Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed. Classical therapeutic approaches and conventional drug discovery programs have been aimed at designing ligands and small molecule drugs capable of interacting and inactivating the function of a particular protein. In addition to traditional drugs , many efIn addition to ASOs, significant progress has been made in the field of RNAs that has led to the emergence of other entities capable of interacting specifically with mRNAs  as well as targeting other kinds of RNA molecules which are not translated into proteins  . Both strategies enabled the modulation of the RNA function by modifying gene expression levels and consequently the identification of novel DNA and RNA entities as promising therapeutic platforms and potential targets for drug development ,5. The fThis extensive research focuses on developing strategies to obtain more stable oligonucleotides along with functionalizing materials with the potential to facilitate gene delivery. It has resulted in the launching of numerous nucleic acid-based therapeutic candidates into the clinic ,11,12,13Several mechanisms used by cells have been extensively studied which deal with the increase or decrease of protein production. This process is termed gene regulation and can be triggered by different pathways:Antisense technology. This therapeutic strategy is based on hybridizing target mRNAs with oligonucleotides through Watson-Crick base-pair interactions [ractions ,16. ASOsRNA interference (RNAi). Fire and Mello’s discovery in the nematode Caenorhabditis elegans in which double-stranded RNAs (dsRNAs) can trigger the silencing of up-regulated genes was reported in 1998 and was finally awarded the Nobel Prize in Physiology or Medicine in 2006 [ in 2006 . The RNAAfter confirming synthetic siRNAs can also trigger the RNAi process in mammalian cells , the impmiRNA technology. A miRNA molecule is a small ncRNA made up of an oligonucleotide track containing about 21–23 nucleotides and is expressed in plants and animals. MiRNAs are involved in a good number of physiological processes like RNA silencing and post-transcriptional regulation of gene expression after binding at the 3′-untranslated region (3′-UTR) of specific mRNAs [ic mRNAs . NcRNAs ic mRNAs . There aIn general, antisense technology has shown to be one of the most employed strategies for targeting miRNAs. This is produced by designing oligonucleotides (anti-miRs) fully complementary to miRNA leading to the formation of ASO:RNA duplexes with high stability. The use of miRNA mimics is similar to the siRNA molecule. The pre-formed duplex is loaded into RISC and causes the inhibition of the endogenous target mRNA after transfection. To improve this delivery, anti-miRs and miRNA mimics have been chemically modified in order to improve stability and biodistribution properties and therefore enhance their therapeutic applications ,25. SuchTaking into account the importance of the gene expression regulation and its potential in the search for novel therapeutic agents, this article has placed special emphasis on the design of covalent strategies for delivering siRNA and miRNA conjugates reported in the last seven years. In the case of siRNA delivery, this review has been divided according to the non-viral vector used . For a more detailed view on the field of chemical modifications and the strategies carried out for launching siRNAs into the clinic, several articles have been recently published ,30,31. ICell-penetrating peptides—siRNA conjugates. Cell-penetrating peptides (CPPs) are a family of short cationic peptides that have been employed as delivery vehicles for a good number of small molecules [olecules  and macrolecules . CPPs maolecules  and haveolecules  and contolecules . HoweverA detailed study recently reported by Pärnaste et al. involving Transportan10 and a series of CPPs containing hydrophobic residues   were comCovalent strategies have also been used for conjugating siRNA oligonucleotides with CPPs to overcome the polyplex dissociation risk in the bloodstream. This strategy helped obtain novel CPP-siRNA conjugates with promising silencing activities both in vitro and in vivo in the absence of transfection agents. Consequently, Tat , PenetraGV1001 is a peptide which corresponds to residues 611–626 (EARPALLTSRLRFIPK) of the human telomerase reverse transcriptase (hTERT) protein. Initially, this peptide was used for stimulating an immune response against several cancers in an injectable cancer vaccine and is currently found under clinical studies ,49,50. IThe combination of liposomes with siRNA conjugates has been also reported to increase both stability and delivery efficacy of such macromolecules. Fan et al. were able to conjugate the peptide KALLAL into two siRNAs that were targeted against MEK1 mRNA (siMek1) and mutant B-RAF mRNA (siMB3), respectively . In part3O4 [myc oncogene, which is overexpressed in several cancer tumours. The siRNA was easily conjugated into the CPP through disulphide bonds to facilitate the dissociation of the corresponding siRNA in the cytosol. The authors reported in vitro gene silencing efficiencies as well as in vivo targeted delivery experiments in HT-1080 xenograft murine models mediated by CPP-siRNA conjugates when formulated into TML, TSL [myc expression levels. Furthermore, this strategy allowed them to retard the tumour progression in vivo. These therapeutic strategies could open up new prospects in the synthesis of novel siRNA materials involving the covalent combination of CPPs.Related to this approach, Liu et al. have carried out extensive research for improving the gene delivery capacities of CPP-siRNA conjugates by combining thermal and magnetic dual-responsive liposomes (TMLs) with magnetic fluid Fe3O4 . Additio3O4  in order3O4 . These sTML, TSL  and polyTML, TSL , respectCarbohydrate—siRNA conjugates. The covalent combination between carbohydrates and siRNA oligonucleotides has afforded promising nucleic acid conjugates with excellent properties not only in stability but also in promoting gene delivery. Carbohydrates participate in a good number of biological processes such as signal trafficking, cell surface recognition through lectins and carbohydrate-binding proteins [proteins ,62. The proteins ,64. Galaproteins . The preRenilla luciferase. Such modifications were recognized by the RNAi machinery when they were formulated with lipofectamine. Interestingly, these carbohydrate conjugates exhibited resistance to 5′-exonuclease up to 72 h after incubation with bovine spleen phosphodiesterase (BSP). The authors also evaluated the capacity of the conjugates to impart cellular uptake without using lipofectamine. These experiments only revealed that the double-tailed permethylated carbohydrate siRNA conjugate was able to moderately inhibit gene expression up to ~20%.In addition to the 3′-termini, Gal derivatives were introduced at the 5′-termini using phosphoramidite approaches. A small series of carbohydrate derivatives fully modified with OMe moieties were incorporated at the 5′-termini of the siRNA passenger strand. This strategy reported by Vengut-Climent et al.  gave risN-acetylgalactosamine  and Gal residues [2) and triantennary GalNAc residues  [Other kind of carbohydrate architectures based on one, two and four Gal residues have been proposed by Aviñó et al. with the aim of modifying the 5′-termini of the siRNA passenger strand. The authors carried out the synthesis of a small series of carbohydrate-siRNA conjugates which were designed for inhibiting tumour necrosis factor-α (TNF-α) production and also for enhancing the cellular internalization of siRNAs . TNF-α presidues . Extensiresidues . In partresidues ,72. NairGalNAc3) . These CGalNAc3)  and mousGalNAc3) . InteresThese results encouraged the same authors to explore the effect of the triantennary GalNAc residues at the 3′-termini but containing sequential GalNAc moieties (up to three residues) through non-nucleosidic linkers  Figure B. To accBesides obtaining non-nucleosidic building blocks, the same authors carried out structure-activity studies by attaching three consecutive GalNAc residues at the 2′- and 3′-position of the ribosugar and the nucleobase, respectively . This meThese results highlight that potent silencing activities exhibited by the majority of GalNAc-siRNA conjugates  is determined by modifying the 3′-termini of the siRNA passenger strand and consequently leaving unvaried the 5′-termini of the guide strand ,76,77. TE)-vinylphosphonate (VP) has proven to be a stable phosphate group mimic that has afforded important stability to GalNAc-siRNA conjugates in endocytic processes [To increase the stability of phosphate groups at the 5′-termini of the siRNA guide strand,  with potential to respond under intracellular conditions. Additionally, the authors prepared a conventional disulphide linker covalently linked to a siRNA molecule (conjugate_2) for comparison purposes. The amount of siRNA release from conjugate_1 and _2 