Effects of synaptic activity on dendritic spine motility of developing cortical layer v pyramidal neurons

It is increasingly clear that dendritic spines play an important role in compartmentalizing post-synaptic signals and that their dynamic morphological properties have functional consequences. Here, we examine this issue using two-photon microscopy to characterize spine motility on layer V pyramidal neurons in acute slices of the developing mouse cortex. In this system, all spine classes except filopodia become less dynamic as development proceeds. General manipulations of activity (TTX or KCl treatment) do not alter spine dynamics, although increased glutamatergic transmission (AMPA or NMDA treatment) stabilizes developing cortical spines. These effects on spine dynamics do not appear to be related to AMPA or NMDA receptor expression as assessed with immunolabeling, as there is no correlation between spine motility and AMPA (GluR1/2) or NMDA (NR1/NR2B) receptor subunit expression on a spine by spine basis. These results indicate that activity through glutamatergic synapses is important for regulating spine motility in the developing mouse cortex, and that the relative complement of receptors, while different across morphological classifications, cannot account for differences in dynamic structural changes in dendritic spines.     

Adults' perceived prevalence of enteric fever predicts laboratory-validated incidence of typhoid fever in children

This study was undertaken to develop a model to predict the incidence of typhoid in children based on adults' perception of prevalence of enteric fever in the wider community. Typhoid cases among children, aged 5-15 years, from epidemic regions in five Asian countries were confirmed with a positive Salmonella Typhi culture of the blood sample. Estimates of the prevalence of enteric fever were obtained from random samples of adults in the same study sites. Regression models were used for establishing the prediction equation. The percentages of enteric fever reported by adults and cases of typhoid incidence per 100,000, detected through blood culture were 4.7 and 24.18 for Viet Nam, 3.8 and 29.20 for China, 26.3 and 180.33 for Indonesia, 66.0 and 454.15 for India, and 52.7 and 407.18 for Pakistan respectively. An established prediction equation was: incidence of typhoid (1/100,000= -2.6946 + 7.2296 x reported prevalence of enteric fever (%) (F=31.7, p<0.01; R2=0.992). Using adults' perception of prevalence of disease as the basis for estimating its incidence in children provides a cost-effective behavioural epidemiologic method to facilitate prevention and control of the disease.     

Effect of tibial tubercle elevation on patellofemoral compressive force in patellofemoral arthrosis

In this study, the effect of tibial tubercle elevation on the patellofemoral compressive force (PFCF) was investigated in patients with patellofemoral arthrosis. Fifteen (11 women and 4 men) patients who had undergone tibial tubercle elevation were included in the study. The average follow-up was 4.5 years. The mean age of the patients was 59 years (range 34–71 years). They were able to maintain a normal pain-free daily life. Maximal isometric quadriceps force (Q) was calculated by equating the moment generated by this force to the moment of the force measured at the ankle by a modified hand dynamometer. PFCF was calculated from the quadriceps and patellar tendon forces. Statistical analyses were then applied to the measured and calculated quantities. The mean quadriceps force in the operated knee decreased by 20%. Likewise, the mean PFCF was found to be reduced by 30% in the operated knees as compared with the asymptomatic contralateral knees. The above differences in Q and PFCF between the operated knee and the asymptomatic knee were statistically significant (P < 0.05). Therefore, the results of this study do not agree with the previously held view that Q and PFCF increase due to the removal of pain after the elevation operation. It is our contention that comparison of the forces measured preoperatively in a painful joint with the forces that can be attained postoperatively in the pain-free joint can lead to errors in biomechanical evaluations.     

Pollen NAD(P)H oxidases and their contribution to allergic inflammation

This article provides an overview of NADPH oxidase and its role in allergic inflammation. A background and historical perspectives of NADPH oxidase are first provided, followed by a detailed overview of mammalian NADPH oxidase subunits and their functional organization. Plant NADPH oxidase, the authors' discovery of NADPH oxidase in pollens, and their contribution to allergic inflammation are then discussed, concluding with a discussion of future directions and outstanding questions that require attention.     

