An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Blood-Nanoparticle Interactions Create a Brain Delivery Superhighway for Doxorubicin.

Purpose: This study investigated the brain targeting mechanism of doxorubicin-loaded polybutyl cyanoacrylate (PBCA) nanoparticles, particularly their interactions with the blood-brain barrier (BBB). The BBB protects the brain from drugs in the bloodstream and represents a crucial obstacle in the treatment of brain cancer. Methods: An advanced computer model analyzed the brain delivery of two distinct formulations, Doxil® and surfactant-coated PBCA nanoparticles. Computational learning was combined with in vitro release and cell interaction studies to comprehend the underlying brain delivery pathways. Results: Our analysis yielded a surprising discovery regarding the brain delivery mechanism of PBCA nanoparticles. While Doxil® exhibited the expected behavior, accumulating in the brain through extravasation in tumor tissue, PBCA nanoparticles employed a unique and previously uncharacterized mechanism. They underwent cell hitchhiking, resulting in a remarkable more than 1000-fold increase in brain permeation rate compared to Doxil® (2.59 × 10-4 vs 0.32 h-1). Conclusion: The nonspecific binding to blood cells facilitated and intensified interactions of surfactant-coated PBCA nanoparticles with the vascular endothelium, leading to enhanced transcytosis. Consequently, the significant increase in circulation time in the bloodstream, coupled with improved receptor interactions, contributes to this remarkable uptake of doxorubicin into the brain.

Modeling the Drug delivery Lifecycle of PLG Nanoparticles Using Intravital Microscopy.

Polylactide-co-glycolide (PLG) nanoparticles hold immense promise for cancer therapy due to their enhanced efficacy and biodegradable matrix structure. Understanding their interactions with blood cells and subsequent biodistribution kinetics is crucial for optimizing their therapeutic potential. In this study, three doxorubicin-loaded PLG nanoparticle systems are synthesized and characterized, analyzing their size, zeta potential, morphology, and in vitro release behavior. Employing intravital microscopy in 4T1-tumor-bearing mice, real-time blood and tumor distribution kinetics are investigated. A mechanistic pharmacokinetic model is used to analyze biodistribution kinetics. Additionally, flow cytometry is utilized to identify cells involved in nanoparticle hitchhiking. Following intravenous injection, PLG nanoparticles exhibit an initial burst release (<1 min) and rapidly adsorb to blood cells (<5 min), hindering extravasation. Agglomeration leads to the clearance of one carrier species within 3 min. In stable dispersions, drug release rather than extravasation remains the dominant pathway for drug elimination from circulation. This comprehensive investigation provides valuable insights into the interplay between competing kinetics that influence the lifecycle of PLG nanoparticles post-injection. The findings advance the understanding of nanoparticle behavior and lay the foundation for improved cancer therapy strategies using nanoparticle-based drug delivery systems.

SpatialDM for rapid identification of spatially co-expressed ligand-receptor and revealing cell-cell communication patterns.

Cell-cell communication is a key aspect of dissecting the complex cellular microenvironment. Existing single-cell and spatial transcriptomics-based methods primarily focus on identifying cell-type pairs for a specific interaction, while less attention has been paid to the prioritisation of interaction features or the identification of interaction spots in the spatial context. Here, we introduce SpatialDM, a statistical model and toolbox leveraging a bivariant Moran's statistic to detect spatially co-expressed ligand and receptor pairs, their local interacting spots (single-spot resolution), and communication patterns. By deriving an analytical null distribution, this method is scalable to millions of spots and shows accurate and robust performance in various simulations. On multiple datasets including melanoma, Ventricular-Subventricular Zone, and intestine, SpatialDM reveals promising communication patterns and identifies differential interactions between conditions, hence enabling the discovery of context-specific cell cooperation and signalling.

Fecal Microbiota Transplantation Revealed a Pain-related Gut Microbiota Community in Ovariectomized Mice.