was investigated both in extracellular milieu  and intracellular environments  (1) as practically inhibited the siRNA release (8%) under extracellular reductive conditions whereas the siRNA containing the disulphide group (conjugate_2) displayed a considerable siRNA release (38%) at the same experimental conditions. When GSH concentration was increased up to 1 mM, the integrity of conjugate_1 was scarcely affected (20% of siRNA release). Finally, total siRNA release was achieved when glutathione S-transferase was added to the GSH solution mimicking the conditions of intracellular environments. To further investigate the potential and stability of this polymer-siRNA construct, the authors carried out gene silencing studies by incubating three siRNA conjugates  in the presence of lipofectamine. The authors found significant inhibition activities for conjugate_1 which reduced luciferase expression up to 40% whereas gene knockdown efficiencies were halved in the other constructs  B,C. Intenstructs D.In a related topic, Takemoto et al. engineered an acid pH-responsive polymer-siRNA conjugate capable of enhancing siRNA delivery . The autl-ornithine and l-phenylalanine (in a ratio of 4:1) [a and acid cleavable acetal linkers [Other polymers with endosomolytic capabilities have been proven to be efficient vehicles for siRNA delivery. Poly(amido amine) (PAA)-based polymer derivatives ,95 conta of 4:1)  and (iv) linkers . d,l-lactic-co-glycolic acid) (PLGA) and linear polyethyleneimine (LPEI) have been also reported [Hybrid micelles based on a mixture of a siRNA covalently conjugated to hydrophobic poly self-assembled micelles for increasing the delivery of siRNA oligonucleotides . RelatedLipid-siRNA conjugates. Lipids are also one of the most used modifications that have been used for being covalently introduced to nucleic acids. For example, bile and fatty acids, cholesterol and a good number of hydrophobic residues have become the main hydrophobic residues to improve important properties of nucleic acids  [rofiles) ,103,104.Cholesterol (Chol) has been one of the first hydrophobic residues that has been used in RNA interference with the aim of modifying the 3′- or 5′-termini of a siRNA passenger strand to increase the transfection capacity of siRNAs ,104. RecFurther studies and following similar synthetic strategies, the same authors evaluated the efficacy of Chol-siRNA conjugates to reduce PPIB and Htt mRNA expressions by adding a 5′-phosphonate (5′-VP) moiety at 5′-termini of the guide strand . As descThe development of new delivery strategies has helped to improve efficiently the ability of siRNA-Chol conjugates to reach specific tissues and organs in vivo. SNALP and iNOP platforms are part of these technologies which are based on lipid-based systems and generally used as vehicles for improving the delivery of siRNA molecules into the liver ,108. TheE. coli β-galactosidase mRNA. These studies confirmed that Chol hydrophobic residues introduced at the 5′-termini of the sense strand showed superior knockdown efficiencies when compared with the rest of the siRNA modifications and unmodified siRNA molecules. These potent silencing activities in vivo and cellular uptake mechanism were further characterized [In addition to using phosphoramidites for introducing Chol residues at the 5′-termini, several strategies have been described for the introduction of hydrophobic lipid derivatives at the 3′-termini. For example, the use of a pyrrolidine linker was selected to incorporate Chol at the 3′-termini of the siRNA sense strand . This recterized . Wolfrumcterized .Taking the effect of HDL into consideration on gene silencing and transport of macromolecules, Ding et al. found promising in vitro and in vivo activities of siRNA-Chol against hepatocellular carcinoma (HCC), which is one of the most common cancer disease worldwide . SpecifiIn addition to Chol, other natural products have been covalently conjugated into siRNAs  and nuclα-tocopherol (TP) is a natural hydrocarbonated molecule that has been explored as a suitable carrier for siRNAs. As described with Chol and other natural compounds, TP has a tendency to be transferred to LDL lipoproteins in the liver . In this2 integrin (CR3). However, this cellular uptake was less efficient in the presence of CR3-receptor knockout cells.Other small molecules have been described as scaffolds for introducing chemically modified siRNAs at the 3′-termini. Thus, Grijalvo et al. used a protected form of glycerol for modifying the passenger strand with hydrophobic residues containing neutral and cationic lipids ,124. TheO-DMT-6-N-Fmoc-2-hydroxy-methylhexane with docosahexaenoic acid (DHA) [Htt) mRNA and CyclophilinB, a binding protein that is involved in some neurodegenerative disorders [Htt mRNA expression .The continuing interest on the search of a therapeutic platform for the treatment of neurodegenerative diseases allowed Nikan et al. to derivatize 1-id (DHA)   . The resisorders . To detepression C. In vivl-serine-based phosphocholine derivative containing a DHA pendent group. This strategy allowed to modify the 3′-termini of a siRNA passenger strand, obtaining the corresponding siRNA-DHA-PC conjugates designed for silencing Htt mRNA expression in mouse brain. The authors observed that siRNA conjugates containing the combination DHA-PC were able to mediate cellular uptake in mouse primary cortical neurons and also promoted silencing activities of Htt mRNA expression (80%) as well as reducing the amount of protein (~71%) in the striatum after one week.In the case of targeting Cyclophilin B, DHA-siRNA conjugates were able to silence mRNA expression up to 80% without inducing neuronal toxicity and innate immune response. To increase the effect of the siRNA-DHA conjugate in the central nervous system (CNS) distribution but maintaining its lack of toxicity, the same authors proposed to incorporate additional chemical modifications based on lysophosphatidylcholine (LPC) moieties in order to favour siRNA delivery to the brain in vivo ,130. TheThese silencing activities were comparable with the siRNA conjugate only modified with DHA (siRNA-DHA conjugate) but increasing neuronal toxicity and stimulating astrogliosis after a single intrastriatal injection (50 μg of siRNA-DHA-PC).Extracellular vesicles. In addition to LDL lipoproteins which have become promising non-viral carriers as described before [to-cell communication process. Depending on size and composition, EVs can be classified into exosomes and microvesicles. Examples of exosomes as carriers for the delivery of siRNA molecules have been previously reported both in vitro and in vivo [d before , other ed before . EVs are in vivo ,133. O’LAptamer-siRNA chimeras. Aptamers are synthetic oligonucleotides isolated from combinatorial libraries that are able to recognize and bind to specific target molecules with high affinity [affinity . Aptameraffinity . Aptameraffinity  and theraffinity . Thus, aaffinity ,138 and affinity ,139 amonaffinity .There are multiples approaches for conjugating siRNAs with aptamers as described by Zhou and Rossi in an excellent review . In thism) when conjugated with the 4-1BB aptamer.Berezhnoy et al. carried out a detailed study by conjugating 4-1BB aptamer with multiple siRNA targets by using a Durascribe T7 polymerase, which introduced 2′-fluoro-modified pyrimidines in the RNA backbone . The autNovel aptamer-siRNA chimeras based on bivalent aptamers targeting PSMA were efficiently engineered to evaluate their efficacy for treating prostate cancer in vitro and in vivo (102) . In partThe rapid evolution experienced in the field of RNA interference has allowed to develop novel strategies to increase siRNA delivery involving nanoparticles . In addibis-maleimidoethane, DTME) and this resulted in obtaining either multimeric or dimeric antisense siRNAs. Final hybridization of the resulting antisense siRNA strands with MUC1 aptamer-siRNA chimeras gave rise to the formation of multimeric (Comb-Apt-siRNA) and dimeric (Di-Apt-siRNA) chimeras, respectively. Interestingly, the presence of multiple ligands helped to promote excellent cellular internalizations for Comb-Apt-siRNA in MCF-7 expressing MUC1 cells when compared to fluorescently labelled dimeric and monomeric chimeras with POPO-3 dye. These internalization studies were also carried out in the presence of HepG2 cells (negative control). The authors confirmed that internalization mechanisms were mediated by clathrin-dependent endocytosis. Additionally, cell viability and gene silencing studies targeting GFP and Bcl-2 were also studied. In both cases, Comb-Apt-siRNA was able to mediate gene expression inhibition with better efficiencies than the rest of the prepared aptamers. To facilitate endosome escape and therefore increase the suppression of MCF-7 and HepG2 cell proliferation by inhibiting Bcl-2 mRNA expression, the authors combined the aptamer chimeras with LPEI before transfection experiments. This strategy improved remarkably the ability of