Correction: Spontaneous mutations and mutational responses to penicillin treatment in the bacterial pathogen Streptococcus pneumoniae D39.

[This corrects the article DOI: 10.1007/s42995-024-00220-6.].

Extended pegRNAs enhance the editing capability of Prime editing.

Genome editing is highly valuable in biomedical research. Despite their versatility, current Prime editing (PE) techniques are limited to short sequence alterations [up to ~44 base pairs (bp)], and exhibit inconsistent or low efficiency across genomic loci, particularly when faced with poly-T sequences. To address these challenges, we developed an extended PE (exPE) technique that can potentially execute any precise genome editing. By harnessing RNA polymerase II (Pol II) promoters to transcribe extended PE guide RNAs (expegRNAs), exPE substantially improves editing efficiency and overcomes the challenges posed by poly-T sequences. Compared with conventional PE, exPE achieves up to a 14-fold increase in the efficiency of base conversions and short insertions, and, remarkably, up to a 259-fold improvement in regions with poly-T sequences. Uniquely, exPE enables seamless insertion of gene-sized DNA fragments into genomes, potentially correcting nearly 90% of human genetic variants, thereby broadening its applications in genetic research and therapy.

Causal associations between vitamin D and postpartum depression: A bidirectional mendelian randomization study.

Background: Previous epidemiological studies have reported associations between vitamin D and postpartum depression (PPD); however, the findings are inconsistent. This study employs bidirectional Mendelian Randomization (MR) to investigate the causal link between serum 25-hydroxyvitamin D [25(OH)D] levels and PPD. By utilizing genetic data from cohorts, this research aims to provide a more robust understanding of the potential relationship between vitamin D and PPD, addressing a critical gap in the current literature. Methods: A bidirectional MR analysis was conducted to investigate the genetic association between serum 25(OH)D and PPD using summary statistics extracted from GWAS datasets. The study included data from 15,668 patients with PPD and 376,755 healthy controls of European ancestry. The GWAS data for 25(OH)D were obtained from two studies within the UK Biobank, encompassing 496,946 and 79,366 participants. The primary analysis employed the inverse-variance weighted (IVW) method, while supplementary MR estimates were derived through the MR-Egger and weighted median (WME) methods. Furthermore, sensitivity analyses were implemented to ensure robustness and reliability, including Cochran's Q test, MR-PRESSO, MR-Egger intercept test, and the leave-one-out test. Results: The MR study revealed no substantial genetic correlation between serum 25(OH)D levels and PPD (OR = 1.065, 95%CI = 0.878-1.293, P = 0.522 for set A; OR = 0.978, 95 % CI = 0.669-1.430, P = 0.910 for set B). Additionally, in the reverse analysis, we did not observe a significant causal impact of PPD on serum 25(OH)D (OR = 1.001, 95%CI = 0.974-1.028, P = 0.951 for set A; OR = 1.011, 95%CI = 0.992-1.031, P = 0.261 for set B). The results obtained from MR-Egger and WME analyses concord with those derived from the IVW method. Conducting leave-one-out tests did not identify any single nucleotide polymorphism that might have influenced the MR results, confirming the robustness and reliability of the findings. Conclusions: The results suggest the absence of a causal link between vitamin D concentrations and PPD. Inconsistent observations in previous observational studies may be attributed to residual confounding.

Spontaneous mutations and mutational responses to penicillin treatment in the bacterial pathogen Streptococcus pneumoniae D39.

Bacteria with functional DNA repair systems are expected to have low mutation rates due to strong natural selection for genomic stability. However, our study of the wild-type Streptococcus pneumoniae D39, a pathogen responsible for many common diseases, revealed a high spontaneous mutation rate of 0.02 per genome per cell division in mutation-accumulation (MA) lines. This rate is orders of magnitude higher than that of other non-mutator bacteria and is characterized by a high mutation bias in the A/T direction. The high mutation rate may have resulted from a reduction in the overall efficiency of selection, conferred by the tiny effective population size in nature. In line with this, S. pneumoniae D39 also exhibited the lowest DNA mismatch-repair (MMR) efficiency among bacteria. Treatment with the antibiotic penicillin did not elevate the mutation rate, as penicillin did not induce DNA damage and S. pneumoniae lacks a stress response pathway. Our findings suggested that the MA results are applicable to within-host scenarios and provide insights into pathogen evolution. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00220-6.

