Shikonin, a natural naphthoquinone phytochemical, exerts anti-leukemia effects in human CBF-AML cell lines and zebrafish xenograft models.

Core binding factor acute myeloid leukemia (CBF-AML) stands out as the most common type of adult AML, characterized by specific chromosomal rearrangements involving CBF genes, particularly t(8;21). Shikonin (SHK), a naphthoquinone phytochemical widely employed as a food colorant and traditional Chinese herbal medicine, exhibits antioxidant, anti-inflammatory, and anti-cancer activities. In this study, we aim to investigate the antileukemic effects of SHK and its underlying mechanisms in human CBF-AML cells and zebrafish xenograft models. Our study revealed that SHK reduced the viability of CBF-AML cells. SHK induced cell cycle arrest, promoted cell apoptosis, and induced differentiation in Kasumi-1 cells. Additionally, SHK downregulated the gene expression of AML1-ETO and c-KIT in Kasumi-1 cells. In animal studies, SHK showed no toxic effects in zebrafish and markedly inhibited the growth of leukemia cells in zebrafish xenografts. Transcriptomic analysis showed that differentially expressed genes (DEGs) altered by SHK are linked to key biological processes like DNA repair, replication, cell cycle regulation, apoptosis, and division. Furthermore, KEGG pathways associated with cell growth, such as the cell cycle and p53 signaling pathway, were significantly enriched by DEGs. Analysis of AML-associated genes in response to SHK treatment using DisGeNET and the STRING database indicated that SHK downregulates the expression of cell division regulators regarding AML progression. Finally, we found that SHK combined with cytarabine synergistically reduced the viability of Kasumi-1 cells. In conclusion, our findings provide novel insights into the mechanisms of SHK in suppressing leukemia cell growth, suggesting its potential as a chemotherapeutic agent for human CBF-AML.

A Narrative Review: Repurposing Metformin as a Potential Therapeutic Agent for Oral Cancer.

Oral cancer, particularly oral squamous cell carcinoma (OSCC), is a significant global health challenge because of its high incidence and limited treatment options. Major risk factors, including tobacco use, alcohol consumption, and specific microbiota, contribute to the disease's prevalence. Recently, a compelling association between diabetes mellitus (DM) and oral cancer has been identified, with metformin, a widely used antidiabetic drug, emerging as a potential therapeutic agent across various cancers, including OSCC. This review explores both preclinical and clinical studies to understand the mechanisms by which metformin may exert its anticancer effects, such as inhibiting cancer cell proliferation, inducing apoptosis, and enhancing the efficacy of existing treatments. Preclinical studies demonstrate that metformin modulates crucial metabolic pathways, reduces inflammation, and impacts cellular proliferation, thereby potentially lowering cancer risk and improving patient outcomes. Additionally, metformin's ability to reverse epithelial-to-mesenchymal transition (EMT), regulate the LIN28/let-7 axis, and its therapeutic role in head and neck squamous cell carcinoma (HNSCC) are examined through experimental models. In clinical contexts, metformin shows promise in enhancing therapeutic outcomes and reducing recurrence rates, although challenges such as drug interactions, complex dosing regimens, and risks such as vitamin B12 deficiency remain. Future research should focus on optimizing metformin's application, investigating its synergistic effects with other therapies, and conducting rigorous clinical trials to validate its efficacy in OSCC treatment. This dual exploration underscores metformin's potential to play a transformative role in both diabetes management and cancer care, potentially revolutionizing oral cancer treatment strategies.

Nine months of bedaquiline, linezolid, levofloxacin, clofazimine, and cycloserine chemotherapy for rifampicin/multidrug-resistant tuberculosis: a multicenter, randomized, open-label non-inferiority trial in China.

