Confirmation of recent heroin abuse: Accepting the challenge

Confirmation or exclusion of recent heroin consumption is still one of the major challenges for forensic and clinical toxicologists. A great variety of biomarkers is available for heroin abuse confirmation, including various opium alkaloids (eg, morphine, codeine), street heroin impurities (eg, 6‐acetylcodeine [6‐AC], noscapine, papaverine) as well as associated metabolites (eg, 6‐monoacetylmorphine [6‐MAM], morphine glucuronides). However, the presence of most of these biomarkers cannot solely be attributed to a previous heroin administration but can, among other things, also be due to consumption of poppy seed products (‘poppy seed defense’), opium preparations or specific medications, respectively. A reliable allocation is of great importance in different contexts, for instance in the case of DUID (driving under the influence of drugs) investigations, in driving licence re‐granting processes, in workplace drug testing (WDT), as well as in post‐mortem identification of illicit opiate use. Additionally, differentiation between illicit street heroin abuse and pharmaceutical heroin administration is also important, especially within the frame of heroin‐assisted treatments. Therefore, analysis of multiple biomarkers is recommended when illicit opiate consumption is assumed to obtain the most reliable results possible. Beyond that, interpretation of positive opiate test results requires a profound insight into the great variety of biomarkers available and their validity regarding the alleged consumption. This paper aims to provide an overview of the wide variety of heroin abuse biomarkers described in the literature and to review them regarding their utility and reliability in daily routine analysis.     

Geminivirus–Host Interactions: Action and Reaction in Receptor-Mediated Antiviral Immunity

In plant−virus interactions, the plant immune system and virulence strategies are under constant pressure for dominance, and the balance of these opposing selection pressures can result in disease or resistance. The naturally evolving plant antiviral immune defense consists of a multilayered perception system represented by pattern recognition receptors (PRR) and resistance (R) proteins similarly to the nonviral pathogen innate defenses. Another layer of antiviral immunity, signaling via a cell surface receptor-like kinase to inhibit host and viral mRNA translation, has been identified as a virulence target of the geminivirus nuclear shuttle protein. The Geminiviridae family comprises broad-host range viruses that cause devastating plant diseases in a large variety of relevant crops and vegetables and hence have evolved a repertoire of immune-suppressing functions. In this review, we discuss the primary layers of the receptor-mediated antiviral immune system, focusing on the mechanisms developed by geminiviruses to overcome plant immunity.     

野菊花总黄酮提取方法比较及其抗氧化活性研究

目的研究不同提取工艺的野菊花中总黄酮的量及其抗氧化活性。方法采用经典回流提取法、超声提取法、组织破碎提取法提取野菊花中黄酮类成分,以芦丁为对照品,通过紫外-可见分光光度法测定黄酮类成分的量,并用流动注射化学发光法测定野菊花中黄酮类成分的抗氧化活性。结果经典回流提取法、超声提取法、组织破碎提取法所提取的总黄酮的量分别为12.60、11.02、10.95 mg/g,采用流动注射化学发光法测得3种不同提取方法的总黄酮提取物的IC50分别是2.67、3.43、5.13μg/m L。结论经典回流提取法提取得到的野菊花总黄酮的量最高,且抗氧化活性最强,说明野菊花中总黄酮的量和抗氧化活性具有一致性。     