HM_ADET: a hybrid model for automatic detection of eyelid tumors based on photographic images.

BACKGROUND: The accurate detection of eyelid tumors is essential for effective treatment, but it can be challenging due to small and unevenly distributed lesions surrounded by irrelevant noise. Moreover, early symptoms of eyelid tumors are atypical, and some categories of eyelid tumors exhibit similar color and texture features, making it difficult to distinguish between benign and malignant eyelid tumors, particularly for ophthalmologists with limited clinical experience. METHODS: We propose a hybrid model, HM_ADET, for automatic detection of eyelid tumors, including YOLOv7_CNFG to locate eyelid tumors and vision transformer (ViT) to classify benign and malignant eyelid tumors. First, the ConvNeXt module with an inverted bottleneck layer in the backbone of YOLOv7_CNFG is employed to prevent information loss of small eyelid tumors. Then, the flexible rectified linear unit (FReLU) is applied to capture multi-scale features such as texture, edge, and shape, thereby improving the localization accuracy of eyelid tumors. In addition, considering the geometric center and area difference between the predicted box (PB) and the ground truth box (GT), the GIoU_loss was utilized to handle cases of eyelid tumors with varying shapes and irregular boundaries. Finally, the multi-head attention (MHA) module is applied in ViT to extract discriminative features of eyelid tumors for benign and malignant classification. RESULTS: Experimental results demonstrate that the HM_ADET model achieves excellent performance in the detection of eyelid tumors. In specific, YOLOv7_CNFG outperforms YOLOv7, with AP increasing from 0.763 to 0.893 on the internal test set and from 0.647 to 0.765 on the external test set. ViT achieves AUCs of 0.945 (95% CI 0.894-0.981) and 0.915 (95% CI 0.860-0.955) for the classification of benign and malignant tumors on the internal and external test sets, respectively. CONCLUSIONS: Our study provides a promising strategy for the automatic diagnosis of eyelid tumors, which could potentially improve patient outcomes and reduce healthcare costs.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E. coli.

Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30-50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00185-y.

An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data.

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00182-1.

Prognostic and therapeutic implications of iron-related cell death pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is diverse in terms of its molecular abnormalities and clinical outcomes. Iron homeostasis and cell death pathways play crucial roles in cancer pathogenesis, including AML. The objective of this study was to examine the clinical significance of genes involved in iron-related cell death and apoptotic pathways in AML, with the intention of providing insights that could have prognostic implications and facilitate the development of targeted therapeutic interventions. Gene expression profiles, clinical information, and molecular alterations were integrated from multiple datasets, including TCGA-LAML and GSE71014. Our analysis identified specific molecular subtypes of acute myeloid leukemia (AML) displaying varying outcomes, patterns of immune cell infiltration, and profiles of drug sensitivity for targeted therapies based on the expression of genes involved in iron-related apoptotic and cell death pathways. We further developed a risk model based on four genes, which demonstrated promising prognostic value in both the training and validation cohorts, indicating the potential of this model for clinical decision-making and risk stratification in AML. Subsequently, Western blot analysis showed that the expression levels of C-Myc and CyclinD1 were significantly reduced after CD4 expression levels were knocked down. The findings underscore the potential of iron-related cell death pathways as prognostic biomarkers and therapeutic targets in AML, paving the way for further research aimed at understanding the molecular mechanisms underlying the correlation between iron balance, apoptosis regulation, and immune modulation in the bone marrow microenvironment.

The divergence of mutation rates and spectra across the Tree of Life.