BACKGROUND: We concurrently developed a prospective study to assess clinical outcomes among patients receiving 9-month bedaquiline (BDQ)-containing regimens, aiming to provide valuable data on the use of this short-course regimen in China. METHODS: This open-label, randomized, controlled, multicenter, non-inferiority trial was conducted at sixteen hospitals, and enrolled participants aged 18 years and older with pulmonary rifampicin/multidrug tuberculosis. Participants were randomly assigned, in a 1:1 ratio. Individuals within the standard-regimen group received 6 months of BDQ, linezolid, levofloxacin, clofazimine, and cycloserine plus 12 months of levofloxacin, and any three potentially effective drugs from clofazimine, cycloserine pyrazinamide, ethambutol and protionamide, whereas individuals within shorter-regimen group received 9 months of BDQ, linezolid, levofloxacin, clofazimine and cycloserine. The primary outcome was the percentage of participants with a composite unfavorable outcome (treatment failure, death, treatment discontinuation, or loss to follow-up) by the end of the treatment course after randomization in the modified intention-to-treat population. The noninferiority margin was 10%. This trial was registered with www.chictr.org.cn , ChiCTR2000029012. RESULTS: Between Jan 1, 2020, and Dec 31, 2023, 264 were screened and randomly assigned, 132 of 264 participants were assigned to the standard-regimen group and 132 were assigned to the shorter-regimen. Thirty-three (12.55%) of 264 participants were excluded from the modified intention-to-treat analysis. As a result, 231 participants were included in the modified intention-to-treat analysis (116 in the standard-regimen group and 115 in the shorter-regimen group).In the modified intention-to-treat population, unfavorable outcomes were reported in 19 (16.5%) of 115 participants for whom the outcome was assessable in the shorter-regimen group and 26 (22.4%) of 116 participants in the standard care group (risk difference 5.9 percentage points (97.5% CI - 5.8 to 17.5)). One death was reported in the standard-regimen group. The incidence of QTcF prolongation in the shorter-regimen group (22.6%, 26/115) was similar to the standard-regimen group (24.1%, 28/116). CONCLUSIONS: The 9-month, all-oral regimen is safe and efficacious for the treatment of pulmonary rifampicin/multidrug-resistant tuberculosis. The high incidence of QTc prolongation associated with the use of BDQ highlights the urgent need of routine electrocardiogram monitoring under treatment with BDQ-containing regimens in the Chinese population.

Interaction of the gut microbiota and brain functional connectivity in late-life depression.

BACKGROUND: Increasing evidence suggests an important role of the gut microbiome in the pathogenesis of mental disorders, including depression, along the microbiota-gut-brain axis. We sought to explore the interactions between gut microbe composition and neural circuits in late-life depression (LLD). METHODS: We performed fecal 16S ribosomal RNA (rRNA) sequencing and resting-state functional magnetic resonance imaging in a case-control cohort of older adults with LLD and healthy controls to characterize the association between gut microbiota and brain functional connectivity (FC). We used the Hamilton Depression Rating Scale (HAMD) to assess depressive symptoms. RESULTS: We included 32 adults with LLD and 16 healthy controls. At the genus level, the relative abundance of Enterobacter, Akkermansiaceae, Hemophilus, Burkholderia, and Rothia was significantly higher among patients with LDD than controls. Reduced FC within mood regulation circuits was mainly found in the frontal cortex (e.g., the right superior and inferior frontal gyrus, right lateral occipital cortex, left middle frontal gyrus, and left caudate) among patients with MDD. Group-characterized gut microbes among controls and patients showed opposite correlations with seed-based FC, which may account for the aberrant emotion regulation among patients with LDD. The abundance of Enterobacter (dominant genus among patients with LLD) was positively correlated with both HAMD scores (r = 0.49, p = 0.0004) and group-characterized FC (r = -0.37, p < 0.05), while Odoribacter (dominant genus among controls) was negatively correlated with both HAMD scores (r = -0.30, p = 0.04) and group-characterized FC. LIMITATIONS: The study's cross-sectional design and small sample size limit causal inferences; larger longitudinal studies are required for detailed subgroup analyses. CONCLUSION: We identified significant correlations between LDD-characterized gut microbes and brain FC, as well as depression severity, which may contribute to the pathophysiology of depression development among patients with LLD. Specific microbes were linked to altered brain connectivity, suggesting potential targets for treating LLD.

