Advances in structural-guided modifications of siRNA.

To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery.

A pyruvate transporter in the apicoplast of apicomplexan parasites.

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

Spinal myeloid sarcoma presenting as initial symptom in acute promyelocytic leukemia with a rare cryptic PLZF::RARα fusion gene: a case report and literature review.

Background: Acute promyelocytic leukemia (APL) is rarely caused by the PLZF::RARα fusion gene. While APL patients with PLZF::RARα fusion commonly exhibit diverse hematologic symptoms, the presentation of myeloid sarcoma (MS) as an initial manifestation is infrequent. Case presentation: A 61-year-old patient was referred to our hospital with 6-month history of low back pain and difficulty walking. Before this admission, spine magnetic resonance imaging (MRI) conducted at another hospital revealed multiple abnormal signals in the left iliac bone and vertebral bodies spanning the thoracic (T11-T12), lumbar (L1-L4), and sacral (S1/S3) regions. This led to a provisional diagnosis of bone tumors with an unknown cause. On admission, complete blood count (CBC) test and peripheral blood smear revealed a slightly increased counts of monocytes. Immunohistochemical staining of both spinal and bone marrow (BM) biopsy revealed positive expression for CD117, myeloperoxidase (MPO), and lysozyme. BM aspirate showed a significant elevation in the percentage of promyelocytes (21%), which were morphologically characterized by round nuclei and hypergranular cytoplasm. Multiparameter flow cytometry of BM aspirate revealed that blasts were positive for CD13, CD33, CD117, and MPO. Through the integrated application of chromosome analysis, fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR), and Sanger sequencing, it was determined that the patient possessed a normal karyotype and a rare cryptic PLZF::RARα fusion gene, confirming the diagnosis of APL. Conclusion: In the present study, we report the clinical features and outcome of a rare APL patient characterized by a cryptic PLZF::RARα fusion and spinal myeloid sarcoma (MS) as the initial presenting symptom. Our study not only offers valuable insights into the heterogeneity of APL clinical manifestations but also emphasizes the crucial need to promptly consider the potential link between APL and MS for ensuring a timely diagnosis and personalized treatments.

A novel coupling process to replace the traditional multi-stage anammox process-sulfur autotrophic denitrification coupled anammox system.

A novel coupling process to replace the traditional multi-stage anammox process-sulfur autotrophic denitrification (SAD) coupled anaerobic ammonium oxidation (anammox) system was designed, which solved problems of nitrate produced in anammox process and low nitrate conversion rate caused by nitrite accumulation in SAD process. Different filter structures (SAD filter and anammox granular sludge) were investigated to further explore the excellent performance of the novel integrated reactor. The results of sequential batch experiments indicated that nitrite accumulation occurred during SAD, which inhibited the conversion of nitrate to dinitrogen gas. When SAD filter and anammox granular sludge were added to packed bed reactor simultaneously, the nitrate removal rate increased by 37.21% and effluent nitrite concentration decreased by 100% compared to that achieved using SAD. The stratified filter structure solved groove flow. Different proportion influence of SAD filter and anammox granular sludge on the stratified filter structure was evaluated. More suitable ratio of SAD filter to anammox granular sludge was 2:1. Proteobacteria (57.26%), Bacteroidetes (20.12%) and Chloroflexi (9.95%) were the main phyla. The dominant genera of denitrification functional bacteria were Thiobacillus (39.80%), Chlorobaculum (3.99%), norank_f_PHOs-HE36 (2.90%) and Ignavibacterium (2.64%). The dominant genus of anammox bacterium was Candidatus_Kuenenia (3.05%).

Treadmill Exercise Enhances Post-Stroke Functional Recovery in Mice via the CX3CL1/CX3CR1 Signaling Pathway.