自体骨髓来源内皮祖细胞移植对移植静脉桥血管再内皮化及内膜增生的影响

目的:已有实验研究证明,移植骨髓来源内皮祖细胞可以促进球囊损伤后血管内皮的修复及抑制内膜的增生.那么移植骨髓来源内皮祖细胞对自体静脉移植术后静脉血管内皮修复和内膜增生是否起同样的作用?观察自体骨髓来源内皮祖细胞移植对自体静脉移植术后静脉桥血管再内皮化及内膜增生的作用。方法:实验于2007-01/08在北华大学附属医院内科实验室完成。实验室级别:P2级。①实验材料:6～8月龄雄性新西兰大白兔由解放军第二军医大学实验动物中心提供.体质量(2.5±0.5)kg,清洁级,实验过程中对动物处置符合动物伦理学标准。②实验方法:抽取成年兔骨髓分离制备骨髓单个核细胞,体外扩增法培养出骨髓来源内皮祖细胞.在培养第7天通过Ac-Dil-LDL、FITC-BS-1双染色及流式细胞仪检测CD34、CD133、FIK-1表达进行细胞鉴定,应用DAPI荧光染料体外标记培养7 d的骨髓来源内皮祖细胞、将成年新西兰大白兔23只随机分为细胞移植组(n=13)和对照组(n=10)进行自体左颈外静脉、颈总动脉移植术,在建模后第3天将DAPI标记的骨髓来源内皮祖细胞悬液经耳缘静脉植入细胞移植组动物体内,埘照组植入等量的PBS液③实验评估:细胞移植后4周.观察兔静脉移植段血管性及内膜厚度。结果:23只免均进入结果分析,无脱落:①通过体外扩增法培养的骨髓来源内皮组细胞,培养至7 d可见AC-Dil-LDI及FITC-BS-1双染色阳性细胞并表达CD34、CD133及FIK-1。②在呈绿荧光染色的血管内皮中可见有部分不均匀的散发蓝色荧光(DAPI标记细胞的细胞核)。③细胞移植组兔静脉血管内皮有DAPI标记的细胞.且内膜厚度明显低于对照组(P<0.05)结论:自体骨髓来源的内皮祖细胞移植可以促进损伤部位血管内皮的修复.并抑制内膜的过度增生。     

Structuring a safer donor-replacement program

BACKGROUND: Replacement donors are more likely than volunteer donors to have positive or abnormal tests for transfusion-transmissible disease. In an effort to increase the donor pool, workers sought to identify a safer replacement-donor subgroup that may be acceptable for routine donations. STUDY DESIGN AND METHODS: In a retrospective review and cohort study, the replacement-donor effect was separated from the new- donor effect. The relative effect the replacement donor has on the risk of transfusion-transmissible diseases, donor retention, and frequency of returning donations was then quantified by comparison against the effect of repeat volunteer donors. RESULTS: The replacement donor had 3.1 times the risk and 0.72 times the donor retention rate and made 0.81 times as many returning donations as the repeat volunteer donor. The figures for the new-donor effect were similar. The two risks were additive, making a new replacement donor particularly hazardous. If replacement donations only from repeat replacement donors were considered, the donor risk and the number of donations per returning donor were made comparable to those for the general (combined) volunteer donor. CONCLUSION: The negative effect of the replacement donor is similar in magnitude to that of the new volunteer donor. A replacement-donation program targeting repeat replacement donors has an acceptable risk profile and may be a valuable adjunct to the collection of blood from general volunteer donors.     

Characterization of the novel GlyT1 PET tracer [18F]MK‐6577 in humans

Decreased glutamatergic neurotransmission is hypothesized to be involved in the pathophysiology of schizophrenia. Inhibition of glycine transporter Type‐1 (GlyT1) reuptake is expected to increase the glutamatergic neurotransmission and may serve as treatment for cognitive and negative symptoms of schizophrenia. In this article, we present human data from a novel GlyT1 PET tracer, [¹⁸F]MK‐6577. In the process of developing a GlyT1 inhibitor therapeutic, a PET tracer can assist in determining the dose with a high probability of sufficiently testing the mechanism of action. This article reports the human PET studies with [¹⁸F]MK‐6577 for measuring GlyT1 receptor availability at baseline in normal human subjects and occupancy with a GlyT1 inhibitor, MK‐2637. Studies were also performed to measure radiation burden and the baseline test‐retest (T‐RT) variability of the tracer. The effective dose from sequential whole‐body dosimetry scans in three male subjects was estimated to be 24.5 ± 2.9 µSV/MBq (mean ± SD). The time–activity curves from T‐RT scans modeled satisfactorily using a two tissue compartmental model. The tracer uptake was highest in the pons (V_T = 6.7 ± 0.9, BP_ND = 4.1 ± 0.43) and lowest in the cortex (V_T = 2.1 ± 0.5, BP_ND = 0.60 ± 0.23). V_T T‐RT variability measured in three subjects was <12% on average. The occupancy scans performed in a cohort of 15 subjects indicated absence of a reference region. The in vivo potency (Occ₅₀) of MK‐2637 was determined using two methods: A: Lassen plot with a population input function (Occ₅₀ = 106 nM, SE = 20 nM) and B: pseudo reference tissue model using cortex as the pseudo reference region (Occ₅₀ = 141 nM, SE = 21 nM). Synapse 69:33–40, 2015. © 2014 Wiley Periodicals, Inc.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces. While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

