Manganese threonine chelate—a new enteric contrast agent for MRI: a pilot study on rats

Enteric contrast agents are important in gastrointestinal MRI. However, no currently available agent is well established as the standard of care. In this study, in vitro relaxivities of manganese threonine chelate (Mn‐Thr), a common nutritional food supplement, were measured at 1.5 T and 3 T with further investigation of its efficacy and safety in vivo as an enteric contrast agent. According to the calculated relaxivities, T₁W and T₂W TSE sequences of Mn‐Thr solutions at different concentrations were acquired, and the optimal concentration for dark lumen imaging on both T₁W and T₂W images was determined in vitro. To validate the optimal concentration in vivo, eight Sprague‐Dawley rats were randomly divided into two groups. Each group received rectal injection of either 2.00 g/L (about 3.80 mM) Mn‐Thr or saline as an enteric contrast agent and underwent MRI. After a time interval of one week, the same procedures were repeated with the alternative contrast agent. Animals were sacrificed after the second MRI. Tissue manganese quantification and histopathological examination were obtained. Qualitative MR image quality assessments were performed and compared between Mn‐Thr and saline. Measured T₁ and T₂ relaxivities of Mn‐Thr were significantly higher than those of MnCl₂ in vitro (p < 0.05). At the concentration of 2.00 g/L (about 3.80 mM), Mn‐Thr produced a dark lumen on T₁W and T₂W images both in vitro and in vivo. Compared with saline, Mn‐Thr showed significantly more homogenous luminal signal and increased bowel wall conspicuity in image quality assessments. Tissue manganese concentrations were not significantly different between two groups. Histopathological examinations were normal in both groups. Our data suggest that Mn‐Thr possesses favorable paramagnetic properties and can create a homogenous dark lumen on T₁W and T₂W images without obvious side effects in healthy rats. As a commercially available nutritional food supplement, Mn‐Thr appears to be a promising enteric contrast agent for MRI.     

Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.     

