did not affect the pharmacokinetic profile of coadministered paclitaxel. In the xenograft model of the P-gp-overexpressing LCC6MDR cell line, EC31 reversed P-gp-mediated paclitaxel resistance and inhibited tumor growth by 27.4 to 36.1% (p < 0.001). Moreover, it also increased the intratumor paclitaxel level in the LCC6MDR xenograft by 6 fold (p < 0.001). In both murine leukemia P388ADR and human leukemia K562/P-gp mice models, the cotreatment of EC31 and doxorubicin significantly prolonged the survival of the mice (p < 0.001 and p < 0.01) as compared to the doxorubicin alone group, respectively. Our results suggested that EC31 was a promising candidate for further investigation on combination therapy for treating P-gp-overexpressing cancers.

Workplace empowerment, psychological empowerment and work-related wellbeing in southeast Asian employees: a cross-sectional survey.

There is a growing body of research examining the nature and correlates of salutogenic factors in the workplace and employee wellbeing, and the role of empowerment therewithin. A paucity of research has distinguished between structural and psychological forms of empowerment in the workplace and examined how they independently and collectively relate to employee wellbeing. Much of the existing research has examined such considerations in western samples, with limited exploration of eastern working populations. The aim of this study is to investigate the association between structural empowerment (SE) and employee self-reported work-related wellbeing (operationalized as psychological wellbeing and job satisfaction), and the postulated mediating role of psychological empowerment (PE). With a sample of 324 southeast Asian employees from a single organization, this study used a cross-sectional case study design using self-report measures to examine the relationships between SE, PE and employee job satisfaction and psychological wellbeing. PE was postulated to mediate the relationship between SE and work-related wellbeing outcomes of job satisfaction and psychological wellbeing. Mediation analyses revealed that SE was positively associated with PE which, in turn, completely mediated the positive relationships between SE, and job satisfaction and psychological wellbeing. The results of this study contribute empirically and practically in the following ways: (i) it expands upon previous research on employee empowerment, (ii) contributes to the developing field of positive occupational health psychology and (iii) highlights the crucial role of organizations in creating sources of SE and PE.

In this study, we explore two forms of empowerment at work and investigate their relationship with employee wellbeing. Workplace empowerment (WE) comes from organizational structures, policies and practices that ensure power and resources are shared across employees, and, in turn, facilitate their accomplishment and development at work. Psychological empowerment (PE) derives from the meaning, competence, perceived control and influence employees feel they have at work and on work outcomes. Limited research has investigated whether WE and PE interact with each other, and whether this, in turn, relates to employees' motivation, health and wellbeing. This is the aim of this study. We collected data from 334 employees in a large organization in southeast Asia using an online survey. Both WE and PE were observed to be important factors in relationship to employees' satisfaction at work and psychological wellbeing. We found the relationship between WE and employees' self-reported job satisfaction and psychological wellbeing was facilitated through their PE. This study contributes to the small, but growing, body of literature examining empowerment in the workplace. Our findings highlight the importance of workplace health promotion initiatives to ensure employee empowerment is cultivated both within individuals and their work environments.

Discovery of a Flavonoid FM04 as a Potent Inhibitor to Reverse P-Glycoprotein-Mediated Drug Resistance in Xenografts and Improve Oral Bioavailability of Paclitaxel.