Owing to advances in genome sequencing, genome stability has become one of the most scrutinized cellular traits across the Tree of Life. Despite its centrality to all things biological, the mutation rate (per nucleotide site per generation) ranges over three orders of magnitude among species and several-fold within individual phylogenetic lineages. Within all major organismal groups, mutation rates scale negatively with the effective population size of a species and with the amount of functional DNA in the genome. This relationship is most parsimoniously explained by the drift-barrier hypothesis, which postulates that natural selection typically operates to reduce mutation rates until further improvement is thwarted by the power of random genetic drift. Despite this constraint, the molecular mechanisms underlying DNA replication fidelity and repair are free to wander, provided the performance of the entire system is maintained at the prevailing level. The evolutionary flexibility of the mutation rate bears on the resolution of several prior conundrums in phylogenetic and population-genetic analysis and raises challenges for future applications in these areas.

Promoting AMPK/SR-A1-mediated clearance of HMGB1 attenuates chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of chemotherapy with poorly understood mechanisms and few treatments. High-mobility group box 1 (HMGB1)-induced neuroinflammation is the main cause of CIPN. Here, we aimed to illustrate the role of the macrophage scavenger receptor A1 (SR-A1) in HMGB1 clearance and CIPN resolution. METHODS: Oxaliplatin (L-OHP) was used to establish a CIPN model. Recombinant HMGB1 (rHMGB1) (his tag) was used to evaluate the phagocytosis of HMGB1 by macrophages. RESULTS: In the clinic, HMGB1 expression and MMP-9 activity were increased in the plasma of patients with CIPN. Plasma HMGB1 expression was positively correlated with the cumulative dose of L-OHP and the visual analog scale. In vitro, engulfment and degradation of rHMGB1 increased and inflammatory factor expression decreased after AMP-activated protein kinase (AMPK) activation. Neutralizing antibodies, inhibitors, or knockout of SR-A1 abolished the effects of AMPK activation on rHMGB1 engulfment. In vivo, AMPK activation increased SR-A1 expression in the dorsal root ganglion, decreased plasma HMGB1 expression and MMP-9 activity, and attenuated CIPN, which was abolished by AMPK inhibition or SR-A1 knockout in the CIPN mice model. CONCLUSION: Activation of the AMPK/SR-A1 axis alleviated CIPN by increasing macrophage-mediated HMGB1 engulfment and degradation. Therefore, promoting HMGB1 clearance may be a potential treatment strategy for CIPN. Video abstract.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway.

Opioids, such as morphine, are the most potent drugs used to treat pain. Long-term use results in high tolerance to morphine. High mobility group box-1 (HMGB1) has been shown to participate in neuropathic or inflammatory pain, but its role in morphine tolerance is unclear. In this study, we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days. We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1. HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1ß production by increasing Toll-like receptor 4 receptor expression in microglia, thereby inducing morphine tolerance. Glycyrrhizin, an HMGB1 inhibitor, markedly attenuated chronic morphine tolerance in the mouse model. Finally, compound C (adenosine 5'-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin (heme oxygenase-1 inhibitor) alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1ß production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tolerance, and alleviated morphine tolerance in the mouse model. These findings suggest that morphine induces HMGB1 release via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway, and that inhibiting this signaling pathway can effectively reduce morphine tolerance.