Polyketide synthases (PKSs) are large enzymatic machines that synthesize structurally diverse natural products, many of which are used as antibiotics, immunosuppressants, anticancer agents, and other types of medicines. In bacteria, a substantial fraction of polyketides is synthesized by multimodular PKSs, where each module consists of a set of domains that collectively catalyze one round of elongation and chemical modification of the growing polyketide chain (1) (Fig. 1A). The homologous modules of each PKS operate in a defined assembly-line manner. The emergence of this multimodular architecture and its subsequent diversification has led to an astounding complexity and variety of polyketide natural products. However, the underlying evolutionary processes that drive the evolution of assembly-line PKSs are not well understood (2).Open in a separate windowFig. 1.Gene conversion in assembly-line PKSs. (A) Typical architecture of an assembly-line PKS, exemplified by closely related angolamycin synthase (ANGS) and tylactone synthase (TYLS), which contain the same set of biosynthetic domains and produce the same polyketide, tylactone. Domains: KS, ketosynthase; KS^Q, decarboxylative ketosynthase; AT, acyltransferase; DH, dehydratase; DHt, inactive dehydratase; ER, enoyl-reductase; KR, ketoreductase; KR°, ketoreductase-inactive epimerase; ACP, acyl carrier protein; TE, thioesterase. (B) Phylogenetic tree of protein sequences of angolamycin and tylactone synthase modules. Some orthologous modules clade together, as is expected for closely related PKSs that only recently diverged from a common ancestor through point mutations. However, in many cases paralogous modules clade more closely together, which can be explained by gene conversion between these modules. Sequence alignment performed using ClustalOmega (10); phylogenetic tree constructed using RAxML (30). (C) Gene conversion is a process in which a DNA sequence is nonreciprocally transferred from one homologous region to another and was implicated in the evolution of assembly-line PKSs (6, 7).According to one model, present-day assembly-line PKSs mainly arose through successive duplications of a parent module, whereafter each prototypical multimodular PKS evolved into a family of distinct but functionally related contemporary PKSs. However, this model has several discordances. For instance, it predicts that orthologous modules of closely related PKSs should cluster together in phylogenetic trees, which is often not the case (Fig. 1B). An alternative model proposes that assembly-line PKSs arose through recombination between different modules in a mosaic-like manner (3), whereafter present-day PKSs evolved through a combination of point mutations, recombination, and gene conversion (2).Gene conversion is a process in which a DNA sequence is nonreciprocally transferred from one homologous region to another, thereby homogenizing these homologous sequences (Fig. 1C). It is common in eukaryotic genomes, where it frequently occurs during mitosis, meiosis, and double-strand-break repair and has not only been implicated in the evolution of many gene families but also identified as the mechanism causing certain genetic diseases (4). In bacteria, gene conversion has been described only in a few systems, but its overall evolutionary role in prokaryotic genomes is not well understood (5). Gene conversion has also been implicated in assembly-line PKS evolution (6, 7), although its extent, role, and mechanism remain unclear.We recently proposed that extensive gene conversion between paralogous modules of assembly-line PKSs could explain why paralogous modules are often more similar to each other than orthologous ones (Fig. 1B) (2). In this work we sought to quantify the prevalence of gene conversion in assembly-line PKSs and investigate whether it might confer an evolutionary advantage. We discovered not only that gene conversion is widespread in assembly-line PKSs but also that it is frequently associated with the presence of a genetic element which recodes PKS genes and undergoes gene conversion. This association is particularly strong within Streptomyces bacteria, suggesting a major role in the diversification of assembly-line PKSs and possibly other gene families.     

The metabolite 1 alpha,25-dihydroxyvitamin D3 enhances lymphokine- mediated activation of the oxidative metabolism of the U937 cell line and phosphorylation of a 48-kD respiratory burst-associated protein

U937 cells respond to a variety of stimuli with increased differentiation as manifested by reduced growth, increased adherence, increased expression of several surface receptors, and increased capacity for phagocytosis and formation of reactive oxygen intermediates. In the present study the effects of lymphocyte conditioned media, recombinant interferon-gamma (IFN-gamma), and 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the ability to form reactive oxygen intermediates by U937 cells were measured by using the luminol-dependent luminescence (LDL) assay. Neither 1,25(OH)2D3 alone nor IFN-gamma alone enhanced competence for phorbol myristate acetate- stimulated LDL. Cells were capable of moderate LDL after exposure to lymphocyte conditioned media, and this was enhanced by 1,25(OH)2D3 (10(- 8) mol/L) and other vitamin D metabolites at higher concentrations. This effect was not secondary to accelerated production of myeloperoxidase, which is important in the LDL assay. Enhanced phorbol myristate acetate-stimulated phosphorylation of a 48-kd substrate was observed in 32P-labeled intact cells treated with 1,25(OH)2D3 alone or in combination with IFN-gamma. Treatment of cells with IFN-gamma or lymphocyte conditioned media did not alter phosphorylation. These results support the concept that 1,25(OH)2D3 plays a role in phagocyte differentiation and activation beyond the effects of lymphokines. Protein kinase C-mediated phosphorylation reactions may be necessary for the ability of U937 cells to reduce O2 and required for maximal activity under some conditions of incubation.