Higher sensitivity to pain is a common clinical symptom in postmenopausal females. The gut microbiota (GM) has recently been identified as participating in various pathophysiological processes and may change during menopause and contribute to multiple postmenopausal symptoms. Here, we investigated the possible correlation between GM alteration and allodynia in ovariectomized (OVX) mice. Results showed that OVX mice exhibited allodynia from 7 weeks after surgery compared with sham-operated (SHAM) mice by comparing pain-related behaviors. Fecal microbiota transplantation (FMT) from OVX mice induced allodynia in normal mice while FMT from SHAM mice alleviated allodynia in OVX mice. Microbiome 16S rRNA sequencing and linear discriminant analysis revealed alteration of the GM after OVX. Furthermore, Spearman's correlation analysis showed associations between pain-related behaviors and genera, and further verification identified the possible pain-related genera complex. Our findings provide new insights into the underlying mechanisms of postmenopausal allodynia, and suggest pain-related microbiota community as a promising therapeutic target. PERSPECTIVE: This article provided the evidence of gut microbiota playing essential roles in postmenopausal allodynia. This work intended to offer a guidance for further mechanism investigation into gut-brain axis and probiotics screening for postmenopausal chronic pain.

Can HER2 1+ Breast Cancer Be Considered as HER2-Low Tumor? A Comparison of Clinicopathological Features, Quantitative HER2 mRNA Levels, and Prognosis among HER2-Negative Breast Cancer.

Background: Human epidermal growth factor receptor 2 (HER2)-low tumor is a new entity defined as HER2 immunohistochemistry (IHC) 1+ or 2+/fluorescence in situ hybridization (FISH)-negative. We aimed to evaluate whether HER2 mRNA levels tested by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) could better define HER2-low tumors. Patients and methods: Consecutive breast cancer patients with hormonal receptor-positive, HER2-negative diseases, and HER2 mRNA results were included. Clinicopathologic features, HER2 mRNA expression level, and prognosis were compared among HER2 0, 1+ and 2+/FISH− groups. Concordance of the HER2 category between qRT-PCR and IHC/FISH was analyzed for each group. Results: 2296 patients were included: 368 (16.0%) HER2 0, 911 (39.7%) 1+, and 1017 (44.3%) 2+/FISH− tumors. HER2 1+ cases shared similarities with HER2 0 tumors in terms of clinicopathologic features (all p > 0.05), whereas IHC 2+/FISH− cases were less often non-IDC (p = 0.045), node-negative (p = 0.044), and Ki-67 < 14% (p <0.001). The mRNA expression was similar between HER2 0 and 1+ cases (p = 0.063), and both were lower than 2+/FISH− cases (p < 0.001). A poor concordance rate was found between IHC/FISH and qRT-PCR for HER2 0 and HER2-low cases (Cohen's kappa 0.126, p < 0.001). No survival difference was observed among these groups, whether stratified by HER2 IHC/FISH status or mRNA level (all p > 0.05). Conclusions: HER2 1+ cases had similar clinicopathological features to HER2 0 breast cancers, and both were different from HER2 2+/FISH− cases. HER2 mRNA levels were comparable between HER2 0 and 1+ tumors, and both were significantly lower than IHC 2+/FISH− tumors. Neither IHC nor qRT-PCR may be optimal to quantify HER2-low expression, especially for HER2 1+ patients.

Accuracy of ultrasonographic changes during neoadjuvant chemotherapy to predict axillary lymph node response in clinical node-positive breast cancer patients.

Purpose: To evaluate whether changes in ultrasound features during neoadjuvant chemotherapy (NAC) could predict axillary node response in clinically node-positive breast cancer patients. Methods: Patients with biopsy-proven node-positive disease receiving NAC between February 2009 and March 2021 were included. Ultrasound (US) images were obtained using a 5-12-MHz linear array transducer before NAC, after two cycles, and at the completion of NAC. Long and short diameter, cortical thickness, vascularity, and hilum status of the metastatic node were retrospectively reviewed according to breast imaging-reporting and data system (BI-RADS). The included population was randomly divided into a training set and a validation set at a 2:1 ratio using a simple random sampling method. Factors associated with node response were identified through univariate and multivariate analyses. A nomogram combining clinical and changes in ultrasonographic (US) features was developed and validated. The receiver operating characteristic (ROC) and calibration plots were applied to evaluate nomogram performance and discrimination. Results: A total of 296 breast cancer patients were included, 108 (36.5%) of whom achieved axillary pathologic complete response (pCR) and 188 (63.5%) had residual nodal disease. Multivariate regression indicated that independent predictors of node pCR contain ultrasound features in addition to clinical features, clinical features including neoadjuvant HER2-targeted therapy and clinical response, ultrasound features after NAC including cortical thickness, hilum status, and reduction in short diameter ≥50%. The nomogram combining clinical features and US features showed better diagnostic performance compared to clinical-only model in the training cohort (AUC: 0.799 vs. 0.699, P=0.001) and the validation cohort (AUC: 0.764 vs. 0.638, P=0.027). Conclusions: Ultrasound changes during NAC could improve the accuracy to predict node response after NAC in clinically node-positive breast cancer patients.