multivalent Comb-Apt-siRNA chimeras to mediate siRNA delivery into target cancer cells and therefore reduce the viability in MCF-7 cells. As expected, no significant effect of the constructs was observed in the case of HepG2 cells. In addition to introducing relevant siRNA sequences like Bcl-2, the same authors were able to engineer a dual drug delivery system made of the aforementioned multivalent aptamers but containing an additional intercalated chemotherapeutic drug  [Novel architectures based on aptamer-siRNA chimeras have been described for improving receptor-specificity and therefore mediating efficiently cellular uptake via endocytosis . Yoo et in, DOX) . Thus, tsulfo--butyrate; sulfo-SMPB) linker (Aptamer AS1411 has proved to be an extraordinary targeting ligand for highly cell surface-expressed nucleolin (NCL) in the plasma membrane. This important property has been used to engineer efficient delivery systems to facilitate the internalization of a good number of therapeutic drugs, antisense and siRNA oligonucleotides into cancer cells ,158,159.) linker A. The au) linker B,C. InteC. elegans [MiRNA molecules are often expressed in a good number of cells and tissues  and also elegans . Ten yea elegans . Since tantagomiRs” (anti-miRs), locked nucleic acids (LNAs), peptide nucleic acids (PNAs), which are able to interact specifically with target oncogenic miRNAs as well as the introduction of miRNAs or miRNA mimics in order to reconstitute tumour suppressor activity have been opened up novel pathways for the search of alternative cancer-based therapies [The process of miRNA regulation has become an important issue for the discovery of new drugs in last years. A good number of approaches such as the synthesis of “herapies ,165,166.AntagomiR conjugates. Synthetic ASOs have proved to be effective molecules for targeting ncRNAs confirming their practicability of regulating miRNAs in vitro and in vivo [ in vivo . This ab in vivo . By virtO-methyl modified RNA bases and phosphorothioate modifications at the two ends of the oligonucleotide chain (3′- and 5′-) was engineered by Krützfeldt et al. in 2007 [The first antagomiR conjugate containing 2′- in 2007 . Followi in 2007  at the 3 in 2007 . Silenci in 2007 . This an in 2007 .Locked nucleic acids (LNAs). In addition to phosphorothioate conjugates, locked nucleic acids (LNAs) have also shown an important approach for targeting miRNAs because of their extraordinary affinity and specificity to interact with RNA complementary sequences as well as displaying high thermal stabilities [endo conformation. This special feature has allowed LNA technology to be an important tool for antisense gene silencing processes and more recently in the search of miRNA-based therapeutics by designing efficient antagomiRs (LNA-antimiRs) for silencing miRNA expressions both in vitro and in vivo [bilities ,184. The in vivo ,186. Imp in vivo . The autAs with antimiRs-based drugs modified with GalNAc in which exhibited potent inhibition activities against HCV, Santaris Pharma developed a 15-mer LNA-antimiR oligonucleotide complementary to miR-122 (Miravirsen) which was only modified with phosphorothioate linkages. This drug showed remarkable in vitro antiviral activity  and alsoThe versatility of LNA technology have been also demonstrated by targeting other potential miRNA targets involved in cancer diseases. Nedaeinia et al. carried out antisense gene silencing studies with the aim to find inhibitors for miR-21, which is related to various cancers  . The autN--succinimide ester, which is a non-degradable short linker and (iii) PEG24, a non-degradable long linker which favours the circulation of NPs without affecting aggregation processes in bloodstream . In addition to LNAs, peptide nucleic acids (PNAs) have been also used for regulating gene expression [N-(2-aminoethyl)glycine residues [pression ,198. Theresidues . Just asresidues . PNAs arresidues . To imprresidues ,203.6-Penetratin), four Lys residues and cystamine (Cys) containing terminal-free thiol groups [PNAs were used as antimiRs for the first time in 2008, when Fabani and Gait were able to target miR-122 in human and rat liver cells, as a proof of concept . The autl groups  have deml groups  This miRl groups  and the l groups . The autOther important target that has been currently studied for cancer therapy is the miRNA-221/22 cluster. This miRNA cluster is involved in glioblastoma progression when is upregulated. This produces cell proliferation by targeting PUMA (a p53-upregulated apoptosis modulator)  and otheN-(2-aminoethyl)-glycine backbone bearing homo-arginine and arginine side chains, respectively [The therapeutic potential of PNAs as antimiR molecules has been also demonstrated for targeting miRNA-210. This miRNA has been found upregulated in several cells and has been involved in cardiac diseases , variousectively . As obseTo avoid undesirable enzymatic degradations and provide fully protection to PNA conjugates from proteolysis, Manicardi et al. proposed to introduce eight cationic residues (Arg) within the PNA backbone containing α- and γ-PNAs without altering the inhibition activity of such PNA-antimiRs. The silencing activity was also evaluated on K562 cells. Interestingly, the new conjugates exhibited resistance when incubated in the presence of serum and peptidases. Furthermore, PNA conjugates were able to impart cellular uptake. This internalization was located entirely in the cytoplasm. Even though all chiral PNA-antimiRs prepared by the authors were able to inhibit the activity of miRNA-210, silencing activities differed according to the modifications introduced into the PNA-antimiR conjugates, obtaining the best inhibition results in the case of γ-PNA-antimiR-210. Although this PNA behaviour is not fully understood, the authors suggested that the presence of side modifications in the PNA backbone might produce differences both in the mechanism of cellular entry of PNA-antimiRs and the recognition with their target miRNA-210 (RNA binding). These important results would allow chemically modified PNA molecules, not only at the end of the sequence but also in their backbone and thus obtaining more potent PNA-antimiR-based drugs.Other approaches. Gold nanoparticles (AuNPs) have provided important contributions as vehicles for transporting small molecule drugs and other kinds of payloads  due to their unique optical properties, stability in the bloodstream and low cytotoxicity [toxicity ,220. Numtoxicity ,222. Thetoxicity . The strtoxicity . Hao et toxicity . Both ncOther nanoconstructs have been proposed by Kim et al. for targeting miR-29b which has been considered a key for regulating the expression of Mcl-1 protein as well as apoptosis . The str2-PEG-SH), a thiolated DNA oligonucleotide with a spacer of 10 adenine bases was also introduced. Finally, the AuNP was decorated with anti-miR-712 which was hybridized with the aforementioned DNA sequence  has helped to improve other kind of parameters such as stability and pharmacokinetic profiles. Disulphide chemistry is one of the most used covalent strategies in which siRNA molecules have been preferentially linked to CPPs, polymers and aptamers. Thus, once transfected, the presence of this cleavable linker under high concentrations of glutathione in tumour cells promotes the cleavage of the linker and therefore releasing the anticipated siRNAs. Alternatively, the versatility afforded by covalent approaches has also allowed the incorporation of tailed-made small molecules into nucleic acids. For example, excellent therapeutic results have been obtained with the synthesis of a series of siRNA conjugates decorated with the triantennary GalNAc residue. Several families containing this residue are found at various clinical statuses, including one (ALN-TTRsc/Revusiran) indicated for the treatment of amyloidotic cardiomyopathies, which is currently found at Phase III of clinical trials. In addition to carbohydrates, important findings have been achieved for the treatment of Huntingtin disease by developing synthetic strategies for attaching lipids, in particular the linkage of docosahexanoic acid with siRNA oligonucleotides.The use of covalent approaches has allowed miRNA technology to accelerate the process of achieving promising therapeutic molecules. LNAs and PNAs are good examples of how the synthesis of nucleic acid analogues can specifically interact with high affinity with up-regulated miRNA molecules in vitro and in vivo. Promising results in vivo have been also obtained wit anti-miRs covalently modified with Chol and GalNAc pendent groups, confirming the suitability of such modifications in therapies based on nucleic acids. While we still have a long way to go, the possibility of surpassing the bottlenecks associated to gene silencing activity by covalently introducing targeting ligands have resulted in a nice approach for improving cellular internalization and an important strategy for revitalizing antisense, siRNA and miRNA-based therapies.


