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Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum

Riboswitches are RNA elements that regulate gene expression in response to their ligand. Although these regulations are thought to be performed without any aid of other factors, recent studies suggested the participation of protein factors such as transcriptional termination factor Rho and RNase in some riboswitch regulations. However, to what extent these protein factors contribute to the regulation was unclear. Here, we studied the regulatory mechanism of the flavin mononucleotide (FMN) riboswitch of Corynebacterium glutamicum which controls the expression of downstream ribM gene. Our results showed that this riboswitch downregulates both ribM mRNA and RibM protein levels in FMN-rich cells. Analysis of mRNA stability and chromatin immunoprecipitation–real-time PCR analysis targeting RNA polymerase suggested the involvement of the mRNA degradation and premature transcriptional termination in this regulation, respectively. Simultaneous disruption of RNase E/G and Rho function completely abolished the regulation at the mRNA level. Also, the regulation at the protein level was largely diminished. However, some FMN-dependent regulation at the protein level remained, suggesting the presence of other minor regulatory mechanisms. Altogether, we demonstrated for the first time that two protein factors, Rho and RNase E/G, play a central role in the riboswitch-mediated gene expression control.

INTRODUCTION
Riboswitches are non-coding RNA elements that control gene expression in response to cellular conditions. They are typically composed of two functional domains, a ligandbinding aptamer and downstream expression platform, which is involved in the genetic control of associated genes (1,2). Riboswitches are classified by their specific ligands; more than 10 small molecule compounds ranging from amino acids to coenzymes are known to act as ligands. A ligand binding to the aptamer during and/or after transcription leads to the formation of the 'on' or 'off' conformation of the expression platform region. In the past decade, many riboswitches have been discovered, and their molecular mechanisms of gene regulation have been extensively studied (3). From these studies, two general mechanisms, control of the translation initiation by an anti-Shine-Dalgarno (SD) sequence and control of the transcription termination by an intrinsic terminator, have been revealed. In the case of control at the translational level, ligand binding typically promotes the sequestration of SD sequences by anti-SD sequences, thus inhibiting ribosome binding to SD sequences. Without a ligand, an anti-anti-SD sequence in the aptamer domain base-pairs with an anti-SD sequence, resulting in the liberation of the SD sequence and translation of the mRNA. In the transcription termination regulation of riboswitches, a ligand bound to the aptamer typically facilitates the formation of a transcriptional terminator stem in the expression platform and promotes premature transcription termination. In the absence of ligand, the formation of a termination stem is inhibited by an antiterminator sequence in the aptamer domain, and transcription elongates into the protein-coding region.
In contrast to these canonical mechanisms, recent studies suggested the participation of protein factors, such as transcription termination factor Rho and RNase, in some riboswitch-based gene regulations (4,5). In both cases, these riboswitches are equipped with anti-SD sequences; therefore, ligand-dependent regulation is thought to be performed at both the transcriptional and translational levels. Thus the regulation pathways of riboswitches are more complicated than had been thought. The redundant regulation pathways in one riboswitch make it difficult to dissect the relative contribution from either pathway in physiological condition. Therefore, to what extent these protein factors contribute to the regulation is unclear. Also, in some putative riboswitches, a lack of cis-acting elements such as anti-SD sequences or the pair of intrinsic terminator and anti-terminator sequences suggests that protein factors like Rho and RNase are involved in the function of these riboswitches (3). However, regulatory mechanism of these riboswitches has not been elucidated and reports that suggest the participation of protein factors in riboswitch regulation are limited in ␥ -proteobacteria such as Escherichia coli and Salmonella enterica (4,5). It is unclear if functional interplay between riboswitches and protein factors is widely distributed.
Corynebacterium glutamicum is a high-GC gram-positive bacterium that is widely used for the industrial production of amino acids, and it is expected to be a versatile biocatalyst for biofuels and biochemicals (6)(7)(8)(9)(10). In addition, this non-pathogenic soil bacterium is of interest as a model organism for the Corynebacterineae, an Actinomycetales suborder that includes medically important pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis (11)(12)(13). With respect to the riboswitches of C. glutamicum, the existence of flavin mononucleotide (FMN), thiamin pyrophosphate, cobalamin, and S-adenosyl methionine riboswitches has been predicted by comparative genomics or RNA-Seq analysis (14)(15)(16). Because the ligand compounds of these riboswitches are used as cofactors for many enzymes, establishing the fundamental characteristics of the metabolic regulation of these cofactors is of great interest.
Previously, we demonstrated that the expression of the C. glutamicum ribM gene, which encodes riboflavin transporter, is regulated by the FMN riboswitch (17). However, an experimental evaluation of the mechanism has not been performed. Here, we report that the FMN riboswitch of C. glutamicum mainly controls ribM gene expression at the mRNA level by regulating the action of two trans-acting protein factors, Rho and RNase E/G. Also, we assessed the relative contributions from either regulation pathways in physiological condition. Although FMN riboswitch of C. glutamicum has been thought to be controlled at translational level by anti-SD sequence from comparative genomics study (15), our results demonstrated that the regulation at the mRNA level by Rho and RNase E/G comprises the basis of this riboswitch regulation. These findings revealed the physiological significance of regulation at the mRNA level by protein factors in riboswitch-mediated gene expression control. Also, our observations raise a question about the control of many other riboswitches that have been thought to be controlled by cis-elements.

