A countrywide survey of hrp2/3 deletions and kelch13 mutation co-occurrence in Ethiopia.

Malaria elimination relies on detection of Plasmodium falciparum Histidine-Rich Proteins 2/3 (HRP2/3) through rapid diagnostic tests (RDTs) and treatment with artemisinin-combination therapies (ACTs). Data from the Horn of Africa suggest increasing hrp2/3 gene deletions and ACT partial resistance kelch13 (k13) mutations. To assess this, 233 samples collected during a national survey from 7 regions of Ethiopia were studied for hrp2/3 deletions by droplet digital dPCR and k13 mutations by DNA sequencing. Approximately 22% of the study population harbored complete hrp2/3 deletions by ddPCR. Thirty-two of 42 of k13 SNPs identified were R622I associated with ACT partial resistance. Both hrp2/3 deletions and k13 mutations associated with ACT partial resistance appear to be co-occurring especially in Northwest Ethiopia. Ongoing national surveillance relying on accurate laboratory methods are required to fully elaborate the genetic diversity of P. falciparum to inform public health policy makers.

Mutations in Plasmodium falciparum Kelch13 (PfK13) dysregulates PI3K/AKT signalling pathway in artemisinin resistance.

BACKGROUND: PfK13 protein mutations are associated with the emergence of artemisinin resistance in Plasmodium falciparum. PfK13 protein is essential for mediating ubiquitination and controlling the PI3K/AKT pathway. Mutant PfK13 variations can interfere with substrate binding, especially with PfPI3K, which raises PfPI3K levels. METHODS: DUET, DynaMut2, mCSM, iStable 2.0, I-Mutant 2.0, and MuPro were utilized to study the protein stability and protein-substrate binding was studied using HADDOCK 2.4 docking algorithm between Wild-type and mutant PfK13 with the helical and catalytic domain of PfPI3K. RESULTS: i-Stable server analysis predicted that seven, out of the nine mutations associated with artemisinin resistance (F446I, Y493H, R539T, I543T, P553L, R561H, C580Y) reduced the protein stability. HADDOCK scores of the catalytic domain underscores the significant impact of the reported mutations on the binding affinity of the PfK13 protein. Further validation through the MM_GBSA technique, the binding free energy (DDG) between the wild-type and the mutant PfK13 protein analysis revealed a loss of interactions resulting from mutations in PfK13. CONCLUSION: The study finding suggest that mutations in the PfK13 cause destabilization in the protein structure and affects the binding of PfPI3K. Although the findings remain preliminary and require further validation, it provides the basis for further research considering the importance of the interaction of PfK13 and PfPI3K to overcome the impact of artemisinin resistance.

High prevalence and risk of malaria among asymptomatic individuals from villages with high prevalence of artemisinin partial resistance in Kyerwa district of Kagera region, north-western Tanzania.

BACKGROUND: Although Tanzania adopted and has been implementing effective interventions to control and eventually eliminate malaria, the disease is still a leading public health problem, and the country experiences heterogeneous transmission. Recent studies reported the emergence of parasites with artemisinin partial resistance (ART-R) in Kagera region with high prevalence (> 10.0%) in two districts of Karagwe and Kyerwa. This study assessed the prevalence and predictors/risk of malaria infections among asymptomatic individuals living in a hyperendemic area where ART-R has emerged in Kyerwa District of Kagera region, north-western Tanzania. METHODS: This was a community-based cross-sectional survey which was conducted in July and August 2023 and involved individuals aged ≥ 6 months from five villages in Kyerwa district. Demographic, anthropometric, clinical, parasitological, type of house inhabited and socio-economic status (SES) data were collected using electronic capture tools run on Open Data Kit (ODK) software. Predictors/risks of malaria infections were determined by univariate and multivariate logistic regression, and the results were presented as crude (cORs) and adjusted odds ratios (aORs), with 95% confidence intervals (CIs). RESULTS: Overall, 4454 individuals were tested using rapid diagnostic tests (RDTs), and 1979 (44.4%) had positive results. The prevalence of malaria infections ranged from 14.4% to 68.5% and varied significantly among the villages (p < 0.001). The prevalence and odds of infections were significantly higher in males (aOR = 1.28, 95% CI 1.08 -1.51, p = 0.003), school children (aged 5-≤10 years (aOR = 3.88, 95% CI 3.07-4.91, p < 0.001) and 10-≤15 years (aOR = 4.06, 95% CI 3.22-5.13, p < 0.001)) and among individuals who were not using bed nets (aOR = 1.22, 95% CI 1.03-1.46, p = 0.024). The odds of malaria infections were also higher in individuals with lower SES (aOR = 1.42, 95% CI 1.17-1.72, p < 0.001), and living in houses without windows (aOR = 2.08, 95% CI 1.46-2.96, p < 0.001), partially open (aOR = 1.33, 95% CI 1.11-1.58, p = 0.002) or fully open windows (aOR = 1.30, 95%CI 1.05-1.61, p = 0.015). CONCLUSION: The five villages had a high prevalence of malaria infections and heterogeneity at micro-geographic levels. Groups with higher odds of malaria infections included school children, males, and individuals with low SES, living in poorly constructed houses or non-bed net users. These are important baseline data from an area with high prevalence of parasites with ART-R and will be useful in planning interventions for these groups, and in future studies to monitor the trends and potential spread of such parasites, and in designing a response to ART-R.