Contralateral S1 nerve root transfer for motor function recovery in the lower extremity among patients with central nervous system injury: study protocol for a randomized controlled trial.

BACKGROUND: Central nervous system injury (CNSI) comprises a series of common diseases that severely affect patients' motor function and quality of life and is associated with high disability and mortality rates. Previous studies have shown that contralateral lumbosacral nerve root transfer significantly improved the function of the paralyzed limb in rat models of CNSI. These studies showed that severing the sacral 1 nerve root (S1) did not damage the function of the ipsilateral lower extremity. Thus, we speculate that contralateral S1 nerve root transfer can improve the recovery of a paralyzed limb. Because no associated rigorously designed randomized controlled trial has evaluated the effectiveness of contralateral S1 nerve transfer thus far, we designed this clinical trial to compare the effects of this new treatment approach with those of traditional treatments in paralyzed patients after chronic CNSI. METHODS: This is a single-center, prospective, randomized controlled trial. Forty patients, who meet the inclusion criteria and have hemiplegia caused by chronic CNSI, will be randomly divided into the surgical or non-surgical group. The treatment effect in the 2 groups will be assessed before and 3, 6, 9, 12, 18, and 24 months after intervention by using numerous scales and resting-state functional magnetic resonance imaging. The primary outcome will be the Fugl-Meyer score for the lower limbs 24 months after treatment. The secondary outcomes include the modified Ashworth spasm scale, the modified Barthel scale, 10-m walking speed measurement results, three-dimensional gait analysis, muscle strength testing, electromyography, and resting-state functional magnetic resonance imaging findings. Safety outcomes and adverse events will be observed simultaneously. DISCUSSION: We expect that the surgery will improve the sensorimotor functions of the paralyzed limb, and the results of this trial will provide high-quality clinical evidence for a new efficient treatment strategy for disability after CNSI. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR1800014414, registration date: 12 January 2018.

Application of vermillion myocutaneous flap in restoration after lip cancer resection.

To investigate the application of the vermillion myocutaneous flap in restoration after lip cancer resection. From July 2012 to December 2019, six patients with lip cancer admitted in our department were included in this study. Total defect of the whole layer of the lip, accounted for 1/3 to 1/2 of the total length of the lip after tumor resection, were repaired by vermillion myocutaneous flap. For the case defect presenting >1/2 of the total length of the lip, a local rotational flap was designed with vermillion myocutaneous flap compound to repair the defect. At present, the patients were followed up for 6 months to 7 years. There was no tumor recurrence. The aesthetic effects and function of lip after operation were ideal, with satisfactory outcomes. Vermillion myocutaneous flap with the labial artery as the axis was an effective method to restore the anatomical structure and function of the lip. In the presence of a defect area of larger than 1/2 of the length of the whole lip, local skin flap can be used for simultaneous repair in order to achieve satisfactory outcome.

Sparks fly between ascorbic acid and iron-based nanozymes: A study on Prussian blue nanoparticles.

Herein we reported Prussian blue nanoparticles (PBNPs) possess ascorbic acid oxidase (AAO)- and ascorbic acid peroxidase (APOD)-like activities, which suppressed the formation of harmful H2O2 and finally inhibited the anti-cancer efficiency of ascorbic acid (AA). This newly revealed correlation between iron and AA could provide new insight for the studies of nanozymes and free radical biology.

Novel Strategy Utilizing Extracellular Cysteine-Rich Domain of Membrane Receptor for Constructing d-Peptide Mediated Targeted Drug Delivery Systems: A Case Study on Fn14.