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Microbial ion-pumping rhodopsins are integral membrane proteins that actively transport ions across membranes upon light stimulation (1). Bacteriorhodopsin (bR) and halorhodopsin (HR) are well-known microbial ion-pumping rhodopsins found in halophilic archaea (2, 3). bR is a light-driven outward proton pump and HR is a light-driven inward anion pump, specific for chloride ion. Microbial ion-pumping rhodopsins possess common structural features consisting of seven α-helices with an all-trans retinal covalently bound to a lysine residue as the chromophore, despite the transport of different ions (4). The retinal undergoes photoisomerization from the all-trans to 13-cis configuration, which initiates the photocycle accompanied by several intermediates to export ions (4, 5). Its light-controllable function is suitable for optogenetics applications for manipulating cells, such as neurons, by changing the ion concentration inside or outside the membrane (6, 7). In fact, microbial rhodopsins, including channelrhodopsins and HRs, are employed as optogenetic tools (8–10).Nonlabens marinus rhodopsin-3 (NM-R3) is a light-driven chloride pump recently discovered in a marine flavobacterium (11). It is a distinct chloride pump from HRs and shows low amino acid sequence homology with HRs (11). To date, HR-type chloride pumps have been found in haloarchaea, marine bacteria, and cyanobacteria, including Halobacterium salinarum, Natronomonas pharaonis, and Mastigocladopsins repens, with sequence identities of 20%, 21%, and 20% to NM-R3, respectively (3, 12–15). Interestingly, NM-R3 has higher sequence identity (36%) to Krokinobacter rhodopsin 2 (KR2), a sodium pump found in Krokinobacter eikastus (16). NM-R3 possesses a unique NTQ motif (Asn98, Thr102, Gln109) in the third helix (helix C), which corresponds to key residues (DTD motif, Asp85, Thr89, Asp96) for proton transport in bR (11, 17, 18) (SI Appendix, Table S1). Asp85 acts as the primary proton acceptor of bR from the protonated Schiff-base (PSB), with assistance from Thr89 and Asp96, which is the proton donor (5, 17, 18). HRs from haloarchaea have a highly conserved TSA (Thr, Ser, Ala) motif, while the Ala residue is replaced by Asp in HR from cyanobacteria (19). In the X-ray crystal structure of NM-R3 (SI Appendix, Fig. S1A), a chloride ion located between the PSB and Asn98 (SI Appendix, Fig. S1B) is stabilized by the positive charge of the PSB (20). The position of this chloride ion is similar to those in the H. salinarum HR and N. pharaonis HR (NpHR) structures except for Thr and Ser, which correspond to Asn98 and Thr102 in NM-R3, respectively (20–22). Several amino acid residues around the retinal, including Arg95, Trp99, Trp201, and Asp231, are highly conserved among ion-pumping rhodopsins. Previous spectroscopic studies suggested that NM-R3 displays a similar sequence of intermediates, with K-, L-, N-, and O-like species, as in other HRs (23) (Fig. 1A). Recently, intermediate structures of NM-R3 obtained by low-temperature trapping X-ray crystallography and serial femtosecond crystallography (SFX) have been reported (24, 25). However, the detailed ion-pump mechanism still remains unclear, due to the lack of dynamic structures of anion transport at atomic resolution.Open in a separate windowFig. 1.TR-visible absorption spectroscopy for microcrystals. (A) Photocycle model of NM-R3 in the 1 M NaCl buffer solution (23). (B) TR difference spectra ΔA upon the 532-nm excitation. The difference was calculated by subtracting the spectrum of NM-R3. (C) Global fitting analysis with two exponentials. The A₁ and A₂ amplitude spectra correspond to the differences of [ΔA_O – ΔA_{10 µs}] and [ΔA_{200 ms} − ΔA_O], respectively. Here, ΔA_O represents the difference spectrum of the O intermediate minus NM-R3. (D) The isomeric forms of the retinal chromophore in bacterial-type rhodopsins.Time-resolved serial femtosecond crystallography (TR-SFX) is a powerful tool for visualizing reactions and motions in proteins at the atomic level (26–28). In SFX, myriads of microcrystals are continuously injected by a sample injector into an irradiation point of X-ray free electron lasers (XFELs) at room temperature, thus providing diffraction patterns before the onset of radiation damage by the intense X-ray pulse. Combined with a visible-light pump laser for reaction initiation, TR-SFX has been applied to light-driven ion pumps to observe the structural dynamics during the ion transfer. While TR-SFX has revealed femto-to-millisecond structural dynamics in light-driven cation pumps, including bR and KR2 (29–31), TR-SFX studies of anion pumps have been limited to early-stage structures adopted at picoseconds after light illumination (32). In addition, although NM-R3 pumps a chloride ion (Cl⁻) as a physiological substrate, it can also transport bromide (Br⁻), iodide (I⁻), and other anions from the extracellular side to the cytoplasmic side (23). I⁻ or Br⁻ serves as a marker for tracking the positions of ions, due to the greater number of electrons, whereas Cl⁻ is less distinguishable in X-ray crystallography. Therefore, TR-SFX experiments using I⁻ or Br⁻ are expected to directly visualize the process of ion transport.Here, we report the conformational alterations in NM-R3 during Br⁻ or I⁻ pumping, obtained by both TR-SFX and time-resolved spectroscopy of crystals. The resulting sequence of movements in NM-R3 demonstrates how the chloride pump transports anions with a large ionic radius and prevents the backflow of anions from the cytoplasmic side.     

Characterization of T cell receptors reactive to HCRTNH2, pHA273-287, and NP17-31 in control and narcolepsy patients

Narcolepsy type 1 (NT1), a disorder caused by hypocretin/orexin (HCRT) cell loss, is associated with human leukocyte antigen (HLA)-DQ0602 (98%) and T cell receptor (TCR) polymorphisms. Increased CD4⁺ T cell reactivity to HCRT, especially DQ0602-presented amidated C-terminal HCRT (HCRT_NH2), has been reported, and homology with pHA_273–287 flu antigens from pandemic 2009 H1N1, an established trigger of the disease, suggests molecular mimicry. In this work, we extended DQ0602 tetramer and dextramer data to 77 cases and 44 controls, replicating our prior finding and testing 709 TCRs in Jurkat 76 T cells for functional activation. We found that fewer TCRs isolated with HCRT_NH2 (∼11%) versus pHA_273–287 or NP_17–31 antigens (∼50%) were activated by their ligand. Single-cell characterization did not reveal phenotype differences in influenza versus HCRT_NH2-reactive T cells, and analysis of TCR CDR3αβ sequences showed TCR clustering by responses to antigens but no cross-peptide class reactivity. Our results do not support the existence of molecular mimicry between HCRT and pHA_273–287 or NP_17–31.