Media and growth conditions
E. coli was grown at 37 • C in Luria-Bertani medium while C. glutamicum was grown aerobically at 33 • C with shaking at 180 rpm. Media were supplemented with 50-g ml −1 antibiotics when necessary. For all analyses, 10-ml overnight pre-cultures of C. glutamicum in A medium (18) was washed twice with 10-ml minimal medium (BTM medium) (19), and the cells were resuspended in BTM medium for subsequent analysis. For all analysis, pre-cultured cells were inoculated in 100-ml BTM medium supplemented with 1% glucose and a limited (0.1 M) or sufficient (1 M) concentration of riboflavin and grown to mid-log phase.

Bacterial strains and plasmids
Strains and plasmids used in this study are listed in Supplementary Table S1. Oligonucleotide primers used in this study are listed in Supplementary Tables S2 and S3. A detailed procedure of construction of strains and plasmids is described in the SI MATERIAL AND METHODS (Supplementary data).

Quantification of cytoplasmic FMN level
Cytoplasmic FMN levels were quantified as described previously (17).

Total RNA purification
After cultivation, cultures were mixed with an equal amount of RNAprotect Bacteria Reagent (Qiagen) and incubated for 5 min at room temperature. Cells were collected by centrifugation (5 min, 6000 × g, 25 • C) and stored at −80 • C. Cells were resuspended in 10-mg/ml lysozyme/TE buffer and incubated for 1.5 h at 37 • C. Total RNA was purified using NucleoSpin R RNA (MACHEREY-NAGEL) according to the manufacturer's instructions. Purified RNA was further treated with DNaseI (TaKaRa), ethanol precipitated and suspended in water.

Northern blot analysis
A detailed procedure of northern blot analysis is described in the SI MATERIAL AND METHODS (Supplementary data). Briefly, digoxigenin (DIG)-labeled anti-sense RNA probes targeting the RFN element (RFN probe) and ribM coding region (CDS probe) were used to detect ribM mRNA. Sense strands of ribM RNA of different lengths (157, 245 and 950 bases) were synthesized by in vitro transcription and were used as an RNA molecular weight marker. Total RNA purified from cultured cells and synthesized sense-strand RNA were analyzed on a 1.25% denaturing formaldehyde agarose gel followed by blotting to nylon membrane. After the hybridization and washing, hybridized RNA probes were detected using anti-DIG-AP, Fab fragments, and CDP-star (Roche Applied Science).

Quantitative reverse transcription-polymerase chain reaction
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed as described previously (20). Total RNA (20 ng) was used as a template for analysis of the ribM, aceA and rneG genes, and 0.4 ng was used for analysis of the 16S rRNA to generate cDNA and for the subsequent PCR. Each qRT-PCR mixture (20 l) contained 500 nM each primer, 10-l Power SYBR green PCR Master Mix (Applied Biosystems) or FastStart SYBR Green Master (Roche Applied Science), 8 units RNase inhibitor and 5 units murine leukemia virus reverse transcriptase (Applied Biosystems). The primers used in these reactions are listed in Supplementary Table S3. Reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) with the following cycle parameters: one cycle of 50 • C for 30 min and 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 30 s. The 16S rRNA result was used as an internal control.