From the Cover: PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Neuropeptides enable interneuronal communication and signaling (1), mediating diverse functions ranging from endocrine stimulation and homeostatic regulation to immune signaling, pain modulation, and circadian rhythm maintenance. At present, over 100 neuropeptides are known in mammals (2). These peptides originate from neurons in the central, enteric, or peripheral nervous systems and within immune organs (3). Canonically, neuropeptides exert their biological function by binding to a cognate receptor (usually a G-coupled protein receptor [GPCR]), triggering a signal transduction pathway that leads to a functional change in the target cell (1). Neuropeptides are typically considered neurotransmitters or neurohormones, but recent work has illuminated their potential roles in modulating immune responses and neuroinflammation (4–8).Human innate and adaptive immunity have evolved via two parallel and complementary paradigms in host defense against microbial invasion: molecular and cellular. Molecular defense mediators are secreted or activated rapidly and locally to directly inhibit pathogens. Prototypic examples include host-defense peptides (HDPs), the acute-phase reactants, and the complement cascade. Cellular defense involves infiltration of professional immune phagocytes (neutrophils and macrophages) and lymphocytes into infected tissues. Cellular infiltration into the central nervous system (CNS) is a double-edged sword, given its anatomically confined space and physiologically delicate context. On one hand cellular defense may be necessary to control or clear certain pathogens. On the other hand, neutrophils and other phagocytes can cause counterproductive damage to tissue parenchyma due to production and release of reactive oxygen species and other cytotoxic constituents from phagolysosomes. Thus, molecular defenses that are rapidly deployable in immediate settings of infection to obviate the need for infiltration of potentially harmful immune cells would be of special relevance in context of the CNS.To explore putative molecular host-defense mediators within the CNS that may have both neuro- and immunomodulatory properties, we used a support vector machine (SVM) trained on HDPs (9, 10) to identify neuropeptides with potential host defense capabilities. Among the human neuropeptides identified as potential HDPs for molecular host defense of the CNS is pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP is a member of the vasoactive intestinal peptide (VIP)/PACAP/secretin family (11) that regulates neurodevelopment (12), metabolism, emotion, mood, and stress responses via GPCRs (13). PACAP is known to interact with the immune system (14, 15) and modulate T helper type 1 (TH1)/TH2 cytokine production (3). Important previous work on structure activity relationships (SAR) of PACAP have also shown that it possess antimicrobial activity in vitro against a range of organisms (16–18), as well as anti-cancer activity against tumor cell lines. (Interestingly, our use of an SVM classifier that can scan different fragments of the same peptide allows us to identify antimicrobial activity in previously identified metabolites of PACAP as well^* (19)). However, host defense functions, contextual bioactivity, or pathogen-specific inducibility of PACAP or other neuropeptides regarding antimicrobial activity in vivo are not known. More specifically, the role of PACAP in the larger context of innate immunity and its in vivo relevance to antimicrobial defense of the CNS and in other tissues remains unclear, given that antimicrobial activity is strongly dependent on biochemical and physiological context (20, 21, 22). Here, we examine PACAP inducibility in response to infection in the CNS and other tissues, and whether PACAP exerts antimicrobial activity against relevant organisms in the specific biochemical context relevant to those tissues. Bioinformatic and structural analyses showed PACAP to possess almost identical structural similarity to human cathelicidin LL-37, despite having overall low sequence similarity to other known HDPs. Synchrotron X-ray scattering revealed that PACAP can induce negative Gaussian curvature (NGC) in microbial membranes, a general requirement for membrane-permeating antimicrobial processes such as pore formation, blebbing, and other membrane-perturbing events (23–25). Moreover, extending from prior work (18), antimicrobial assays and mechanistic fingerprinting analyses showed that PACAP exerts potent antimicrobial mechanisms against drug-resistant bacteria and fungi via multiple synergistic pathways, including permeabilization, disruption of cellular energetics, and activation of regulated cell death pathways. In mouse models of bacterial or fungal infection, we demonstrated that PACAP is strongly induced up to 50-fold in brain, spleen, or kidney. Further, in media simulating these tissue contexts, PACAP exerted robust microbiostatic and microbicidal efficacy. Taken together, these findings imply that PACAP is an infection-inducible, tissue-specific host-defense effector that affords rapid and contextual antimicrobial host defense in the CNS and periphery. Beyond immediate contributions to better understanding of antimicrobial defense, the present discoveries reveal specific intersections of neurological and immunological systems and establish insights into antiinfective strategies that preserve critical functions of the CNS.     

Associations of the antioxidant capacity and hemoglobin levels with functional physical performance of the upper and lower body limbs

GeroScience - Herein we considered the role of oxidative stress on deficiencies of functional physical performance that could affect a future pre-frailty condition. Using principal component...     

