Receptor Ligand-Free Mesoporous Silica Nanoparticles: A Streamlined Strategy for Targeted Drug Delivery across the Blood-Brain Barrier.

Mesoporous silica nanoparticles (MSNs) represent a promising avenue for targeted brain tumor therapy. However, the blood-brain barrier (BBB) often presents a formidable obstacle to efficient drug delivery. This study introduces a ligand-free PEGylated MSN variant (RMSN25-PEG-TA) with a 25 nm size and a slight positive charge, which exhibits superior BBB penetration. Utilizing two-photon imaging, RMSN25-PEG-TA particles remained in circulation for over 24 h, indicating significant traversal beyond the cerebrovascular realm. Importantly, DOX@RMSN25-PEG-TA, our MSN loaded with doxorubicin (DOX), harnessed the enhanced permeability and retention (EPR) effect to achieve a 6-fold increase in brain accumulation compared to free DOX. In vivo evaluations confirmed the potent inhibition of orthotopic glioma growth by DOX@RMSN25-PEG-TA, extending survival rates in spontaneous brain tumor models by over 28% and offering an improved biosafety profile. Advanced LC-MS/MS investigations unveiled a distinctive protein corona surrounding RMSN25-PEG-TA, suggesting proteins such as apolipoprotein E and albumin could play pivotal roles in enabling its BBB penetration. Our results underscore the potential of ligand-free MSNs in treating brain tumors, which supports the development of future drug-nanoparticle design paradigms.

[Neurologic complications in children with enterovirus 71-infected hand-foot-mouth disease : clinical features, MRI findings and follow-up study].

OBJECTIVE: To explore the clinical and magnetic resonance imaging (MRI) characteristics and the follow-up outcomes of neurologic complications in children with enterovirus 71-infected hand-foot-mouth disease. METHODS: The clinical and MRI manifestations and follow-up outcomes in 35 children, at Second Affiliated Hospital, Wenzhou Medical College from August 2008 to November 2010, hospitalized with neurologic complications of enterovirus 71-infected hand-foot-mouth disease were retrospectively analyzed. RESULTS: Six children with aseptic meningitis presented the clinical symptoms and signs of meningitis. Five of them showed subdural effusion and ventriculomegaly, or both on MRI. At follow-ups, neurologic sequel could not be found. Among 24 cases with brainstem encephalitis, there were myoclonic jerks and tremor, ataxia, or both (grade I disease, n = 12), myoclonus and cranial-nerve involvement (grade II disease, n = 4), and cardiopulmonary failure after brain-stem infection (grade III disease, n = 8). In patients with brainstem encephalitis, lesions were predominantly located at the posterior portions of medulla and pons with hypointensity on T1WI and hyperintensity on T2WI. Cerebellar dentate nucleus, caudate nucleus and lenticular nucleus could also be involved. At follow-ups, the patients with mild symptoms had no neurologic sequel and the lesions within brain stem became small or vanished in most cases. While in the majority of serious patients, neurologic sequel could be found and the lesions located at brain stem became encephalomalacia. Fourteen cases with acute flaccid paralysis presented acute limb myasthenia with tendon reflex and muscular tension decreased. On spinal MRI, the lesions predominantly involved anterior horn regions of spinal cord with hypointensity on T1WI and hyperintensity on T2WI. Most patients improved their muscle strength and most lesions of spinal cord became smaller or vanished during follow-ups. CONCLUSION: MRI is the most effective modality of diagnosis and follow-up for neurologic complications in children with enterovirus 71-infected hand-foot-mouth disease. On MRI, the lesions mainly involve the anterior horn of spinal cord, medulla oblongata and pons. At follow-ups, most patients have no neurologic sequel and the visualized lesions will be absorbed after active treatment.

Efficient Strategy to Design Protease Inhibitors: Application to Enterovirus 71 2A Protease.