Clinical and serological insights into paraneoplastic brachial amyotrophic diplegia.

BACKGROUND: Brachial amyotrophic diplegia (BAD) is typically linked to a neurodegenerative etiology such as amyotrophic lateral sclerosis (ALS). Clinical and serological characterizations of paraneoplastic neurologic syndromes resembling BAD are limited. METHODS: A retrospective chart review of patients with BAD-like presentations was conducted. Clinical/paraclinical features of paraneoplastic BAD and neurodegenerative BAD cases were compared. RESULTS: Between 2017 and 2023, 13 cases of BAD were identified, of these 10 were neurodegenerative BAD (ALS variant), and 3 cases associated with paraneoplastic autoimmunity. An additional paraneoplastic BAD case diagnosed in 2005 was included. LUZP4-IgG was detected in all four paraneoplastic cases, with coexisting KLHL11-IgG in three cases and ANNA1 (anti-Hu)-IgG in one case. Out of the four paraneoplastic cases, two patients had seminoma, while the remaining two had limited cancer investigation. Three patients exhibited bi-brachial weakness as the initial symptom before the onset of brainstem symptoms or seizures. Compared to BAD patients with a neurodegenerative etiology, a higher proportion of paraneoplastic cases had ataxia (75% vs 0%, p = 0.011). Other clinical features only detected in the paraneoplastic BAD group were vertigo (n = 2), hearing loss (n = 2) and ophthalmoplegia (n = 2). Electrodiagnostic studies in these patients revealed cervical myotome involvement, supportive of motor neuronopathy. All paraneoplastic cases but none of the neurodegenerative BAD cases exhibited inflammatory cerebrospinal fluid (CSF) findings (lymphocytic pleocytosis and/or supernumerary oligoclonal bands; p = 0.067). Despite the administration of immunotherapy and/or cancer treatment, none of the paraneoplastic patients reported clinical improvement. DISCUSSION: BAD or bi-brachial neurogenic weakness is a rare phenotypic presentation associated with paraneoplastic autoimmunity. Co-existing features of brainstem dysfunction or cerebellar ataxia should prompt further paraneoplastic evaluation. Common serological and cancer associations among these cases include LUZP4-IgG and KLHL11-IgG, along with testicular germ cell tumors, respectively.

The prevalence of Pfk13 polymorphism in malaria patients treated with artemisinin-based therapy: a systematic review and meta-analysis.