The changes in extreme rainfall associated with a warming climate have drawn significant attention in recent years. Mounting evidence shows that sub-daily convective rainfall extremes are increasing faster than the rate of change in the atmospheric precipitable water capacity with a warming climate. However, the response of extreme precipitation depends on the type of storm supported by the meteorological environment. Here using long-term satellite, surface radar and rain-gauge network data and atmospheric reanalyses, we show that the observed increases in springtime total and extreme rainfall in the central United States are dominated by mesoscale convective systems (MCSs), the largest type of convective storm, with increased frequency and intensity of long-lasting MCSs. A strengthening of the southerly low-level jet and its associated moisture transport in the Central/Northern Great Plains, in the overall climatology and particularly on days with long-lasting MCSs, accounts for the changes in the precipitation produced by these storms. The central United States has exhibited increased extreme precipitation. Here, using satellite, radar, and rain-gauge data, Feng et al. show that springtime total and extreme rainfall trends are linked to increased intensity and frequency of long-lived Mesoscale Convective Systems. The observed rate of increase for sub-daily extreme convective precipitation has been found to be as much as doubling of the 7% per °C increase in precipitable water from the Clausius–Clapeyron relation13Over North America, some of the world's most intense MCSs commonly occur east of the Rocky Mountains789111315Here, we use the high-resolution  gridded precipitation data set from the North American Land Data Assimilation System (NLDAS) and hourly rain gauge data from National Climatic Data Center (NCDC). We identify MCSs by characteristics of the spatial and temporal contiguousness of their precipitation field embodied in the NLDAS data set (see Methods). Our method is analogous to others that utilize satellite infrared brightness temperature data to track MCSs−1 (roughly 20–40%) per decade increases in total MCS rainfall. Spatially, a majority of the increase in MCS rainfall occurs in areas north of the climatological maximum rainfall from MCSs. Examination of the trends in total MCS rainfall during summer  have become more frequent in the past 36 years. On the other hand, the 95th percentile hourly rainfall intensities from non-MCS precipitation have consistently decreased across the central United States (not shown). These findings of increased frequency and intensity of extreme hourly MCS rainfall reveal that previously reported increases of heavy rainfall frequency78To investigate whether changes in the intensity of MCS precipitation  also contribute to the observed increase in total MCS rainfall, we examined the NCDC hourly rain gauge data that is separated into MCS and non-MCS precipitation (see Methods section). To examine the role of the large-scale environments connected with the longer lifetimes and increased precipitation intensity of MCSs, we perform composite and trend analysis using the North American Regional Reanalysis (NARR)15The formation of large MCSs over the Great Plains in springtime is typically associated with baroclinic waves, which favour the development and/or enhancement of the GPLLJ and thus contribute to moisture convergence and vertical motion forcing for the convective systemsThe GPLLJ maximizes at night18Our analysis shows that more intense and more frequent longer-lasting MCSs occur along with the trend towards warmer springtime over the central United States. This result has significant implications regarding future extreme rainfall occurrence in this convectively dominated region and season and in other regions frequented by MCSs. Climate model projections suggest that low-level moisture flux will further intensify in a warming climate2529http://ldas.gsfc.nasa.gov/nldas/NLDAS2forcing.php#AppendixC, available from 1979 to 2014). The NLDAS data set utilizes a combination of ground-based rain gauges, radar, and satellite observations over the United States to produce a 1-hourly 12-km resolution precipitation data set. The hourly precipitation data from rain gauge stations are obtained from the National Climatic Data Center . Of the ∼2,600 NCDC hourly precipitation stations, 418 sites (east of the Rockies starting at 110°W) were retained. These records have less than 10% missing data between 1979 and 2013. A globally-merged half-hourly 4-km satellite IR brightness temperature data set  is used to track cloud systems and identify mesoscale convective systems (MCSs) based on cloud size and durationThe gridded precipitation data over North America were obtained from the NASA North American Land Data Assimilation System (NLDAS) meteorological forcing data set (The 15-year satellite MCS database was used to develop a precipitation feature (PF) algorithm based on NLDAS precipitation data alone by deriving the precipitation characteristics of MCSs. The PF algorithm was then used to construct an independent MCS database, which was validated against the satellite MCS database. The MCS database constructed by the PF algorithm provides consistent MCS rainfall compared with the satellite MCS database, with a majority of the normalized monthly MCS rainfall bias within 15% (see more detail description in ‘Developing the precipitation feature method'). The PF algorithm was then applied to the full NLDAS data set to construct a 36-year MCS database from 1979 to 2014. The days with land-falling tropical cyclones in the United States are excluded from the analysis to remove any potential impact of tropical cyclones on precipitation. After MCSs are identified, the total hourly precipitation is separated into MCS and non-MCS (isolated convection or weak stratiform) precipitation at each grid point.TIR) data set  is used to track individual cloud systemsTIR<241 K). A cloud system with a cold cloud shield area exceeding 6 × 104 km2 that persists at least 4 h is defined as an MCS12−1. Note that the PF and cold cloud shield associated with the MCS have reasonable correspondence in the spatial and temporal evolution throughout most of the life cycle of the MCS. Such correspondence has been explored in previous works using a combination of satellite-based cold cloud shields and ground-based radar and/or rain gauge data set to study the structure and evolution of MCSs31323334The satellite IR brightness temperature (x axis) and a particular percentile value of these three parameters (y axis).To develop a PF algorithm to identify MCSs, the same feature-tracking algorithm derived from analysis of satellite data is adapted to the NLDAS precipitation data set to track PFs rather than cold cloud shields. A PF database is then constructed from the tracked PFs to identify MCSs. Consistent with the satellite definition, we require an MCS to have a PF major axis length exceeding 200 km and persist for at least 4 h. In addition, a tracked PF's maximum area, maximum areal mean rain-rate, and maximum rain-rate skewness must exceed the least square fit values for its corresponding lifetime to be considered as an MCS. In other words, in addition to the minimum size and duration criteria, these three lifetime-integrated PF parameters must be satisfied for an object to be considered an MCS. The least square fit values  and intense contiguous rainfall feature exceeding the aforementioned PF parameter thresholds is considered an MCS. Some large weather systems producing widespread  and/or long lasting rainfall  are sometimes referred to as synoptic events7PSat and PPF are grid-level monthly accumulated MCS rainfall from satellite-based and PF-based MCS database, respectively. Results show that majority of the monthly rainfall errors are smaller than ±15% and are evenly distributed around 0, suggesting that the PF-based MCS database does not have systematic biases compared with the satellite-based MCS database. Over all 15 warm seasons, the spatial distribution of the amount and percentage of MCS rainfall between the satellite-based and PF-based MCS databases are shown in 11where http://ldas.gsfc.nasa.gov/nldas/NLDAS2forcing.php#AppendixC). The hourly radar/satellite precipitation products are divided by their respective daily totals to create hourly temporal disaggregation weights to represent proportion of the daily total precipitation that fell within each hour. These hourly weights are then multiplied by the daily gauge-only Climate Prediction Center (CPC) precipitation analysis to obtain the hourly NLDAS precipitation fields. Before 1996, the CPC daily contiguous United States (CONUS) gauge dataThe PF parameters obtained in To account for this change, the PF parameter fitting functions derived using data after 1996  are adjuWe assume that MCS properties do not change significantly between the two 5-year periods (1991–1995 and 1996–2000). In general, for NLDAS data before1996, the MCS definition is adjusted to have larger areal extent, smaller areal mean rain-rate, and similar pixel-level rain-rate skewness compared with data after 1996. Note that this change does not affect the calculation of accumulated rainfall on temporal scales of one day and longer , as the The MCS database produced in this study, along with all relevant data presented are available from the authors upon request.How to cite this article: Feng, Z. et al. More frequent intense and long-lived storms dominate the springtime trend in central US rainfall. Nat. Commun.7, 13429 doi: 10.1038/ncomms13429 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-11


