The siEGFR nanoplexes for the enhanced brain glioma treatment: Endoplasmic reticulum biomimetic strategy to induce homing effect and non-degradable intracellular transport.

The epidermal growth factor receptor (EGFR) plays a pivotal role in tumor progression and is an essential therapeutic target for treating malignant gliomas. Small interfering RNA (siRNA) has the potential to selectively degrade EGFR mRNA, yet its clinical utilization is impeded by various challenges, such as inefficient targeting and limited escape from lysosomes. Our research introduces polyethylene glycol (PEG) and endoplasmic reticulum membrane-coated siEGFR nanoplexes (PEhCv/siEGFR NPs) as an innovative approach to brain glioma therapy by overcoming several obstacles: 1) Tumor-derived endoplasmic reticulum membrane modifications provide a homing effect, facilitating targeted accumulation and cellular uptake; 2) Endoplasmic reticulum membrane proteins mediate a non-degradable "endosome-Golgi-endoplasmic reticulum" transport pathway, circumventing lysosomal degradation. These nanoplexes demonstrated significantly enhanced siEGFR gene silencing in both in vitro and in vivo U87 glioma models. The findings of this study pave the way for the advanced design and effective application of nucleic acid-based therapeutic nanocarriers.

An efficient and universal In silico screening strategy for acquisition of high-affinity Aptamer and its application in analytical utility.

Numerous aptamers against various targets have been identified through the technology of systematic evolution of ligands by exponential enrichment (SELEX), but the affinity of these aptamers are often insufficient due to the limitations of SELEX. Therefore, a more rational in silico screening strategy (ISS) was developed for efficient screening of high affinity aptamers, which took shape complementarity and thermodynamic stability into consideration. Neuron specific enolase (NSE), a tumor marker, was selected as the target molecule. In the screening process, three aptamer candidates with good shape complementarity, lower ΔG values, and higher ZDOCK scores were produced. The dissociation constant (Kd) of these candidates to NSE was determined to be 10.13 nM, 14.82 nM, and 2.76 nM, respectively. Each of them exhibited higher affinity to NSE than the parent aptamer (Kd = 23.83 nM). Finally, an antibody-free fluorescence aptasensor assay, based on the aptamer with the highest affinity, P-5C8G, was conducted, resulting in a limit of detection (LOD) value of 1.8 nM, which was much lower than the parental aptamer (P, LOD = 12.6 nM). The proposed ISS approach provided an efficient and universal strategy to improve the aptamer to have a high affinity and good analytical utility.

Influence of COVID-19 pandemic on the epidemiology of Mycoplasma pneumoniae infections among hospitalized children in Henan, China.

Background: Increasing reports have indicated that non-pharmaceutical interventions to control the COVID-19 pandemic may also have an effect on the prevalence of other pathogens. Mycoplasma pneumoniae is an important atypical pathogen prevalent in children with high rates of macrolide resistance. The aim of this study was to investigate the epidemiological characteristics of M. pneumoniae infection in children before and during the COVID-19 pandemic. Methods: In this study, M. pneumoniae detection results were extracted from Henan Children's Hospital from 2018 to 2021. The epidemiological characteristics of pediatric M. pneumoniae infection were analyzed. Results: We found that the highest positive rate of M. pneumoniae infection was 11.00 % in 2018, 14.01 % in 2019, followed by 11.24 % in 2021 and 8.75 % in 2020 (p < 0.001). Most tested children had respiratory system manifestations, and pneumoniae was the most common diagnosis (53.23 %). An increase in the number of positive cases was observed with an increase in age, with a higher number of cases among children over 6 years old. No positive cases were identified among children aged 1-28 days. The decrease in the positive rate among children aged between1-6 years old in 2020 and 2021 was found to be statistically significant (p < 0.001). The pre-pandemic period demonstrated a higher incidence rate in the fall, whereas the summers and winters exhibited a significantly higher positive rate during the pandemic period (p < 0.001). Different regions in Henan also showed different epidemic patterns. Conclusions: In summary, strict pandemic measures influenced the spread of M. pneumoniae to some extent and changed demographic characteristics, including age, season and regional distribution. Continuous monitoring is required for the control and prevention of related diseases.