Quantitative analysis of RibM protein expression level
Relative expression level of RibM protein was measured by western blot analysis as described previously (17). Strains harboring a modified ribM gene encoding C-terminally FLAG-tagged RibM protein were used. Briefly, cells were disrupted by vigorous shaking with zirconia/silica beads (BIO SPEC). After centrifugation to eliminate intact cells and debris, the supernatant was collected as the cell-free extract. Total protein in the soluble fraction of the cellfree extract was determined by the Bio-Rad protein assay (Bio-Rad Laboratories) using bovine serum albumin as a standard. Cell-free extracts were analyzed on 12% polyacrylamide gels. After transfer to Immobilon-P polyvinylidene fluoride membranes (Millipore), FLAG-tagged proteins were detected with anti-FLAG M2 monoclonal antibody (Sigma). Peroxidase-coupled anti-mouse IgG antibody was used as secondary antibody, and chemiluminescence reagents (GE Healthcare) were used to develop the blots.
Semi-quantitative analysis of the relative RibM protein level was conducted using cell-free extract of strain TK05 cultured with 1-M riboflavin for 9 h as a standard (17). A calibration curve was obtained using standard samples containing different amounts of total soluble protein. The calibration curve was used to estimate the RibM-FLAG expression content of samples. Relative expression levels of RibM protein were calculated by normalizing values to that obtained in −FMN cells of the ribA ribM-FLAG strain (TK05).

Analysis of mRNA stability by the Rifampicin chase assay
After cultivation to mid-log phase, 1-ml culture was mixed with an equal amount of RNAprotect Bacteria Reagent (Qiagen) for a reference sample (0 min). Immediately after the sampling of the reference sample, Rifampicin (Wako) was added at 200 g/ml, and samples were taken at 1, 2, 3, 4 and 6 min for strains with an intact rneG gene and at 1, 2, 4 and 8 min for the rneg ribA ribM-FLAG strain (TK12). After incubation for 5-15 min at room temperature, cells were collected by centrifugation (5 min, 6000 × g, 25 • C) and stored at −80 • C. The purification of total RNA and qRT-PCR analysis was performed as described above.

Chromatin immunoprecipitation-qPCR analysis targeting RNA polymerase
The detailed chromatin immunoprecipitation (ChIP) procedure is described in the SI MATERIAL AND METHODS (Supplementary data). Briefly, C. glutamicum strains harboring the rpoC gene with a FLAG-tagged coding sequence were grown to mid-log phase. Formaldehyde was added to cross-link chromosomal DNA and RNA polymerase (RNAP) complexes. Cross-linking was quenched by the addition of glycine, and cells were then collected. Cell disruption and genomic DNA shearing were conducted using a Bioruptor UCD-250 (Cosmo Bio). After the preparation of the cell-free extract, an aliquot was stored for the purification of total genomic DNA sample. FLAG-tagged RNAP cross-linked with chromosomal DNA fragments was immunoprecipitated using anti-FLAG M2 agarose (Sigma).
The total genomic DNA sample and ChIP DNA sample were purified from the cell-free extract and immunoprecipitated fraction, respectively.
Reference DNA (4 ng) and 4-l ChIP DNA were used as a template for qPCR analysis. Each qPCR mixture (20 l) contained 500 nM each primer and 10-l FastStart SYBR Green Master (Roche Applied Science). The primers used in these reactions are listed in Supplementary Table S3. Reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) with the following cycle parameters: one cycle of 50 • C for 2 min and 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 30 s. Cycle threshold values were obtained using automated settings and were used to calculate the concentration factor and relative RNAP occupancies (see the legend of Figure  3).

Rho-function knock-down
The Rho-function knock-down system was developed as described in the Supplementary data. C. glutamicum strains carrying pCRC104 were grown to mid-log phase. During cultivation, the expression of mutant Rho protein was induced for 3 h by 1-mM Isopropyl ␤-D-1thiogalactopyranoside (IPTG).