ß-Cyclodextrin-optimized supramolecular nanovesicles enhance the droplet/foliage interface interactions and inhibition of succinate dehydrogenase (SDH) for efficient treatment of fungal diseases.

BACKGROUND: Plant fungal diseases present a major challenge to global agricultural production. Despite extensive efforts to develop fungicides, particularly succinate dehydrogenase inhibitors (SDHIs), their effectiveness is often limited by poor retention of fungicide droplets on hydrophobic leaves. The off-target losses and unintended release cause fungal resistance and severe environmental pollution. RESULTS: To update the structure of existing SDHIs and synchronously realize the efficient utilization, we have employed a sophisticated supramolecular strategy to optimize a structurally novel SDH inhibitor (AoH25), creating an innovative supramolecular SDH fungicide (AoH25@ß-CD), driven by the host-guest recognition principle between AoH25 and ß-cyclodextrin (ß-CD). Intriguingly, AoH25@ß-CD self-assembles into biocompatible supramolecular nanovesicles, which reinforce the droplet/foliage (liquid-solid) interface interaction and the effective wetting and retention on leaf surfaces, setting the foundation for enhancing fungicide utilization. Mechanistic studies revealed that AoH25@ß-CD exhibited significantly higher inhibition of SDH (IC50 = 1.56 µM) compared to fluopyram (IC50 = 244.41 µM) and AoH25 alone (IC50 = 2.29 µM). Additionally, AoH25@ß-CD increased the permeability of cell membranes in Botryosphaeria dothidea, facilitating better penetration of active ingredients into pathogenic cells. Further experimental outcomes confirmed that AoH25@ß-CD was 88.5% effective against kiwifruit soft rot at a low-dose of 100 µg mL-1, outperforming commercial fungicides such as fluopyram (52.4%) and azoxystrobin (65.4%). Moreover, AoH25@ß-CD showed broad-spectrum bioactivity against oilseed rape sclerotinia, achieving an efficacy of 87.2%, outstripping those of fluopyram (48.7%) and azoxystrobin (76.7%). CONCLUSION: This innovative approach addresses key challenges related to fungicide deposition and resistance, improving the bioavailability of agricultural chemicals. The findings highlight AoH25@ß-CD as a novel supramolecular SDH inhibitor, demonstrating its potential as an efficient and sustainable solution for plant disease management.

Chiral aggregates of rod-coil molecules inside nanopores as efficient nanoreactors for asymmetric synthesis.

An important subject of porous organic materials is their capacity to access enantioselectivity due to their high surface area, controllable pore size, and ease of functionalization. However, recyclability of enantio-separation is a challenge, mainly due to the complex procedures of recovery and refreshing from enantiomers. For the first time, we combined nanochannel technology and supramolecular chiral assembly to achieve efficient enantioselectivity. Fine-designed amphiphilic chiral rod-coil molecules 1-3 were immobilized to SBA-15 pore walls to form SA-M1-3 (abbreviation for amino-functionalized SBA-15 connected to molecules 1-3), which commenced chiral aggregation inside the channels. The experimental results indicated that the strong π-π stacking interaction between the rigid terphenyl groups, as well as hydrophilic-hydrophobic interaction of the amphiphiles, assisted in chiral arrangement in aqueous solution, and was accompanied by amplification of chirality. As a result, porous chiral channels exhibiting enhanced efficiency in asymmetric synthesis were manufactured, where enantioselectivity can be controlled by the initial structural design of amphiphiles that induce chiral aggregation behaviors. The chiral centers of SA-M1 and SA-M2 are located on hydrophobic and hydrophilic coils, respectively, while SA-M3 possesses both chiral coils. The SA-M materials proceeded with chiral aggregation and behaved efficiently for enantioselectivity. SA-M3, which contained the most chiral centers, showed the most optimal enantioselectivity with an enantiomeric excess (ee) value up to 71.75%, which occurred because of the strongly driven chiral aggregation of the hydrophobic and hydrophilic chiral coils. The covalent hybrid structures of the SA-M materials can be easily refreshed simply through washing, and exhibited excellent recyclability with negligible loss of efficiency. Therefore, the SA-M materials have the ability to provide sustainable and reliable application value for enantiomer separation.