Molecular typing of Mycoplasma pneumoniae and its correlation with macrolide resistance in children in Henan of China.

BACKGROUND/PURPOSE: As a major causative pathogen of community-acquired pneumonia, Mycoplasma pneumoniae (M. pneumoniae) can cause both upper and lower respiratory tract inflammation as well as extrapulmonary syndromes, especially in infants and the elderly. The emergence of macrolide-resistance has significant effects on the treatment of relevant diseases in children. This study aimed to analyze the genotypes and the macrolide resistance-associated mutations in M. pneumoniae sampled from the pediatric patients in Henan, China. METHODS: A segment of gene on the 23S rRNA was amplified and sequenced to detect the mutations related to macrolide resistance. Molecular typing was performed by the method named multiple locus variable-number tandem repeat analysis (MLVA) for macrolide-susceptible and macrolide-resistant specimens. RESULTS: Among the M. pneumoniae-positive samples, 95.7% (111/116) had macrolide-resistant mutation, and all of them consisted of the A2063G mutation. There were only two MLVA types identified in this study, type 4-5-7-2 (51/92, 55.4%) and type 3-5-6-2 (41/92, 44.6%). CONCLUSION: There was no correlation between MLVA types and macrolide resistance (P â€‹> â€‹0.05).

A novel loop-mediated isothermal amplification-lateral flow dipstick method for Helicobacter pylori detection.

Introduction: To eradicate Helicobacter pylori (H. pylori) and reduce the risk of gastric cancer, a sensitive, specific, convenient, and simple detection method is needed. This study aimed to establish a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for H. pylori detection. Methods: LAMP primer design software was used to design primers for the conserved sites of the H. pylori ureB gene. UreB-FIP-labeled biotin was used for LAMP amplification, and FAM-labeled probes were specifically hybridized with LAMP amplification products, which were then detected by LFD. In addition, a clinical study was conducted to assess LAMP-LFD in 20 fecal samples. Results: The results of the optimization indicated that H. pylori could be specifically detected by LFD without cross-reaction with other non-H. pylori bacteria when the LAMP was performed at 65°C for 60 min. The lower limit of the detection method was 102 copies/µL, which was 100 times the sensitivity of polymerase chain reaction (PCR). H. pylori-positive fecal samples were detected by LAMP-LFD in 13/20 patients. Discussion: In conclusion, a new LAMP-LFD assay has been fully established and confirmed for H. pylori detection. The entire process can be completed in approximately 1.5 h, with the advantages of strong specificity, high sensitivity, and simple operation. This study provides a novel potential method for the detection of H. pylori in the clinical settings of primary hospitals and low-resource countries.

Molecular beacon based real-time PCR p1 gene genotyping, macrolide resistance mutation detection and clinical characteristics analysis of Mycoplasma pneumoniae infections in children.

BACKGROUND: Mycoplasma pneumoniae can be divided into different subtypes on the basis of the sequence differences of adhesive protein P1, but the relationship between different subtypes, macrolide resistance and clinical manifestations are still unclear. In the present study, we established a molecular beacon based real-time polymerase chain reaction (real-time PCR) p1 gene genotyping method, analyzed the macrolide resistance gene mutations and the relationship of clinical characteristics with the genotypes. METHODS: A molecular beacon based real-time PCR p1 gene genotyping method was established, the mutation sites of macrolide resistance genes were analyzed by PCR and sequenced, and the relationship of clinical characteristics with the genotypes was analyzed. RESULTS: The detection limit was 1-100 copies/reaction. No cross-reactivity was observed in the two subtypes. In total, samples from 100 patients with positive M. pneumoniae detection results in 2019 and 2021 were genotyped using the beacon based real-time PCR method and P1-1 M. pneumoniae accounted for 69.0%. All the patients had the A2063G mutation in the macrolide resistance related 23S rRNA gene. Novel mutations were also found, which were C2622T, C2150A, C2202G and C2443A mutations. The relationship between p1 gene genotyping and the clinical characteristics were not statistically related. CONCLUSION: A rapid and easy clinical application molecular beacon based real-time PCR genotyping method targeting the p1 gene was established. A shift from type 1 to type 2 was found and 100.0% macrolide resistance was detected. Our study provided an efficient method for genotyping M. pneumoniae, valuable epidemiological monitoring information and clinical treatment guidance to control high macrolide resistance.