Expression of ribM mRNA is increased under FMNdepletion conditions
Previously, we demonstrated that the expression of RibM protein is upregulated under FMN-depleted conditions (17). Although translational regulation was predicted from comparative genomics study (15), a prediction of the secondary structure of the C. glutamicum FMN riboswitch suggested that a putative SD sequence forms a stem even in the ligand-free state ( Figure 1A). This prompted us to examine whether the FMN riboswitch regulation is controlled by some mechanism other than SD sequestration. Therefore, we carried out northern blot analysis using RNA probes for the RFN element (an aptamer region of FMN riboswitch; RFN probe) or RibM proteincoding sequence (CDS probe) with RNA samples isolated from C. glutamicum strain TK02 ( ribA) ( Figure 1B). Because ribA gene encodes bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase which catalyzes the first step of riboflavin synthesis, a ribA disruptant will not make FMN and it is therefore possible to better control cellular FMN levels in this mutant. The cultivation of ribA disruptant to mid-log phase in the presence of a limited (0.1 M) concentration of riboflavin resulted in decreased cytoplasmic FMN levels without affecting growth. In contrast, supplementing riboflavin at a sufficient (1 M) concentration rescued the cytoplasmic FMN level (Supplementary Table S4). Thus, in this study, ribA-deleted cells cultured with limited and sufficient riboflavin concentrations are called FMN-deprivation (−FMN) and FMN-rich (+FMN) cells, respectively. Interestingly, northern analysis showed that transcripts including the ribM coding region were markedly increased in −FMN cells ( Figure 1B, filled arrowhead). This fragment is slightly longer than the control RNA and encompasses the nucleotides from the transcription start site to the C-terminus of the RibM proteincoding region ( Figure 1B, lanes 3 and 9), indicating that the RNA includes the entire ribM CDS with an untranslated region. qRT-PCR was performed to compare the mRNA expression level using primers targeting the ribM coding region ( Figure 1C and Supplementary Table S5). The expression of ribM mRNA increased ∼100-fold in −FMN cells. These results were consistent with the increase of RibM protein. Interestingly, in northern analysis, although we did not observe any signal with the CDS probe in +FMN cells (Figure 1B, lane 11), the RFN probe detected a short transcript that corresponds to the length of the RFN element ( Figure  1B, lane 5, open arrowhead), indicating that the regulation of ribM gene expression is executed after transcription initiation.
We previously found that mutations in the aptamer region of the FMN riboswitch lead to constitutive expression of RibM protein (17) and referred to these mutations as constitutive active mutation (CAmut). When the ribA CAmut strain (TK07) was analyzed, northern analysis showed the presence of similar signals between −FMN and +FMN cells; this pattern resembles that of −FMN cells of the intact FMN riboswitch strain ( Figure 1B). Thus, regulation at the mRNA level was abolished by CAmut. The signal intensity was slightly decreased in the CAmut strain, and the decreased ribM mRNA level was also confirmed by qRT-PCR analysis ( Figure 1C). The origin of this slight decrease is discussed below.