Artemisinin (ART) combination therapy is the main treatment for malaria. Pfk13 mutations (or K13 mutations, Kelch 13) are associated with ART resistance. This study aims to conduct a systematic review and meta-analysis of the prevalence of K13 mutations with ART resistance in malaria-endemic countries. An electronic search of studies in 2018 and a manual search in 2020 were performed to identify relevant studies. The risk of bias was assessed using the National Institutes of Health (NIH) quality assessment tool for observational cohort and cross-sectional studies. Data analysis was performed using R 4.1.0. Heterogeneity was estimated using the statistic I2 and Cochran Q test. A total of 170 studies were included in our review. Of these, 55 studies investigated the prevalence of K13 mutations in Southeast Asia. The meta-analysis showed that Southeast Asia had the highest prevalence of K13 mutations, whereas Africa, South America, Oceania, and other Asian countries outside Southeast Asia had a low prevalence of K13 mutations. The C580Y mutation was the most common in Southeast Asia with 35.5% (95%CI: 25.4-46.4%), whereas the dominant mutation in Africa was K189T (22.8%, 95%CI: 7.6-43.2%). This study revealed the emergence of ART resistance associated with K13 mutations in Southeast Asia. The diversity of each type of K13 mutation in other regions was also reported.

Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

Anti-aging mechanism and effect of treatment with raw and wine-steamed Polygonatum sibiricum on D-galactose-induced aging in mice by inhibiting oxidative stress and modulating gut microbiota.

Background: Polygonatum sibiricum (PS) is a traditional Chinese medicine (TCM) first recorded in Mingyi Bielu. The book documents that PS can nourish five internal organs, be taken for a long time, relax the body and prolong lifespan. Presently, PS is widely used in TCM to prevent premature graying of hair. Based on TCM theory and clinical trials, the wine steaming processed product from PS provides a better effect. However, no published study has elucidated the anti-aging mechanism. Purpose: The study aim was to investigate the anti-aging mechanism of PS and its wine steaming processed product in mice, specifically focusing on the effect of D-galactose (D-gal) surrounding the intestinal flora and the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2-antioxidant response elements (Keap1/Nrf2/ARE) pathway. Methods: The chemical components in Raw PS (RPS) and Wine-steamed PS (WPS) were identified by ultra-performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). An aging model using Kunming mice was established through intraperitoneally injected D-gal. Concentrations of RPS and WPS at 5, 10, or 15 g/kg/day levels were administered intragastrically, respectively. The body weight, liver and spleen indexes, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) activities in serum and brain tissue were recorded. Hematoxylin and eosin (HE) stained brain tissue was histopathologically examined. The expressions of Keap1, Nrf2 and heme oxygenase 1 (HO-1) in the brain tissue at the mRNA and protein levels were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Moreover, an Illumina Hiseq platform was used for 16S ribosomal RNA (16S rRNA) high-throughput sequencing to evaluate the proportions of intestinal flora in aging mice. Results: The proportions of saccharides, flavonoids, and triterpene acids were different between RPS and WPS. In the aging model mice, WPS outperformed RPS in improving body weight and mental state by increasing the spleen index, SOD and GSH-PX activities, decreasing the liver index and MDA activities, and restoring the histopathological morphology in D-gal-induced aging mice. At the mRNA levels, RPS and WPS significantly reduced the expression of Keap1 and increased the expressions of Nrf2 and HO-1. The trend in protein expressions was similar to that of the mRNA results, and WPS had a stronger effect than RPS. Fecal microbiota analysis showed that RPS and WPS restored intestinal microbiota proportions to normal levels. Conclusion: The results demonstrated that PS and its WPS had a positive effect in relieving oxidative stress in aging mice. WPS outperformed RPS, which might be related to the activation of the Keap1/Nrf2/ARE pathway and regulation of intestinal flora.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted. CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

High frequency of artemisinin partial resistance mutations in the great lake region revealed through rapid pooled deep sequencing.

In Africa, the first Plasmodium falciparum Kelch13 (K13) artemisinin partial resistance mutation 561H was first detected and validated in Rwanda. Surveillance to better define the extent of the emergence in Rwanda and neighboring countries as other mutations arise in East Africa is critical. We employ a novel scheme of liquid blood drop preservation combined with pooled sequencing to provide a cost-effective rapid assessment of resistance mutation frequencies at multiple collection sites across Rwanda and neighboring countries. Malaria-positive samples (n=5,465) were collected from 39 health facilities in Rwanda, Uganda, Tanzania, and the Democratic Republic of the Congo (DRC) between May 2022 and March 2023 and sequenced in 199 pools. In Rwanda, K13 561H and 675V were detected in 90% and 65% of sites with an average frequency of 19.0% (0-54.5%) and 5.0% (0-35.5%), respectively. In Tanzania, 561H had high frequency in multiple sites while it was absent from the DRC although 675V was seen at low frequency. Conceringly candidate mutations were observed: 441L, 449A, and 469F co-occurred with validated mutations suggesting they are arising under the same pressures. Other resistance markers associated with artemether-lumefantrine are common: P. falciparum multidrug resistance protein 1 N86 at 98.0% and 184F at 47.0% (0-94.3%) and P. falciparum chloroquine resistance transporter 76T at 14.7% (0-58.6%). Additionally, sulfadoxine-pyrimethamine-associated mutations show high frequencies. Overall, K13 mutations are rapidly expanding in the region further endangering control efforts with the potential of engendering partner drug resistance.