Development of local injectable, bone-targeting nanocarriers of triptolide for treatment of bone-only metastasis.

Triptolide (TP) is known for its diverse pharmacological activities but also its delivery and toxicity issues. This study aimed at exploiting TP's anticancer effects at lower risk of systemic toxicity by developing local-injectable "bone-targeting TP nanoparticle" (TPN) for bone-only metastasis treatment. The lipid/oil-based TPNs decorated with alendronate (ALE) achieved size of 70.4-111.2 nm with good dispersion stability. The drug encapsulation efficiency reached 97 % and drug release profiles were in biphasic, controlled manner lasting for 5 days in medium with serum proteins and calcium. TPNs were more cytotoxic than free TP against MDA-MB-231 breast cancer cells (IC50: 16.40 ± 0.80 nM vs 25.45 ± 1.83 nM, P < 0.05) but less cytotoxic against MC3T3-E1 osteoblasts (P < 0.05). When combined with paclitaxel or docetaxel, low dose TPN (containing 10 nM) significantly increased the effectiveness of the two chemotherapy drugs against MDA-MB-231 (IC50 values decreased from 7.3 nM to 2.5 nM for docetaxel; from 4.6 nM to 1.1 nM), indicating potent chemosensitization effects. Retardation of in vitro cancer cell migration by TPN was also observed in the standard scratch assay. ALE decoration significantly enhanced the TPN affinity for both calcium hydroxyapatite and porcine bone chip models, which led to enhancement in TP retention in the bones up to 8.1-fold versus free drug. Overall, TPN demonstrated good potential as a local-injectable, bone-targeted nanotherapy tailored for eradication of bone-only metastasis at reduced risk of systemic toxicity.

HPDL mutations identified by exome sequencing are associated with infant neurodevelopmental disorders.

BACKGROUND: Recent research found that biallelic HPDL variants can cause neurodevelopmental disorder with progressive spasticity and brain white matter abnormalities (NEDSWMA), with only a few reports. Clinical phenotypic information on individuals with damaging HPDL variants may also be incomplete. The phenotype of NEDSWMA is characterized by severe neurodevelopmental delay, brain atrophy, and spasticity in infancy. METHODS: Exome sequencing was used in the proband and his parents to identify the underlying genetic cause. Candidate mutations were validated by classic Sanger sequencing. The clinical presentation of the infant who carried HPDL variants was summarized. RESULTS: We identified a novel compound heterozygous variants in HPDL, c.995delC (p.T332Mfs) and c.1051C>T (p.Q351*) in the patient a 6-month-old boy presenting with global developmental delay, seizures, hypertonia, and limb spasticity. Brain magnetic resonance imaging (MRI) showed thin corpus callosum, ventriculomegaly, white matter volume reduction, bilateral frontotemporal subarachnoid widening, and sulcus deeping. CONCLUSION: Our results provided important information for the associations of variants in HPDL with the neurodevelopmental disorder in infants, and broaden the genetic spectrum of HPDL-related disease. This is the second report of the HPDL mutation causing infant neurodevelopmental disorders in a Chinese population.

Estrogen Suppresses Cytokines Release in cc4821 Neisseria meningitidis Infection via TLR4 and ERß-p38-MAPK Pathway.

Estrogen has long been known to possess immune-modulatory effects in diseases, and multiple pathological conditions show great sex disparities. However, the impact of estrogen in Neisseria meningitidis infection has not been determined. The present study aimed to investigate the role of estrogen in N. meningitidis infection and the molecular mechanism. We selected 35 N. meningitidis isolates representing different clonal complexes (cc), serogroups, and isolation sources to infect the HBMEC cell line. Results showed that the expression of estrogen receptor (ER) ß in N. meningitidis-infected cells was downregulated compared with that in normal cells. The expression of ERß induced by invasive isolates was lower than that in carriers. Serogroup C isolates induced the lowest expression of ERß compared with serogroup A and B isolates. We used four cc4821 N. meningitidis isolates to infect two kinds of host cells (human brain microvascular endothelial cells and meningeal epithelial cells). The results showed that 17 ß-estradiol (E2) could inhibit the release of inflammatory factors interleukin (IL)-6, IL-8, and tumor necrosis factor-α after N. meningitidis infection via TLR4. E2 could inhibit the activation of the p38-MAPK signal pathway induced by N. meningitidis infection through binding to ERß, and significantly inhibit the release of inflammatory factors in N. meningitidis-infected host cells. This study demonstrated that estrogen plays a protective role in N. meningitidis infection. ERß is potentially associated with the release of inflammatory cytokines in N. meningitidis infection, which sheds light on a possible therapeutic strategy for the treatment of invasive diseases caused by N. meningitidis.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics.

