Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins  and proteoglycans . Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both  Matricryptins are biologically active fragments released from extracellular matrix (ECM) proteins and glycosaminoglycans by proteases  collagen prolylyl hydroxylase  is composed of two membrane associated proteins (the protective protein/cathepsin A and neuraminidase-1) and of the elastin-binding protein, an inactive spliced variant of lysosomal β-galactosidase . These trials include phase I (Lin et al., Clinical trials of endostatin mostly focus on solid tumors in association with cytotoxic drugs (fundus oculi angiogenesis diseases (Zhang et al., in vivo and exerts anti-fibrotic activity (Nishimoto et al., Several matricryptins such as the propeptide of lysyl oxidase, which is a tumor suppressor (Min et al., SV drafted the Section Receptors and Co-receptors of Matricryptins and Table The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, two groups have reported that FMRP is important in the replication stress (RS) response and may play a role in meiosis  in response to RS, rather than double strand break (DSB), is suppressed when endogenous FMRP is down-regulated. This suppression can be rescued by exogenous expression of wild-type FMRP but not by its nucleosome-binding-deficient mutant T102A or Y103L. Similar phenomena were also observed by Zhang et al. in a Drosophila S2 cells, while Alpatov et al. demonstrated that Fmrp levels in total lysate of mouse embryonic fibroblasts (MEFs) reduces slightly upon aphidicolin (APH) treatment. Using fractionation and immunofluorescence (IF) data, they both conclude that FMRP is recruited to chromatin upon RS.Zhang et al. found that dFmr1 increases at both the mRNA and protein levels in replication-stressed Due to the nature of FMRP, such a fractionation strategy may not be suitable for researching the intracellular localization of the protein. It has been well-established that FMRP is tightly associated with the ribosome and the rough endoplasmic reticulum (RER)  to facilitate IF detection of nuclear FMRP. Zhang et al. demonstrated that dFmr1 accumulate in an S2 nucleus treated with combination of hydroxyurea (HU) and LPB, but not with HU or LPB alone, and that the dFmr1 signal concentrates in the Hoechst dull staining area. In MEFs, Fmrp staining is proximal to DAPI-condensed chromocenters, reminiscent of the centromere localization of PARP-1, which has been reported to interact with FMRP , indicating that FMRP chromatin function is independent of its canonical mGluR function. However, as reported by Reeve et al.  is necessary to clarify its locus on chromatin (if ChIP-grade anti-FMRP is available). The data will provide deeper insight into the chromatin function of FMRP and also provide supporting information about whether or what histone modification recruits FMRP st year since the property of FMRP protein was initially characterized. As a cytoplasmic protein also functioning in chromatin, FMRP opens a new chapter of its story (Fig. This year marks the 21


Together these diverse perspectives spin an intricate web of ion channel regulation in the nervous system.Ion channels are complex hetero-oligomeric structures characterized by large, dynamic interaction networks, or “interactomes.” In addition to directing channel localization, density and ion fluxes, these complexes facilitate downstream signaling events. Moreover, pathological modulation of these networks contributes to neurological dysfunction. Our contributors to this Research Topic, Described by Fan et al.  as a “muSeveral other contributions shed light on the new insights into the function and composition of interactomes of various voltage-gated channels, regulated leak channels, and so called large pore channels.+ channel regulatory proteins are growing in complexity in terms of function and type. Known to regulate activation and trafficking of muscarinic receptor-activated Kir3 channels, Zylbergold et al. , by the so-called auxiliary subunits, dipeptidyl peptidase-like proteins (DPLPs) and Kv4 channel interacting proteins (KChIPs). While these were amongst the first identified interactors  and low voltage-activated Ca2+ channels (Cav3) functionally interact with other conductances to regulate signal processing in the cerebellum.Traditionally viewed as auxiliary subunits, Kd et al.  provide d et al.  focus spd et al.  describes et al.  review hElusive until recently, understanding of this regulated leak channel whose loss in mice is lethal (Lu et al., Permeable to ions and small metabolites like ATP, Panx1 channels gained early notoriety as “death pores” in ischemic stroke and seizure (Thompson et al., 2+ entry. They describe foundational work implicating STIM1 as the Ca2+ sensor in this process critical for maintaining neurotransmission. Further they outline key physical interactions between STIM1 with Ca2+-release activated channels and voltage-gated Ca2+ channels that coordinate the activation and inhibition of these types of channels, respectively. Finally, two papers by Wilson et al. (2+ channels.A number of contributions underscore the capacity of “promiscuous” intracellular proteins to modulate a variety of ion channels and receptors through physical interaction. Reviewed by Donnelier and Braun , cysteinn et al. ,b focus Adding further complexity to ion channel networks is consideration of lipid membrane composition and lipid second messengers. In the sole lipidome-oriented contribution, Raboune et al.  identifyWhile daunting, elucidating these macromolecular intricacies has a translational silver lining: while difficult to identify and unravel, the myriad interaction loci revealed by studying these interactions present unique opportunities for discrete, and potentially safer therapeutic intervention. For example, selective blockade at key interaction loci with cell-permeable peptides now provides an infinite number of ways in which interactomes can be discretely modulated to improve health outcomes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Depression is a heterogeneous mood disorder that has been classified and treated in a variety of ways. Although, a number of synthetic drugs are being used as standard treatment for clinically depressed patients, but they have adverse effects that can compromise the therapeutic treatments and patient's compliance. Unlike, synthetic medications, herbal medicines are widely used across the globe due to their wide applicability and therapeutic efficacy associated with least side effects, which in turn has initiated the scientific research regarding the antidepressant activity. This review is mostly based on the literature of the last decade, aimed at exploring the preclinical profile of plant-based alkaloids (the abundant secondary metabolite) as an emerging therapy for depression. Depression is a state of low mood and aversion to activity that can affect a person's thoughts, behavior, feelings and sense of well-being Sandra, . People The current antidepressant drugs are facing many challenges as mentioned by Alexander and Preskorn , in his Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral (McNaught and Wilkinson, Psychotria myriantha Mull. which exhibited antidepressant-like effect when studied on a 5-HT system in rat hippocampus (Farias et al., Annona cherimolia, including 1,2-dimethoxy-5,6,6a,7-tetrahydro-4H-dibenzoquinoline-3,8,9,10-tetraol, anonaine, liriodenine, and nornuciferine. The results showed that repeated treatment with this plant produced an antidepressant-like action in mice (Martínez-Vázqueza et al., Rhazya stricta. Acute administration of the lyophilized extract of R. stricta resulted in a significant antidepressant-like effect in experimental animals (Ali et al., Mitagyna spicosa. Mitagynine i.p injection significantly reduced the immobility time of mice in both forced swim test and tail suspension test without any significant effect on locomotor activity (Idayu et al., Ziziphus apetala which showed strong activity against 11-β-hydroxysteroid dehydrogenase inhibition in vitro (Han et al., Aconitum baicalens exhibited an antidepressant-like effect in an animal model of depression (Nesterova et al., Boerhaavia diffusa Linn. It showed significant antidepressant activity in unstressed and stressed mice in different models (Dhingra and Valecha, Evodia fructus and found that it could reverse the decreases of sucrose preference, a number of crossing, 5-HT, and Na level and also increase immobility time (Jiang et al., Sceletium tortuosum have antidepressant property in animal studies (Loria et al., Piper nigrum. Its antidepressant activity has been observed in mice exposed to both chronic and acute stress. It caused a significant change in both immobility and swimming times (Wattanathorn et al., Piper laetispicum. When tested in the forced swim test, it caused a significant dose-dependent decrease in mobility at various test doses and thus possessed antidepressant activity (Yao et al., Dactylicapnos scandens Hutch, had an antidepressant effect in mice. It dose-dependently reduced the immobility time in the tail suspension test and thus could be effective for the moderate state of depression (Xu et al., The antidepressant effect of various plant alkaloids has been reported in the literature Table . Braziliin vitro and, therefore, could be the possible mechanism for its antidepressant effect (Han et al., Aconitum baicalense, improved serotonergic system activity in an animal model of depression (Nesterova et al., in vitro assays (Xu et al., Strictosidinic acid probably exhibited antidepressant effect due to an inhibition of monoamine oxidase activity (Farias et al., Our review on the basis of available literature suggested that alkaloids could play a potential role as natural antidepressants. Keeping in mind their abundance in nature, the alkaloids could be an economical source of healing the depressive disorder. The available therapeutic agents fail to produced effect in all patients; approximately 30–40% failure has been reported to first-line antidepressant drugs accompanied by the very slow onset of action. Several alkaloids are in clinical practice and producing outstanding results in different therapeutic classes. These reported alkaloids though evoked antidepressant effects in various animal studies, but still deficient in clinical evidence. In conclusion, enough scientific evidence gathered in our review supported that the plant-based alkaloids can serve as leads for antidepressant drug discovery. It is key to subject these alkaloids to further clinical studies for efficacy, potency, and safety to ensure their clinical status.SP carried out all the literature survey and written an initial draft of the review and AK draw the structures of compounds and help in review-draft. HK supervised the overall project and finalized the final version of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brueelia-complex (Bush et al., 2015Brueelia-complex and 30 outgroup taxa selected from across the order Phthiraptera. Also included are phylogenetic reconstructions based on Bayesian inference analyses of combined COI and EF-1α sequences for Brueelia-complex species and outgroup taxa.Data is presented in support of a phylogenetic reconstruction of one of the largest, and most poorly understood, groups of lice: the


International experience and evidence-based practices have shown that reduction in variability through use of protocolized perioperative care improves surgical outcomes and reduces costs to patients and healthcare systems. In this series of Expert Opinions, we provide consensus recommendations for the various components of perioperative care to aid with the development of enhanced recovery after surgery protocols. Early ambulation, early feeding, and early return of bowel function are linked with quicker recovery, earlier discharge, and improved satisfaction. Postoperative nausea and vomiting (PONV) is a common complication following abdominal surgical procedures with patient , anesthetic (use of opioids and volatile anesthetics), and surgical  factors contributing to its incidence and severity. Nausea and vomiting should be viewed as existing on a continuum, and sequelae and patient discomfort vary based on patient experience, surgical procedure, and recovery profile. Guidelines for its avoidance and treatment are based on evidence-based literature, expert opinion, and professional association recommendations [ASPAN’S evidence-based clinical practice opinion for the prevention and/or management of PONV/PDNV. J Perianesth Nurs 2006 [Gan et al. 2007] Gan et al. 2014. Gan et al. 2003. Use of volatile anestheticsIntraoperative use of opioidsSurgical/anesthetic timeSmoking (tobacco use decreases PONV risk)Modifiable risk factorsFemale sexYoung age (<50)History of PONVSurgical location Surgical technique (laparoscopic)Non-modifiable risk factors Apfel et al. 2012a Prophylaxis of PONV is based on a number of risk factors . Prophylaxis and treatment should rely on modification of anesthetic technique, if possible, and the use of medications that work at a variety of receptor sites allowing for full multimodal benefit. Non-pharmacologic techniques include ensuring appropriate analgesia and hydration, acupuncture/acupressure, and aromatherapy. Pharmacologic techniques include medications that work at different receptor sites:All patients should receive PONV prophylaxis during the perioperative period. The numbers of medications used for treatment and prophylaxis should be determined by the number of modifiable and non-modifiable risk factors. Medications used should represent different mechanisms of action in an attempt to achieve multimodal benefit Tramèr 2001.