RNase E/G-dependent degradation of ribM mRNA controlled by the FMN riboswitch
Since the steady-state mRNA level is controlled by the balance between de novo synthesis and degradation, decreased mRNA level in +FMN cells suggests that synthesis and/or degradation efficiency is changed in response to cytoplasmic FMN level. To examine whether ribM mRNA degradation is enhanced in +FMN cells, ribM mRNA stability was measured in −FMN and +FMN cells of ribA ribM-FLAG strain ( Figure 2A). As expected, the ribM mRNA decay was accelerated in +FMN cells. This suggests that the decrease of the steady-state ribM mRNA level in +FMN cells is attributed to the destabilization of ribM mRNA. To evaluate the role of RNase in the modulation of ribM mRNA stability, we attempted to disrupt the RNase gene. In E. coli, RNase E is a primary endoribonuclease in mRNA decay (21). In the C. glutamicum genome, there is one RNase E homolog (rneG), and its gene product, RNase E/G, has been reported to degrade mRNA (22). Therefore, we analyzed ribM mRNA stability in the rneG disruptant. The stability was markedly increased with the disruption of the rneG gene in +FMN conditions, while the effect of rneG disruption was very small in −FMN cells (Figure 2A). Consequently, FMN-dependent regulation of ribM mRNA stability was abolished in the rneG disruptant. The results in the ribA strain that neither the stability of known RNase E/G target (aceA mRNA) (22) nor the rneG mRNA level was modulated by cytoplasmic FMN level ruled out the possibility that RNase E/G activity was decreased in −FMN cells (Supplementary Figure S1). These results suggest that RNase E/G specifically degrades the FMN-bound form of ribM mRNA. Consistent with this, a 6-fold increase in the steady-state level of ribM mRNA was observed in rneG-deleted cells under +FMN conditions but not under −FMN conditions by qRT-PCR ( Figure 2B and Supplementary Table S5). However, a large part of the FMN-dependent regulation of ribM mRNA remained. Indeed, in northern blot analysis, the signal in +FMN cells was undetectable even in the rneG disruptant ( Figure 2C,  lane 12). Unexpectedly, in contrast to the mRNA level, the RibM protein level in +FMN cells was not increased by rneG deletion. Instead, the RibM protein level in −FMN cells was slightly decreased with rneG deletion ( Figure 2B). Thus, RNase E/G clearly takes part in the FMN riboswitch function. However, regulation at both the mRNA and protein levels remained in the rneG disruptant.
In E. coli, a translating ribosome lowers the accessibility of the RNA degradosome to the mRNA, resulting in increased mRNA stability (23). To examine whether the reduction in mRNA level in +FMN cells is a secondary effect of the decreased translation efficiency, a putative SD sequence of the ribM gene (AGGGGA) was mutated to AGGCCT to lower the ribosome affinity (SDcct mutant). As expected, the mutations markedly decreased the RibM protein level, which could not be detected even in −FMN cells ( Figure 1C, less than 0.002-fold compared with the intact SD strain). However, in contrast to protein levels, ribM mRNA levels of the SDcct strain were nearly identical to those of the intact SD strain in both +FMN and −FMN cells ( Figure 2B and Supplementary Table S5) and decay of ribM mRNA was only slightly accelerated by SD mutations under −FMN conditions ( Supplementary Figure S2A). This slight difference in ribM mRNA stability is probably due to the stabilization effect by efficient translation which occurs only in intact SD strain. Absence of such difference in +FMN conditions suggests that translation is inhibited in +FMN cells of intact SD strain. These results imply the existence of FMN-dependent translation inhibition. In the CAmut strain, mRNA stability control was not observed, and the stability was almost the same as that of −FMN cells of the intact FMN riboswitch strain (Supplementary Figure S2B). These results are consistent with the hypothesis that the regulation of ribM mRNA stability depends on the FMN-dependent conformational change. Also, the result that CAmut did not destabilize ribM mRNA indicates that the decreased mRNA level in the CAmut strain ( Figure 1B and C) is not due to the altered mRNA stability caused by the mutations.

Premature transcriptional termination occurs in +FMN cells
The results presented thus far imply the presence of ribM gene regulatory mechanisms other than mRNA degradation. In riboswitch-based gene regulation, the control of premature transcriptional termination is one of the general mechanisms. To test the possibility that premature transcriptional termination occurs in +FMN cells, we compared the transcription elongation efficiency of +FMN and −FMN cells. For this purpose, we measured the amount of ribM gene DNA fragment purified by ChIP with RNAP. Because the occupancy of RNAP on specific genome regions Nucleic Acids Research, 2015, Vol. 43, No. 1 525 is correlated with the transcription efficiency of the region (24), this analysis enables us to clarify whether the transcription efficiency is modulated in ribM. Using the ribA rpoC-FLAG strain, DNA associated with RNAP in vivo was co-immunoprecipitated, and the amount of purified DNA was measured by quantitative PCR (qPCR). To monitor the local occupancy of RNAP within the ribM gene, five primer pairs were designed ( Figure 3A). The results were normalized to the RFN1 region to compare the transcription elongation efficiency. In −FMN cells, RNAP occupancies were constant throughout the entire coding region of ribM, indicating that most transcription elongated to the end of the ribM gene, as expected. In contrast, in +FMN cells, RNAP occupancies in the coding region decreased to 40-60% that of the RFN1 region ( Figure 3B). These results strongly indicate that premature transcription termination occurs selectively in +FMN cells. In the ribA CAmut rpoC-FLAG strain (TK14), we expected that RNAP occupancies in the coding region would not be decreased even in +FMN cells and that FMN-dependent regulation of RNAP occupancies would be abolished because CAmut fixes the conformation of the FMN riboswitch in the 'on' state. Unexpectedly, RNAP occupancies in the CDS decreased in −FMN cells of the CAmut strain. These results suggest that mutations in the RFN element may raise some termination sites or transcription pausing sites that enhance termination events, which leads to the decreased expression of ribM mRNA in CAmut strain ( Figure 1C). However, we also observed FMN-dependent regulation of RNAP occupancies being abolished by CAmut ( Figure 3C). These results suggest that the FMN-dependent response of RNAP elongation depends on an FMN-dependent conformational change of the FMN riboswitch.