Researchers navigate the ocean of biomarkers searching for proper targets and optimal utilization of them. Emergency medicine builds up the front line to maximize the utility of clinically validated biomarkers and is the cutting edge field to test the applicability of promising biomarkers emerging from thorough translational researches. The role of biomarkers in clinical decision making would be of greater significance for identification, risk stratification, monitoring, and prognostication of the patients in the critical- and acute-care settings. No doubt basic research to explore novel biomarkers in relation to the pathogenesis is as important as its clinical counterpart. This special issue includes five selected research papers that cover a variety of biomarker- and disease-related topics.The paper by N. Shi et al. demonstrated that the plasma microRNA-127 (miR-127) level was significantly downregulated in acute pancreatitis (AP) patients with respiratory failure compared with the healthy volunteers and those without respiratory failure. It was the first study that integrated miR-127 and the inflammatory injuries of the pancreas and the lung. miR-127 might serve as a potential marker for the identification of AP with lung injury.The paper by L.-J. Chen et al. explored the association between plasma miRNA-24-3p (miR-24) expression and coagulation factor X (FX) and XII (FXII) levels in major trauma and trauma-induced coagulopathy (TIC) patients. In their study, miR-24 was overexpressed in major trauma and TIC patients, and miR-24 expression correlated with FX level negatively, suggesting the possibility that miR-24 might inhibit the synthesis of FX during TIC.P. Lochner et al. measured osteopontin (OPN: biomarker for inflammation) and neurofilament heavy chain  in patients with acute optic neuritis and sex- and age-matched healthy controls. They demonstrated that OPN and NfH are elevated in these patients, supporting the presence of underlying inflammation and axonal injury as well as the prognostic utility of examining biomarkers in optic neuritis.The paper by B. Morawiec et al. addressed the prognostic value of copeptin in patients admitted with chest pain and suspected acute coronary syndrome (ACS). They concluded that copeptin appears to be an independent predictor of long-term mortality in patients with suspected ACS. Copeptin may be also considered as an early marker for identifying high-risk patients who would develop heart failure.The last paper by C. Spiekermann et al. explored S100 proteins A8 and A9 (S100A8/A9) as a promising diagnostic marker for peritonsillar abscess (PTA). Using a combination of S100A8/A9 levels and characteristic symptoms of PTA, they also developed a PTA score as an objective and appropriate tool to differentiate between peritonsillitis and PTA.We hope that this special issue would bridge basic research and clinical practice on biomarkers, facilitate the scientific development in this research field, and eventually contribute to the unrivaled value of disease markers.Patrizia CardelliMina HurSalvatore Di Somma


Central Pattern Generator (CPG) circuits are neural networks that generate rhythmic motor patterns. These circuits are typically built of half-center oscillator subcircuits with reciprocally inhibitory connections. Another common property in many CPGs is the remarkable rich spiking-bursting dynamics of their constituent cells, which balance robustness and flexibility to generate their joint coordinated rhythms. In this paper, we use conductance-based models and realistic connection topologies inspired by the crustacean pyloric CPG to address the study of asymmetry factors shaping CPG bursting rhythms. In particular, we assess the role of asymmetric maximal synaptic conductances, time constants and gap-junction connectivity to establish the regularity of half-center oscillator based CPGs. We map and characterize the synaptic parameter space that lead to regular and irregular bursting activity in these networks. The analysis indicates that asymmetric configurations display robust regular rhythms and that large regions of both regular and irregular but coordinated rhythms exist as a function of the asymmetry in the circuit. Our results show that asymmetry both in the maximal conductances and in the temporal dynamics of mutually inhibitory neurons can synergistically contribute to shape wide regimes of regular spiking-bursting activity in CPGs. Finally, we discuss how a closed-loop protocol driven by a regularity goal can be used to find and characterize regular regimes when there is not time to perform an exhaustive search, as in most experimental studies. Central Pattern Generators (CPGs) are neural networks that control muscle function by generating rhythmic patterns , Ks is the intracellular Ca-uptake rate constant and ρ is the endogenous Ca buffer capacity.The calcium stationary [This Hodgkin-Huxley type model displays the dynamical richness observed in the spiking-bursting activity of several neuron types  consisting of alternating bursting between the two neurons. White spaces represent regions were irregular spiking-bursting activity exists. One can observe that this map is nearly symmetric, i.e., regions that lead to regularized spiking-bursting rhythms correspond to values of maximal conductances g12 and g21 that do not differ much beyond 0.02 μS. Thus, for temporal symmetric fast inhibitory synapses, regularization occurs for closely symmetric maximal conductance values.As a departing point, we have considered a half-center oscillator topology with symmetric fast temporal dynamics in the synapse model , we obtain a k1 vs k2 map with reduced regularity bands  and at the end of the protocol the new set of values Cv < 5%). Noting the color code, one can observe that the changes in Figure k1 and k2 simultaneously or one at a time, for example only k2 to unveil the regularity bands shown in Figures This close-loop protocol allows different configurations and optimizations. The experimenter can choose to modify time constants Using realistic conductance-based models of half-center oscillator based CPG circuits, in this paper we have addressed the study of synaptic asymmetry to shape robust alternating spiking-bursting rhythms. In particular, we focused on the analysis of asymmetry of synaptic maximal conductances and time constants in the reciprocal inhibition of half center oscillators. We have mapped the regimes of regular and irregular coordinated rhythms as a function of these parameters. We have shown that asymmetry both in the maximal conductances and in the temporal dynamics of mutually inhibitory neurons, including modulation by gap-junction connectivity, can synergistically contribute to shape large regions of regular spiking-bursting regimes in central pattern generator circuits. Regular rhythms resulting from realistic asymmetric synaptic configurations display specific phase relationships that reflect the balance among the distinct sources of asymmetry.Irregularity regimes are typically disregarded both in experimental and theoretical CPG research. One reason for this is that regular rhythms, as recorded in experimental setups, are more easily associated with observable rhythmic CPG motor functions. Several theoretical studies have shown that regular regimes could be more efficient for specific motor tasks  and ONRG grant N62909-14-1-N279.We acknowledge support from MINECO/FEDER DPI2015-65833-P and TIN-2012-30883 (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.