To validate that treadmill exercise promotes neurofunctional recovery post ischemic stroke and to specifically explore the role of the CX3CL1/CX3CR1 signaling pathway in this treadmill-mediated recovery process. C57BL/6 J mice were used to establish a middle cerebral artery occlusion (MCAO) model. From days 5 to 28 post-stroke, the experimental group did 10-min treadmill sessions twice daily at 12 r/min; the control group remained inactive. On day 6 post-stroke, mice received three intraperitoneal injections of Bromodeoxyuridine (BrdU) or PBS. On days 1, 3, and 5 post-stroke, mice received intracerebroventricular injections of exogenous recombinant CX3CL1, CX3CL1 antagonist, or PBS. The modified neurological severity score (mNSS) and the corner test were used to assess sensorimotor function, and the morris water maze (MWM) test was employed to evaluate cognitive function. Western blot detected CX3CL1 and CX3CR1 protein expression, while immunofluorescence observed these proteins, neurogenesis in the subventricular zone (SVZ), rostral migratory stream (RMS), and dentate gyrus (DG), along with Iba1 and CD68 co-expression. ELISA quantified IL-1ß, IL-4, and IL-10 levels. Treadmill exercise significantly improved neurofunctional recovery in MCAO mice, enhanced neurogenesis in the RMS and SVZ, and increased the expression of CX3CL1 and CX3CR1. The CX3CL1/CX3CR1 axis enhanced the impact of treadmill exercise on neurofunctional recovery, promoting neurogenesis in the RMS and SVZ, and reducing inflammation. Additionally, this axis also enhanced neurogenesis and suppressed microglial activation in the DG induced by treadmill exercise. This study demonstrates the CX3CL1/CX3CR1 pathway as critical for treadmill-induced post-stroke recovery, indicating its potential target for exercise mimetics in rehabilitation.

Depleting profibrotic macrophages using bioactivated in vivo assembly peptides ameliorates kidney fibrosis.

Managing renal fibrosis is challenging owing to the complex cell signaling redundancy in diseased kidneys. Renal fibrosis involves an immune response dominated by macrophages, which activates myofibroblasts in fibrotic niches. However, macrophages exhibit high heterogeneity, hindering their potential as therapeutic cell targets. Herein, we aimed to eliminate specific macrophage subsets that drive the profibrotic immune response in the kidney both temporally and spatially. We identified the major profibrotic macrophage subset (Fn1+Spp1+Arg1+) in the kidney and then constructed a 12-mer glycopeptide that was designated as bioactivated in vivo assembly PK (BIVA-PK) to deplete these cells. BIVA-PK specifically binds to and is internalized by profibrotic macrophages. By inducing macrophage cell death, BIVA-PK reshaped the renal microenvironment and suppressed profibrotic immune responses. The robust efficacy of BIVA-PK in ameliorating renal fibrosis and preserving kidney function highlights the value of targeting macrophage subsets as a potential therapy for patients with CKD.

The NS2B-PP1α-eIF2α axis: Inhibiting stress granule formation and Boosting Zika virus replication.

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.

A Hydroxylamine-Mediated Amidination of Lysine Residues That Retains the Protein's Positive Charge.

Lysine-specific peptide and protein modification strategies are widely used to study charge-related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge-related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge-related protein properties, such as liquid-liquid phase separation behaviour of α-synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine-containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.

Novel diamine-scaffold based N-acetylgalactosamine (GalNAc)-siRNA conjugate: synthesis and in vivo activities.

GalNAc-conjugated siRNA has shown remarkable potential in liver-targeted delivery in recent years. In general, tetrahydroxymethylmethane or other branching clusters constitute the basis of GalNAc's structure, which yields trivalent or tetravalent ligands. A novel diamine-scaffold GalNAc conjugate was synthesized and evaluated for its efficiency in siRNA administration. It exhibits comparable siRNA delivery effectiveness to a GalNAc NAG37 phase II clinical drug candidate targeting ANGPTL3. In addition, it exhibits more powerful silencing activity when connected to the 3'-end of the sense strand with an additional PS-linkage instead of a PO linkage between the ligand and the oligomer compared to a GalNAc L96 standard targeting TTR. Taken together, the incorporation of a diamine-scaffold into the GalNAc conjugate structure has potential in the field of gene therapy.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors.

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

External Validation of the International IgA Nephropathy Prediction Tool in Older Adult Patients.