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis

The global yield of bananas—one of the most important food crops—is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant–nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques ¹H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant’s defense system, and derived strategies for improving banana resistance are highlighted.Bananas and plantains (Musa spp.) are among the world’s most important food and cash crops, with a global production of about 138 million tons in 2010. These crops are part of a well-balanced human diet and are a major food staple for more than 400 million people in the tropics (1, 2). About 82% of the world’s banana production is consumed locally, particularly in India, China, and many African countries (Table S1) (1, 2). Export of bananas to the northern hemisphere represents an important source of employment in countries such as Costa Rica, Ecuador, Colombia, and the Philippines (Table S2) (1, 2). Banana yields are severely hampered by fungi, insects, and plant-parasitic nematodes. The burrowing nematode, Radopholus similis (Cobb, 1893) Thorne, 1949, is the key nematode pathogen, causing yield losses up to 75% (3). R. similis is found in all major banana-producing regions of the world; its best-known hosts are bananas, black pepper, Citrus spp. (4), and coffee (5). R. similis causes extensive root lesions that can lead to toppling of banana plants (6).Plant-parasitic nematodes have been effectively managed through the use of nematicides. However, their high toxicity has adverse effects on humans and their toxic residues are known to accumulate through nontarget organisms in the food chain (7). After the withdrawal of many effective nematicides, such as methyl bromide, from the market (8), organophosphate and carbamate nematicides are still intensively applied to banana and therefore continue to threaten the health of agricultural workers and the environment (9). Although several biological control approaches, including the application of both single and multiple control organisms—such as Fusarium oxysporum, Paecilomyces lilacinus, Trichoderma atroviride isolates, and Bacillus firmus—have proved promising under greenhouse conditions, the control they confer to banana plants most probably does not protect plants for more than one cycle in the field, and most of these organisms have yet to be tested under field conditions (10).The in-depth investigation of the plant–nematode interactions at the cellular and molecular level could lead to the development of more rational and efficient control strategies (11). The production of toxic, herbivore-deterrent or -repellent secondary metabolites, which is typical for many plant defense systems, is particularly interesting in this context. Musa cultivars resistant to R. similis have been identified, especially the cultivar Yangambi km5 (Ykm5) (12). Histochemical and ultrastructural investigations of lesions caused by R. similis in Ykm5 revealed the accumulation of phenolic compounds in response to infection (13). Unfortunately, many of these studies were based solely on histochemical staining methods and did not identify the chemical structures of nematicidal secondary metabolites (7, 14, 15). Initial phytochemical analyses of R. similis-infected roots of the Musa cultivar Pisang sipulu identified the phenylphenalenone anigorufone (1) as a phytoalexin produced in response to nematode damage and confirmed earlier suggestions of the significant role of phytoalexins in the plant defense system (16). Phenylphenalenones are a group of special phenylpropanoid-derived natural products (17), which are known as Musaceae phytoalexins (18). The activity of phenylalanine ammonia lyase (EC 4.3.15), the entry-point enzyme of the phenylpropanoid pathway, is correlated to the biosynthesis of specific phenylpropanoids involved in defense and was substantially induced in nematode infected roots of Ykm5 (19). Phenylphenalenone-related compounds show biological activity against bacteria, fungi, algae, and diatoms (18, 20–22). The formation of these compounds has been elicited in banana leaves by Mycosphaerella fijiensis (Black Sigatoka leaf streak disease), in the fruit peels by Colletotrichum musae (anthracnose disease), and in roots and rhizomes by F. oxysporum f. sp. cubense (Panama disease) and R. similis (16, 18, 21, 23).     

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear‐ and anxiety‐like behaviors in female rats

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E₂) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole‐cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E₂. Our results show that E₂ increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E₂ as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E₂ persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E₂ treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α‐ and β‐adrenoreceptors. In summary, our results implicate E₂ in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E₂ may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.     

