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Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16. Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses. The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16. EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials. ClinicalTrials.gov NCT01268787.

This technical report describes a method to clear fixed brain tissues while allowing for fluorescent dye tracing and retaining cellular morphology. The authors demonstrate the utility of the technique by obtaining a wiring diagram for sister mitral cells. We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8–37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8–37–cholestanol, but not unconjugated CGRP8–37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8–37–cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund’s adjuvant more effectively than unconjugated CGRP8–37. Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

DNA–protein crosslinks (DPCs) induced by aldehydes interfere with replication and transcription. Hereditary deficiencies in DPC repair and aldehyde clearance processes cause progeria, including Ruijs–Aalfs syndrome (RJALS) and AMeD syndrome (AMeDS) in humans. Although the elimination of DPC during replication has been well established, how cells overcome DPC lesions in transcription remains elusive. Here we show that endogenous aldehyde-induced DPC roadblocks are efficiently resolved by transcription-coupled repair (TCR). We develop a high-throughput sequencing technique to measure the genome-wide distribution of DPCs (DPC-seq). Using proteomics and DPC-seq, we demonstrate that the conventional TCR complex as well as VCP/p97 and the proteasome are required for the removal of formaldehyde-induced DPCs. TFIIS-dependent cleavage of RNAPII transcripts protects against transcription obstacles. Finally, a mouse model lacking both aldehyde clearance and TCR confirms endogenous DPC accumulation in actively transcribed regions. Collectively, our data provide evidence that transcription-coupled DPC repair (TC-DPCR) as well as aldehyde clearance are crucial for protecting against metabolic genotoxin, thus explaining the molecular pathogenesis of AMeDS and other disorders associated with defects in TCR, such as Cockayne syndrome. Three studies identify a transcription-coupled DNA–protein crosslink repair pathway that depends on the Cockayne syndrome proteins and the proteasome.

The present study aimed to investigate the optimal inactivants and inactivation conditions for preparing inactivated vaccines of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Mycoplasma inactivation was performed using formaldehyde, thimerosal, β-propiolactone (BPL), and binary ethylenimine (BEI) and compared. The results showed that M. hyopneumoniae was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.0008 % thimerosal for 12 h at 37 °C, with 0.02 % BPL for 24 h or 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. M. hyorhinis was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.004 % thimerosal for 24 h or 0.02 % thimerosal for 12 h at 37 °C, with 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. Next, the immunogenicity of the mycoplasmas after inactivation was evaluated by immunizing BALB/c mice. Immunization of mice with a high dose (106 color-changing units [CCU] per dose) of M. hyopneumoniae and M. hyorhinis vaccines inactivated with all inactivants led to high levels of serum IgG antibodies. M. hyopneumoniae vaccines inactivated with formaldehyde induced significantly higher titers of antibodies than vaccines inactivated with other inactivants, whereas M. hyorhinis vaccines inactivated with BEI induced significantly higher titers of antibodies than vaccines inactivated with thimerosal. However, in mice immunized with a low dose of mycoplasmas (104 CCU per dose), only M. hyopneumoniae vaccines inactivated with formaldehyde and BEI and M. hyorhinis vaccines inactivated with formaldehyde, BPL, and BEI led to significant levels of serum IgG antibodies. Among these groups, the antibody levels in the formaldehyde-inactivated vaccine group were higher than those in the other inactivant groups. This study provides a reliable basis for inactivation during large-scale production of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis inactivated vaccines.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Nutrient disorder and presence of disease-causing agents in soilless media negatively influence the growth of muskmelon. To combat these issues, use of environmentally-friendly sanitation techniques is crucial for increased crop productivity. The study was conducted under greenhouse and field conditions to investigate the effect of two different sanitation techniques: steaming and formalin fumigation on various media’s characteristics and their impact on muskmelon yield. Media: jantar, guar, wheat straw and rice hull and peat moss of 10% air-filled porosity and sanitized with formalin and steaming. Steaming of guar, jantar, and wheat straw increased the phosphorus (P) and potassium (K) concentrations by 13.80–14.86% and 6.22–8.45% over formalin fumigation. Likewise, P and K concentrations in muskmelon were higher under steaming. Steaming significantly inhibited the survival of Fusarium wilt sp. melonis , root knot nematode sp. meloidogyne and nitrifying bacteria in media than formalin fumigation. In conclusion, steaming decreased the prevalence of nitrifying bacteria and pathogens which thus improved the NO 3 − –N:NH 4 + –N ratios, P and K nutritional balance both in the media and muskmelon transplants. Hence, steaming as an environment-friendly approach is recommended for soilless media. Further, optimization of steaming for various composts with different crops needs to be investigated with steaming teachnique.