Biotransformation of flavonoid dimer FD18 resulted in an active metabolite FM04. It was more druggable because of its improved physicochemical properties. FM04 (EC50 = 83 nM) was 1.8-fold more potent than FD18 in reversing P-glycoprotein (P-gp)-mediated paclitaxel (PTX) resistance in vitro. Similar to FD18, FM04 chemosensitized LCC6MDR cells towards multiple anticancer drugs by inhibiting the transport activity of P-gp and restoring intracellular drug levels. It stimulated the P-gp ATPase by 3.3-fold at 100 µM. Different from FD18, FM04 itself was not a transport substrate of P-gp and presumably, it cannot work as a competitive inhibitor. In the human melanoma MDA435/LCC6MDR xenograft, the co-administration of FM04 (28 mg/kg, I.P.) with PTX (12 mg/kg, I.V.) directly modulated P-gp-mediated PTX resistance and caused a 56% (*, p < 0.05) reduction in tumor volume without toxicity or animal death. When FM04 was administered orally at 45 mg/kg as a dual inhibitor of P-gp/CYP2C8 or 3A4 enzymes in the intestine, it increased the intestinal absorption of PTX from 0.2% to 14% in mice and caused about 57- to 66-fold improvement of AUC as compared to a single oral dose of PTX. Oral co-administration of FM04 (45 mg/kg) with PTX (40, 60 or 70 mg/kg) suppressed the human melanoma MDA435/LCC6 tumor growth with at least a 73% (***, p < 0.001) reduction in tumor volume without serious toxicity. Therefore, FM04 can be developed into a novel combination chemotherapy to treat cancer by directly targeting the P-gp overexpressed tumors or potentiating the oral bioavailability of P-gp substrate drugs.

Characterization of a Potent, Selective, and Safe Inhibitor, Ac15(Az8)2, in Reversing Multidrug Resistance Mediated by Breast Cancer Resistance Protein (BCRP/ABCG2).

Overexpression of breast cancer resistance transporter (BCRP/ABCG2) in cancers has been explained for the failure of chemotherapy in clinic. Inhibition of the transport activity of BCRP during chemotherapy should reverse multidrug resistance. In this study, a triazole-bridged flavonoid dimer Ac15(Az8)2 was identified as a potent, nontoxic, and selective BCRP inhibitor. Using BCRP-overexpressing cell lines, its EC50 for reversing BCRP-mediated topotecan resistance was 3 nM in MCF7/MX100 and 72 nM in S1M180 in vitro. Mechanistic studies revealed that Ac15(Az8)2 restored intracellular drug accumulation by inhibiting BCRP-ATPase activity and drug efflux. It did not down-regulate the cell surface BCRP level to enhance drug retention. It was not a transport substrate of BCRP and showed a non-competitive relationship with DOX in binding to BCRP. A pharmacokinetic study revealed that I.P. administration of 45 mg/kg of Ac15(Az8)2 resulted in plasma concentration above its EC50 (72 nM) for longer than 24 h. It increased the AUC of topotecan by 2-fold. In an in vivo model of BCRP-overexpressing S1M180 xenograft in Balb/c nude mice, it significantly reversed BCRP-mediated topotecan resistance and inhibited tumor growth by 40% with no serious body weight loss or death incidence. Moreover, it also increased the topotecan level in the S1M180 xenograft by 2-fold. Our results suggest that Ac15(Az8)2 is a promising candidate for further investigation into combination therapy for treating BCRP-overexpressing cancers.

LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate.

BACKGROUND & AIMS: In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. METHODS: We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. RESULTS: Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. CONCLUSIONS: LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

SALL4 promotes tumor progression in breast cancer by targeting EMT.

Sal-like protein 4 (SALL4) is overexpressed in breast cancer and might contribute to breast cancer progression, but the molecular mechanism remains unknown. Here, we found that within a group of 371 ethnic Chinese breast cancer patients, SALL4 was associated with lower grade (P = .002) and progesterone receptor positivity (P = .004) for overall cases; lower Ki67 (P = .045) and high vimentin (P = .007) for luminal cases. Patients with high SALL4 expression in lymph node metastasis showed a significantly worse survival than those with low expression. Knockout of SALL4 in a triple-negative breast cancer cell line MDA-MB-231-Red-FLuc-GFP led to suppressed ability in proliferation, clonogenic formation, migration, and mammosphere formation in vitro, tumorigenicity and lung colonization in vivo. On the other hand, overexpression of SALL4 enhanced migration and mammosphere formation in vitro and tumorigenicity in vivo. Mechanistically, there was a positive correlation between SALL4 expression and mesenchymal markers including Zinc finger E-box binding homeobox 1 (ZEB1), vimentin, Slug, and Snail in vivo. Chromatin immunoprecipitation experiment indicated that SALL4 can bind to the promoter region of vimentin (-778 to -550 bp). Taken together, we hypothesize that SALL4 promotes tumor progression in breast cancer by inducing the mesenchymal markers like vimentin through directly binding to its promoter. Increased SALL4 level in metastatic lymph node relative to the primary site is an important poor survival marker in breast cancer.