Neutrophil extracellular traps as a unique target in the treatment of chemotherapy-induced peripheral neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of chemotherapy and remains a huge clinical challenge. Here, we explore the role of microcirculation hypoxia induced by neutrophil extracellular traps (NETs) in the development of CIPN and look for potential treatment. METHODS: The expression of NETs in plasma and dorsal root ganglion (DRG) are examined by ELISA, IHC, IF and Western blotting. IVIS Spectrum imaging and Laser Doppler Flow Metry are applied to explore the microcirculation hypoxia induced by NETs in the development of CIPN. Stroke Homing peptide (SHp)-guided deoxyribonuclease 1 (DNase1) is used to degrade NETs. FINDINGS: The level of NETs in patients received chemotherapy increases significantly. And NETs accumulate in the DRG and limbs in CIPN mice. It leads to disturbed microcirculation and ischemic status in limbs and sciatic nerves treated with oxaliplatin (L-OHP). Furthermore, targeting NETs with DNase1 significantly reduces the chemotherapy-induced mechanical hyperalgesia. The pharmacological or genetic inhibition on myeloperoxidase (MPO) or peptidyl arginine deiminase-4 (PAD4) dramatically improves microcirculation disturbance caused by L-OHP and prevents the development of CIPN in mice. INTERPRETATION: In addition to uncovering the role of NETs as a key element in the development of CIPN, our finding provides a potential therapeutic strategy that targeted degradation of NETs by SHp-guided DNase1 could be an effective treatment for CIPN. FUNDING: This study was funded by the National Natural Science Foundation of China81870870, 81971047, 81773798, 82271252; Natural Science Foundation of Jiangsu ProvinceBK20191253; Major Project of "Science and Technology Innovation Fund" of Nanjing Medical University2017NJMUCX004; Key R&D Program (Social Development) Project of Jiangsu ProvinceBE2019732; Nanjing Special Fund for Health Science and Technology DevelopmentYKK19170.

An automated, fully-integrated nucleic acid analyzer based on microfluidic liquid handling robot technique.

On-site nucleic acid testing (NAT) plays an important role for disease monitoring and pathogen diagnosis. In this work, we developed an automated and fully-integrated nucleic acid analyzer by combining the automated liquid handling robot technique with the microfluidic droplet-based real-time PCR assay technique. The present analyzer could achieve multiple operations including sample introduction, nucleic acid extraction based on magnetic solid-phase extraction, reverse transcription and, sample droplet generation, PCR amplification, real-time and dual fluorescence detection of droplet array. A strategy of constructing an integrated compact and low-cost system was adopted to minimize the analyzer size to 50 × 45 × 45 cm (length × width × height), and reduce the instrument cost to ca. $900 with a single analysis cost less than $5. A simple chip was also designed to pre-load reagents and carry oil-covered PCR reaction droplets. We applied the analyzer to identify eight types of influenza pathogens in human throat swabs, and the results were consistent with the colloidal gold method.

NET-Triggered NLRP3 Activation and IL18 Release Drive Oxaliplatin-Induced Peripheral Neuropathy.

Oxaliplatin is an antineoplastic agent frequently used in the treatment of gastrointestinal tumors. However, it causes dose-limiting sensorimotor neuropathy, referred to as oxaliplatin-induced peripheral neuropathy (OIPN), for which there is no effective treatment. Here, we report that the elevation of neutrophil extracellular traps (NET) is a pathologic change common to both cancer patients treated with oxaliplatin and a murine model of OIPN. Mechanistically, we found that NETs trigger NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and the subsequent release of IL18 by macrophages, resulting in mechanical hyperalgesia. In NLRP3-deficient mice, the mechanical hyperalgesia characteristic of OIPN in our model was reduced. In addition, in the murine model, treatment with the IL18 decoy receptor IL18BP prevented the development of OIPN. We further showed that eicosapentaenoic acid (EPA) reduced NET formation by suppressing the LPS-TLR4-JNK pathway and thereby abolished NLRP3 inflammasome activation and the subsequent secretion of IL18, which markedly prevented oxaliplatin-induced mechanical hyperalgesia in mice. These results identify a role for NET-triggered NLRP3 activation and IL18 release in the development of OIPN and suggest that utilizing IL18BP and EPA could be effective treatments for OIPN.

Development and Preclinical Evaluation of a Near-Infrared Fluorescence Probe Based on Tailored Hepatitis B Core Particles for Imaging-Guided Surgery in Breast Cancer.