One strategy to counter viruses that persistently cause outbreaks is to design molecules that can specifically inhibit an essential multifunctional viral protease. Herein, we present such a strategy using well-established methods to first identify a region present only in viral (but not human) proteases and find peptides that can bind specifically to this "unique" region by maximizing the protease-peptide binding free energy iteratively using single-point mutations starting with the substrate peptide. We applied this strategy to discover pseudosubstrate peptide inhibitors for the multifunctional 2A protease of enterovirus 71 (EV71), a key causative pathogen for hand-foot-and-mouth disease affecting young children, along with coxsackievirus A16. Four peptide candidates predicted to bind EV71 2A protease more tightly than the natural substrate were experimentally validated and found to inhibit protease activity. Furthermore, the crystal structure of the best pseudosubstrate peptide bound to the EV71 2A protease was determined to provide a molecular basis for the observed inhibition. Since the 2A proteases of EV71 and coxsackievirus A16 share nearly identical sequences and structures, our pseudosubstrate peptide inhibitor may prove useful in inhibiting the two key pathogens of hand-foot-and-mouth disease.

Critical Features for Mesoporous Silica Nanoparticles Encapsulated into Erythrocytes.

Mesoporous silica nanoparticles (MSNs) hold great potential as a versatile platform for biomedical applications, especially drug delivery. However, evidence shows that MSNs even when PEGylated are rapidly cleared from the bloodstream by the monocyte phagocytic system. Erythrocytes, also called red blood cells (RBCs), can serve as biocompatible carriers of various bioactive substances, including drugs, enzymes, and peptides. In this work, we synthesize a series of fluorescent PEGylated MSNs with different synthetic diameters ranging from 10 to 200 nm and investigate the size effect on their encapsulation in human RBCs (hRBCs) by a hypotonic dialysis-based method. According to fluorescence images and flow cytometry analyses, we demonstrated that a hydrodynamic diameter below 30 nm is critical for efficient MSN encapsulation. Confocal microscopy and scanning electron microscopy images further confirmed that PEGylated MSNs were successfully embedded inside RBC. PEGylation serves an important role not only for stabilizing MSNs in biological milieu but also for reducing significant hemolysis caused by bare MSNs and thus for successful encapsulation. In addition to PEGylation, we further introduce positively charged functional groups onto the MSNs to show that nanoparticle-encapsulated hRBCs could serve as depots for delivering biological molecules through electrostatic attraction or chemical conjugation with MSNs. Also, we verify the existence of CD47 membrane protein, a marker of self, on the nanoparticle-encapsulated hRBCs and assess its ability of circulation in the blood, which could act as a circulation reservoir for delivering pharmacological substances through an osmosis-based method with MSNs.

Identification of Norovirus and Human Parechovirus in Patients With Hand, Foot and Mouth Disease Syndrome.

BACKGROUND: Hand, foot and mouth disease (HFMD) is caused mostly by enteroviruses. However, other viral agents also can cause similar syndromes, and hence, the infections they cause are often misdiagnosed clinically. To determine non-enterovirus etiologic agents in HFMD-like cases, we screened enterovirus-negative samples collected from the patients who were clinically diagnosed as HFMD in China. METHODS: Two hundred enterovirus-negative samples were collected previously in Wenzhou city of Zhejiang province, China. Both high throughput sequencing and RT-PCR were used to screen viral agents. In addition, their clinical features were analyzed. RESULTS: Norovirus (NoV) and human parechovirus (HPeV) were identified from 22 (11.00%) and 9 (4.50%) samples, respectively. In addition, the complete genome sequences were recovered from 4 NoV-positive samples, and the VP1/3Dpol gene sequences were recovered from 5 HPeV-positive samples. Phylogenetic analyses of the NoV sequences revealed that they were closely related to those circulated in other regions of China. Notably, 4 genotypes of HPeVs, including HPeV-1, HPeV-4, HPeV-5 and HPeV-14, were found, indicating high genetic diversity of the virus. Frequent recombination between various genotypes was also observed in the HPeVs. Although most of the patients presented with the clinical features of HFMD, 4 patients infected with NoV GII.4 and 3 patients infected with HPeV-1 (1) and HPeV-4 (2) were characterized with diarrhea. Finally, tonsillitis, convulsion and granulocytopenia were observed in 1 NoV GII.4 patient, while liver dysfunction was found in 1 NoV GII.17 patient. CONCLUSIONS: These data reveal the variety of agents in the cases clinically diagnosed as HFMD.

The tongue features associated with type 2 diabetes mellitus.