Synthesis and in-vitro anti-leishmanial activity of (4-arylpiperazin-1-yl)(1-(thiophen-2-yl)-9H-pyrido[3,4-b]indol-3-yl)methanone derivatives.

In the present study, we have reported synthesis and biological evaluation of a series of fifteen 1-(thiophen-2-yl)-9H-pyrido[3,4-b]indole derivatives against both promastigotes and amastigotes of Leishmania parasites responsible for visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis. Among these reported analogues, compounds 7b, 7c, 7f, 7g, 7i, 7j, 7m, 7o displayed potent activity (15.55, 7.70, 7.00, 3.80, 14.10, 9.25, 3.10, 4.85µM, respectively) against L. donovani promastigotes than standard drugs miltefosine (15.70µM) and pentamidine (32.70µM) with good selectivity index. In further, in-vitro evaluation against amastigote forms, two compounds 7g (8.80µM) and 7i (7.50µM) showed significant inhibition of L. donovani amastigotes. Standard drug amphotericin B is also used as control to compare inhibition potency of compounds against both promastigote (0.24µM) and amastigote (0.05µM) forms.

A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo.

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 µM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 µM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 µM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

Optimization of permethyl ningalin B analogs as P-glycoprotein inhibitors.

In the present study, a total of 9 novel permethyl ningalin B analogs have been synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Among these derivatives, compound 12 with dimethoxy groups at rings A and B and tri-substitution at ring C with ortho-methoxyethylmorpholine, meta-bromo and para-benzyloxy groups displays the most potent P-gp modulating activity with EC50 of 423 nM to reverse paclitaxel resistance. It is non-toxic towards L929 fibroblast with IC50 greater than 100 µM and with selective index greater than 236. Its mechanism to reverse P-gp mediated drug resistance is by virtue of inhibiting transport activity of P-gp, restoring intracellular drug accumulation and eventually chemosensitizing the cancer cells to anticancer drug again. Moreover, compound 12 showed better solubility (405 ng/mL) than hit compound 1 in phosphate buffer (pH 4.0). In summary, our study demonstrates that permethyl ningalin B derivative 12 is non-toxic and efficient P-gp inhibitor that is a potential candidate to be used clinically to reverse P-gp mediated cancer drug resistance.

In vitro and in vivo efficacy of novel flavonoid dimers against cutaneous leishmaniasis.

Treatment of leishmaniasis by chemotherapy remains a challenge because of limited efficacy, toxic side effects, and drug resistance. We previously reported that synthetic flavonoid dimers have potent antipromastigote and antiamastigote activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Here, we further investigate their leishmanicidal activities against cutaneous Leishmania species. One of the flavonoid dimers (compound 39) has marked antipromastigote (50% inhibitory concentrations [IC50s], 0.19 to 0.69 µM) and antiamastigote (IC50s, 0.17 to 2.2 µM) activities toward different species of Leishmania that cause cutaneous leishmaniasis, including Leishmania amazonensis, Leishmania braziliensis, Leishmania tropica, and Leishmania major. Compound 39 is not toxic to peritoneal elicited macrophages, with IC50 values higher than 88 µM. In the mouse model of cutaneous leishmaniasis induced by subcutaneous inoculation of L. amazonensis in mouse footpads, intralesional administration of 2.5 mg/kg of body weight of compound 39.HCl can reduce footpad thickness by 36%, compared with that of controls values. The amastigote load in the lesions was reduced 20-fold. The present study suggests that flavonoid dimer 39 represents a new class of safe and effective leishmanicidal agent against visceral and cutaneous leishmaniasis.