First evidence of protein G-binding protein in the most primitive vertebrate: Serum lectin from lamprey (Lampetra japonica)

The intelectins, a recently identified subgroup of extracellular animal lectins, are glycan-binding receptors that recognize glycan epitopes on foreign pathogens in host systems. Here, we have described NPGBP (novel protein G-binding protein), a novel serum lectin found in the lamprey, Lampetra japonica. RT-PCR yielded a 1005 bp cDNA sequence from the lamprey liver encoding a 334 amino acid secretory protein with homology to mammalian and aquatic organism intelectins. Gene expression analyses showed that the NPGBP gene was expressed in the blood, intestines, kidney, heart, gill, liver, adipose tissue and gonads. NPGBP was isolated by protein G-conjugated agarose immunoprecipitation, and SDS–PAGE analyses showed that NPGBP migrated as a specific band (∼35 and ∼124 kDa under reducing and non-reducing conditions, respectively). These results suggested that NPGBP forms monomers and tetramers. NPGBP gene expression was induced by in vivo bacterial stimulation, and NPGBP showed different agglutination activities against pathogenic Gram-positive bacteria, Gram-negative bacteria and fungi. The induction of NPGBP suggested that it plays an important role in defense against microorganisms in the internal circulation system of the lamprey. When incubated with an unrelated antibody, the specific binding between NPGBP and protein G was competitively inhibited, indicating that NPGBP and the Fc region of Ig bind to the same site on protein G. We thus assume that the tertiary structure of NPGBP is similar to that of the Fc region of Ig. Additionally, NPGBP can effectively promote endothelial cell mitosis. These findings suggest that NPGBP plays a role in the immune defense against microorganisms, and this study represents one of the few examples of the characterization and functional analysis of an aquatic organism intelectin.     

Effects of pirfenidone on experimental head injury in rats

Traumatic brain injury (TBI) continues to be a significant public healthcare concern. Neuroinflammation that occurs in the secondary phase of TBI leads to cognitive and physical dysfunction. A number of therapeutic modalities have been evaluated in an attempt to find a suitable treatment. The only drug approved for the treatment of idiopathic pulmonary fibrosis, pirfenidone, has been evaluated for its antifibrotic, anti-inflammatory, and anti-oxidant properties for various disorders, but this is the first study to examine its effects in an experimental TBI model. Twenty-four Wistar rats were randomly divided into three groups: control, trauma, and pirfenidone. The two latter groups underwent experimental diffuse cortical injury mimicking TBI. Neurological assessment was performed using the Garcia test, histological analysis was performed to examine neuroprotective and anti-inflammatory effects, and biochemical analyses of neuron-specific enolase (NSE), S-100B, caspase-3, and thiobarbituric acid reactive substances were performed. The pirfenidone group had a better Garcia test score (P=0.001), an increased anti-inflammatory effect (P<0.001), and an enhanced neuroprotective effect (P=0.007) along with decreased NSE, S100B, and TBARS levels compared to the trauma group. However, pirfenidone did not show a beneficial effect on caspase-3 levels. Pirfenidone may help decrease mortality and morbidity rates after TBI through its anti-inflammatory and antioxidant effects.     

厚朴超临界CO_2提取工艺优化及提取物抗氧化活性研究

目的优选超临界CO_2提取厚朴有效成分的工艺并探讨厚朴超临界CO_2提取物的抗氧化活性。方法采用HPLC法测定厚朴超临界CO_2提取物中厚朴酚与和厚朴酚的含量,正交试验优选厚朴超临界CO_2提取工艺,MTT法检测提取物抗氧化活性。结果厚朴酚优化工艺为萃取压力25 MPa,萃取温度55℃,CO_2用量30 kg;和厚朴酚最佳提取工艺压力15 MPa、萃取温度50℃、CO_2用量25 kg。厚朴超临界CO_2提取物具有抗氧化活性,且分离参数不同,抗氧化活性有显著差异。结论在所优选的提取工艺条件下,厚朴酚、和厚朴酚的提取效率较高,重复性较好,工艺稳定可行,提取物具有良好的抗氧化活性。