Purpose: Tumor-free surgical margin is crucial but challenging in breast-conserving surgery (BCS). Fluorescence imaging is a promising strategy for surgical navigation that can reliably assist the surgeon with visualization Of the tumor in real-time. Notably, finding an optimized fluorescent probe has been a challenging research topic. Herein, we developed a novel near-infrared (NIR) fluorescent probe based on tailored Hepatitis B Core virus-like protein (HBc VLP) and presented the preclinical imaging-guided surgery. Methods: The RGD-HBc160 VLP was synthesized by genetic engineering followed encapsulation of ICG via disassembly-reassembly. The applicability of the probe was tested for cell and tissue binding capacities through cell-based plate assays, xenograft mice model, and MMTV-PyVT mammary tumor transgenic mice. Subsequently, the efficacy of RGD-HBc160/ICG-guided surgery was evaluated in an infiltrative tumor-bearing mouse model. The protein-induced body's immune response was further assessed. Results: The prepared RGD-HBc160/ICG showed outstanding integrin αvß3 targeting ability in vitro and in vivo. After intravenous administration of probe, the fluorescence guidance facilitated more complete tumor resection and improved overall survival Of the infiltrative tumor-bearing mice. The probe also showed the excellent capability to differentiate between benign and malignant breast tissues in the mammary tumor transgenic mice. Interestingly, the ingenious tailoring of HBc VLP could not only endow its tumor-targeting ability towards integrin αvß3 but also significantly reduce the humoral and cellular immune response. Conclusion: The RGD-HBc160/ICG holds promise as an effective tool to delineate tumor margin. These results have translational potential to achieve margin-negative resection and improve the stratification of patients for a potentially curative.

Photoacoustic molecular imaging-escorted adipose photodynamic-browning synergy for fighting obesity with virus-like complexes.

Photodynamic therapy and adipose browning induction are two promising approaches to reverse obesity. The former strategy acts rapidly and locally, whereas the latter has a more gradual and widespread effect. Despite their complementarity, they have rarely been combined and imaged non-invasively in vivo. Here we introduce an adipose-targeting hepatitis B core protein complex that contains a traceable photosensitizer (ZnPcS4 (zinc phthalocyanine tetrasulfonate)) and a browning agent (rosiglitazone) that allows simultaneous photodynamic and browning treatments, with photoacoustic molecular imaging. After intravenous injection in obese mice, the complex binds specifically to white adipose tissues, especially those rich in blood supply, and drives adipose reduction thanks to the synergy of ZnPcS4 photodynamics and rosiglitazone browning. Using photoacoustic molecular imaging, we could monitor the changes induced by the treatment, which included complex activity, lipid catabolism and angiogenesis. Our findings demonstrate the anti-obesity potential of our feedback-based synergic regimen orchestrated by the targeted hepatitis B core complex.

FUT4siRNA augments the chemosensitivity of non-small cell lung cancer to cisplatin through activation of FOXO1-induced apoptosis.

BACKGROUND: Increased fucosylation is associated with the chemoresistance phenotype. Meanwhile, fucosyltransferase IV (FUT4) amounts are frequently elevated in lung cancer and may be related to increased chemoresistance. METHODS: In the present work, FUT4's role in cisplatin-induced apoptosis was assessed in A549 and H1975 cells, respectively. To clarify whether the FUT4 gene attenuates chemosensitivity in tumor cells, we constructed FUT4siRNA and evaluated its effects on cisplatin-induced apoptosis and cell growth inhibition. Cell viability, apoptosis, migration and invasion assay were conducted to investigate cisplatin sensitivity. The activation of EGFR/AKT/FOXO1 signaling were measured by western blot. The translocation of FOXO1 was assessed by IFC using Laser Scanning Confocal Microscope. RESULTS: We found that FUT4 knockdown dose-dependently increased cisplatin-associated cytotoxicity. Furthermore, FUT4 silencing induced apoptosis and inhibited proliferation in A549 and H1975 cells by suppressing Akt and FOXO1 phosphorylation induced by cisplatin administration, which resulted in nuclear translocation of FOXO1. CONCLUSION: These results suggested FUT4 might control chemoresistance to cisplatin in lung cancer by suppressing FOXO1-induced apoptosis.