Expansion of artemisinin partial resistance mutations and lack of histidine rich protein-2 and -3 deletions in Plasmodium falciparum infections from Rukara, Rwanda.

BACKGROUND: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed. METHODS: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. RESULTS: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. CONCLUSIONS: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Apicomplexa micropore: history, function, and formation.

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.

TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone­induced MC3T3­E1 cells by activating the Keap1/Nrf2 pathway.

Steroid-induced osteonecrosis of the femoral head (ONFH) is a serious complication caused by long-term or excessive use of glucocorticoids. The present study aimed to ascertain the effects of tripartite motif-containing protein 21 (TRIM21) on the process of steroid-induced ONFH and its hidden action mechanism. TRIM21 expression in dexamethasone (Dex)-treated mouse MC3T3-E1 preosteoblast cells was examined using reverse transcription-quantitative PCR and western blotting. The Cell Counting Kit-8 (CCK-8) method and lactate dehydrogenase release assay were used to respectively measure cell viability and injury. Flow cytometry analysis was used to assay cell apoptosis. Caspase 3 activity was evaluated using a specific assay, while alkaline phosphatase and Alizarin red S staining were used to evaluate osteogenesis. 2,7-dichloro-dihydrofluorescein diacetate fluorescence probe was used to estimate reactive oxygen species generation. Specific assay kits were used to appraise oxidative stress levels. In addition, the expression of apoptosis-, osteogenic differentiation- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-associated proteins was assessed using western blotting. In Nrf2 inhibitor (ML385)-pretreated MC3T3-E1 cells exposed to Dex, cell apoptosis, osteogenesis and oxidative stress were detected again as aforementioned. Results revealed that TRIM21 expression was raised in Dex-induced MC3T3-E1 cells and TRIM21 deletion improved the viability and osteogenic differentiation, whereas it hampered the oxidative stress and apoptosis in MC3T3-E1 cells with Dex induction. In addition, silencing of TRIM21 activated Keap1/Nrf2 signaling. Moreover, ML385 partially abrogated the effects of TRIM21 depletion on the oxidative stress, apoptosis and osteogenic differentiation in MC3T3-E1 cells exposed to Dex. In conclusion, TRIM21 silencing might activate Keap1/Nrf2 signaling to protect against steroid-induced ONFH.

Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Rhizoma Paridis saponins attenuate Gram-negative bacteria-induced inflammatory acne by binding to KEAP1 and modulating Nrf2 and MAPK pathways.

Acne vulgaris represents a chronic inflammatory condition, the pathogenesis of which is closely associated with the altered skin microbiome. Recent studies have implicated a profound role of Gram-negative bacteria in acne development, but there is a lack of antiacne agents targeting these bacteria. Polyphyllins are major components of Rhizoma Paridis with great anti-inflammatory potential. In this study, we aimed to evaluate the antiacne effects and the underlying mechanisms of PPH and a PPH-enriched Rhizoma Paridis extract (RPE) in treating the Gram-negative bacteria-induced acne. PPH and RPE treatments significantly suppressed the mRNA and protein expressions of interleukin (IL)-1ß and IL-6 in lipopolysaccharide (LPS)-induced RAW 264.7 and HaCaT cells, along with the intracellular reactive oxygen species (ROS) generation. Furthermore, PPH and RPE inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in LPS-induced RAW 264.7 cells. Based on molecular docking, PPH could bind to kelch-like ECH-associated protein 1 (KEAP1) protein. PPH and RPE treatments could activate nuclear factor erythroid 2-related factor 2 (NRF2) and upregulate haem oxygenase-1 (HO-1). Moreover, RPE suppressed the mitogen-activated protein kinase (MAPK) pathway. Therefore, PPH-enriched RPE showed anti-inflammatory and antioxidative effects in vitro, which is promising for alternative antiacne therapeutic.