Modification of marine natural product ningalin B and SAR study lead to potent P-glycoprotein inhibitors.

In this study, new marine ningalin B analogues containing a piperazine or a benzoloxy group at ring C have been synthesized and evaluated on their P-gp modulating activity in human breast cancer and leukemia cell lines. Their structure-activity relationship was preliminarily studied. Compounds 19 and 20 are potent P-gp inhibitors. These two synthetic analogues of permethyl ningalin B may be potentially used as effective modulators of P-gp-mediated drug resistance in cancer cells.

A Targeted and Protease-Activated Genetically Encoded Melittin-Containing Particle for the Treatment of Cutaneous and Visceral Leishmaniasis.

Intracellular infections are difficult to treat, as pathogens can take advantage of intracellular hiding, evade the immune system, and persist and multiply in host cells. One such intracellular parasite, Leishmania, is the causative agent of leishmaniasis, a neglected tropical disease (NTD), which disproportionately affects the world's most economically disadvantaged. Existing treatments have relied mostly on chemotherapeutic compounds that are becoming increasingly ineffective due to drug resistance, while the development of new therapeutics has been challenging due to the variety of clinical manifestations caused by different Leishmania species. The antimicrobial peptide melittin has been shown to be effective in vitro against a broad spectrum of Leishmania, including species that cause the most common form, cutaneous leishmaniasis, and the most deadly, visceral leishmaniasis. However, melittin's high hemolytic and cytotoxic activity toward host cells has limited its potential for clinical translation. Herein, we report a design strategy for producing a melittin-containing antileishmanial agent that not only enhances melittin's leishmanicidal potency but also abrogates its hemolytic and cytotoxic activity. This therapeutic construct can be directly produced in bacteria, significantly reducing its production cost critical for a NTD therapeutic. The designed melittin-containing fusion crystal incorporates a bioresponsive cathepsin linker that enables it to specifically release melittin in the phagolysosome of infected macrophages. Significantly, this targeted approach has been demonstrated to be efficacious in treating macrophages infected with L. amazonensis and L. donovani in cell-based models and in the corresponding cutaneous and visceral mouse models.

Fexinidazole optimization: enhancing anti-leishmanial profile, metabolic stability and hERG safety.

The lack of adequate anti-leishmanial therapies has led to the continued suffering of millions of people from developing nations. Moreover, optimism for a therapeutic intervention by fexinidazole was dashed due to the inability to maintain cures and control unwanted side effects. To solve these shortcomings, the structural elements of fexinidazole responsible for anti-leishmanial activity and toxicities were explored. Accordingly, a systematic analog design approach was taken for the synthesis of 24 novel analogs. We established the structural features important for activity and identified modifications that improved the hERG receptor safety and liver microsomal metabolic stability. Compared to fexinidazole, the S-configured imidazolooxazole analog 51 exhibited 25-fold greater potency against miltefosine resistant L. donovani amastigotes, greater metabolic stability and little hERG receptor inhibition. Replacement of the toxicophore nitro group for a cyano group resulted in a complete loss of anti-leishmanial activity. The SAR findings should be useful in the further development of this important class of anti-leishmanial agents.

Repurposing cetylpyridinium chloride and domiphen bromide as phosphoethanolamine transferase inhibitor to combat colistin-resistant Enterobacterales.

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales.

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis.

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

Identification of Binding Sites in the Nucleotide-Binding Domain of P-Glycoprotein for a Potent and Nontoxic Modulator, the Amine-Containing Monomeric Flavonoid FM04.

We have previously discovered an amine-containing flavonoid monomer FM04 as a potent P-glycoprotein (P-gp) inhibitor (EC50 = 83 nM). Here, a series of photoactive FM04 analogues were synthesized and used together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the FM04-binding sites on P-gp. Point mutations around the photo-crosslinked sites were made for verification. Together with the results from mutational studies, molecular docking, and molecular dynamics simulations, it was found that FM04 can interact with Q1193 and I1115 in the nucleotide-binding domain 2 (NBD2) of human P-gp. It was proposed that FM04 can inhibit P-gp in 2 novel mechanisms. FM04 can either bind to (1) Q1193, followed by interacting with the functionally critical residues H1195 and T1226 or (2) I1115 (a functionally critical residue itself), disrupting the R262-Q1081-Q1118 interaction pocket and uncoupling ICL2-NBD2 interaction and thereby inhibiting P-gp. Q1118 would subsequently be pushed to the ATP-binding site and stimulate ATPase.