Purpose: The International IgA Nephropathy Prediction Tool (IIgAN-PT) can predict the risk of End-stage renal disease (ESRD) or estimated glomerular filtration rate (eGFR) decline ≥ 50% for adult IgAN patients. Considering the differential progression between older adult and adult patients, this study aims to externally validate its performance in the older adult cohort. Patients and Methods: We analyzed 165 IgAN patients aged 60 and above from six medical centers, categorizing them by their predicted risk. The primary outcome was a ≥50% reduction in estimated glomerular filtration rate (eGFR) or kidney failure. Evaluation of both models involved concordance statistics (C-statistics), time-dependent receiver operating characteristic (ROC) curves, Kaplan-Meier survival curves, and calibration plots. Comparative reclassification was conducted using net reclassification improvement (NRI) and integrated discrimination improvement (IDI). Results: The study included 165 Chinese patients (median age 64, 60% male), with a median follow-up of 5.1 years. Of these, 21% reached the primary outcome. Both models with or without race demonstrated good discrimination (C-statistics 0.788 and 0.790, respectively). Survival curves for risk groups were well-separated. The full model without race more accurately predicted 5-year risks, whereas the full model with race tended to overestimate risks after 3 years. No significant reclassification improvement was noted in the full model without race (NRI 0.09, 95% CI: -0.27 to 0.34; IDI 0.003, 95% CI: -0.009 to 0.019). Conclusion: : Both models exhibited excellent discrimination among older adult IgAN patients. The full model without race demonstrated superior calibration in predicting the 5-year risk.

Label-Free Light Scattering Imaging with Purified Brownian Motion Differentiates Small Extracellular Vesicles in Cell Microenvironments.

Small extracellular vesicles (sEVs) are heterogeneous biological nanoparticles (NPs) with wide biomedicine applications. Tracking individual nanoscale sEVs can reveal information that conventional microscopic methods may lack, especially in cellular microenvironments. This usually requires biolabeling to identify single sEVs. Here, we developed a light scattering imaging method based on dark-field technology for label-free nanoparticle diffusion analysis (NDA). Compared with nanoparticle tracking analysis (NTA), our method was shown to determine the diffusion probabilities of a single NP. It was demonstrated that accurate size determination of NPs of 41 and 120 nm in diameter is achieved by purified Brownian motion (pBM), without or within the cell microenvironments. Our pBM method was also shown to obtain a consistent size estimation of the normal and cancerous plasma-derived sEVs without and within cell microenvironments, while cancerous plasma-derived sEVs are statistically smaller than normal ones. Moreover, we showed that the velocity and diffusion coefficient are key parameters for determining the diffusion types of the NPs and sEVs in a cancerous cell microenvironment. Our light scattering-based NDA and pBM methods can be used for size determination of NPs, even in cell microenvironments, and also provide a tool that may be used to analyze sEVs for many biomedical applications.

Copper-Catalyzed 1,2-Sulfonyletherification of 1,3-Dienes.

A selective three-component 1,2-sulfonyl etherification of aryl 1,3-dienes enabled by copper catalysis to afford biologically interesting alkenyl 1,2-sulfone ether derivatives through C-S and C-O bond formation is described. The protocol proceeds with the sulfonyl chloride and alcohols under simple, mild, and base-free conditions, providing a straightforward route to sulfonylated allyl ether compounds with broad functional group tolerance and excellent chemo- and regioselectivity. Mechanistic studies indicate that the selective alkene difunctionalization includes a key copper-mediated single-electron transfer process.

Targeting DUSP5 suppresses malignant phenotypes of BRAF-mutant thyroid cancer cells and improves their response to sorafenib.

PURPOSE: The role of dual-specificity phosphatase-5 (DUSP5) in BRAF-mutant thyroid cancers remains unclear. The aims of this study are to investigate the role of DUSP5 in BRAF-mutant thyroid cancer cells, explore its value in the diagnosis and evaluate therapeutic potential of targeting DUSP5 combined with sorafenib for BRAF-mutant thyroid cancer patients. METHODS: The role of DUSP5 in thyroid cancer cells was determined by a series of in vitro and in vivo experiments. Underlying mechanisms were explored by western blotting analysis. The diagnostic value of combination detection of DUSP5 expression and BRAFV600E mutation was evaluated using ROC curve. RESULTS: Knocking down DUSP5 in BRAF-mutant thyroid cancer cells significantly inhibited colony formation, cell migration and invasion, meanwhile, induced cell cycle arrest and cell apoptosis. Moreover, inhibition of DUSP5 improved the anti-tumor efficacy of sorafenib both in vitro and in vivo. Besides, combination detection of DUSP5 expression and BRAFV600E mutation showed much more accuracy in preoperative diagnosis of thyroid cancer. CONCLUSIONS: Our data demonstrate an oncogenic role of DUSP5 in BRAF-mutant thyroid cancer cells, and combined analysis of its expression and BRAFV600E mutation can accurately diagnose thyroid cancer. In addition, inhibition of DUSP5 improves the response of BRAF-mutant thyroid cancer cells to sorafenib.