Rho-factor contributes to the FMN riboswitch-mediated control of transcription elongation
To test the possibility that Rho-factor is involved in the premature transcriptional termination observed in ribM, we performed ChIP-qPCR analysis in the rho disruptant (Figure 3D). In −FMN cells, RNAP occupancies in the coding region were similar to those in the RFN1 region, as observed in the rho-intact strain ( Figure 3D). However, in contrast to the rho-intact strain, deletion of the rho gene resulted in RNAP occupancies in the protein-coding region that were more than 70% of those of the RFN1 region in +FMN conditions ( Figure 3D). Thus, the FMN-dependent decrease of RNAP occupancies was partially relieved by deletion of the rho gene. This result strongly indicates that Rho-factor contributes to the premature transcription termination of ribM in +FMN cells.
Then we assessed the effect of rho disruption on ribM gene expression. In −FMN cells of the rho gene disruptant, in addition to the ribM transcript detected in the ribA strain, longer transcripts were detected ( Figure 2C,  lanes 13 and 14, open arrowheads). This suggests that Rhodependent transcription termination occurs at the end of the ribM gene and that the disruption of the rho gene diminished termination at the proper site. Interestingly, ribM transcripts were also detected in +FMN cells, indicating that expression of ribM mRNA was increased by rho disrup-  Figure 2C, lane 14). Quantitative analysis showed that, in +FMN cells, disruption of the rho gene increased both the mRNA and protein levels more than 10-fold, while in −FMN cells, no such increase was observed ( Figure 3E and Supplementary Table S5). To test the possibility that rho gene expression is increased in +FMN cells, leading to enhanced transcription termination, we examined the expression level of Rho protein (Supplementary Figure S3). Western blot analysis showed that the expression level of Rho protein decreased rather than increased in +FMN cells (Supplementary Figure S3, lanes 3 and 4). Although the cause of this decrease is unclear, this result rules out the possibility. Taken together, these results indicate that Rho selectively recognizes the FMN-bound form of the FMN riboswitch and terminates transcription.

Inhibition of Rho function in the rneG strain completely abolished the FMN-dependent regulation of ribM mRNA
The results presented thus far indicated that both RNase E/G and Rho contribute to FMN riboswitch-dependent regulation of ribM gene expression. These observations prompted us to construct double deletion mutant of rneG and rho. However, we could not obtain such a strain, probably owing to a synthetic lethal effect. Therefore, we de-veloped a Rho-function inhibition system in which Rho function is inhibited by overexpressing a dominant-negative mutant of Rho protein (see SI and Supplementary Figure S3). Although the effect of the knock-down of Rho function (RhoKD) on ribM gene expression is smaller to that of rho gene deletion (Figures 3E, 4B and Supplementary Table S5), signals in northern blot analysis of +FMN cells were detected in the RhoKD strain ( Figure 4A, lanes  10 and 12). In accordance, qRT-PCR analysis showed that RhoKD had 10-fold increased ribM mRNA expression in +FMN cells but not in −FMN cells ( Figure 4B and Supplementary Table S5). Similar effects were also observed in RibM protein expression levels. Then, we tested the effect of rneG gene deletion in the RhoKD background. Deletion of the rneG gene diminished the differences in the patterns of northern blot signals between +FMN and −FMN cells. Although the signal intensities seems slightly stronger in +FMN cells than in −FMN cells (ratio of signal intensity of whole lane was 1:1.5 (lane 13:lane 14)), qRT-PCR analysis showed that simultaneous disruption of the rneG gene and Rho function almost completely diminished the FMN-dependent regulation of ribM mRNA levels ( Figure  4B). These results strongly indicate that FMN-dependent regulation of ribM mRNA was mainly controlled by RNase E/G and Rho. In addition to these observations, northern blot analysis revealed that a signal corresponding to the size of the ribM gene was decreased in the rneG ribA RhoKD strain ( Figure 4A, gray arrowhead). Instead, a strong signal was observed at ∼3 kb ( Figure 4A, filled arrowhead). Although the signal intensity was weak, a band with similar mobility was observed in −FMN cells of the rho ribA strain ( Figure 2C, lane 13). These results suggest that longer transcripts produced under Rho-functiondeficient conditions were processed by RNase E/G. With respect to the short fragment corresponding to the size of the RFN element observed in +FMN cells ( Figure 4A, open arrowhead); although the signal was markedly diminished by the simultaneous disruption of rneG and Rho function, a band with the same mobility was detected in the rneG ribA RhoKD strain. Some exoribonucleases may work after FMN-dependent premature transcription termination by Rho and/or FMN-dependent cleavage by RNase E/G.
With respect to the RibM protein level in +FMN cells, as expected, deletion of the rneG gene increased RibM expression about 2-fold in the RhoKD background ( Figure  4B and Supplementary Table S5). This result indicates that RNase E/G-dependent degradation of ribM mRNA contributed to the repression of RibM protein expression in +FMN cells. In contrast, in −FMN cells, deletion of the rneG gene in the RhoKD background decreased RibM protein expression to about one-third the level in rneG-intact cells ( Figure 4B). This result is similar to that observed in strains harboring intact Rho function ( Figure 2B). Considering that ribM mRNA levels in −FMN cells were not changed as a result of rneG deletion, the translational efficiency may be decreased in rneG strains. Although further investigations are required to uncover the role of RNase E/G, differences in the RibM protein expression level in +FMN and −FMN conditions were considerably reduced with the simultaneous disruption of Rho function and the rneG gene, indicating these two factors plays a key role in the FMN riboswitch function in C. glutamicum. Besides these mechanisms regulating the mRNA level, minor FMNdependent regulation of the RibM protein level remained even in the absence of regulation at the mRNA level, implying the existence of translational regulation of this riboswitch ( Figure 4B).