Narcolepsy type 1 (NT1) is caused by a loss of hypocretin/orexin (HCRT) neurons in the mediolateral hypothalamus (1–3), with recent data suggesting reversion of the human and animal phenotype with orexin agonists. The disease is strongly associated with human leukocyte antigen (HLA) DQB1*06:02/DQA1*01:02 (98% vs. 25%) (DQ0602) and displays weaker genetic associations with other immune loci, thus suggesting autoimmunity (4–9), although not meeting all criteria for being classified as an autoimmune disease (10). Like other autoimmune diseases, NT1 presents with increased comorbidity with other autoimmune conditions and asthma (11–13).Onset of NT1 is often abrupt and seasonal, and association with both Streptococcus pyogenes (14, 15) and influenza A infections (16) suggests that it may be triggered by winter infections. Most strikingly, prevalence of NT1 increased several folds in mainland China and Taiwan following the 2009 to 2010 “swine flu” H1N1 influenza pandemic (pH1N1) (4, 17, 18), although association with the pandemic is less clear in other countries (19). Vaccination with the pH1N1 vaccine Pandemrix has also been associated with an elevated relative risk for developing narcolepsy of 5- to 14-fold in children and adolescents and 2- to 7-fold in adults (18, 20–22). As Pandemrix is an AS03-adjuvanted vaccine containing the artificially produced reassortant strain X-179A, a mix of A/Puerto Rico/8/1934 (PR8), an old H1N1 strain derived from pre-2009 seasonal H1N1, and the key H1N1 2009 surface proteins hemagglutinin (HA) and neuraminidase (NA) (23), flu proteins are likely critically involved in triggering NT1. Evidence showing that HLA and T cell receptor (TCR) genetic associations are universal (9, 24–27) is also consistent with a flu trigger, as influenza A infections occur on a global basis (28). Importantly, however, even with Pandemrix vaccination in Europe, only ∼1 in 16,000 vaccinated children developed NT1, thus demanding the consideration of additional factors to fully explain the initiation of NT1 (29).Unlike in other autoimmune diseases, autoantibodies against HCRT cell proteins, HCRT itself (30–32), or other targets such as TRIB2 (33, 34) or HCRT receptor 2 (35–38) have not been consistently found. This has led to the suggestion that HCRT cell loss may be primarily T cell mediated, with limited or no involvement of autoantibodies. Consistent with this hypothesis, mounting evidence suggests involvement of CD4⁺ T cell reactivity to HCRT in NT1 (39–41), notably toward amidated fragments of the secreted, mature peptide (HCRT_54–66-NH2 and HCRT_86–97-NH2, homologous peptides collectively denoted as HCRT_NH2) (42), as critical factors in the development of the disease. Furthermore, CD8⁺ mediation of HCRT cell death has also been shown to cause NT1 in an animal model (43) and Pedersen and colleagues (44) recently highlighted the presence of CD8⁺ T cell responses against intracellular proteins contained in HCRT neurons in narcolepsy patients. Of additional interest is the observation that the TCR polymorphisms associated with NT1 are quantitative trait loci for TRAJ24 (decreasing), TRAJ28, and TRBV4-2 (increasing) usage in peripheral T cells in both controls and patients (29). A significant L to F coding polymorphism located within the antigen-binding complementarity-determining region (CDR) 3 loop of TRAJ24 expressing TCRs is also associated with NT1. Altogether, this suggests that T cell responses involving TRAJ24- or TRAJ28- and TRBV4-2–bearing TCRs may be bottleneck responses in a causative autoimmune T cell response, leading to HCRT cell death (4, 14, 17–19, 45).Based on the evidence provided above, our group hypothesized that a CD4⁺ T cell–mediated response directed against specific flu epitopes could lead to molecular mimicry with HCRT itself, potentially HCRT_NH2, subsequently recruiting CD8⁺ cytotoxic T cells and leading to HCRT cell death. To test this hypothesis, we screened 135 DQ0602 tetramers binding peptides originating from Pandemrix, wild-type 2009 H1N1, and two autoantigens (HCRT and RFX4) for the presence of antigen-restricted CD4⁺ T cells (42). After this systematic survey, it was established that CD4⁺ T cell populations recognizing influenza pHA_273–287 (pH1N1 specific) and PR8 (H1N1 pre-2009 and H2N2)-restricted NP_17–31 epitopes were increased in NT1 versus DQ0602 controls. Supporting this finding, this difference was also present in post-Pandemrix cases versus controls and was stronger in recent onset cases (42). Additionally, studies of single cells recognizing these peptides revealed that TCR clones carrying TRBV4-2 and TRAJ24 were retrieved from both HCRT_NH2 and pHA_273–287 tetramers (42), suggesting involvement of these clones in molecular mimicry and disease pathophysiology. Similarly, Jiang et al. (39) isolated TRAJ24-positive cells recognizing DQ0602 bound to HCRT_87–100 tetramer, many of which expressed perforin and granzyme-B, suggesting a terminally differentiated effector T cell (T_EMRA) phenotype. In one case, a TRAJ24 clone isolated from a narcoleptic patient showed elevated TCR reactivity toward HCRT_87–97-NH2 when transfected in Jurkat 76 (J76) cells, thus implying a role for TRAJ24 reactivity toward DQ0602-HCRT in narcolepsy autoimmunity (39).Here, we extend prior work from our group by doubling the number of patients and controls and increasing the representation of TRAJ24F narcolepsy susceptibility–associated alleles in these subjects. Results validated an increased frequency of pHA_273–287 and HCRT_54–66-NH2 tetramer-positive CD4⁺ T cells in NT1, while also testing isolated T cell clones for potential activation by their cognate ligands when expressed in J76 cells. Importantly, we also analyzed TCR CDR3αβ sequences in this larger dataset and conducted expression profiling of the corresponding T cells, providing insights into T cell characteristics in narcolepsy.     