During the Oligo‐Miocene, major phases of phosphogenesis occurred in the Earth's oceans. However, most phosphate deposits represent condensed or allochthonous hemipelagic deposits, formed by complex physical and chemical enrichment processes, limiting their applicability for the study regarding the temporal pacing of Miocene phosphogenesis. The Oligo‐Miocene Decontra section located on the Maiella Platform  is a widely continuous carbonate succession deposited in a mostly middle to outer neritic setting. Of particular interest are the well‐winnowed grain to packstones of the middle Miocene Bryozoan Limestone, where occurrences of authigenic phosphate grains coincide with the prominent carbon isotope excursion of the Monterey event. This unique setting allows the analysis of orbital forcing on phosphogenesis, within a bio, chemo, and cyclostratigraphically constrained age‐model. LA‐ICP‐MS analyses revealed a significant enrichment of uranium in the studied authigenic phosphates compared to the surrounding carbonates, allowing natural gamma‐radiation (GR) to be used as a qualitative proxy for autochthonous phosphate content. Time series analyses indicate a strong 405 kyr eccentricity forcing of GR in the Bryozoan Limestone. These results link maxima in the GR record and thus phosphate content to orbitally paced increases in the burial of organic carbon, particularly during the carbon isotope maxima of the Monterey event. Thus, phosphogenesis during the middle Miocene in the Mediterranean was controlled by the 405 kyr eccentricity and its influence on large‐scale paleoproductivity patterns. Rare earth element data were used as a tool to reconstruct the formation conditions of the investigated phosphates, indicating generally oxic formation conditions, which are consistent with microbially mediated phosphogenesis. Phosphogenesis during the Monterey event forced by 405 kyr eccentricity controlled paleoproductivityGamma radiation is directly linked to phosphogenesis via U enrichment of authigenic phosphateTrace elements show the phosphates formed under microbially mediated and well‐oxygenated conditions This also presupposes the need for sections with well‐established correlations to both global chronostratigraphy as well as global climatic records. Furthermore, phosphogenesis needs to be studied with sufficiently high temporal resolution to estimate changes in the amount of phosphate formed over time, in order to accurately correlate them with established palaeoecological and paleoclimatological records. To that end the Decontra section, which is a well‐established shallow‐marine reference section, with a robust orbitally tuned stratigraphic framework [Föllmi, Mutti and Bernoulli, Hubert et al., Crosby and Bailey, Föllmi, Filippelli, Delaney, Schenau et al., Slomp et al., van der Zee et al., Paytan and McLaughlin, Slomp and Van Cappellen, Recent studies found that most occurrences of phosphogenesis in the present day and ancient oceans were directly mediated by microbial activity, irrespective of the marine setting in which they formed [The present work deals with three questions regarding phosphogenesis in the Decontra section during the MMCO and Monterey event: (1) Determine the phosphate contents on a sufficiently high‐resolution to correlate them with global climatic records; (2) Apply selected rare earth element (REE) proxies to reconstruct the formation history of the phosphates, in order to put them into relation to the already established paleoenvironmental history of the section; (3) Find the dominant uranium‐bearing phase using electron microprobe and LA‐ICP‐MS analyses and understand the proxy‐relationship between these phases and existing gamma log data.2Vecsei and Sanders, Crescenti et al., Mutti et al., Vecsei et al., Vecsei and Sanders, Carnevale et al., The late Oligocene to late Miocene Decontra section lies on the northern slope of the Orfento river valley  southeast of the village Decontra on the Maiella mountains in Central Italy  The Cerratina cherty Limestone—a 35 m thick succession of hemipelagic wackestones to packstones dominated by planktic foraminifers containing chert nodules, phosphatized foraminiferal tests, and sponge spicules. The first occurrence of the planktic foraminifer Praeorbulina sp. in the upper part of this unit indicates an age of <16.2 Ma; (3) The 32 m thick Bryozoan Limestone dominated by winnowed bryozoan grainstones with abundant planktonic and benthic foraminifers; (4) The 3 m thick Orbulina Limestone with abundant Orbulina sp.; (5) The Lithothamnium Limestone characterized by abundant red algal fragments overlying the Orbulina Limestone with a sharp contact. The base of the Lithothamnium Limestone is a 1.5 m thick horizon containing abundant Heterostegina fragments [see Reuter et al., Vecsei and Sanders, Carnevale et al., Reuter et al., Auer et al., Vecsei and Sanders, Cornacchia et al., Reuter et al. [Within the section five lithostratigraphic units are described Figure : (1) Ther et al.  and the inifera;  The CerrMutti and Bernoulli, Reuter et al., Mutti and Bernoulli, Reuter et al., Reuter et al., Auer et al., Orbulina Limestone, is interpreted as the Ser4/Tor1 sequence boundary [Reuter et al., Orbulina Limestone to ∼15.24–∼11.92 Ma, indicating generally low but continuous sedimentation within this part of the section [Auer et al., The Decontra section is also well known for occurrences of authigenic phosphate, particularly within the middle Miocene Bryozoan Limestone, where a prominent phosphatic hardground occurs [3Reuter et al., Auer et al., 2O5 content and check for impurities in the limestone  as well as JLS‐1  were measured as unknowns and reproduced within the reported errors for major components including P2O5.The gamma‐ray measurements were carried out in the field in 2012 using a portable “GS‐512” gamma‐ray spectrometer , and are reported in total counts (TC) [2O5 was subsequently correlated with the reported gamma‐ray values from the field measurements. Comparison was done by averaging the gamma‐ray measurements in the vicinity of the sampling spot. The results of this comparison together with the correlation coefficient were plotted in a standard cross plot  of the samples were prepared and documentation of phosphatic grains was performed by backscattered electron (BSE) imaging, using a Jeol Superprobe JXA‐8200 electron microprobe (EMP) at the Eugen F. Stumpfl Electron Microprobe Laboratory, UZAG . Figure .Williams et al., [Fryer et al., Using the BSE images, suitable phosphates were selected for LA‐ICP‐MS analyses. LA‐ICP‐MS analyses were performed at the NAWI Graz Central Lab “Water, Minerals and Rocks” (University of Graz and Graz University of Technology), in order to obtain concentrations for U, Th, and selected rare earth element  610 of the National Institute of Standards and Technology, Gaithersburg, MD, USA. Values for the SRMs reported by m et al.  were appJochum et al., Evans and Müller, Caragnano et al., Williams et al., http://georem.mpch-mainz.gwdg.de) [Jochum et al., While the NIST SRM glasses are not matrix‐matched to the analyzed carbonate and phosphate material, they offer distinct benefits compared to various matrix‐matched standards . Several analytical studies using biogenic carbonates and authigenic apatite found that the analytical advantages offered by the NIST SRM glasses outweigh the disadvantages of standardization using an unmatched matrix, which is largely based on the much better known trace element concentration of the NIST reference glasses compared to other standards [Koenig et al., Electron microprobe (EMP) analyses were subsequently carried out in close proximity to the LA‐ICP‐MS craters, after repolishing the samples. Since the grains are complex and often highly porous mineral aggregates of calcium fluor apatite and calcium carbonate, EMP analysis did not yield results close to the theoretical value of fluor apatite and calcite. Thus, all calculations of analyzed phosphates as well as calcium carbonates utilize a consistent CaO concentration of 55 wt. % as the internal standard value [e.g., 3.1Prior to interpretation, the measurements were evaluated for mixed signals of phosphate grains and surrounding carbonate rock based on their major elements . This preevaluation excluded several spots from the subsequent data analyses .McLennan, Alibo and Nozaki, Shields and Stille, Haley et al., Garnit et al., REE concentrations for each spot were transformed into shale‐normalized REE concentrations using the Post Archean Australian Sedimentary Rocks (PAAS) standard [German and Elderfield, Bau and Dulski, Morad and Felitsyn, Shields and Stille, Garnit et al., German and Elderfield [De Baar et al. [PAAS normalized REE concentrations were then used to calculate the Ce anomaly, a useful tool for the characterization of paleoredox conditions [derfield  defined r et al. .  [Hammer et al., The REDFIT power spectrum for the Bryozoan Limestone shown in Figure r et al.  and used44.1Amphestigina, Elphidium, and miliolids) and corallinaceans are common [Reuter et al., Mutti and Bernoulli, Reuter et al., Thin section analysis of the 27 samples taken within the Bryozoan Limestone reveals well‐winnowed carbonatic grainstones, predominantly composed of bryozoan and echinoid derived skeletal fragments containing a variable amount of planktonic and benthic foraminifera. In the lowermost 2 m of the unit below, the microbially formed phosphatic hardground, notable occurrences of benthic foraminifera  , showing that potassium is not a major gamma‐ray source for the sediment .Results of the XRF analysis show that all samples are very pure carbonates, with only race amounts of SiO4.2LA‐ICP‐MS and EMP analyses reveal that most investigated phosphatic grains likely represent complex microcrystalline aggregates of phosphate and calcium carbonate, making accurate analysis of the mineral composition difficult. This was further corroborated by the performed electron microprobe analysis of the samples. Nevertheless, matrix standardized results reveal a significant U enrichment of phosphate containing grains in gamma‐ray sources compared to the surrounding purely carbonatic host material. Results of the LA‐ICP‐MS analyses show that the uranium content in the investigated phosphate grains is consistently elevated ranging between 13.5 and 131 ppm, with an average of 48.4 ppm and a median of 43.5 ppm. By comparison, the uranium concentration in the carbonate matrix ranges between 0.17 and 1 ppm, with an average of 0.44 ppm and a median of 0.39 ppm. The standard deviation of the U concentration in the biogenic carbonates is 0.25 ppm. These values result in an average enrichment of roughly 1:111 of uranium in the phosphatic grains compared to the carbonate matrix. Thorium content is comparably low in most analyzed grains, with only three grains showing significantly elevated Th content, in the range of ∼30 ppm , indicating overall weak contribution of 40K to the total gamma ray counts. K2O is furthermore only weakly correlated with gamma‐ray intensity  and both large and small‐scale patterns occur within the gamma‐ray (GR) record of the Decontra section [2O5 and total gamma radiation counts . TOC is generally lost in the sedimentary record, as it is quickly dissolved and refracted by biological processes in most settings. This also explains the comparably low organic carbon content of the investigated grainstones Figure . TOC val5.2Auer et al., 2O5 content of the XRF measurements of discrete rock samples  is assumed to be the predominant U bearing mineral phase within the sediment [s Figure a.Compton et al., The fact that no other significant U sources occur within the sediment, and U concentrations of the carbonates remained relatively constant  in the marine realm [−1) [Auer et al., O'Brien et al., Mutti and Bernoulli, Reuter et al., Auer et al., The comparably low sedimentation rates for the Bryozoan Limestone , the main constituent of marine snow, acts as a transport mechanism for organically bound phosphate to the sediment.Föllmi, Mutti and Bernoulli, Arning et al., Filippelli, Crosby and Bailey, This setting led to a complex interplay of organic matter transport , authigenic/microbial phosphate formation, and uranium incorporation. Release of OM‐derived P in the sediment pore water occurred through degradation of both microbial mats and marine snow through microbial activity within the sediment. Microbes at the sediment water interface used POM as nutrient source, which in turn liberated P and U into a closed biochemically controlled micromilieu at the sediment water interface [Davey and O'toole, Föllmi, Schenau et al., van der Zee et al., Paytan and McLaughlin, Filippelli, Crosby and Bailey, Reuter et al., Auer et al., Thin section and geochemical evidence  within in the sediment, without significant mixing or enrichment from the overlying ocean water and in conjunction with the activity of magnetotactic bacteria [Bau and Dulski, German and Elderfield, German and Elderfield, Bau and Dulski, Alibo and Nozaki, Morad and Felitsyn, Haley et al., Shields and Stille, Garnit et al., Emsbo et al., REE signatures show a negative Ce anomaly in both the carbonate matrix and all investigated phosphatic grains using the Ce/Ce* versus Pr/Pr* plot [Reynard et al., Shields and Stille, Garnit et al., Reynard et al., A slight trend toward more negative La/Sm values as well as enriched La/Yb values occurs in our data. This suggests that both phosphates and carbonates in the Decontra section were affected by substitution processes as well as adsorption processes during early diagenesis [Auer et al., This creates a good explanation for the largely in‐phase variability of both the magnetic susceptibility and GR records of the Decontra section [5.4Filippelli, Mutti and Bernoulli, Reuter et al., Vincent and Berger, Jacobs et al., Abels et al., Mourik et al., Holbourn et al., J. C. Zachos et al., J. Zachos et al. Harzhauser et al., Economically viable phosphorites are generally deposited as lag deposits or hardgrounds formed during episodes of nondeposition or even active sediment removal. Thus, virtually all global phosphorite deposits are generally unrelated to primary P accumulation within the sediment and subsequent phosphogenesis [Föllmi, Filippelli, van der Zee et al., Filippelli, Holbourn et al., Our results indicate that the well‐known link between the carbon and phosphorous cycle [Compton et al., Föllmi, Jacobs et al., John et al., Föllmi et al., Holbourn et al., Diester‐Haass et al., Holbourn et al., Our results provide insights into the pacing and formation requirements of the widespread middle Miocene shallow marine phosphorite deposits [13C record during the Monterey event [Holbourn et al., Diester‐Haass et al., Holbourn et al., Diester‐Haass et al., Holbourn et al., Our model is thus in accordance with the hypothesis that variations in ocean circulation are the cause of the 405 kyr eccentricity‐forcing observed in the δ6Mutti and Bernoulli, Reuter et al., Auer et al., The present study uses LA‐ICP‐MS data of carbonate grainstone as well as authigenic phosphate to characterize microbially mediated phosphogenesis in the middle Miocene Bryozoan Limestone of the Decontra section in the Maiella mountain range . Our results show that: (1) primary microbially controlled phosphogenesis persisted throughout the Bryozoan Limestone in accordance with previous studies [Supporting Information S1Click here for additional data file.Table S1Click here for additional data file.