The HtrA chaperone monitors sortase-assembled pilus biogenesis in Enterococcus faecalis.

Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the OG1RF ΔsrtAΔhtrA background partially restores the observed defects of the ΔsrtAΔhtrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal resistance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.

Esculetin facilitates post-stroke rehabilitation by inhibiting CKLF1-mediated neutrophil infiltration.

Esculetin (ESC) is a coumarin-derived phytochemical prevalent in traditional Chinese medicine that exhibits anti-acute ischemic stroke activities. Our previous studies demonstrate that CKLF1 is a potential anti-stroke target for coumarin-derived compound. In this study we investigated whether CKLF1 was involved in the neuroprotective effects of ESC against photothrombotic stroke in mice. The mice were treated with ESC (20, 40 or 80 mg·kg-1·d-1, i.g.) for two weeks. The therapeutic effect of ESC was assessed using MRI, neurological function evaluation, and a range of behavioral tests on D1, 3, 7 and 14 of ESC administration. We showed that oral administration of ESC dose-dependently reduced the cerebral infarction volume within one week after stroke, improved behavioral performance, and alleviated neuropathological damage within two weeks. Functional MRI revealed that ESC significantly enhanced the abnormal low-frequency fluctuation (ALFF) value of the motor cortex and promoted functional connectivity between the supplementary motor area (SMA) and multiple brain regions. We demonstrated that ESC significantly reduced the protein levels of CKLF1 and CCR5, as well as the CKLF1/CCR5 protein complex in the peri-infarcted area. We showed that ESC (0.1-10 µM) dose-dependently blocked CKLF1-induced chemotactic movement of neutrophils in the Transwell assay, reducing the interaction of CKLF1/CCR5 on the surface of neutrophils, thereby reducing neutrophil infiltration, and decreasing the expression of ICAM-1, VCAM-1 and MMP-9 in the peri-infarct tissue. Knockout of CKLF1 reduced brain infarction volume and motor dysfunction after stroke but also negated the anti-stroke efficacy and neutrophil infiltration of ESC. These results suggest that the efficacy of ESC in promoting post-stroke neural repair depends on its inhibition on CKLF1-mediated neutrophil infiltration, which offering novel perspectives for elucidating the therapeutic properties of coumarins.

[Improving effect of selenium on spermatogenesis in mice with cyclophosphamide-induced spermatogenic impairment and its underlying mechanism].