Diabetes mellitus (DM) is a public problem closely associated with numerous oral complications, such as coated tongue, xerostomia, salivary dysfunction, etc. Tongue diagnosis plays an important role in clinical prognosis and treatment of diabetes in the traditional Chinese medicine (TCM). This study investigated discriminating tongue features to distinguish between type 2 DM and non-DM individuals through non-invasive TCM tongue diagnosis.The tongue features for 199 patients with type 2 DM, and 372 non-DM individuals, serving as control, are extracted by the automatic tongue diagnosis system (ATDS). A total of 9 tongue features, namely, tongue shape, tongue color, fur thickness, fur color, saliva, tongue fissure, ecchymosis, teeth mark, and red dot. The demography, laboratory, physical examination, and tongue manifestation data between 2 groups were compared.Patients with type 2 DM possessed significantly larger covering area of yellow fur (58.5% vs 22.5%, P < .001), thick fur (50.8% vs 29.2%, P < .001), and bluish tongue (P < .001) than those of the control group. Also, a significantly higher portion (72.7% vs 55.2%, P < .05) of patients with long-term diabetics having yellow fur color than the short-term counterparts was observed.The high prevalence of thick fur, yellow fur color, and bluish tongue in patient with type 2 DM revealed that TCM tongue diagnosis can serve as a preliminary screening procedure in the early detection of type 2 DM in light of its simple and non-invasive nature, followed by other more accurate testing process. To the best of our knowledge, this is the first attempt in applying non-invasive TCM tongue diagnosis to the discrimination of type 2 DM patients and non-DM individuals.

Gender- and age-dependent tongue features in a community-based population.

This study, an important groundwork for clinical tongue diagnosis and future traditional Chinese medicine (TCM) research, tested the hypothesis that some tongue features vary significantly between different gender and age groups by utilizing an automatic tongue diagnosis system (ATDS).A cross-sectional study of 1487 participants from a community-based population was performed. Study subjects with ages ranging from 20 to 92 were categorized into 3 groups: <40, 40 to 64, and ≥65 years old, and the subjects were also stratified according to gender. Tongue images were collected at the end of each normal health examination routine to further derive the relevant tongue features of every participant by using the ATDS developed by our team. There were a total of nine tongue features that were identified: tongue shape, tongue color, fur thickness, fur color, saliva, tongue fissure, ecchymosis, teeth mark, and red dot. The corresponding tongue features, demography, and physical/laboratory examination data were compared between different gender and age groups.Our study showed that, compared to females, males had enlarged tongue shape, thicker fur, more fissures and fewer teeth marks (all P < .001), and also had more red tongue color (P = .019), normal saliva (P = .001), more red dots (P = .005) and yellower fur (P = .014). In females, increasing age was associated with more enlarged tongue shape, thicker fur, yellower fur, more saliva, fissures and fewer teeth marks (all P < .001), more ecchymoses (P = .009), and more red tongue color (P = .023). These associations of age with more fissures, fewer teeth marks, fewer red dots (P < .001), median tongue shape (P = .029), and wet saliva (P = .014) were also evident in males, but other relationships were not clearly evident.Even though most of the common tongue features derived from a community-based population are consistent with TCM theory, yet some significantly gender- and age-dependent tongue characteristics were identified. These disparities in tongue features associated with gender or age shall be prudently taken into consideration in clinical tongue diagnosis and future TCM research.

Chinese guidelines for the diagnosis and treatment of hand, foot and mouth disease (2018 edition).

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common infectious disease in childhood caused by an enterovirus (EV), and which is principally seen in children under 5 years of age. To promote diagnostic awareness and effective treatments, to further standardize and strengthen the clinical management and to reduce the mortality of HFMD, the guidelines for diagnosis and treatment have been developed. METHODS: National Health Commission of China assembled an expert committee for a revision of the guidelines. The committee included 33 members who are specialized in diagnosis and treatment of HFMD. RESULTS: Early recognition of severe cases is utmost important in diagnosis and treatment of patients with HFMD. The key to diagnosis and treatment of severe cases lies in the timely and accurate recognition of stages 2 and 3 of HFMD, in order to stop progression to stage 4. Clinicians should particularly pay attention to those EV-A71 cases in children aged less than 3 years, and those with disease duration less than 3 days. The following indicators should alert the clinician of possible deterioration and impending critical disease: (1) persistent hyperthermia; (2) involvement of nervous system; (3) worsening respiratory rate and rhythm; (4) circulatory dysfunction; (5) elevated peripheral WBC count; (6) elevated blood glucose and (7) elevated blood lactic acid. For treatment, most mild cases can be treated as outpatients. Patients should be isolated to avoid cross-infection. Intense treatment modalities should be given for those severe cases. CONCLUSION: The guidelines can provide systematic guidance on the diagnosis and management of HFMD.