Intermittent hepatic pedicle clamping (HPC) is often performed during hepatectomy. Whether it affects the long-term prognosis of hepatocellular carcinoma (HCC) patients is still controversial. This study evaluated the impact of HPC in patients with different stages of HCC. The study included 1401 patients who underwent hepatectomy in the primary cohort with 129 AJCC stage IIIB HCC patients; there were 80 AJCC stage IIIB HCC patients in the validation cohort. In each cohort, patients were placed in the long-term HPC (LTHPC) group or the short-term HPC (STHPC) group based on the cut-off time of HPC estimated by the receiver-operating characteristic (ROC) curve. Although HPC did not show significant effects on the prognosis of stage I–IIIA HCC patients in the primary cohort, 1−, 3−, and 5-year overall survival (OS) and recurrence-free survival (RFS) rates of stage IIIB HCC patients who received LTHPC (HPC time > 12 minutes) were significantly higher than those with STHPC (HPC time ≤ 12 minutes or received no HPC), similar in the validation cohort. Multivariate analysis demonstrated HPC time was an independent protective factor for RFS and OS in stage IIIB HCC patients. Herein, we report that proper HPC improved the postoperative prognosis of stage IIIB HCC patients and served as an independent protective factor. Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. A best test shows the ROC curve that passes through the upper left-hand corner  [2 test (or Fisher's exact test) for two categorical variables. Survival curves were calculated by the Kaplan-Meier method and compared by log-rank test. Multivariate analysis was performed using Cox proportional hazards model. Stratified log-rank tests were used to test the effect of HPC whilst controlling for the effect of another variable. Variables with P < 0.05 on univariate analysis were further evaluated by multivariate analysis.The RFS time was defined as the interval between the operation and the date of diagnosis of the first recurrence. The OS time was defined as the interval between the operation and death. The optimal cut-off time of HPC was assessed by the receiver-operator characteristic (ROC) curve and estimated the area under the curve  –42. Ther


Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and  A miRNA gene is transcribed by RNA polymerase II into a primary miRNA transcript (pri-miRNA), which folds upon itself forming a hairpin structure with a precise sequence and structural motif that is then cleaved by the RNAse enzyme Drosha and its co-factor DGCR8. The processed stem-loop intermediate, known as the precursor miRNA (pre-miRNA), is exported from the nucleus and cleaved by the RNAse enzyme Dicer in the cytosol to produce a ~22 nucleotide (nt) duplex. Upon loading of one strand of the RNA duplex into the RNA-induced silencing complex (RISC), the Argonaute protein in RISC mediates pairing between the miRNA ‘seed’ sequence (nucleotides 2–8 from the 5 prime end of the mature miRNA) and the 3 prime untranslated region (3′UTR) of the target messenger RNA (mRNA), inducing mRNA degradation and protein destabilization123in vivo, however, these efforts have been hindered by the complex genomic organization of miRNA genes. During the evolution of higher organisms, genomic duplication events generated miRNA families comprised of multiple miRNAs with high sequence homology and identical seed regionsmiRNA regulation is an important mechanism to control the expression of numerous key genes involved in development, morphogenesis, and disease pathogenesis56Unfortunately, the current genetic approaches in vertebrates only allow for the mutagenesis of single or clustered miRNA genes, but not of miRNA families originating from non-local duplication. These technologies include classical gene ablation strategies and transcription activator-like effector nucleases (TALEN)-based strategies91013151617in vivo.Here, we optimized a scalable multiplexed CRISPR/Cas9 strategy to induce heritable loss-of-function mutations of miRNA family members. We present a comprehensive survey of mutations within pri-miRNA genes encoding 45 miRNAs. Our data showed 99% of CRISPR/Cas9 mutations alter critical sequences within each hairpin pri-miRNA structure that impairs recognition by the miRNA biogenesis machinery, and thus prevents miRNA family expression 20 sgRNA architecture (where X20 represents the 20 nt gene specific sequence), a design that effectively discriminates off-target sites20-N15, where N15 represents the 15 nt region that anneals with a common universal reverse primer containing crRNA and tracrRNA sequences  and injected together with Cas9 mRNA into zebrafish embryos at the one-cell stage. F0 lesion frequency was tested for each miRNA locus in five individually injected fish using a T7 Endonuclease I assay (T7EI). Finally, we out-crossed F0 adults to generate F1 progeny and tested for miRNA indel transmission and miRNA functionality  to annotate structure and sequence features of the hairpin. Structural features such as the number and position of bulges, stem length29Intrigued by our results on the analysis of miR-24 and let-7 mutations, we reasoned that a comprehensive analysis of our mutagenized miRNA sequences would establish the most successful mutations to functionally perturb miRNA expression in vivo . We firsTo assess the impact of mutations in the miRNA genomic sequence on miRNA biogenesis, we developed an mCherry reporter assay. We cloned the wild type or the mutant pri-miRNA sequence into an mCherry reporter construct, the pTol2-Bact-pri-miRNA-mCherry biogenesis assay vector 31. Upon in vivo heritable mutations in multiple miRNA loci to abrogate the activity of an entire miRNA family and enables the functional analysis of miRNA gene families in biological processes (such as development). In addition, our system can advance the understanding of the critical structural rules of a miRNA gene and how they impact miRNA expression levels.Here, we describe a successful strategy to multiplex the CRISPR/Cas9 system to target miRNA gene families. This method can generate in vivo model. Second, we showed that multiplexed mutations were transmittable to the F1 generation. Finally, we analyzed the impact on miRNA biogenesis of a significant number of mutations within the pri-miRNA genes. Our data showed that the multiplexing strategy simultaneously generates mutations in up to 14 miRNA loci with minimal off-target effects. Additionally, we provide the proof-of-principle that F0 and F1 miRNA family mutants can result in loss of miRNA activity. Indeed, the injection of our CRISPR/Cas9 against the let-7 superfamily or miR-24 was sufficient to disrupt mature miRNA activity leading to derepression of a GFP sensor containing miRNA-binding sites. As our mutagenesis was successfully applied to several different miRNA families, our data represent the largest collection of compound miRNA family mutants generated by CRISPR/Cas9. Our strategy enables the analysis of more complex biological questions related to miRNA function, such as the functional redundancy between miRNAs that share the same seed region, are expressed within the same cell type, or are part of the same signaling pathways. Therefore, our protocol can be applied to address critical questions related to miRNA functional complexity in vivo.Our strategy represents a significant improvement from the preexisting protocols. First, we used multiplexed CRISPR/Cas9 to target multiple non-coding genomic sequences in an We analyzed the effects of a comprehensive number of indels generated in the pri-miRNA genome family sequences on miRNA biogenesis. We discovered that most of the changes affecting predicted nucleotide pairing and overall length of the pri-miRNA stem length are sufficient to impair mature miRNA biogenesis. This may be due in part to the fact that many of our sgRNAs were designed to target mature miRNA sequences within the pri-miRNA. Indeed, sequence regions and features within the stem of the pri-miRNA  would be preferentially disrupted by indels in this region. Interestingly, we found a mutation within a miRNA that can also enhance miRNA expression. We identified a mutation in miR-30c that reduces the size of the apical loop and increases the stem length of the pri-miRNA. Apical loop size has previously been reported to control microprocessor cleavage efficiency in mammalian cells with optimal apical loop size ranges of 3–23 nt14in vivo. In light of studies showing that polymorphisms in human miRNA genes are strongly associated with disease susceptibility such as schizophrenia and diabetes3233Altogether, the application of our strategy will allow not only the generation of models to study miRNA family expression and function, but also the identification of novel and critical sequences within a miRNA gene essential for miRNA biogenesis CF)Zebrafish  yielded similar mutation efficiencies but had higher toxicity.To generate sgRNA we purchased forward primers with the following architecture: 5′-TAATACGACTCACTATA-GG-XGenomic DNA was isolated using a DNeasy Blood and Tissue kit from a clutch of 15–20 embryos at 24 hpf injected with sgRNA and Cas9. Genomic DNA (50 ng/μl) was used to amplify an approximately 200–400 bp region surrounding the intended target see . Mutatioet al. submitted). Briefly, hairpin structures were predicted based on the initial identification of the apical loop position. Primary hairpin stem length was determined by counting the number of bases within the stem starting at the first paired base after the apical loop and stopping at the basal unpaired region. Primary sequence motifs .