The development of proteolysis-resistant d-peptide ligands for targeted drug/gene delivery has been greatly limited, due to the challenge that lies in the chemical synthesis of membrane receptors without altering their structures. In the present research, a novel strategy utilizing self-stabilized extracellular CRD of the membrane receptor was developed to construct d-peptide ligands and their mediated targeted drug delivery systems. Fn14, a cell surface receptor overexpressed in many cancers including pancreatic and triple-negative breast cancers, was selected as the model receptor. Fn14 CRD was synthesized and folded, and used to screen Fn14 binding peptides using phage display (l-peptide) and mirror-image phage display (d-peptide) techniques, respectively. The d-peptide ligand successfully mediated targeted drug delivery to Fn14 positive tumor cells. In addition, the d-peptide possessed better target-binding affinity, stromal barrier permeability, and tumor targeting ability in vivo when conjugated with liposomes. More importantly, d-peptide mediated liposomal paclitaxel delivery significantly inhibited pancreatic tumor growth in a subcutaneous xenograft model and drastically prolonged survival in a lung metastasis of breast cancer mouse model. This study demonstrated that mirror-image phage display based on the CRD of membrane receptor can be a promising strategy to advance active targeted drug delivery via biostable d-peptides.

Inducible epitope imprinting: 'generating' the required binding site in membrane receptors for targeted drug delivery.

We report an inducible epitope imprinting strategy that as a template, a flexible peptide chain can have a disordered-to-ordered conformational change by suitable inducement through a molecular imprinting procedure, and the formed nanoparticles can, in turn, remold the original peptide into the expected conformation and specifically bind to the corresponding protein.

Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display.

Although liver fibrosis is a major public health issue, there is still no effective drug therapy in the clinic. Fibroblast growth factor-inducible 14 (Fn14), a membrane receptor highly specifically expressed in activated hepatic stellate cells (HSCs), is the key driver of liver fibrosis, and thus, it has a great potential as a novel target for the development of effective treatment. Here, we identified a d-enantiomeric peptide ligand of Fn14 through mirror-image mRNA display. This included the chemical synthesis of a d-enantiomer of the target protein (extracellular domain of Fn14), identification of an l-peptide ligand of d-Fn14 using a constructed mRNA peptide library, and identification of a d-enantiomer of the l-peptide, which is a ligand of the natural Fn14 for reasons of symmetry. The obtained d-peptide ligand showed strong binding to Fn14 while maintaining high proteolytic resistance. As a targeting moiety, this d-peptide successfully mediated high selectivity of activated HSCs for liposomal vehicles compared to that of other major cell types in the liver and significantly enhanced the accumulation of liposomes in the liver fibrosis region of a carbon tetrachloride-induced mouse model. Moreover, in combination with curcumin as an encapsulated load, a liposomal formulation conjugated with this d-peptide showed powerful inhibition of the proliferation of activated HSCs and reduced the liver fibrosis to a significant extent in vivo. This Fn14-targeting strategy may represent a promising approach to targeted drug delivery for liver fibrosis treatment. Meanwhile, the mirror-image mRNA display can provide a new arsenal for the development of d-peptide-based therapeutics against a variety of human diseases.

[Novel liposomal drug delivery system actively targeting Cryptococcus neoformans and elimination of infection].

The purpose of this study is to develop a liposomal drug delivery system actively targeting Cryptococcus neoformans and explore its feasibility in therapy of cryptococcal infection. The specific fungi-binding peptide was screened from 12-mer random phage display library, and linked to PEG-DSPE as the functional material of liposomes. The targeting capability of peptide-modified liposomes were investigated by fungi binding assay in vitro and fluorescence imaging in vivo. Itraconazole as a model drug were then encapsulated in the liposomes and were evaluated in pharmacodynamic test in vitro and for therapeutic effects against cryptococcal meningitis complicated with pulmonary cryptococcosis in vivo. The results showed that the peptide (sequence: NNHREPPDHRTS) could selectively recognize Cryptococcus and effectively mediate the corresponding liposomal formulation to accumulate in the infection site in vivo. This peptide-modified liposome has a small particle size (mean diameter of 88.25 ± 2.43 nm) with a homogeneous distribution and high encapsulation efficiency (88.05 ± 0.25 %) of itraconazole. After intravenous administration, the pathogens were obviously eliminated in lung and brain, and the life-span of model mice were significantly prolonged, suggesting a promising potential of this cryptococcosis targeting strategy.