Versatile chondroitin sulfate-based nanoplatform for chemo-photodynamic therapy against triple-negative breast cancer.

Versatile nanoplatform equipped with chemo-photodynamic therapeutic attributes play an important role in improving the effectiveness of tumor treatments. Herein, we developed multifunctional nanoparticles based on chondroitin sulfate A (CSA) for the targeted delivery of chlorin e6 (Ce6) and doxorubicin (DOX), in a combined chemo-photodynamic therapy against triple-negative breast cancer. CSA was chosen for its hydrophilic properties and its affinity to CD44 receptor-overexpressed tumor cells. The CSA-ss-Ce6 (CSSC) conjugate was synthesized utilizing a disulfide linker. Subsequently, DOX-loaded CSSC (CSSC-D) nanoparticles were fabricated, showcasing a nearly spherical shape with an average particle size of 267 nm. In the CSSC-D nanoparticles, the chemically attached Ce6 constituted 1.53 %, while the physically encapsulated DOX accounted for 8.11 %. Both CSSC-D and CSSC nanoparticles demonstrated a reduction-sensitive release of DOX or Ce6 in vitro. Under near-infrared (NIR) laser irradiation, CSSC-D showed the enhanced generation of reactive oxygen species (ROS), improving cytotoxic effects against triple-negative breast cancer 4T1 and MDA-MB-231 cells. Remarkably, the CSSC-D with NIR exhibited the most potent tumor growth inhibition in comparison to other groups in the 4T1-bearing Balb/c mice model. Overall, this CSSC-D nanoplatform shows significant promise as a powerful tool for a synergetic approach in chemo-photodynamic therapy in triple-negative breast cancer.

Relationship Between Urinary Iodine Concentration and the Prevalence of Thyroid Nodules and Subclinical Hypothyroidism.

The aim of the study was to investigate the iodine intake in the resident population in Xi'an and analyze the relationship between iodine nutritional status and the prevalence of subclinical hypothyroidism and thyroid nodules (TNs). A total of 2507 people were enrolled in Xi'an. Venous serum thyroid stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TgAb), urinary iodine concentration (UIC), and thyroid ultrasonography were collected. Patients with abnormal TSH were checked for free thyroxine (FT4) and triiodothyronine (FT3). Adults in Xi'an had median UICs of 220.80 µg/L and 178.56 µg/l, respectively. A sum of 16.78% of people had subclinical hypothyroidism. Both iodine excess and iodine deficit increased the frequency of subclinical hypothyroidism. The lowest was around 15.09% in females with urine iodine levels between 200 and 299 µg/l. With a rate of 10.69%, the lowest prevalence range for males was 100-199 µg/l. In Xi'an, 11.37% of people have TNs. In comparison to other UIC categories, TN occurrences were higher in females (18.5%) and males (12%) when UIC were below 100 µg/l. In conclusion, iodine intake was sufficient in the Xi'an area, while the adults' UIC remains slightly higher than the criteria. Iodine excess or deficiency can lead to an increase in the prevalence of subclinical hypothyroidism. Patients with iodine deficiency are more likely to develop TNs.

Innovative gelatin-based micelles with AS1411 aptamer targeting and reduction responsiveness for doxorubicin delivery in tumor therapy.

Herein, we constructed innovative reduction-sensitive and targeted gelatin-based micelles for doxorubicin (DOX) delivery in tumor therapy. AS1411 aptamer-modified gelatin-ss-tocopherol succinate (AGSST) and the control GSST without AS1411 modification were synthesized and characterized. Antitumor drug DOX-containing AGSST (AGSST-D) and GSST-D nanoparticles were prepared, and their shapes were almost spherical. Reduction-responsive characteristics of DOX release in vitro were revealed in AGSST-D and GSST-D. Compared with non-targeted GSST-D, AGSST-D demonstrated better intracellular uptake and stronger cytotoxicity against nucleolin-overexpressed A549 cells. Importantly, AGSST-D micelles showed more effective killing activity in A549-bearing mice than GSST-D and DOX⋅HCl. It was revealed that AGSST-D micelles had no obvious systemic toxicity. Overall, AGSST micelles would have the potential to be an effective drug carrier for targeted tumor therapy.