Public knowledge of SARS-CoV-2 serological and viral lineage laboratory testing and result interpretation: A GENCOV study cross-sectional survey.

OBJECTIVES: Concepts related to SARS-CoV-2 laboratory testing and result interpretation can be challenging to understand. A cross-sectional survey of COVID-19 positive adults residing in Ontario, Canada was conducted to explore how well people understand SARS-CoV-2 laboratory tests and their associated results. DESIGN AND METHODS: Participants were recruited through fliers or by prospective recruitment of outpatients and hospitalized inpatients with COVID-19. Enrolled participants included consenting adults with a positive SARS-CoV-2 polymerase chain reaction test result. An 11-item questionnaire was developed by researchers, nurses, and physicians in the study team and was administered online between April 2021 to May 2022 upon enrolment into the study. RESULTS: Responses were obtained from 940 of 1106 eligible participants (85% participation rate). Most respondents understood 1) that antibody results should not influence adherence to social distancing measures (n = 602/888, 68%), 2) asymptomatic SARS-CoV-2 infection following test positivity (n = 698/888, 79%), 3) serological test sensitivity in relation to post-infection timeline (n = 540/891, 61%), and 4) limitations of experts' knowledge related to SARS-CoV-2 serology (n = 693/887, 78%). Conversely, respondents demonstrated challenges understanding 1) conflicting molecular and serological test results and their relationship with immune protection (n = 162/893, 18%) and 2) the impact of SARS-CoV-2 variants on vaccine effectiveness (n = 235/891, 26%). Analysis of responses stratified by sociodemographic variables identified that respondents who were either: 1) female, 2) more educated, 3) aged 18-44, 4) from a high-income household, or 5) healthcare workers responded expectedly more often. CONCLUSIONS: We have highlighted concepts related to SARS-CoV-2 laboratory tests and associated results which may be challenging to understand. The findings of this study enable us to identify 1) misconceptions related to various SARS-CoV-2 test results, 2) groups of individuals at risk, and 3) strategies to improve people's understanding of their test results.

Localization of Epigenetic Markers in Leishmania Chromatin.

Eukaryotes use histone variants and post-translation modifications (PTMs), as well as DNA base modifications, to regulate DNA replication/repair, chromosome condensation, and gene expression. Despite the unusual organization of their protein-coding genes into large polycistronic transcription units (PTUs), trypanosomatid parasites also employ a "histone code" to control these processes, but the details of this epigenetic code are poorly understood. Here, we present the results of experiments designed to elucidate the distribution of histone variants and PTMs over the chromatin landscape of Leishmania tarentolae. These experiments show that two histone variants (H2A.Z and H2B.V) and three histone H3 PTMs (H3K4me3, H3K16ac, and H3K76me3) are enriched at transcription start sites (TSSs); while a histone variant (H3.V) and the trypanosomatid-specific hyper-modified DNA base J are located at transcription termination sites (TTSs). Reduced nucleosome density was observed at all TTSs and TSSs for RNA genes transcribed by RNA polymerases I (RNAPI) or RNAPIII; as well as (to a lesser extent) at TSSs for the PTUs transcribed by RNAPII. Several PTMs (H3K4me3, H3K16ac H3K20me2 and H3K36me3) and base J were enriched at centromeres, while H3K50ac was specifically associated with the periphery of these centromeric sequences. These findings significantly expand our knowledge of the epigenetic markers associated with transcription, DNA replication and/or chromosome segregation in these early diverging eukaryotes and will hopefully lay the groundwork for future studies to elucidate how they control these fundamental processes.