•β-propiolactone (BPL) and formaldehyde (FA) were used to inactivate several influenza virus strains.•BPL abolished the infectivity, FA was unable to completely inactivate the virus.•All methods damaged the binding and fusion capacity; BPL caused greater loss than FA.•FA treatments caused the highest reduction in TLR-7 stimulation.•All the observed effects were strain-dependent. The vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. The first step in the production process is virus inactivation with β-propiolactone (BPL) or formaldehyde (FA). Recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. We assessed the effect of inactivation with BPL and FA on 5 different influenza virus strains. The properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (HA) binding ability, fusion capacity and the potential to stimulate a Toll-like receptor 7 (TLR7) reporter cell line were then assessed and compared to the properties of the untreated virus. Inactivation with BPL resulted in undetectable infectivity levels, while FA-treated virus retained very low infectious titers. Hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with BPL-treated virus binding and fusing at a lower degree compared to FA-inactivated samples. On the other hand, BPL-inactivated virus induced higher levels of activation of TLR7 than FA-inactivated virus. The alterations caused by BPL or FA treatments were virus strain dependent. This data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product.

Indoor formaldehyde (CH 2 O) exceeding the recommended level is a severe threat to human health. Few studies have investigated its effect on indoor surface bacterial communities, affecting habitants' health. This study used 20-L glass containers to mimic the indoor environment with bacterial inputs from human oral respiration. The behavior of bacterial communities responding to CH 2 O varied among the different CH 2 O levels. The bacterial community structure significantly changed over time in the 0.054 mg·m −3 CH 2 O group, which varied from the 0.1 mg·m −3 and 0.25 mg·m −3 CH 2 O groups. The Chao1 and Shannon index significantly increased in the 0.054 mg·m −3 CH 2 O group at 6 week, while they remained unchanged in the 0.25 mg·m −3 CH 2 O group. At 12 week, the Chao1 significantly increased in the 0.25 mg·m −3 CH 2 O group, while it remained unchanged in the 0.054 mg·m −3 CH 2 O group. Only a few Operational Taxonomic Units (OTUs) significantly correlated with the CH 2 O concentration. CH 2 O-induced OTUs mainly belong to the Proteobacteria and Firmicutes. Furthermore, bacterial communities formed at 6 or 12 weeks differed significantly among different CH 2 O levels. Functional analysis of bacterial communities showed that inferred genes related to chemical degradation and diseases were the highest in the 0.25 mg·m −3 CH 2 O group at 12 weeks. The development of nematodes fed with bacteria collected at 12 weeks was applied to evaluate the bacterial community's hazards. This showed significantly impaired growth in the 0.1 mg·m −3 and 0.25 mg·m −3 CH 2 O groups. These findings confirmed that CH 2 O concentration and exposure time could affect the indoor bacterial community and formed bacterial communities with a possibly more significant hazard to human health after long-term exposure to high CH 2 O levels.

A new aldehyde-free fixative has been developed and its effect has been compared to traditional formaldehyde fixative in terms of the fixation effect and HE staining of the heart, liver, lung, and kidney. The air in the experimental area was examined to evaluate its impact on the environment and human health. The organs from mice of groups 1-6 were taken respectively (thickness of liver and lung was 3 mm). After the heart and kidney capsule were removed, the organs were longitudinally cut along their maximum surface, and half was taken. Thereafter, the tissue fixation effect was observed by Hematein and Eosin (H＆E) staining and the total protein content of tissue was examined by the ultramicro spectrophotometer. Additionally, the volatility ratio of the new fixative and the traditional formaldehyde is compared. The results showed that there was no significant difference between the fixation effect of the new aldehyde-free fixation and the traditional formaldehyde fixative on mouse organs and the air quality in the experimental area was found to be significantly better when the new aldehyde-free fixative is used than when the traditional formaldehyde fixative is used. Traditional formaldehyde fixative in HE staining can be replaced by the new environment-friendly formaldehyde-free fixative, however further special staining of fixed tissue and immunohistochemical studies are needed.