Matricaria chamomilla L. essential oils and a potent pro-apoptotic molecule  and enhanced the production of tumor growth factor-beta 1 (TGF-β1) on NCTC 2544 keratinocytes, although it did not change the activity of monocytes and dendritic cells , as the major mechanism of tumor immune escape, a crucial hurdle for tumor immunotherapy (Jacobs et al., in vitro cultured keratinocytes (Frikeche et al., +CD25+FoxP3+ regulatory T cells, through TGF-β expression (Baumgartner et al., in situ.Darra et al., reported that the anti-neoplastic action exerted by α-bisabolol, derives fundamentally by its ability in inducing mitochondria-mediated apoptosis in cancer cells (Darra et al., In vitro research usually neglected this issue, as most of investigations based on cell lines obviously never consider the immune microenvironment existing in the in vivo situation. In this perspective, the recent article by Frikeche et al., raises some criticism about the actual role of α-bisabolol as a real, promising chemopreventive molecule.Despite its promising activity as an anti-tumor molecule, α-bisabolol does not possess so different features respect to the widest family of plant-derived anti-inflammatory and chemopreventive polyphenols (Chirumbolo, Alpha bisabolol might affect mitochondrial permeability transition also in non cancer cells (Leanza et al., in vitro cancer lines, such as MiaPaCa, should be considered with caution.As with other phytochemicals, the role of α-bisabolol on cancer therapy should be expanded in future debates, while any further proposals to investigate this organic compound on The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood–brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-β, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-β. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction. As highlighted by recent expression profiling studies, the majority of the genes acutely modulated in the blood of stroke patients are implicated in the regulation of the innate immune system  and specialized cells, activated in the brain or recruited from the periphery, actively participate to the detrimental processes implicated in tissue damage, as well as to the repair and regeneration phases  and cytokine signaling pathways -γ  proteins and other DAMPS, plays an important role in ischemic brain injury , represent promising therapeutic options, effective in reducing the inflammatory response to stroke injury  is a member of the immunoglobulin family of cell surface receptors and has been implicated in the development and progression of stroke. The full-length, membrane-bound RAGE isoform (fl-RAGE) is mainly expressed in neurons and in microglia/macrophages. Up-regulation of this receptor has been documented after both permanent and transient focal cerebral ischemia in rodents  has been reported in rats subjected to either transient or permanent focal brain ischemia -1α have been correlated with favorable long-term outcome  and MIP-1α have been shown to play an important role in promoting tissue damage via recruitment of inflammatory cells  deficiency exacerbates ischemic brain damage by upregulation of proinflammatory cytokines and this Zn finger-containing immunoregulatory protein also participates in LPS- and electroacupuncture-induced ischemic stroke tolerance  occurring in peri-ischemic regions 2–7 days after focal ischemia in mice is coincident with the delayed increase of MMP-9, suggesting its involvement in neurovascular remodeling (Zhu et al., 2+ homeostasis in neurons through activation of EP1 receptors (Kawano et al., After an ischemic insult, the expression of COX-2 is elevated in neurons, vascular cells and neutrophils, as demonstrated both in stroke patients and in animal models (Nogawa et al., in vitro (Kim et al., COX-2 has been implicated in hyperbaric oxygen preconditioning, since pharmacological inhibition of this enzyme abolishes the beneficial effects of the conditioning stimulus in a rat model of transient global cerebral ischemia (Cheng et al., de novo expression of inducible NOS contribute to ischemic tissue damage (Iadecola et al., While elevated expression of inducible NOS is associated with ischemic (Cho et al., NO plays a crucial role in cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats (Horiguchi et al., Although ischemic stroke is a major cause of mortality and long-term disability worldwide (Go et al., All the authors participated in the collection, review, and analysis of the relevant literature, as well as to drafting and revising of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sepsis is an infection-initiated systemic inflammatory syndrome with an estimated incidence of 18 million cases annually worldwide. Despite advances in intensive care and supportive technology, the mortality rate of sepsis still ranges from 15% to 80%, reminding scientists and clinicians that it remains to be a major clinical challenge. The key to winning the “campaign” to combat sepsis is improved understanding of the epidemiology, pathogenesis, and biomarkers of sepsis and discovery of novel therapies. The present special issue shows several encouraging results and provides comprehensive reviews of the latest advances in this field. in vitro, in vivo, and genetic studies on the effects of defensins as well as the corresponding mechanisms within sepsis. Their review, “Defensins and Sepsis,” also points out that the function of defensins reflects both their immunomodulatory and broad-spectrum antimicrobial effects.The effector cells from the innate and adaptive immune systems play a crucial role in sepsis. Dendritic cells, in particular, serve as professional antigen presenting cells and are involved in the aberrant immune response to sepsis. In this special issue, X. Fan et al. discuss the effects of sepsis on the amount, surface molecule expression, cytokine secretion, and T-cell activating function of dendritic cells and the underlying mechanisms in their review “Alterations of Dendritic Cells in Sepsis: Featured Role in Immunoparalysis.” Recent postmortem studies of patients who died of sepsis showed that depletion of CD4 and CD8 lymphocytes is an important characteristic. Thus, knowledge of these circulating lymphocyte abnormalities is relevant for the understanding of sepsis pathophysiology. R. de Pablo et al., who have previously reported on the alteration of B cells, natural killer cells, and T-cell function in septic patients, summarize their latest findings on the role of blood lymphocytes in sepsis and discuss the different kinetic patterns of lymphocyte subsets and their relationship to outcome in their review “Role of Circulating Lymphocytes in Patients with Sepsis.” Both the clinical and basic researches have shown that sepsis-associated immunosuppression is associated with adverse outcomes. A novel heterogeneous population of immature myeloid cells that possess immunosuppressive activities, termed myeloid-derivedsuppressorcells(MDSCs), has gainedmuchattention in recent sepsis studies. D. Lai et al. discuss the complex functions of MDSCs in the pathogenesis of sepsis. Their review “Myeloid-Derived Suppressor Cells in Sepsis” also proposes that the overall role of MDSCs involves much more than simply being an immunosuppressive cell population. These 3 review articles provide a comprehensive analysis of the major important immune cells in sepsis and highlight potential therapeutic targets. As a group who have investigated the function of the family of defensins in sepsis for nearly 10 years, G.-H. Xie et al. summarize theκB-Mediated Inflammatory Response.”Although the international Surviving Sepsis Campaign guidelines have been released for 10 years, sepsis remains a fatal syndrome due to the lack of efficient biomarkers and novel treatments. D. N. Nguyen et al. investigated plasma cortisol levels in septic patients with delirium and coma and found that cortisol is a potential biomarker of brain dysfunction in their article “Cortisol Is an Associated-Risk Factor of Brain Dysfunction in Patients with Severe Sepsis and Septic Shock.” F. Song et al. and P. Madhusudan et al. discuss two important but controversial issues related to the Surviving Sepsis Campaign Guidelines. In a meta-analysis of 12 randomized trials involving 4100 septic patients, “Intensive Insulin Therapy for Septic Patients: A Meta-Analysis of Randomized Controlled Trials,” F. Song et al. reported no benefit and a higher incidence of hypoglycemia with intensive insulin therapy compared with conservative glucose management. P. Madhusudan et al. discuss the current debate on the choice, amount, and end points for fluid resuscitation in sepsis in their review “Fluid Resuscitation in Sepsis: Reexamining the Paradigm.” K. Xie et al. investigated the therapeutic function of hydrogen gas in a septic animal model for several years, and, in their present review, “Hydrogen Gas Presents a Promising Therapeutic Strategy for Sepsis,” they summarize the progress of hydrogen treatment in sepsis. J. Zhou et al. and X. Li et al. explore novel drugs for sepsis from the perspective of the neuroendocrine network in sepsis in their two studies, “Epinephrine Enhances the Response of Macrophages under LPS Stimulation” and “Agmatine Protects against Zymosan-Induced Acute Lung Injury in Mice by Inhibiting NF-In this present special issue about the pathogenesis, biomarkers, and treatment of sepsis, the authors provide comprehensive reviews and attractive research perspectives on the mechanisms of sepsis which we hope will inspire researchers investigating novel biomarkers and therapeutic sepsis targets.


Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise.Shiga toxin-producing  Escherichia coli (STEC) are a group of bacterial organisms that are capable of producing one or more types of Shiga toxin (Stx). STEC are associated with a disease spectrum ranging from diarrhea and hemorrhagic colitis (HC) to the potentially fatal hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). STEC infections are typically food-borne  that has acquired the ability to produce Stx2, typically produced by enterohemorrhagic E. coli (EHEC) rather than EAEC group members. This may have occurred via horizontal gene transfer resulting in a new E. coli virotype dubbed the Enteroaggregative Hemorrhagic E. coli or EAHEC (Bloch et al., E. coli O104:H4 and humans are believed to be the major reservoir for this organism (Wieler et al., E. coli O104:H4 was relatively similar to that caused by other STEC infections some pertinent differences existed. For instance, about a quarter of subjects affected developed HUS during the 2011 outbreak, which is 2–5 fold higher than the rate usually observed for an STEC infection (WHO, Perhaps highlighting the relevance of monitoring these non-O157 serotypes was the emergence of the rather notorious The lack of an effective treatment strategy for an STEC infection has made these agents a prominent public health threat and a burden to the medical community at large. The currently recommended management of an STEC infection mainly relies on supportive therapy and hydration Thorpe, . The useThe debatable use of antimicrobial agents for the treatment of an STEC infection has led to the rise of various alternative treatment approaches Table . These hin vitro and were demonstrated to protect challenged mice when administered intravenously (Nishikawa et al., in vitro and in animals (Kitov et al., in vitro and upon testing in animals (Paton et al., in vitro. This agent, however, was not effective in clinical trials (Trachtman et al., Various agents that mimic Stx receptors and bind them thus reducing their availability to cellular receptors have been developed. Carbosilane dendrimers harboring Gb3 at their termini neutralize Shiga toxins cycl, was shown to protect cells in culture against Stx (Noel et al., Cell permeable agents that can bind Stx2 and potentially interfere with its intracellular trafficking have been reported. These include Ac-PPP-tet (Watanabe-Takahashi et al., E. coli O104:H4 outbreak (Lapeyraque et al., E. coli O104:H4-induced HUS results in no additional benefits (Kielstein et al., Preparations of antibodies that can bind Shiga toxins and neutralize their effects have been reported. Anti-lipopolysaccharide antibodies have shown protective abilities upon laboratory assessment (Paton et al., in vitro or in experimental animal models; however, they have not been evaluated in clinical studies. Worth noting is a study that showed a synergistic effect between green tea extract and an antibiotic, levofloxacin, in the treatment of an STEC-infected mouse model (Isogai et al., Various natural products have been considered as potential therapeutic agents for STEC infections. These have included lactic acid (Pittman et al., The use of antimicrobial agents in treating STEC infections has been controversial and the subject of an ongoing debate. While some studies indicated that the use of particular agents may increase the risk of HUS, others have reported a decrease of this risk upon implementation of antimicrobials. While these observations may be particular to certain agents at some doses, the potential risk of antimicrobial treatment inducing HUS has led to a general contraindication of such agents (Qadri and Kayali, in vitro; these include the quinolones, trimethoprim, and furazolidone (Kimmitt et al., E. coli O104:H4 from the 2011 outbreak in Europe do not display an increase in toxin production upon treatment with meropenem, ciprofloxacin, chloramphenicol, or fosfomycin, unlike E. coli O157:H7 (Corogeanu et al., E. coli O157:H7 and in E. coli O104:H4. A sub-MIC concentration may, after all, be the concentration available locally at the site of infection. We noted that the response is variable depending on the isolate used and the concentration of antimicrobial implemented (Nassar et al., Several antimicrobial agents have been shown to enhance the release or the production of Shiga toxins from STEC cells E. coli O104:H4 during the 2011 outbreak (Geerdes-Fenge et al., E. coli O157:H7 cells remain viable, followed by treatment with gentamicin at a bactericidal concentration. This strategy was effective in decreasing toxin release compared to solely treating the cells with a bactericidal gentamicin concentration (Kanbar et al., E. coli O157:H7 infection mouse model resulted in an improved animal survival rate (Rahal et al., E. coli O104:H4 infected mice similarly resulted in an improved survival rate compared to untreated control mice that were infected with the organism; however, the highest survival rate observed was with mice treated with gentamicin alone, unlike our observations with E. coli O157:H7 (Fadlallah et al., Reconsideration of treating STEC infections with antimicrobial agents has nevertheless gained ground in recent years. Ciprofloxacin was recently reported to decrease the risk of HUS in subjects infected with in vitro beneficial effects of probiotics and that inoculation of animal models with a probiotic prior to an experimental STEC infection has preventative capabilities (Asahara et al., in vitro study showed that cultivation of STEC organisms in the presence of various Bifidobacterium, Pediococcus, and Lactobacillus strains results in a decreased production of Stx2. This was attributed to a decrease in pH due to the acids produced by these agents (Carey et al., Although probiotics may not have a therapeutic benefit in the management of an STEC infection, they may have a relevant preventative utility. Probiotics are probably capable of disrupting host-infectious agent/toxin interactions by occupying cellular receptors themselves, by producing decoy receptors that take up the toxins or by modifying the local milieu, hence making these interactions unfavorable (Corr et al., in vitro (Niu et al., Another preventative measure proposed as a means of controlling STEC is the application of lytic phages. Lytic phages have been shown to reduce STEC numbers Various vaccine approaches have also been attempted including the development of preparations that contain bacterial peptides and virulence factors (Wen et al., In conclusion, despite the passage of more than three decades since STEC organisms were first associated with human clinical illness CDC, , a generThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis  when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This “virtual” nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis.Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called “open mitosis.” In contrast, many fungi undergo a process termed “closed mitosis” in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast  In eukaryotic cells, the nucleus is enclosed by a nuclear envelope (NE) in interphase. The NE is a double membrane structure which separates the nucleoplasm from the cytoplasm. Macromolecules are transported between the nucleus and the cytoplasm across the NE through nuclear pores which are formed by large protein complexes called nuclear pore complexes  occurs (open mitosis) , nuclear and cytoplasmic molecules are mixed similar to open mitosis. Interestingly, both the NE and NPCs are maintained in this phase and thus this phenomenon is called “virtual” NEBD  that regulate its localization  unts Figure . NotablyS. pombe. Interestingly, the metazoan Nup107-160 subcomplex is known to re-localize to the kinetochores upon NEBD and functions in kinetochore-microtubule attachment (Loïodice et al., S. pombe meiosis I. In addition, Nup133, a human homolog of S. pombe Nup132, is required for proper localization of RanGAP1 at the kinetochore in human cells (Zuccolo et al., Our discovery raises the question of how Nup132 regulates both kinetochore assembly and NE permeability during meiosis I in nup132 mutant activates the spindle assembly checkpoint and delays meiosis I progression (Figure Inadequate kinetochore-microtubule attachment in the n Figure . The durn Figure . Alternan Figure . These rS. cerevisiae Nup133 results in Ran being uniformly localized in the cell rather than being enriched in the nucleus (Gao et al., S. pombe and S. cerevisiae, Nup132/Nup133 is required for the uniform distribution of NPCs along the NE (Doye et al., nup132 mutant will help determine whether changes in nuclear permeability during anaphase I involve NPC disassembly or NE rupture.As mentioned above, Nup132 depletion results in the loss of barrier function of the NE during anaphase I (Yang et al., S. pombe may be regulated by phosphorylation or other post-translational modifications of nucleoporins. Further studies are required to elucidate the role of post-translational modifications of Nup132 or other nucleoporins in the regulation of barrier function of NPCs and vNEBD during meiosis in S. pombe.Functional alterations in Nup132 or other nucleoporins may change the barrier function of the NE without inducing NPC disassembly. Post-translational modifications in nucleoporins may alter the barrier function of NPCs. In mammals, nucleoporin Nup50 is phosphorylated by ERK kinase, which in turn changes the affinity of importin β to NPCs (Kosako et al., S. pombe cells produce spores, and a correlation of vNEBD with spore formation has been established (Arai et al., mes1 mutant, which is deficient in blocking the degradation of cyclin Cdc13 (Izawa et al., mes1 mutant shows no vNEBD (Arai et al., tws1 mutant, a meiosis-specific allelic mutant of Cdc2 (MacNeill et al., tws1 mutant, nuclear proteins diffuse to the cytoplasm and RanGAP1 localizes to the nucleus during meiosis I (Arai et al., S. japonicus (Aoki et al., The timing of vNEBD corresponds to meiosis II during which S. pombe produces spores under nutritionally starved conditions. These spores are metabolically inactive until they experience growth-favorable conditions (Shimoda and Nakamura, S. pombe (Nishijima et al., vNEBD occurs specifically during anaphase II and results in the diffusion of nuclear proteins into the cytoplasm and cytoplasmic proteins into the nucleus, which is similar to that observed during open mitosis. vNEBD may allow cytoplasmic proteins to function in the nucleus during or after anaphase II. Occurrence of vNEBD is correlated with spore formation (Arai et al., S. pombe. This provides an example in which the regulated barrier function of the NE plays an important role