Targeting kelch-like (KLHL) proteins: achievements, challenges and perspectives.

Kelch-like proteins (KLHLs) are a large family of BTB-containing proteins. KLHLs function as the substrate adaptor of Cullin 3-RING ligases (CRL3) to recognize substrates. KLHLs play pivotal roles in regulating various physiological and pathological processes by modulating the ubiquitination of their respective substrates. Mounting evidence indicates that mutations or abnormal expression of KLHLs are associated with various human diseases. Targeting KLHLs is a viable strategy for deciphering the KLHLs-related pathways and devising therapies for associated diseases. Here, we comprehensively review the known KLHLs inhibitors to date and the brilliant ideas underlying their development.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993-1998 and 2002-2008.

BACKGROUND: Polymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand. RESULTS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4-99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993-1998 (period 1) compared with 2002-2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68). CONCLUSIONS: The persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021.

BACKGROUND: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. METHODS: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). RESULTS: Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. CONCLUSION: This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Overexpression of KLHL22 correlates with poor prognosis in patients with triple-negative breast cancer.

Background: Kelch-like family member 22 (KLHL22) is a protein-coding gene that is responsible for several Mendelian diseases and has been reported to promote tumorigenesis and aging. The purpose of this study was to investigate its expression in triple-negative breast cancer (TNBC) and its prognostic significance. Methods: Immunohistochemistry (IHC) was performed to examine the expression levels of KLHL22 in 146 patients with TNBC. The Chi-squared test was used to analyze the correlations between KLHL22 expression level and clinicopathological features, and the Kaplan-Meier survival analysis and Cox multivariate regression model were used to analyze the prognostic significance of KLHL22 in patients with TNBC. Results: The results of immunohistochemical analysis showed that the high expression rate of KLHL22 protein in TNBC was 56.85% (83/146). Further analysis revealed a significantly positive correlation (P<0.05) between KLHL22 expression and primary tumor and regional lymph node status, clinical stage, and relapse. Kaplan-Meier survival analysis revealed that patients with low KLHL22 expression had a longer mean survival time than those with high KLHL22 expression (147.93 vs. 90.1 months; P<0.05). In the multivariate analysis, KLHL22 level, P53 expression, and clinical stage were found to be independent prognostic factors for overall survival (P<0.05), while clinical stage and KLHL22 level were independent prognostic factors for progression-free survival (P<0.05). Conclusions: The present study concludes that KLHL22 may serve as a biomarker for poor prognosis in patients with TNBC.

Taurine Chloramine-Mediated Nrf2 Activation and HO-1 Induction Confer Protective Effects in Astrocytes.

Taurine is ubiquitously distributed in mammalian tissues, with the highest levels in the brain, heart, and leukocytes. Taurine reacts with hypochlorous acid (HOCl) to produce taurine chloramine (Tau-Cl) via the myeloperoxidase (MPO) system. In this study, we elucidated the antioxidative and protective effects of Tau-Cl in astrocytes. Tau-Cl increased the expression and nuclear translocation of nuclear factor E2-related factor (Nrf2) and the expression of Nrf2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Nrf2 activity is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1). Tau-Cl decreased the level of the reduced thiol groups of Keap1, resulting in the disruption of the Keap1-Nrf2 complex. Consequently, Tau-Cl rescued the H2O2-induced cell death by enhancing HO-1 expression and suppressing reactive oxygen species. In conclusion, Tau-Cl confers protective effects in astrocytes by disrupting the Keap1-Nrf2 complex, thereby promoting Nrf2 translocation to the nucleus, wherein it binds to the antioxidant response element (ARE) and accelerates the transcription of antioxidant genes. Therefore, in astrocytes, the activation of the Keap1-Nrf2-ARE pathway by Tau-Cl may increase antioxidants and anti-inflammatory mediators as well as other cytoprotective proteins, conferring protection against brain infection and injury.