成人Still病诊疗进展

成人Still病是一类病因未明的全身炎症性疾病,临床表现多样,其诊断和治疗存在一定困难。近年来发现,血红素加氧酶1、钙网蛋白、炎症细胞因子、糖基化终末产物等新型标志物可对成人Still病的活动程度和严重程度进行全面评估,而新研发的生物制剂如肿瘤坏死因子抑制剂、白细胞介素1（IL-1）抑制剂、IL-6抑制剂、重组IL-1...     

Hyperlipidemia Modifies Innate Immune Responses to Lipopolysaccharide via the TLR-NF-κB Signaling Pathway

The objective of this study was to investigate the effect of hyperlipidemia (HLP) on innate immune responses to lipopolysaccharide (LPS). Male New Zealand white rabbits were fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks. In vivo, the rabbits were injected intravenously with LPS for 24 h. In vitro, peripheral mononuclear cells were collected and stimulated (or unstimulated) with LPS for 24 h. Assay results were analyzed with one-way ANOVA or an equivalent non-parametric test. A P value of 0.05 was considered statistically significant. Despite having no influence in body weight, the HFD intake significantly increased serum lipids, C-reactive protein (CRP), nuclear factor (NF)-κB subunit p65, Toll-like receptor (TLR)-4, SR-A and FAS. Although we found increased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-6, CRP, IL-1β, and IL-10 in the ND-fed rabbits, no significant difference was found in the LPS-stimulated production of TNF-α, IL-6, and CRP in the HFD-fed rabbits. The macrophages harvested from the HFD-fed rabbits developed a blunted inflammatory response, with lower mRNA expression of TNF-α, IL-6, CRP, TLR-4, SR-A, FAS, and Bcl-2 than that expressed by the ND group. In the HFD-fed animals, LPS incubation decreased NF-κB subunit p65 expression, whereas the cytoplasmic phosphorylation of the inhibitor of NF-κB protein was enhanced. These data indicate that HLP displayed a form of innate immune paralysis, including reduced pro- and anti-inflammatory cytokine release to external stimulus, which was related to the altered TLR-NF-κB signaling pathway and altered pro- and anti-apoptotic processes in macrophages.     

Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight l/d,l-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites.     

Effect of pharmaceutical care on medication adherence and hospital admission in patients with chronic obstructive pulmonary disease (COPD): a randomized controlled study

Background

Poor adherence leads to a high rate of exacerbation and poor health-related quality of life (HRQoL) in patients with chronic obstructive pulmonary disease (COPD). However, few strategies are acceptable and effective in improving medication adherence. We investigated whether pharmaceutical care by clinical pharmacists could reinforce medication adherence to reduce exacerbation and improve HRQoL.

Methods

A randomized controlled study was carried out at The First Affiliated Hospital of Guangzhou Medical University from February 2012 to January 2014. Non-adherence patients were randomly assigned to receive pharmaceutical care or to usual care. The pharmaceutical care consisted of individualized education and a series of telephone counseling for 6 months provided by clinical pharmacists. Medication adherence was measured by pill counts plus direct interview at 1- and 6-month pharmaceutical care and one-year follow-up. Severe exacerbations were defined as events that led to hospitalization for acute COPD attack. An interview was conducted to investigate hospital admissions and evaluate severe exacerbations at one-year follow-up. HRQoL was measured by St George’s Respiratory Questionnaire at 6 months.

Results

At 6-month pharmaceutical care and one-year follow-up, the pharmaceutical care group exhibited higher medication adherence than the usual care group (73.4±11.1 vs. 55.7±11.9, P=0.016 and 54.4±12.5 vs. 66.5±8.6, P=0.039, respectively). There are 60 acute exacerbations resulted in a hospital admission in the usual group while 37 ones in the pharmaceutical care group during one-year follow-up (P=0.01). Hospital admissions due to acute exacerbation in the pharmaceutical care group were 56.3% less than the usual care group (P=0.01). There was a significant difference in the symptoms and impact subscales respectively at 6-month pharmaceutical care between two groups (P=0.032, P=0.018).

Conclusions

Individualized pharmaceutical care improved medication adherence, reduced hospitalization and elevated HRQoL in patients with COPD.     

Effects of prenatal exposure to ethanol on the cyclin-dependent kinase system in the developing rat cerebellum

Prenatal exposure to ethanol inhibits neurogenesis in the developing cerebellum. Cyclin-dependent kinases (CDKs) are a family of protein kinases that play multiple roles in the regulation of cell proliferation, differentiation and survival. The activity of CDKs is positively regulated by CDK activators, cyclins, and negatively regulated by CDK inhibitors (CDKIs). We hypothesize that impaired cerebellar development induced by gestational ethanol exposure is mediated by disruption of the CDK system. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric control diet (Ct). Cerebella were collected from pups (postnatal day (P) 0 through P21) and examined for CDK, cyclin, or CDKI expression using a quantitative immunoblotting procedure. In Ct-treated rats, the expression of CDK2 and its activator, cyclin A, paralleled the pattern of granule cell proliferation. Prenatal ethanol exposure produced a significant down-regulation of CDK2/cyclin A expression. Although the amounts of CDK4/CDK6 and their activator, cyclin D2, did not oscillate during postnatal development, their expression in Et-treated pups was significantly (P<0.05) higher than in controls. The expression of a CDK inhibitor, p27(Kip), was inversely correlated to proliferation of cerebellar granule progenitors. Prenatal ethanol exposure caused the down-regulation of p27(Kip) between P0 and P21. Thus, prenatal exposure to ethanol disturbed the expression of cell cycle machineries in the postnatal cerebellum. This may account for the teratogenic effects of ethanol on the developing cerebellum.