K-user multicell multiple-input multiple-output (MIMO) Gaussian interference channel. We consider a MIMO interference channel with blind-IA through staggered antenna switching on the receiver. We explore the power allocation and feasibility condition for cooperative cell-edge (CE) mobile users (MUs) by assuming that the channel state information is unknown. The new insight behind the transmission strategy of the proposed scheme is premeditated (randomly generated transmission strategy) and partial cooperative CE MUs, where the transmitter is equipped with a conventional antenna, the receiver is equipped with a reconfigurable multimode antenna (staggered antenna switching pattern), and the receiver switches between preset T modes. Our proposed scheme assists and aligns the desired signals and interference signals to cancel the common interference signals because the received signal must have a corresponding independent signal subspace. The capacity for a K-user multicell MIMO Gaussian interference channel with reconfigurable multimode antennas is completely characterised. Furthermore, we show that the proposed K-user multicell MIMO scheduling and K-user L-cell CEUs partial cooperation algorithms elaborate the generalisation of K-user IA and power allocation strategies. The numerical results demonstrate that the proposed intercell interference scheme with partial-cooperative CE MUs achieves better capacity and signal-to-interference plus noise ratio (SINR) performance compared to noncooperative CE MUs and without intercell interference schemes.In this paper, we characterise the joint interference alignment (IA) and power allocation strategies for a  K-user relay aided multiple-input-single-output (MISO) interference channel are characterized with blind-IA and imperfect channel knowledge schemes, respectively [K-user channel values at the receiver side by using databases to recognize the desired message index values [Interference alignment (IA) is one of the most prominent techniques in wireless communication systems because of their increase in size of wireless network, complexity and challenges encountered . The achectively ,3. Topolectively ,5. Some x values ,7,8,9. Ix values . Authorsx values ,12 reporx values ,14, theyx values  focus onx values . The effx values ,18. In [x values , the antx values ,21.T preset modes. The authors in [L = 2, K = 2) such as (1) mobile user (UE1) located very far (cell edge user) from the base station (BS1) and (2) the mobile user (UE2) located very close to the base station (cell center user). In the existing scheme from [L = 3 cells, K = 5 users) is characterised in T = 2) modes. Since the proposed scheme does not require cooperation at the transmitter, the key idea behind the proposed scheme is to align the interference signals by creating a short-term channel fluctuation pattern at the transmitter side. This new insight behind the proposed scheme is that any vector of a user aligned at one unintended receiver  cannot be aligned at the other unintended receiver. In addition, we consider a multicell MIMO partial-cooperative network and power allocation strategies to improve cell-edge user performance. We completely characterise the K-user, multicell MIMO Gaussian interference channel through the staggered antenna switching pattern in the absence of channel state information (CSI). The base stations (BSs) use the channel fluctuation pattern information to adapt their environment and communicate strategies.In this paper, we propose new IA and power allocation schemes for a Gaussian interference channel where each receiver is equipped with reconfigurable antennas (staggered antenna switching pattern) capable of switching between thors in  considereme from , where tThe novelty of this research is that the interference signal subspace is aligned by considering a randomly generated transmission strategy.K = 5 users) and intercell interference (ICI) leakage (L = 3 cells) among the cells.We propose a partially cooperative cell-edge (CE) mobile users (MUs) scheme and two-tier transmitter beamforming strategy at BSs, to minimize the interuser interference (IUI) leakage , cell-median user (CMU), and cell-edge user (CEU). We also extended our approach to the partial cooperation between CE MUs and with ICI schemes for a multicell MIMO network; the data sharing between cooperative CE MUs and ICI schemes drastically improves the overall system performance.K-user multicell MIMO Gaussian interference channel with reconfigurable antennas at the receiver and considers the effect of the partial cooperation of CE MUs on interference channel scenarios with no CSI knowledge.In addition, our result applies to a downlink K-user multicell MIMO scheduling and K-user L-cell CE MUs cooperation algorithms improve the overall system performance for the K-user interference channel.Numerically, we show that the The capacity for the proposed cooperative CE MUs multimode staggered antenna switching and with ICI schemes achieve better system performance compared to the noncooperative CE MUs and without ICI schemes.The main contributions of this paper are summarized as follows.K-user, L-multicell MIMO Gaussian interference channel through staggered antenna switching, (CEUs) Partial Cooperation and power analysis. The rest of this paper is organised as follows. K, the transmission strategy is predetermined based on the reconfigurable multimode antenna and the receiver switch between T preset modes. Here, we consider a low-cost reconfigurable multimode antenna switch between only two modes The MIMO Gaussian interference channel model for five users per cell  and we also explained IA cases for K = 1, 3, 5 user cases. In our proposed scheme, we consider special cases, such as K = 5 user IA scheme in L = 3 cells CEU cooperation scheme in In this section, we elaborate single cell It is also essential to observe that the receiver can decode in total ten symbols (desired and interference symbols) over the first time slot and we discuss this as follows. We examine each received signal and decode the desired and interference signals; since The channel matrix and the transmit signal vector denoted by From above equation After cancelling the interference symbols that align in the same dimensional subspace, full-rank matrix What remains to be shown is that the We need to observe that receiver 3 can decode its two desired symbols and eight interference symbols by multiplying corresponding beamforming vectors and channel vectors, respectively, as briefly discussed below. The antenna switching pattern for receiver 3 is given in The matrix From the above equation for From the above matrix, the signals that align in the same dimensional subspace are cancelled. After cancelling the interference symbols that align in the same dimensional subspace, the Finally, we need to observe if receiver 5 can decode its two desired symbols and eight interference symbols by multiplying corresponding beamforming vectors and channel vectors. respectively, briefly discussed as follows.We examine each received signal and decode the desired and interference signals. From the above equation for K = 1,3,5 users and L = 1 cell is illustrated in From the above matrix, we observe that the signals that align in the same dimensional subspace are cancelled. After cancelling the interference symbols that align in the same dimensional subspace, the In this section, we focus on the multicell MIMO network by considering the partial cooperation among CE MUs. Since we focus on CE cooperation, we consider the frequency reuse technique to use the same frequency after a certain distance in the wireless system. BS cooperation involves the distribution of control, transmission of data signals, CSI, and precoders through wired backhaul links to synchronise transmitters and MUs. The BSs use this information to adapt their environment and communication strategies to the desired channel conditions. We investigate the multiuser partial cooperation in a multicell environment scenario to improve CEU performance. The signal-to interference-plus-noise ratio (SINR) determines the transmission rate of each MU. We consider partial-cooperative transmission among CEUs for three BSs  exchange between the users and BSs. However, full cooperation between the CCUs, CMUs, and CEUs users typically generates high complexity and imposes a large load on backhaul links. We consider only the cooperation between CEUs to improve cost efficiency, exchanging large information and high complexity at BSs. We consider the CEUs’ cooperation to the neighbouring CEUs because The throughput of individual CEUs is considered as quations  and (43)Therefore, it is sensible for the CEUs to decide whether to perform the cooperation downlink MIMO transmission scheme between adjacent CEUs.Frequency reuse (FR) is the reuse of the same frequency after a certain distance in cellular wireless systems. In general, a limited frequency bandwidth is divided into many subgroups on identifying the user location from the BS, where each group containing a few subcarriers is in turn assigned to adjacent cells. The fractional FR (FFR) and soft FR (SFR) techniques reduce the ICI coordination and aid in improving the spectral efficiency of CEU performance.SINR for k MUs in the L cell is given byKth MUs and In general, the FFR and SFR techniques divide a cell into inner and outer regions, and a different FR factor is used for each region. However, the distance from the BS to the CEUs can cause bad channel conditions and low quality of service (QoS); to solve these problems, locations of cell-centre (CC), cell-median (CM) and cell-edge (CE) users must be identified. In the downlink MIMO Gaussian interference channel, the In the proposed scheme, base station (transmitter) is stationary and the mobile users (receiver) are in motion, and the efficient user pairing method always chooses the best users pair based on user location identification. In order to identify the user location based on distance from BSs, we split the users into three main groups, i.e., cell-center users (CCUs), cell-median users (CMUs), and cell-edge users (CEUs) with the help of median equation, in order to pair the users efficiently, as shown in The major issue is to consider the interference from neighbouring cells because the CE users are significantly affected by ICI. Hence, the ICI signals must be identified and aligned to overcome the problem of low QoS and low throughput. The users near the BSs are considered as CCUs, the users with an average interference are considered as CMUs and the users with a higher interference from the neighbouring cells are considered as CEUs. We split the users into three main groups, i.e., CCUs, CMUs, and CEUs with the help of median, in order to pair the users efficiently. The CMUs are identified from the equation below:By considering the CMU equation in Equation , CE userIn this section, we focus on the cooperative CE MUs and the power analysis. For Consider The optimal power allocation for the partial cooperation of CEUs from cell 1 and cell 3 are Consider The total capacity achieved for partial cooperation of CE users is given below:The optimal power allocation for CEU partial cooperation in cell 1 and cell 2 is Consider The total capacity achieved for partial cooperation of CE users is given below:K = 5 users in L = 3 cells in The optimal power allocation for CEU partial cooperation in cell 2 and cell 3 is K-user multiuser and multicell MIMO scheduling Algorithm 1 deliberates switching pattern and IA by cancelling similarly aligned dimensional subspace signals for K-user and L-cell edge user (CEUs) partial cooperation for Algorithm 1K-user Multiuser & Multicell MIMO Scheduling.1:for example: 2:Beamforming vector: Two symbols per user 3:Received signal vector for receiver 1: 4:Aligning linear independent symbols: 5:Cancelling similar dimensional subspace signals:6:end for.K-user.Repeat the same approach for In this section, we explain the general case joint IA and power allocation for K-user multicell MIMO Gaussian interference channel through staggered antenna switching and power analysis for CE MUs in the absence of CSI. We restrict the total number of K-user communication pairs to be less than the number of transmitter K-user MIMO Gaussian interference channel, where the channel matrix and the transmit signal vector denoted by K independent users transmit n symbols is expressed as K-user We characterise the K-users in a round-robin fashion. We can prove that the proposed algorithm converges for the partial cooperation of K-user and L-cell CEUs and allocates maximum power for all CEUs.Algorithm 2 iterates over all L cells.Algorithm 2K-user and L-cell  Cell-Edge Users (CEUs) Partial Cooperation.1:for example: Cells 2:Partial cooperation between CEUs into adjacent CEUs3:   Total capacity of partial cooperative users:  4:Optimal power allocation for partial CEUs5:   for fixed 6:end for.K-edge user.Repeat the same approach for To produce the In this section, we present the simulation parameters in In In this study, we aligned the interference signals using multimode staggered antenna mode switching on the receiver. A key insight behind the proposed alignment scheme is that any vector aligned at one undesired receiver cannot be aligned at another unintended receiver because the similar dimensional subspace signals are cancelled to align the desired and interference signals in an efficient manner. Moreover, we computed the power allocation and feasibility condition for cooperative CE MUs in the absence of CSI knowledge. The new approach (randomly generated transmission strategy) effectively aligned and eliminated the similar dimensional subspace signals. Importantly, the cooperation between CEUs for a multicell MIMO Gaussian interference channel eliminates ICI and improves the overall system performance drastically. In addition, the numerical results showed that the proposed intercell interference scheme with partially-cooperative CE MUs enhances the capacity and SINR performance compared to noncooperative CE MUs and without intercell interference schemes, respectively.


Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo. Supercoiling is a transition from the relaxed state of the DNA double helix to one that is more under- or over-wound Fig. a. In DNANucleosome core particles connected by linker DNA are the fundamental unit of chromatin structure Fig. . Arrays In eukaryotes most DNA supercoiling is generated by the transcription machinery  DNA Champoux . EukaryoThe presence of unconstrained supercoiling in the linker DNA introduces free energy into the double helix Fig.  which prUnderstanding how the free energy of DNA supercoiling influences DNA–protein interactions through changes in DNA structure is key to identifying the effects of DNA supercoiling on chromatin architecture. Protein–DNA interactions that require changes to the structure of the DNA double helix, including melting and bending, are generally facilitated by an under-wound DNA structure  of the human At promoter regions an under-wound DNA structure may facilitate the formation of a disrupted, alternative or melted DNA structure that influences DNA protein binding. The TATA-box DNA sequence element is predicted to be sensitive to under-wound DNA supercoiling, and the structure of TATA-box binding protein bound to DNA identifies that the DNA has an under-wound structure  dependent manner. This influences the accessibility of linker DNA, which provides a reservoir of unconstrained DNA supercoils, and the capacity of supercoiling to promote/inhibit DNA–protein interactions.The next level of chromatin organisation above the nucleosome array is the higher-order fibre Fig. , which iThe higher-order chromatin fibre is influenced by the underlying distribution of nucleosomes on the nucleosome array and by other DNA–protein interactions. A fibre containing regular repeats of the Widom-601 nucleosome positioning sequence has a uniform structure which has been determined by X ray crystallography  gene. Measuring under-wound DNA with a biotin psoralen probe and visualising by immunofluorescence with streptavidin-green fluorescent protein, these same authors demonstrated that DNA becomes under-wound prior to expression of the HSP70 gene. This result indicates that transcription-generated DNA supercoiling primes large-scale chromatin domains prior to productive gene expression and together with other results supports a role for unconstrained DNA supercoiling in the structure of large-scale chromatin domains in vivo.The large-scale influence of DNA supercoiling on chromatin structure and genome regulation can also be observed cytologically, through changes in the compaction of large-scale chromatin structures. Fluorescence in situ hybridisation studies on human tissue culture cells revealed that under-wound DNA supercoil domains are cytologically decompacted compared to gene-poor over-wound domains and that this decompaction is lost in the presence of transcription inhibitor or DNA-nicking reagents  and condensin Hirano , but furUnderstanding the influence of DNA supercoiling on chromatin structure and gene regulation is in its infancy, despite almost 30 years of research following the publication of the twin-supercoil domain model (Liu and Wang In this model there is the potential for local amplification of unrestrained DNA supercoiling and gene expression in regions containing multiple active genes Fig. . At the To further understand how DNA supercoiling influences protein–DNA interaction, chromatin structure and gene regulation it is necessary to determine the properties of promoters and regulatory elements that are sensitive to DNA supercoiling. The FUSE element of c-myc has been the classic example of a supercoil sensitive protein-binding sequence element, and the TATA-box sequence has been implicated as supercoil sensitive at promoters, but it is unclear how prevalent supercoil-dependent regulation is in eukaryotic genomes. A recently described single-stranded DNA sequencing technique for mapping melted DNA structure in human cells provides a starting point for understanding the prevalence of supercoil-sensitive sites in the genome (Kouzine et al. Drosophila polytene chromosomes (Kouzine et al. Above the scale of individual genes, the importance of DNA supercoiling on chromatin structure in vivo has so far been determined by the addition of transcription inhibitors, topoisomerase inhibitors or DNA-nicking agents or through heat shock in Taking these approaches to understand how transcription and DNA sequence function together, through DNA supercoiling, to facilitate protein binding and chromatin structure will offer fresh insight into the role of DNA structure in the chromatin fibre. The model we propose identifies DNA supercoiling as a key factor regulating general principles of chromatin architecture, in addition to individual protein–DNA interactions, by transmitting information about gene regulation from the site of transcription through the chromatin fibre and over large-scale domains.


Toxoplasma infections constitute a worldwide public health problem responsible for significant morbidity. Symptoms of acute infection by the agent, the apicomplexan parasite Toxoplasma gondii, can range from mild to life-threatening depending upon the immune status of the host. Toxoplasma infections are often unrecognized in immune competent hosts but infection can lead to the long-term establishment of cysts in the brain and thus a persistent infection. Such tissue cysts are associated with altered behavior and cognition in mouse models. Furthermore, serological evidence of Toxoplasma infection has been associated with an increased risk of recent onset psychosis, schizophrenia, and other psychiatric disorders in humans. Adjunct treatment of Toxoplasma-seropositive individuals afflicted with schizophrenia with effective anti-parasitic medications could be beneficial by eliminating the parasite cysts, thereby possibly alleviating psychiatric symptoms. However, currently available medications are ineffective against the cyst form of the organism responsible for persistent infection. Others have published the anti-Toxoplasma potential of derivatives of the bis-amidine pentamidine. We designed and synthesized a panel of novel bis-amidines and explored their anti-Toxoplasma efficacy.We designed and synthesized an array of twelve novel bis-amidines. These compounds were examined for in vitro activity against T. gondii tachyzoites first using a colorimetric assay employing the β-gal producing strain RH-2F to examine the effect of the compounds over 5 days of parasite growth in human fibroblast host cells. This was followed by several immunofluorescence-based assays to determine if compounds can directly act on tachyzoites (red/green invasion assay), on an established infection of host cells (replication assay), and whether the compounds are parasiticidal, ie. can rid the host cells of tachyzoite infection after one dose (recovery assay). A rodent model of acute toxoplasmosis was established for examining in vivo efficacy of lead compounds.Four of these compounds proved highly efficacious in vitro with 50% inhibitory (IC50) values ranging from 200 nM to 1.3 µM. Therapeutic indices based on the ratio between the median cell cytotoxic dose and the IC50 ranged from 4 to 84. These four compounds all inhibited tachyzoite invasion and significantly inhibited in vitro replication over a 24-hour period at nanomolar concentrations. Additionally, two of the compounds evaluated thus far appear to be parasiticidal in vitro at 1 - 2 µM.Our results suggest that bis-amidines can be designed to be effective against experimental Toxoplasma infections. The parasiticidal activity of some of the compounds make them serious candidates for further drug development. Further experiments to determine 1) in vivo efficacy in mouse models of persistent infection, and 2) synergy with antipsychotics and mood stabilizers will investigate the possibility that these compounds could be used as adjunct treatment in Toxoplasma-positive individuals suffering with schizophrenia and other psychiatric disorders.