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

[Clinical characteristics and genetic analysis of a Chinese pedigree affected with mitochondrial DNA depletion syndrome due to compound heterozygous variants of RRM2B gene].

OBJECTIVE: To analyze the clinical characteristics and pathogenic gene in a Chinese pedigree affected with mitochondrial DNA depletion syndrome 8A (MTDPS8A). METHODS: Whole exome sequencing was carried out for the patient. Sanger sequencing was used to verify the results, and PolyPhen-2 and PROVEAN software were used to predict the impact of amino acid changes on the function of the protein. RESULTS: The patient, a two-month-old female, was admitted to the hospital for poor milk intake and poor mental response. Her clinical manifestations included feeding difficulty, shortness of breath and low muscle tone. Auxiliary laboratory test indicated that the infant was underdeveloped with abnormal liver, kidney, and heart functions accompanied by hyperlacticacidemia. She responded poorly to treatment and eventually died. Sequencing revealed that the child has carried compound heterozygous missense variants of the RRM2B gene, namely c.16delA (p.R6Gfs*22) and c.175G>C (p.A59P), which were respectively inherited from her father and mother, and both were newly discovered pathologic variants. CONCLUSION: The c.16delA and c.175G>C compound heterozygous variants of the RRM2B gene probably underlay the pathogenesis of MTDPS8A. Above finding has strengthened the understanding of the clinical feature and genetic etiology of this disease and expanded the mutation spectrum of the RRM2B gene.

Epimutation of MMACHC compound to a genetic mutation in cblC cases.

BACKGROUND: Methylmalonic aciduria (MMA) combined with homocystinuria, cobalamin(cbl)C deficiency type (OMIM 277400), is the most common autosomal recessive inherited disorder of intracellular cobalamin metabolism caused by mutations in the MMACHC gene (OMIM 609831), of which more than 100 mutations have been identified to date. In this study, we only identified a coding mutation in one allele at the MMACHC gene locus, and no large fragments deletion or duplication were found. Up to now, only three epimutation cblC cases were reported. We hypothesized whether the MMACHC was hypermethylated. METHODS: To address this hypothesis, the entire coding region and adjacent splice sites of the panel genes involved in metabolic diseases were sequenced using the Illumina HiSeq X platform, followed by confirmation via Sanger sequencing in their parents and brothers. Methylation analysis of the MMACHC was performed using an EpiTect Bisulfite Kit and methylation-specific PCR (MSP) to investigate the role of epimutations in cblC disease. RESULTS: We identified a clearly pathogenic single heterozygous c.658_660del, p. (K220del) mutation, which was also identified in the mother. Analysis of the MMACHC indicated a heterozygous epimutation consisting of 34 hypermethylated CpG sites in a CpG island encompassing the promoter and first exon of the MMACHC, which was also identified in the father. Furthermore, we identified a single heterozygous c.*2C>T mutation in the sixth exon of the PRDX1 (OMIM 176763) in patients and their fathers, which was the only sequence variation that segregated with the MMACHC methylation. Neither c.658_660del and epimutation in MMACHC nor c.*2C>T in PRDX1 was discovered in her brother. CONCLUSION: We report compound heterozygotes in MMACHC for a genetic mutation and an epimutation in cblC cases. To our best knowledge, this is the first report of two cblC cases from China caused by compound heterozygous mutations with a coding mutation in one allele and an epimutation in the other at the MMACHC locus.

SARS-CoV-2 vaccine candidates in rapid development.