Biosafety evaluations of well-dispersed mesoporous silica nanoparticles: towards in vivo-relevant conditions.

This study aimed to investigate how mesoporous silica nanoparticles (MSNs), especially focussing on their surface functional groups, interacted with Raw 264.7 macrophages, as well as with zebrafish embryos. Upon introducing nanoparticles into a biological milieu, adsorption of proteins and biomolecules onto the nanoparticle surface usually progresses rapidly. Nanoparticles bound with proteins can result in physiological and pathological changes, but the mechanisms remain to be elucidated. In order to evaluate how protein corona affected MSNs and the subsequent cellular immune responses, we experimented in both serum and serum-deprived conditions. Our findings indicated that the level of p-p38 was significantly elevated by the positively charged MSNs, whereas negatively charged MSNs resulted in marked ROS production. Most significantly, our experiments demonstrated that the presence of protein efficiently mitigated the potential nano-hazard. On the other hand, strongly positively charged MSNs caused 94% of the zebrafish embryos to die. In that case, the toxicity caused by the quaternary ammonium ligands on the surface of those nanoparticles was exerted in a dose-dependent manner. In summary, these fundamental studies here provide valuable insights into the design of better biocompatible nanomaterials in the future.

Fourteen types of co-circulating recombinant enterovirus were associated with hand, foot, and mouth disease in children from Wenzhou, China.

BACKGROUND: Although hand, foot, and mouth disease (HFMD) is a major public concern in China, the prevalence and clinical symptoms associated with the different agents of HFMD in this country remain poorly understood. OBJECTIVES: We investigated the clinical and molecular characteristics of enteroviruses in patients with HFMD from Wenzhou, China. STUDY DESIGN: Patients with laboratory-confirmed HFMD admitted to the Yuying Children's Hospital in Wenzhou, China during 2013 were included in this study. Viral RNA sequences were amplified using RT-PCR, determined by sequencing, and compared by phylogenetic analysis. RESULTS: A total of 955 clinically diagnosed HFMD cases were determined using PCR, with whole viral genomes obtained for each enterovirus type. 14 types of enterovirus belonging to two viral species were identified. Notably, Coxsackievirus A6 (CV-A6) was the most common species detected (77.8%), followed by EV-A71 (8.2%) and CV-A10 (8.1%). Phylogenetic analysis revealed multiple independent introductions of these viruses into Wenzhou. In addition, the enterovirus observed in Wenzhou had a recombinant history, with two or three recombination breakpoints. Although the illness associated with CV-A6 was milder than that of EV-A71, CV-A6 infection caused more widespread rash, larger blisters, and subsequent skin peeling and/or nail shedding. CONCLUSION: Our study revealed the co-circulation of 14 types of enteroviruses in a single location - Wenzhou, China - with CV-A6 virus the predominant agent of HFMD. This work highlights the need to perform larger-scale surveillance to fully understand the epidemiology of enteroviruses in China and the wider Asia-Pacific region.

Msx homeobox gene family and craniofacial development.

Vertebrate Msx genes are unlinked, homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development. Inductive interactions mediated by the Msx genes are essential for normal craniofacial, limb and ectodermal organ morphogenesis, and are also essential to survival in mice, as manifested by the phenotypic abnormalities shown in knockout mice and in humans. This review summarizes studies on the expression, regulation, and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice. Key words: Msx genes, craniofacial, tooth, cleft palate, suture, development, transcription factor, signaling molecule.

An immunosensor based on magnetic relaxation switch and polystyrene microparticle-induced immune multivalency enrichment system for the detection of Pantoea stewartii subsp. Stewartii.