OBJECTIVE: To investigate the effect of selenium on cyclophosphamide (CTX)-induced spermatogenic impairment (SI) in mice and its underlying mechanism. METHODS: We equally randomized 36 male KM mice into 3 SI model and 3 control groups, the first 3 treated by intraperitoneal injection of CTX at 100 mg/kg (the SI model control group), CTX plus SI model control group, selenium deficient model group (－Se SI), selenium supplemented model group (+Se SI), while latter 3 by intraperitoneal injection of normal saline (the normal control), selenium deficiency control group (－Se control), selenium addition control group (+Se control), respectively, all once a week for 6 successive weeks. Then we observed the histopathological changes in the testes of all the mice by HE staining, obtained the sperm count in the epididymides, determined the expressions of glutathione peroxidase 4 (GPx4) and SLC7A11 proteins by Western blot and ferroptosis-related genes by RT-qPCR, and examined the changes in the expressions of ferroptosis-related proteins and genes in the GC2-spd cells treated with ferroptosis inhibitors and inducers in combination with different concentrations of inorganic sodium selenite (SeS) and organic selenomethionine (SeM). RESULTS: Compared with the normal controls, the SI model mice showed significantly decreased testicular and prostatic organ coefficients, reduced spermatogenic layers, increased voids, decreased serum ferritin concentration (P<0.05), and elevated transferrin concentration (P<0.05). The organ coefficients were significantly higher in the +Se SI and +Se control than in the －Se SI and －Se control groups (P<0.05, P<0.01), with evident pathological improvement of the testis tissue in the +Se controls. The expressions of the GPx4 and solute carrier family 7 members 11（SLC7A11） genes in the testis were dramatically down-regulated in the SI model controls (P<0.01), but up-regulated in the +Se SI and +Se control compared with those in the －Se SI and －Se control group (P<0.01 and P<0.05), but there were no statistically significant differences between their protein expressions. The results of in vitro GC2 spd cell experiments indicated that the GPx4 gene and GPx4 protein levels in the - Se group were significantly lower than those in the normal control group (P<0.05), while the SLC7A11 gene level decreased (P<0.01). Different doses of SeS and SeM significantly increased the GPx4 protein expression compared to the average Se group. Low doses of SeM promoted a significant increase in GPx4 gene levels, while high doses of SeS increased the expression levels of SLC7A11 gene and SLC7A11 protein (P<0.05, P<0.01). The Se group showed a significant decrease in the levels of acsl4 and ptgs2 genes compared to the normal control group. SeM promoted the expression of acsl4, while SeS promoted the expression of ptgs2 and fth1 (P<0.01, P<0.05). The intervention results of GC2 spd showed that the Erastin group had a decrease in ptgs2 compared to the normal control group, while the SeS+Erastin and SeM+Erastin groups had an increase in ptgs2 gene expression compared to the Erastin group. However, the ptgs2 expression of Fer-1 was lower than that of the normal control group, and the ptgs2 gene level of SeS+Fer-1 and SeM+Fer-1 groups was lower than that of Fer-1 group (P<0.05); The gene quantity of GPx4 in the SeM+Erastin and SeM+Fer-1 groups increased compared to the Erastin and Fer-1 groups (P<0.01, P<0.05); SeM+Erastin and SeS+Erastin showed a decrease in SLC7A11 compared to the Erastin group, as well as SeM+Fer-1 and SeS+Fer-1 groups compared to the Fer-1 group, accompanied by an increase in acsl4 and fth1 (P<0.01). CONCLUSION: Selenium deficiency causes the reduction of the SLC7A11 and GPx4 gene levels, disorder of ferroptosis-related genes and down-regulation of the GPx4 protein expression in the mouse testis and spermatocytes. Selenium can promote the expression of GPx4, up-regulate the level of SLC7A11, and improve spermatogenesis in the testis of the mouse with SI. There are differences between organic SeM and inorganic SeS in regulating the ferroptosis pathway-related genes.

Evaluating artificial intelligence-enhanced digital urine cytology for bladder cancer diagnosis.