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still spreading globally. The scientific community is attempting to procure an effective treatment and prevention strategy for COVID-19. A rising number of vaccines for COVID-19 are being developed at an unprecedented speed. Development platforms include traditional inactivated or live attenuated virus vaccines, DNA or RNA vaccines, recombinant viral vector vaccines, and protein or peptide subunit vaccines. There are 23 vaccines in the clinical evaluation stage and at least 140 candidate vaccines in preclinical evaluation. In this review, we describe research regarding basic knowledge on the virus, updates on the animal models, current landscape of vaccines in clinical evaluation and updated research results on vaccine development. Safe and effective COVID-19 vaccines require further investigation.

Comparative genomics of Helicobacter pullorum from different countries.

BACKGROUND: Helicobacter pullorum commonly colonized in the gastrointestinal tract of poultry and caused gastroenteritis. This bacterium could be transmitted to humans through contaminated food and caused colitis and hepatitis. Currently, the genetic characteristics of the H. pullorum were not recognized enough. In this study, the genomes of 23 H. pullorum strains from different counties were comparatively analyzed. Among them, H. pullorum 2013BJHL was the first isolated and reported in China. RESULTS: The genomes of the studied strains were estimated to vary from 1.55 to 2.03 Mb, with a GC content of ~ 34%. 4064 pan genes and 1267 core genes were obtained from the core-pan genome analysis using the Roary pipeline. Core genome SNPs (cg-SNPs) were obtained using Snippy4 software. Two groups were identified with the phylogenetic analysis based on the cg-SNPs. Some adhesion-related, immune regulation, motility-related, antiphagocytosis-related, toxin-related and quorum sensing related genes were identified as virulence factors. APH(3')-IIIa, APH(2'')-If, and AAC(6')-Ie-APH(2'')-Ia were identified as antibiotic resistance genes among the H. pullorum genomes. cat, SAT-4 and tetO genes were only identified in 2013BJHL, and tet(C) was identified in MIT98-5489. MIC determination revealed that the 2013BJHL showed acquired resistance to ciprofloxacin, nalidixic acid, tetracycline, gentamicin, streptomycin and erythromycin, only sensitive to ampicillin. The antibiotic resistance genetic determinants on the 2013BJHL genome correlate well with observed antimicrobial susceptibility patterns. Two types of VI secretion system (T6SS) were identified in 52.2% (12/23) the studied strains. CONCLUSION: In this study, we obtained the genetic characteristics of H. pullorum from different sources in the world. The comprehensive genetic characteristics of H. pullorum were first described. H. pullorum showed highly genetic diversity and two sub-types of T6SSs were first identified in H. pullorum. 2013BJHL was found to be multidrug resistant as it was resistant to at least three different antibiotic classes.

Incorporation of docosahexaenoic acid (DHA) enhances nanodelivery of antiretroviral across the blood-brain barrier for treatment of HIV reservoir in brain.

Although the newer antiretroviral (ARV) drugs are highly active against the human immunodeficiency virus (HIV) in the body compartment, they often fail to effectively tackle the HIV reservoir in the brain because of inefficient penetration to the blood-brain barrier (BBB). In this study, we investigated the potential benefits of incorporating docosahexaenoic acid (DHA), an omega-3 fatty acid essential for brain development, in lipid nanocarriers for facilitating the BBB passage of an ARV darunavir. The resulting nanocarriers (nanoARVs) containing 5-15% DHA were 90-140 nm in size, had high darunavir payload (~11-13% w/w), good stability and minimal cellular toxicity, and could be further decorated with transferrin (Tf) for Tf-receptor targeting. In BBB models of hCMEC/d3 cells, nanoARVs with higher DHA content achieved increased nanocarrier uptake and up to 8.99-fold higher darunavir permeation than free darunavir. In animals, nanoARVs were able to achieve 3.38-5.93-fold increase in brain darunavir level over free darunavir. Tf-conjugated nanoARVs also achieved significantly higher anti-HIV activity than free darunavir (viral titer 2 to 2.6-fold higher in latter group). Comparison of DHA incorporation and Tf-receptor targeting showed that while both strategies could enhance the cellular uptake and brain accumulation of the nanocarriers, DHA was more effective (P < 0.05) for improving BBB permeation and brain accumulation of the darunavir payload. Substituting DHA with another oil noticeably reduced the cellular uptake of nanoARVs. Overall, this proof-of-concept study has supported the development of DHA-based nanoARVs as an effective, safe yet technically simple strategy to enhance brain delivery of darunavir and potentially other lipophilic ARVs for treatment of HIV reservoir.