A rapid, sensitive, and simple immunosensor has been developed for the detection of Pantoea stewartii subsp. Stewartii (Pss). This immunosensor combines magnetic relaxation switch (MRS) assay with polystyrene microparticle-induced immune multivalency enrichment system. Comparing to conventional enzyme-linked immunosorbent assay (ELISA), the immunosensor developed in this study provides higher sensitivity and requires less analysis time. The detection limit of Pss obtained by immunosensor was determined to be 10(3)cfu/mL, 50 times lower than that by ELISA (5×10(4)cfu/mL), while the analysis time required by immunosensor is 30min much shorter than that by ELISA. The average recoveries studied with Pss at various spiking levels ranged from 85.5% to 93.4% with a relative standard deviation (RSD) below 6.0%. No cross-reaction with the other five strains was found, demonstrating a good specificity of Pss detection. The results showed that the MRS immunosensor combined with PS-induced immune multivalency enhancement system is a promising platform for the determination of large biological molecules due to its high sensitivity, specificity, homogeneity, and speed.

Weak microwave can alleviate water deficit induced by osmotic stress in wheat seedlings.

The aim of the investigation is to determine the effect of microwave pretreatment of wheat seeds on the resistance of seedlings to osmotic stress. Changes in biophysical, physiological and biochemical characters were measured. The results showed: (1) The magnetic field intensity and seeds temperature increased progressively with microwave pretreatments of 5, 10, 15, 20 s and 25 s compared with controls. Although each microwave pretreatment resulted in an increase in alpha-amylase activity and photon emission intensity, the increase of alpha-amylase activity and photon emission intensity was maximal at a microwave pretreatment of 10 s. (2) Osmotic stress induced by PEG treatment enhanced the concentration of malondialdehyde, while decreasing the activities of nitricoxide synthase, catalase, peroxidase, superoxide dismutase and the concentration of nitric oxide, ascorbic acid, glutathione in the seedlings compared with controls. However, compared to osmotic stress alone, in the seedlings treated with microwave irradiation plus osmotic stress the concentration of malondialdehyde decreased, while the activities of nitricoxide synthase, catalase, peroxidase, superoxide dismutase and the concentration of nitric oxide, ascorbic acid and glutathione increased. These results suggest that a suitable dose of microwave radiation can enhance the capability to eliminate free radicals induced by osmotic stress in wheat seedlings resulting in an increase in resistance to osmotic stress.

Making a tooth: growth factors, transcription factors, and stem cells.

Mammalian tooth development is largely dependent on sequential and reciprocal epithelial-mesenchymal interactions. These processes involve a series of inductive and permissive interactions that result in the determination, differentiation, and organization of odontogenic tissues. Multiple signaling molecules, including BMPs, FGFs, Shh, and Wnt proteins, have been implicated in mediating these tissue interactions. Transcription factors participate in epithelial-mesenchymal interactions via linking the signaling loops between tissue layers by responding to inductive signals and regulating the expression of other signaling molecules. Adult stem cells are highly plastic and multipotent. These cells including dental pulp stem cells and bone marrow stromal cells could be reprogrammed into odontogenic fate and participated in tooth formation. Recent progress in the studies of molecular basis of tooth development, adult stem cell biology, and regeneration will provide fundamental knowledge for the realization of human tooth regeneration in the near future.

Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice.

Cleft palate is a congenital disorder arising from a failure in the multistep process of palate development. In its mildest form the cleft affects only the posterior soft palate. In more severe cases the cleft includes the soft (posterior) and hard (anterior) palate. In mice a number of genes show differential expression along the anterior-posterior axis of the palate. Mesenchymal heterogeneity is established early, as evident from Bmp4-mediated induction of Msx1 and cell proliferation exclusively in the anterior and Fgf8-specific induction of Pax9 in the posterior palate alone. In addition, the anterior palatal epithelium has the unique ability to induce Shox2 expression in the anterior mesenchyme in vivo and the posterior mesenchyme in vitro. Therefore, the induction and competence potentials of the epithelium and mesenchyme in the anterior are clearly distinct from those in the posterior. Defective growth in the anterior palate of Msx1-/- and Fgf10-/- mice leads to a complete cleft palate and supports the anterior-to-posterior direction of palatal closure. By contrast, the Shox2-/- mice exhibit incomplete clefts in the anterior presumptive hard palate with an intact posterior palate. This phenotype cannot be explained by the prevailing model of palatal closure. The ability of the posterior palate to fuse independent of the anterior palate in Shox2-/- mice underscores the intrinsic differences along the anterior-posterior axis of the palate. We must hitherto consider the heterogeneity of gene expression and function in the palate to understand better the aetiology and pathogenesis of non-syndromic cleft palate and the mechanics of normal palatogenesis.