BACKGROUND: This study evaluated the diagnostic effectiveness of the AIxURO platform, an artificial intelligence-based tool, to support urine cytology for bladder cancer management, which typically requires experienced cytopathologists and substantial diagnosis time. METHODS: One cytopathologist and two cytotechnologists reviewed 116 urine cytology slides and corresponding whole-slide images (WSIs) from urology patients. They used three diagnostic modalities: microscopy, WSI review, and AIxURO, per The Paris System for Reporting Urinary Cytology (TPS) criteria. Performance metrics, including TPS-guided and binary diagnosis, inter- and intraobserver agreement, and screening time, were compared across all methods and reviewers. RESULTS: AIxURO improved diagnostic accuracy by increasing sensitivity (from 25.0%-30.6% to 63.9%), positive predictive value (PPV; from 21.6%-24.3% to 31.1%), and negative predictive value (NPV; from 91.3%-91.6% to 95.3%) for atypical urothelial cell (AUC) cases. For suspicious for high-grade urothelial carcinoma (SHGUC) cases, it improved sensitivity (from 15.2%-27.3% to 33.3%), PPV (from 31.3%-47.4% to 61.1%), and NPV (from 91.6%-92.7% to 93.3%). Binary diagnoses exhibited an improvement in sensitivity (from 77.8%-82.2% to 90.0%) and NPV (from 91.7%-93.4% to 95.8%). Interobserver agreement across all methods showed moderate consistency (κ = 0.57-0.61), with the cytopathologist demonstrating higher intraobserver agreement than the two cytotechnologists across the methods (κ = 0.75-0.88). AIxURO significantly reduced screening time by 52.3%-83.2% from microscopy and 43.6%-86.7% from WSI review across all reviewers. Screening-positive (AUC+) cases required more time than negative cases across all methods and reviewers. CONCLUSIONS: AIxURO demonstrates the potential to improve both sensitivity and efficiency in bladder cancer diagnostics via urine cytology. Its integration into the cytopathological screening workflow could markedly decrease screening times, which would improve overall diagnostic processes.

Antimicrobial efficacy and amino acid substitutions associated with susceptibility to the tellurium compound AS101 against Haemophilus influenzae and Haemophilus parainfluenzae.

The tellurite toxicity in Haemophilus influenzae and H. parainfluenzae remains unclear. To understand the potential of tellurite as a therapeutic option for these bacteria, we investigated the antimicrobial efficacy of AS101, a tellurium compound, against H. influenzae and H. parainfluenzae and the molecular basis of their differences in AS101 susceptibility. Through broth microdilution, we examined the minimum inhibitory concentration (MIC) of AS101 in 51 H. influenzae and 28 H. parainfluenzae isolates. Whole-genome sequencing was performed on the H. influenzae isolates to identify genetic variations associated with AS101 susceptibility. The MICs of AS101 were ≦ 4, 16-32, and ≧ 64 µg/mL in 9 (17.6%), 12 (23.5%), and 30 (58.8%) H. influenzae isolates, respectively, whereas ≦ 0.5 µg/mL in all H. parainfluenzae isolates, including multidrug-resistant isolates. Time-killing kinetic assay and scanning electron microscopy revealed the in vitro bactericidal activity of AS101 against H. parainfluenzae. Forty variations in nine tellurite resistance-related genes were associated with AS101 susceptibility. Logistic regression, receiver operator characteristic curve analysis, Venn diagram, and protein sequence alignment indicated that Val195Ile substitution in TerC, Ser93Gly in Gor (glutathione reductase), Pro44Ala/Ala50Pro in NapB (nitrate reductase), Val307Leu in TehA (tellurite resistance protein), Cys105Arg in CysK (cysteine synthase), and Thr364Ser in Csd (Cysteine desulfurase) were strongly associated with reduced AS101 susceptibility, whereas Ser155Pro in TehA with increased AS101 susceptibility. In conclusions, the antimicrobial efficacy of AS101 is high against H. parainfluenzae but low against H. influenzae. Genetic variations and corresponding protein changes relevant to AS101 non-susceptibility in H. influenzae were identified.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

In vitro and in vivo studies on exogenous polyamines and α-difluoromethylornithine to enhance bone formation and suppress osteoclast differentiation.

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

Spatial and Single-Cell Investigations Illuminate Theragnostic value and Immune Landscape of Mitochondrial Dynamin-Like GTPase in Breast Cancer.