Enhanced eradication of intracellular and biofilm-residing methicillin-resistant Staphylococcus aureus (MRSA) reservoirs with hybrid nanoparticles delivering rifampicin.

Osteomyelitis carries a high risk of recurrence even after extended, aggressive antibiotic therapy. One of the key challenges is to eradicate the reservoirs of methicillin-resistant Staphylococcus aureus (MRSA) inside the host bone cells and their biofilms. Our goal is to develop rifampicin loaded lipid-polymer hybrid nanocarriers (Rf-LPN) and evaluate if they can achieve enhanced rifampicin delivery to eradicate these intracellular and biofilm-residing MRSA. After optimization of the composition, Rf-LPN demonstrated size around 110 nm in diameter that remained stable in serum-supplemented medium, drug payload up to 11.7% and sustained rifampicin release for 2 weeks. When comparing Rf-LPN with free rifampicin, moderate but significant (p < 0.05) improvement of the activities against three osteomyelitis-causing bacteria (USA300-0114, CDC-587, RP-62A) in planktonic form were observed. In comparison, the enhancements in the activities against the biofilms and intracellular MRSA by Rf-LPN were even more substantial. The MBEC50 values against USA300-0114, CDC-587, and RP-62A were 42 vs 155, 70 vs 388, and 265 ng/ml vs over 400 ng/ml, respectively, and up to 18.5-fold reduction in the intracellular MRSA counts in osteoblasts was obtained. Confocal microscope images confirmed extensive accumulation of Rf-LPN inside the biofilm matrix and MRSA-infected osteoblasts. Overall, in this proof-of-concept study we have developed and validated the strategy to exploit the nanoparticle-cell and nanoparticle-biofilm interactions with a new rifampicin nanoformulation for prevention of osteomyelitis recurrence and chronicity caused by the elusive MRSA.

Lipid-polymer hybrid nanoparticles carrying linezolid improve treatment of methicillin-resistant Staphylococcus aureus (MRSA) harbored inside bone cells and biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent pathogen causing osteomyelitis. The tendency of MRSA to evade standard antibiotic treatment by hiding inside bone cells and biofilms is a major cause of frequent osteomyelitis recurrence. In this study, we developed a lipid-polymer hybrid nanoparticle loading the antibiotic linezolid (LIN-LPN), and focused on evaluating if this new nanoantibiotic can achieve significant in vitro activities against these intracellular and biofilm-embedded MRSA. The optimal LIN-LPN formulation demonstrated both high linezolid payload (12.0% by weight of nanoparticles) and controlled release characteristics (gradually released the entrapped antibiotic in 120 h). Although it achieved lower activities against bacteria including USA300-0114, CDC-587, RP-62A in planktonic form, it was substantially superior against the intracellular MRSA reservoir inside osteoblast cells. The differences of intracellular activities between LIN-LPN and linezolid were 87.0-fold, 12.3-fold, and 12.6-fold in CFU/ml (p < 0.05 or < 0.01) at 2 µg/ml, 4 µg/ml, and 8 µg/ml linezolid concentrations, respectively. LIN-LPN also suppressed the MRSA biofilm growth to 35-60% of the values achieved with free linezolid (p < 0.05). These enhanced intracellular and anti-biofilm activities of LIN-LPN were likely contributed by the extensive accumulation of LIN-LPN inside the MRSA-infected osteoblasts and biofilms as revealed in the confocal microscope images. The study thus validates the feasibility of exploiting the good nanoparticle-host cell and nanoparticle-biofilm interactions for improving the antibiotic drug activities against the poorly accessible bacteria, and supports LIN-LPN as a new alternative therapy for preventing the recurrence of MRSA-mediated bone infections.

Genomic and biological characterization of a pandemic norovirus variant GII.4 Sydney 2012.

Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.