for regulating meiotic processes in eukaryotes.The phenomenon of vNEBD suggests the importance of “opening” the gate between the nucleus and cytoplasm during meiosis. Nucleoporins and RanGAP1 may play key roles during open meiosis without physically breaking down the NE in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gan-Cao, coming from extracts of dried roots of Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata, of which chemical analyses were performed (Zhang and Ye, A recent paper by Glycyrrhiza species (Fiore et al., According to these authors, 18-β-glycyrrhetinic acid and glycyrrhizin possess an anti-viral potential, assessing previous evidence about the antiviral properties of Glycyrrhiza extracts contributes in hampering a full comprehension of their beneficial effects. Licocalchone A downregulates the inflammation-induced P450 1B1 while isoliquiritigenin exerts an opposite effect, then differentially influencing the role of estrogens in chronic inflammation and in carcinogenesis (Dunlap et al., Molecular interactions between 18-β-glycyrrhetinic acid and glycyrrhizin modulates the inhibition of several drug-metabolizing enzymes and efflux transporters (Feng et al., The authors did not address either clinical trial about licorice activity against microbes.Glycyrrhiza-derived purified substances, such as glabridin, are recently addressed as promising tools to prevent bacterial infections and against parasites (Cheema et al., Moreover, the role exerted by licorice in blood pressure may influence immunity and DCs functionality, as the equilibrium between the inflammatory response induced by T cell and T cell suppressor responses is critical for the regulation of blood pressure levels (Rodríguez-Iturbe et al., in vitro, yet surely clinical evidence is needed.This evidence supports the idea that the introduction of a whatsoever can be considered as a beneficial phytochemical from raw plants in the highly complex network of signaling interactions between cells and organs, even considering the different individual ability coming from genetics and phenotypic polymorphism or from epigenetics and lifestyle, cannot allow physicians, as well as researchers, to foresee the “real” effect of that substance in the whole organism. An interesting hypothesis is that phytochemicals, as toxic substances, produced by plants to protect themselves are beneficial only in a restricted range of doses. In this perspective, there is interesting evidence about the role of low doses of phytochemicals, particularly flavonoids, on stress response and immune reactions The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCDC151), which has been shown to be essential in motile cilia of many animals and other vertebrates but its effects in humans was not observed until currently. We observed a novel nonsense mutation in a homozygous state in the CCDC151 gene (NM_145045.4:c.925G>T:p.[E309*]) in a clinically diagnosed PCD patient from a consanguineous family of Arabic ancestry. The variant was absent in 238 randomly selected individuals indicating that the variant is rare and likely not to be a founder mutation. Our finding also shows that given prior knowledge from model organisms, even a single whole-exome sequence can be sufficient to discover a novel causal gene.Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder characterized by impaired ciliary function that leads to subsequent clinical phenotypes such as chronic sinopulmonary disease. PCD is also a genetically heterogeneous disorder with many single gene mutations leading to similar clinical phenotypes. Here, we present a novel PCD causal gene, coiled-coil domain containing 151 ( Primary ciliary dyskinesia  is a genetically heterogeneous ciliopathy inherited in an autosomal-recessive fashion [Bush et al., DNAI1, DNAH5, DNAH11, RPGR, and OFD1) had been identified and these accounted for just 40% of the cases [Zariwala et al., CCDC151 deficiency as a further cause of PCD.Understanding the genetic aetiology of PCD has been challenging. In 2007, only five genes , and a long history of wet cough with light-green colored sputum  and his unaffected brother were studied . The parents’ blood samples were also collected for further analysis. Details on all methods used in this study can be found in the Supp. Materials and Methods. Summary statistics from WES of proband can be found in Supp. Results.CCDC151 gene and the other was a frameshifting insertion in the ZNF595 gene (p.N201fs). The stop gain was absent in dbSNP, Exome Variant Server (EVS), our internal database (which includes his unaffected sibling), and 1000 Genomes Project databases, whereas the insertion was present (in a homozygous state) in many of our previously whole-exome sequenced controls (of Arabic ancestry) and also had a total MAF of 8.6% in EVS. The filtering process is depicted in Figurehttp://databases.lovd.nl/shared/variants/0000040146#04563).Initially, all LRoH regions identified by Plink [Purcell et al., PCR was used to amplify a region 221 bp long (harboring the stop gain) in the proband, the unaffected brother, and the parents, which was then subsequently sequenced (using Sanger sequencing method) and digested with AvrII enzyme to confirm the variant status  is in the local population, a buccal swab sample from 238 randomly selected individuals of Saudi Arabian ancestry  were collected . The PCR amplicons produced (using primers in Supp. To deduce how common the c.925G>T:p.(E309*) variant in CCDC151 gene was first identified as a potential human ciliome gene by Ostrowski et al. (CCDC151 was among the 110 proteins (with Accession no: BAB01602) identified by one-dimensional polyacrylamide gel electrophoresis analysis of human ciliary axonemes . More recently, Jerber et al. -dependent cilia in Drosophila. In the same analysis, they reported that Ccdc151 was expressed in tissues with motile cilia in zebrafish, and morpholino-induced depletion of the gene product lead to, similar to human PCD phenotypes, left–right asymmetry defects [Jerber et al., Ccdc151 is strongly expressed in (and was restricted to) motile-ciliated tissues, where it is required for dynein arm assembly and for the transport of the docking complex Ccdc114  [Jerber et al., Ccdc151 is important in the control of IFT-dependent dynein arm assembly in many animals [Jerber et al., Ccdc151 in IMCD3 mouse cells resulted in a deregulated ciliary length [Jerber et al., Chlamydomonas ODA10 gene that is the homolog of the mouse Ccdc151 gene  was calculated to be approximately 0.0009 . Additional evidence from the absence of mutation in EVS and internal database , GERP scores [Davydov et al., CCDC151 carried out by Jerber et al.  Φ mutations in autosomes were quantified in three healthy individuals (two of Arabic and one of European ancestry); and this figure was found to be approximately 60 on average. However, a very large proportion of these were in a heterozygous state and in olfactory, taste- and hearing-related genes. The amount of Φ mutations that fell in the entire ciliome database (i.e. list 1 and 2) was on average one. The ciliome database contains within it 2,004 genes related to all types of cilia , but a crude estimate on the number of “respiratory cilia”-related genes (not including the known PCD genes) was estimated to be approximately 100 . As aforementioned, known PCD genes explain around 70% of PCD, which leaves the other 30% to these 100 genes. Thus, the probability of one of these Φ mutations falling in one of the second-tier candidate PCD genes in an autozygous region , certain combinations of ultrastructural defects and/or sterility (or subfertility) are far from being completely understood [Vincensini et al., CCDC151 to cilia [Jerber et al., CCDC151 were not observed (until now) to associate these findings to the human model. Here, we report a homozygous nonsense mutation  in CCDC151 in a patient who has been clinically diagnosed with PCD. The variant was screened in 238 unrelated individuals, and it was found to be absent in all of them indicating that the mutation is not a founder mutation and may have occurred relatively recently and/or is tribal specific. Nevertheless, CCDC151 adds to the already identified 25 genes  in which mutations are known to cause PCD and indicates the important role CCDC151 plays in human respiratory ciliary function. Our findings also show that given prior knowledge from an animal model, even a single WES (with high read depth) can be adequate when pinpointing a novel causal gene.To conclude, although animal models have strongly linked


C) to trigger intracellular signals appears central to neurodegeneration pathways, yet the physiological significance of such signals is rather puzzling. For instance, PrPC deregulation disrupts phenomena as diverse as synaptic transmission in mammals and cell adhesion in zebrafish. Although unrelated, the key proteins in these events -the NMDA receptor (NMDAR) and E-cadherin, respectively- are similarly modulated by the Src family kinase (SFK) Fyn. These observations highlight the importance of PrPC-mediated Fyn activation, a finding reported nearly two decades ago. Given their complex functions and regulation, SFKs may hold the key to intriguing aspects of PrP biology such as its seemingly promiscuous functions and the lack of strong phenotypes in knockout mice. Here we provide a mechanistic perspective on how SFKs might contribute to the uncertain molecular basis of neuronal PrP phenotypes affecting ion channel activity, axon myelination and olfactory function. In particular, we discuss SFK target proteins involved in these processes and the role of tyrosine phosphorylation in the regulation of their activity and cell surface expression.The ability of the cellular prion protein (PrP Yes, there are two paths you can go by but in the long run There's still time to change the road you're on(R. Plant and J. Page)C on neuronal cell surfaces is required for pathogenic prions and aβ oligomers to trigger cellular damage  within lipid rafts, thereby stimulating neurite outgrowth  anchor, PrP is unlikely to physically associate with cytosolic SFKs. Nevertheless, co-immunoprecipitation data indicate that, in epithelial cells, PrP and SFKs interact at least indirectly within a larger protein complex (Morel et al., C triggered Fyn activation and hyperphosphorylation of the NR2B subunit of the NMDAR at tyrosine residue 1472 (Um et al., C promotes embryonic cell adhesion by stabilizing E-cadherin at the cell-surface (Málaga-Trillo et al., C as a modulator of cell-cell adhesion in zebrafish and synaptic transmission in mammals converge at a common mechanism, namely the ability of SFKs to control the internalization of trans-membrane