Background: As we delve into the intricate world of mitochondrial inner membrane proteins, particularly Optic Atrophy types 1 and 3 (OPA1/3), we uncover their pivotal role in maintaining mitochondrial dynamic equilibrium and fusion, crucial for cellular energy production and synthesis. Despite extensive scrutiny, the significance of OPA1/3 in breast cancer (BRCA) and its interplay with the immune microenvironment remain elusive. Materials and Methods: We meticulously sourced BRCA data from renowned repositories such as The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Gene Expression Omnibus (GEO), and the Human Protein Atlas (HPA), leveraging cutting-edge techniques including single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and pharmacogenomics. Through multifaceted data analysis, we endeavored to unravel the intricate role and potential value of OPA1/3 in BRCA tumorigenesis and progression. Results: Our investigation reveals a conspicuous upregulation of OPA1/3 expression in BRCA, correlating with dismal prognoses. Kaplan-Meier plot analysis underscores that heightened OPA1/3 levels are associated with poor survival rates. Both clinical specimens and biobank biopsies corroborate the elevated expression of OPA1/3 in breast cancer patients. Moreover, scRNA-seq unveils a strong correlation between OPA1/3 and macrophage infiltration in the BRCA immune milieu, alongside its association with the cellular communication network involving CXCL, TGFb, VEGF, and IL16. Conclusion: In light of these findings, OPA1/3 emerges as a promising contender for therapeutic targeting and as a potential diagnostic, prognostic, and survival biomarker in BRCA. The implications of our study underscore the pressing need to explore these novel biomarkers to enhance patient outcomes.

Multi-omic profiling of breast tumor microenvironment uncovers a role of mitochondrial calcium gatekeepers.

In this study, we aimed to elucidate the role of mitochondrial calcium uptake 1/2 (MiCU1/2) in breast cancer (BRCA) by employing a comprehensive multi-omics approach. Unlike previous research, we utilized a novel web application tailored for whole tumor tissue, single-cell, and spatial transcriptomics analysis to investigate the association between MiCU1/2 and the tumor immune microenvironment (TIME). Our gene set enrichment analysis (GSEA) provided insights into the primary biological effects of MiCU1/2, while our CRISPR-based drug screening repository identified potential effective drugs. Our study revealed that high MiCU1/2 expression serves as an independent diagnostic biomarker, correlating with advanced clinical status and indicating poorer recurrence-free survival (RFS) rates in BRCA patients. Additionally, spatial transcriptome analysis highlighted the heightened expression of MiCU1/2 in tumors and its relevance in surrounding immune cells. Furthermore, using the CIBERSORT algorithm, we discovered a positive correlation between MiCU1/2 levels and macrophage infiltration, underscoring their potential impact on immune infiltration. We also identified expression patterns of immune-related genes associated with responses against various immune cell types, including CXCL, MIF, GDF, SPP1, and IL16. Finally, our pharmacogenomic screening identified potential small molecule drugs capable of effectively targeting breast cancer cells with elevated MiCU1/2 expression. Overall, our study establishes MiCU1/2 as a promising novel biomarker for BRCA diagnosis and prognostic prediction, as well as a potential therapeutic target, highlighting the importance of exploring these pathways to advance patient care and outcomes in BRCA treatment.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth.

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Sensory ASIC3 channel exacerbates psoriatic inflammation via a neurogenic pathway in female mice.

Psoriasis is an immune-mediated skin disease associated with neurogenic inflammation, but the underlying molecular mechanism remains unclear. We demonstrate here that acid-sensing ion channel 3 (ASIC3) exacerbates psoriatic inflammation through a sensory neurogenic pathway. Global or nociceptor-specific Asic3 knockout (KO) in female mice alleviates imiquimod-induced psoriatic acanthosis and type 17 inflammation to the same extent as nociceptor ablation. However, ASIC3 is dispensable for IL-23-induced psoriatic inflammation that bypasses the need for nociceptors. Mechanistically, ASIC3 activation induces the activity-dependent release of calcitonin gene-related peptide (CGRP) from sensory neurons to promote neurogenic inflammation. Botulinum neurotoxin A and CGRP antagonists prevent sensory neuron-mediated exacerbation of psoriatic inflammation to similar extents as Asic3 KO. In contrast, replenishing CGRP in the skin of Asic3 KO mice restores the inflammatory response. These findings establish sensory ASIC3 as a critical constituent in psoriatic inflammation, and a promising target for neurogenic inflammation management.