The isolation and genome sequencing of a novel cyanophage S-H68 from the Bohai Sea, China.

Cyanobacteria, also known as bule-green algae, are capable of photosynthesis and have a fixed carbon and nitrogen effect. The virus that specifically infects cyanobacteria is called the cyanophage. Cyanophages play a key role in building microbial communities. However, only a small number of cyanophages have been reported so far. In this study, a novel Synechococcus cyanophage S-H68 was isolated from the Bohai Sea of China. Transmission electron microscope observations showed that S-H68 has an icosahedral head, 66 ± 1 nm in diameter, and a tail with a length of 107 ± 1 nm, and should be grouped into the family Siphoviridae. To better understand the genetic diversity of this cyanophage, the complete genome was characterized. It consists of 79,639 -bp -length double-stranded DNA with a GC content of 59.8% and is predicted to have 117 open reading frames (ORFs) with an average length of 655 nucleotides. Using the BLASTN tool in the NCBI database for genome comparison, there was no significant similarity between S-H68 and other known cyanophages. So the present study added a new Siphoviridae cyanophage to the marine phage dataset.

Rescue of HSP70 in Spinal Neurons Alleviates Opioids-Induced Hyperalgesia via the Suppression of Endoplasmic Reticulum Stress in Rodents.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opioids. Endoplasmic reticulum (ER) stress has been shown to contribute to neuropathic or inflammatory pain, but its roles in opioids-induced hyperalgesia (OIH) are elusive. Here, we provide the first direct evidence that ER stress is a significant driver of OIH. GRP78, the ER stress marker, is markedly upregulated in neurons in the spinal cord after chronic morphine treatment. At the same time, morphine induces the activation of three arms of unfolded protein response (UPR): inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1), protein kinase RNA-like ER kinase/eukaryotic initiation factor 2 subunit alpha (PERK/eIF2α), and activating transcription factor 6 (ATF6). Notably, we found that inhibition on either IRE1α/XBP1 or ATF6, but not on PERK/eIF2α could attenuate the development of OIH. Consequently, ER stress induced by morphine enhances PKA-mediated phosphorylation of NMDA receptor subunit 1(NR1) and leads to OIH. We further showed that heat shock protein 70 (HSP70), a molecular chaperone involved in protein folding in ER, is heavily released from spinal neurons after morphine treatment upon the control of KATP channel. Glibenclamide, a classic KATP channel blocker that inhibits the efflux of HSP70 from cytoplasm to extracellular environment, or HSP70 overexpression in neurons, could markedly suppress morphine-induced ER stress and hyperalgesia. Taken together, our findings uncover the induction process and the central role of ER stress in the development of OIH and support a novel strategy for anti-OIH treatment.

Theranostic Quercetin Nanoparticle for Treatment of Hepatic Fibrosis.

The progression of hepatic fibrosis can lead to cirrhosis and hepatic failure, but the development of antifibrotic drugs have faced the challenges of poor effectiveness and targeted specificity. Herein, a theranostic strategy was carried to encapsulate a natural medicine (Quercetin, QR) into hepatitis B core (HBc) protein nanocages (NCs) for imaging and targeted treatment of hepatic fibrosis. It was noted that nanoparticles (RGD-HBc/QR) with surface-displayed RGD targeting ligand exhibit a rather high selectivity toward activated HSCs via the binding affinity with integrin αvß3, and an efficient inhibition of proliferation and activation of hepatic stellate cells (HSCs) in vitro and in vivo. Once encapsulated in quercetin-gadolinium complex and/or labeled with the NIR fluorescent probes (Cy5.5), the resulting nanoparticles (RGD-HBc/QGd) show great potential as NIR fluorescent and magnetic resonance imaging contrast agents for hepatic fibrosis in vivo. Therefore, the multifunctional integrin-targeted nanoparticles could selectively deliver QR to the activated HSCs, and may provide an effective antifibrotic theranostic strategy.