proteins by phosphorylating their endocytic signals and/or the corresponding binding molecules. Given the functional diversity of SFK target proteins, we reasoned that altered SFK activity may account for at least some of the distinct neuronal phenotypes observed in PrP knockout and transgenic mice (Figure At first glance, some of these findings have no apparent connection with each other, aside from the common involvement of SFKs. However, closer scrutiny reveals mechanistic similarities pertinent to our understanding of PrP function. This is particularly manifest in two of the aforementioned models, where the downstream targets of PrP turned out to be transmembrane proteins modulated by tyrosine phosphorylation. In the study by the Strittmatter lab, binding of aβ oligomers to PrPC clearly shows that the latter can influence the activity of ion channels (Um et al., C suppresses NMDAR activity and glutamate excitotoxicity via the inhibition of NR2D subunits (Khosravani et al., 2+-mediated excitotoxicity (Solomon et al., 2δ-1 subunit and retain it at the ER, thus disrupting its delivery to the plasma membrane. Notably, while the molecular mechanisms of these two phenotypes seem unrelated, they are both consistent with the ability of SFKs to regulate the activity of ligand- and voltage-gated ion channels (Salter and Kalia, 2δ-1 during biosynthesis is the most likely explanation for its reduced levels at the plasma membrane (Senatore et al., The NMDAR-mediated glutamate excitotoxicity induced by aβ oligomers and PrPv1.3 voltage-gated potassium channels are expressed along the dendrodendritic synapse and are negatively regulated by Src phosphorylation (Fadool and Levitan, v1.3 null mice have modified action potentials and deregulated potassium currents, resulting in structurally altered olfactory bulbs and a heightened sense of smell (Fadool et al., 2+ influx that triggers inhibitory GABA release (Chen et al., v1.3 channels, and NMDARs provides testable explanations for the olfactory defects of PrP knockout mice.Unique among mouse PrP knockout phenotypes was the finding of impaired olfactory behavior (Le Pichon et al., Re-examination of a mild, late-onset phenotype in PrP knockout mice led to the discovery of defects in the maintenance of peripheral myelin (Bremer et al., As with the olfactory phenotype of Le Pichon et al., copious evidence suggests a potential involvement of SFKs in the PrP-CDP phenotype. Comprehensive reviews on the role of Fyn during myelination are available elsewhere (Kramer-Albers and White, v1.5 and Kv2.1 potassium channels in Schwann cells is controlled by the interplay between Src, Fyn, PTPα, and PTPε (Sobko et al., in vivo, this regulation might be relevant for re-myelination but not for OPC development (De Biase et al., Finally, voltage- and ligand-gated ion channels constitute further regulatory targets of SFKs during myelination. For instance, the phosphorylation state of KC in the proliferation and differentiation of various stem cell lineages (reviewed in Martin-Lannerée et al., Altogether, the common regulation of ion channels, olfaction and myelination by PrP and SFKs suggests that this phenomenon may extend to some of the other subtle PrP phenotypes mentioned above. For instance, it would be interesting to see if the role of PrPC to elicit intracellular signals at the cell surface is central to its multiple roles in health and disease. Second, the specificity of these signals is largely determined by extrinsic factors such as the plasma membrane microenvironment and the availability of cell- and tissue-specific protein partners. Third, the ubiquitous expression and functional redundancy of SFKs, along with the diversity of their downstream targets offer plausible explanations for some of the striking facts of PrP biology, such as its functional promiscuity, the viability of PrP knockout mice, and the diversity of PrP transgenic/mutant phenotypes. Based on these considerations, we propose that PrPC acts as a gatekeeper between neuronal survival and toxicity by controlling the activity and/or endocytic trafficking of ion channels, synaptic proteins and cell adhesion molecules via SFKs. Testing these complex scenarios should prove a formidable but rewarding endeavor.Several key observations emerge from the present analysis. First, the ability of PrPThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent advances in brain connectivity research suggest that the human brain operates as a complex but economic global network. Novel approaches from graph theory have been applied to a variety of neuroimaging studies and have achieved great success. However, the brain network architecture and plasticity patterns vary across different brain developmental periods, learning activities, and disease effects. Translation of descriptive imaging levels into clinical and other exploitation crucially depends on the potential to induce and monitor plastic changes in the network of interest. This special issue is intended to stimulate the continuing efforts in understanding the architecture and plasticity patterns of brain networks. It also aims to broaden the attention not only to the researchers who are currently working on these areas but to the general scientific audience who are interested in the brain structures and functions as well.This special issue includes eleven papers that presented an up-to-date progress of MR neuroimaging methods on brain network architecture and plasticity investigation employing various neuroimaging modalities: structural MRI, diffusion MRI, and functional MRI. In these papers, there are five studies focused on brain diseases, including Alzheimer's disease, mild cognitive impairment, mild traumatic brain injury, and major depressive disorder. Two studies investigated the brain plasticity changes after visual or hearing deprivations. Also, there are two studies that assessed volunteers after mediation or cognitive training, and, finally, the remaining two studies are on the methodological development for brain network construction.Brain Diseases. In the paper entitled “Abnormal Resting-State Functional Connectivity Strength in Mild Cognitive Impairment and Its Conversion to Alzheimer's Disease” by Y. Li et al., an investigation was performed for individuals diagnosed with mild cognitive impairment. With a 2-year follow-up, those individuals converted to Alzheimer's disease were compared with nonconverters for their alterations on functional connectivity strength and seed-based functional connections.The paper entitled “Topological Properties of Large-Scale Cortical Networks Based on Multiple Morphological Features in Amnestic Mild Cognitive Impairment” by Q. Li et al. investigated the topological properties of cortical networks based on geometric measures  change in aMCI patients compared with normal controls.The paper entitled “Multilevel Deficiency of White Matter Connectivity Networks in Alzheimer's Disease: A Diffusion MRI Study with DTI and HARDI Models” by T. Wang et al. conducted an evaluation of how the fiber tractography method could influence the construction of brain white matter network and eventually affect the extraction of brain network properties, as well as group comparison using Alzheimer's disease patients as examples.In the paper entitled “Compensation through Functional Hyperconnectivity: A Longitudinal Connectome Assessment of Mild Traumatic Brain Injury” by A. Iraji et al., patients with mild traumatic brain injury were studied for possible brain functional alterations. Brain functional network was constructed through predefined seeds in a 358-landmark mask and a groupwise clustering algorithm was employed to identify general patterns of functional hyperconnectivity.The paper entitled “Reorganization of Anatomical Connectome following Electroconvulsive Therapy in Major Depressive Disorder” by J. Zeng et al. presented a study for patients with major depressive disorder that were treated by electroconvulsive therapy. The plasticity of white matter pathway was evaluated.Sensory Deprivation. In the paper entitled “Alterations of Regional Spontaneous Brain Activity and Gray Matter Volume in the Blind” by A. Jiang et al., authors investigated how the early blindness shapes the regional spontaneous brain activity and gray matter volume in visual areas compared to sighted controls. A correlation analysis is also performed between the onset age of blindness and the brain structure and functions.In the paper entitled “Functional Reorganizations of Brain Network in Prelingually Deaf Adolescents” by W. Li et al., authors used resting-state fMRI to study prelingually deaf adolescents to access the possible brain network reorganizations due to their experience.Training Influence. The paper entitled “State and Training Effects of Mindfulness Meditation on Brain Networks Reflect Neuronal Mechanisms of Its Antidepressant Effect” by C.-C. Yang et al. designed a longitudinal analysis investigating resting-state fMRI both before and after 40 days of meditation training. Differences in functional connectivity both between states (rest versus meditation) and between timepoints (before versus after training) were assessed and reveal functional specificity of plastic reconfiguration of key networks implied in MDD.The paper entitled “The Exercising Brain: Changes in Functional Connectivity Induced by an Integrated Multimodal Cognitive and Whole-Body Coordination Training” by T. Demirakca et al. investigated the impact of “life kinetik” training on brain plasticity in terms of an increased functional connectivity during resting-state functional magnetic resonance imaging (rs-fMRI). The training is an integrated multimodal training that combines motor and cognitive aspects and challenges the brain by introducing new and unfamiliar coordinative tasks.Methodology Development. The paper entitled “Closely Spaced MEG Source Localization and Functional Connectivity Analysis Using a New Prewhitening Invariance of Noise Space Algorithm” by J. Zhang et al. proposed a prewhitening invariance of noise space as a new method for localizing closely spaced and highly correlated cortical sources under real magnetoencephalography (MEG) noise, to facilitate the source-level functional connectivity analysis.The paper entitled “Node Detection Using High-Dimensional Fuzzy Parcellation Applied to the Insular Cortex” by U. Vercelli et al. investigated a fuzzy parcellation scheme that partitions insular cortex into a number of regions based on the variances of their functional signals. Furthermore, the identified 12 clusters located in the insular cortex are found to best correlate with distinct brain areas that subserve different brain circuits/functions.


Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a functional miRNA/mRNA target pair be validated by fulfilling four well-defined experimental criteria. This review summarizes our current knowledge of miRNA biology, miRNA/mRNA target prediction algorithms, validated miRNA/mRNA target data bases, and outlines several experimental methods by which miRNA/mRNA targets can be authenticated. In addition, a case study of human endoglin is presented as an example of the utilization of these methodologies. MicroRNAs (miRNAs) are an endogenous family of single-stranded 20-25 nucleotide non-coding RNAs that play a critical role in posttranscriptional gene regulation by acting as guide molecules for the miRNA-induced silencing complex (miRISC) to inhibit gene expression by targeting specific mRNAs for translational inhibition and/or degradation . It is now clear that the expression levels of miRNAs vary widely; some are ubiquitously expressed, while others are expressed in a tissue- and/or cell-specific manner, and many show spatiotemporal expression patterns . Importal., 2009; Dombkowal., 2009; Friedmaal., 2009; Gurtan al., 2009). Computal., 2009). Therefal., 2009; Fabian al., 2009). Althoual., 2009; Bartel,al., 2009; Selbachal., 2009), recental., 2009; Mendellal., 2009).The vast majority of functional miRNAs are produced by a canonical multistep biogenic process which is initiated in the nucleus and is completed in the cytoplasm Figure 1  and is retained within the miRISC Figure 1  is found . ImportWith few exceptions, MREs are primarily located in the 3′-untranslated region (3′-UTR) of mRNAs and once recognized, mature miRNAs imperfectly base pair with MREs following a set of rules which have been experimentally and computationally identified  and/or deadenylation, decapping, and subsequent decay by a number of silencing factors that are scaffolded to this complex by TNRC6A/GW182 Figure 1 . There http://microrna.gr/microT)        even these two algorithms would benefit from more current data input. To begin to address this critical question, several recent review articles have compared and contrasted many of the miRNA/mRNA target algorithms currently available , Targetal., 2009), Pictaral., 2006), and Elal., 2007). The laal., 2007) provideal., 2011; Grimsonal., 2011), DIANA-al., 2013; Reczko al., 2013), and thal., 2010) recentlal., 2010) and Taral., 2010; Grimsonal., 2010) have beIt is also significant to note that updated algorithms identify up to 60 % of all available miRNA/mRNA targets and provide only one valid target in approximately every three predicted targets . It is Given the above review of miRNA biology and miRNA/mRNA target prediction algorithms, we propose the following “work flow” scheme Figure 2 for the For a case study we have chosen to analyze the human endoglin gene (ENG) for potential MREs. Endoglin  is a homodimeric co-receptor for transforming growth factor beta (TGFβ) and is known to play a regulatory role in TGFβ signaling  or miRNA of interest has been chosen, it must then be analyzed by miRNA/mRNA target prediction algorithms Figure 2. Both thwww.ensembl.org)  and in the 3′-UTR, the seed sequence binding type, whether or not the predicted MRE is conserved , what sal., 2013). This ial., 2013; Wang etal., 2013) and ∼70al., 2013). Importal., 2013; Park etal., 2013; Sandberal., 2013; Tan et al., 2013). TherefInterestingly, the human ENG gene generates two distinct mRNAs through alternative splicing, which results in isoforms that differ in a portion of their CDS and 3′-UTR . As a result, Table 1Since the mRNA isoform which encodes S-endoglin harbors the longest 3′-UTR, the Diana-microT-CDS algorithm will only utilize this sequence for computing miRNA/endoglin mRNA target interactions. When this analysis is performed, a total of 259 (threshold set to 0.4) miRNAs are predicted to interact with the human S-endoglin mRNA isoform at 797 individual MREs, with 186 target sites located in the CDS and 611 sites in the 3′-UTR (data not shown). Table 1In contrast to Diana-microT-CDS, the TargetScan algorithm results include the identified miRNAs and the predicted location of MREs in the 3′-UTR but not in the CDS. However, this tool does allow the user to analyze any annotated splice variant for a given gene. For example, TargetScan will analyze both L-endoglin and S-endoglin 3′-UTRs. The TargetScan results also include the number and type of seed match of conserved and poorly conserved miRNA binding sites, and a total context score (predicted efficacy of targeting) . Interehttp://www.microrna.gr/tarbase) . DIANA-al., 2006). http://mirtarbase.mbc.nctu.edu.tw/)   and > 500,000 interactions derived from high-throughput experiments  . At leaal., 2014) and miRal., 2009), are al-throughput sequencing of RNA isolated by crosslinking immunoprecipitation) experimental approach involves the transfection of a given miRNA mimic into a cell line of choice followed by ultraviolet (UV) cross-linking to generate AGO/miRNA/RNA cross-linked regions  Table 7, human eUCUCUAAGGAAGCGCAUUUC 3′, the partially complementary motifs are underlined) was identified 40 nts downstream from the transcription initiation start site. Given that this predicted MRE is harbored in the 5′-UTR region of the human endoglin mRNA isoforms, miR-522-3p/endoglin mRNA interactions would not be identified by the target algorithms discussed above since they are not programmed to analyze this region. Furthermore, with the tendency of miR-522-3p to interact with noncanonical MRE sequences, Tan et al.  Table . These ial. (2014) demonstIn conclusion, it is important to note that although only two of the ten DIANA-TarBase v7.0 cataloged experimentally identified miRNA/human endoglin mRNA interactions Table 7 Selbach, there iFor this review article we subjected the human endoglin mRNA to Diana-microT-CDS and TargetScan miRNA target analyses and examined DIANA-TarBase v7.0 data sets of cataloged and published experimentally supported miRNA/human endoglin mRNA interactions (see above). Given that the majority of the identification and cataloging of miRNA/mRNA target interactions by DIANA-TarBase v7.0 result from high-throughput techniques  withouthttp://www.ncbi.nlm.nih.gov/pubmed). Six publications were identified  demonstal., 2014) . Shyu eal., 2014) speculaal., 2014). Therefal., 2014; Vasudeval., 2014), it is miRNA profiling expression experiments utilizing ovarian cancer cells and ovarian cancer clinical samples demonstrated that a number of miRNAs were aberrantly expressed, including miR-370-3p, which was down-regulated in these studies . ImportTijsen et al. (2014) Table  focused Again, it is important to note that the miR-15/107 family members, miR-16-5p, miR-103a-3p, and miR-107 were identified to interact with human endoglin mRNAs by the HITS-CLIP technique Table 7  demonstGiven that endoglin has been established to play a regulatory role in TGFβ signaling . This fOnce the plethora of information from prediction algorithms, published validations of miRNA/mRNA interactions, and manual sequence inspections of miRNA binding sites has been assembled, prioritization of specific miRNA/mRNA target interactions to investigate can more effectively proceed. Among the number of putative miRNA/human endoglin mRNA interactions documented above, the remainder of this review article will focus on miR-370 Table 8 since thClearly a given miRNA and its target mRNA must be co-expressed in order for the miRNA to regulate the expression of a given biological target. Therefore, miRNA and target mRNA co-expression experimental studies should be performed first Figure 2, since tCo-expression is typically demonstrated by simply performing Northern blot analysis or quantitative real-time PCR (qPCR) using total RNA isolated from a specific cell type or tissue, and probes or primers specific for a given miRNA and mRNA target . We recin situ hybridization and immunohistochemical experiments utilizing paraffin-embedded, formalin-fixed tissues to address the question of co-expression . Additiovo, 2010; Sansom ovo, 2010).As described in the “Analysis of the Published Literature” section above Table 8, recent Importantly, Chen et al. (2014) initiatAfter the demonstration of co-expression of the miRNA and target mRNA of interest, the physical interaction of a specific miRNA with a candidate MRE harbored in a target mRNA needs to be confirmed Figure 2. The majFor construction of chimeric luciferase reporter constructs, the predicted MRE sequence from the target gene, most often located in the 3'-UTR but also in the 5'-UTR and CDS see , is clohttp://www.mirbase.org) and a passenger strand that is split in two separate antisense chemically synthesized LNA modified RNA oligonucleotide strands . After transfection into cells, the segmented nature of the passenger strand ensures that only the mature miRNA (guide strand) is loaded into the RISC with no resulting miRNA activity from the passenger strand. Regardless of the chemical makeup of the mimic utilized, transfection of a miRNA mimic into cells will increase the proportion of RISC containing this particular miRNA and therefore, gain-of-function studies can assess the biological consequences (i.e. repression of luciferase reporter levels/activity) resulting from an increase in the activity of the mimicked miRNA . In contrast, miRNA inhibitors, which are utilized for loss-of-function experiments, are chemically synthesized, single-stranded, modified antisense RNA oligonucleotides which are designed to bind with and form highly stable heteroduplexes with the complementary endogenous miRNAs when introduced into cells  engineeAlthough the ability of miRNAs to repress the activity of a chimeric luciferase reporter gene is a useful screening device, it remains a surrogate assay for testing the effects of miRNAs on their putative mRNA targets. Therefore, after confirming the physical interaction of a miRNA with a candidate MRE harbored in target mRNAs by reporter assays, we recommend that miRNA gain- and loss-of-function transfection experiments also be performed to validate miRNA-mediated post-transcriptional regulation of target genes of interest Figure 2. Experimental manipulation of endogenous miRNA activity by miRNA mimic and miRNA inhibitor transfection should correspond to predictable changes in target protein levels  . TherefAlthough gain- and loss-of-function experiments are powerful, it is important to remember that results can be confounded by side effects of transfection . Under in vivo . Recallal., 2014). These al., 2014). Alternal., 2014). Therefal., 2014; Helwak al., 2014; Martin al., 2014; Tan et al., 2014). Chen et al., (2014) demonstAfter miRNA gain- and loss-of-function transfection experiments have confirmed that a given miRNA mimic and inhibitor mediate the inverse protein expression of a target gene of interest, it is finally necessary to demonstrate that this regulation equates to changes in biological function Figure 2. Dependiin vivo gain- and loss-of-function experiments in mice or rats . For ex al, 2014; Montgom al, 2014), by inf al, 2014; Hsu et  al, 2014; Huang e al, 2014), or by  al, 2014; Kota et al, 2014; Miyazak al, 2014). In con al, 2014; McClure al, 2014), by inf al, 2014; Seeger  al, 2014; Tijsen  al, 2014), or by  al, 2014; Baigude al, 2014).Chen et al. (2014) demonstmiRNAs are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes  should equate to altered biological function. To date only a small proportion of miRNA/mRNA target interactions have been functionally validated. The unique experimental outline described here can be applied to the validation of any miRNA/mRNA interaction. As relevant targets are identified, the biological functions of a specific miRNA can be unraveled and assist in development of miRNA therapeutics.The authors declare that they have no conflict of interest.We express our appreciation to Ms. Emily Keeler for generating the figures and tables.