DISCUSSION
With respect to the relationship between the translation efficiency and mRNA stability, many studies reported (23) that ribosome association with mRNA for translation prevents the mRNA from RNase attack, resulting in increased stability. Conversely, when translation is prematurely terminated by a non-sense mutation or amino-acid starvation, the untranslated region of RNA is degraded more rapidly than that being translated. Similar phenomena have been observed in some riboswitches that operate at the translational level. In the vitamin B12 riboswitch of the E. coli btuB gene, sequestration of the SD sequence occurs in response to its ligand, adenosylcobalamin (25). A precise analysis using a reporter system demonstrated that the inhibition of translation initiation by ligand binding is accompanied by a decrease in mRNA stability. Thus, ribosome-free RNA is subjected to ribonuclease-dependent degradation (25,26). A similar consequence was observed in the THI-box riboswitch of thiM (4,27). In both cases, it is thought that the modulation of mRNA stability is a secondary effect of the translational regulation.
In this study, we demonstrated that the FMN riboswitch of C. glutamicum ribM also decreased the stability of ribM mRNA in an FMN-dependent manner. However, it should be noted that this riboswitch is essentially different from that of btuB and thiM because the FMN-dependent regulation of ribM mRNA stability occurred even in strains with defective translation (Supplementary Figure S2A). Although the inhibition of translation slightly decreased the ribM mRNA stability in −FMN cells, the effect was too small to affect the steady-state level of ribM mRNA (Figure 2B). Analysis using an rneG disruptant showed that the deletion of rneG eliminated the FMN-dependent regulation of ribM mRNA stability, demonstrating the importance of RNase E/G in the regulation of mRNA stability ( Figure  2A). From these observations, we conclude that the FMN riboswitch of C. glutamicum ribM directly controls the stability of mRNA in response to its ligand. This situation is similar to that of the lysine riboswitch of E. coli lysC. It has been recently reported that, in addition to translational regulation, the lysC riboswitch directly controls mRNA stability by altering the accessibility to the degradosome in response to lysine (4). Thus, the ribM and lysC riboswitches may utilize a similar mechanism to control mRNA stability. However, the effect of mRNA stability control on the regulation of the protein expression level is different between the ribM and lysC riboswitches. In the lysC riboswitch, the deletion of the RNase E cleavage site had only minor effects on the ligand-dependent regulation of translational fusion. In contrast, in the ribM riboswitch, the deletion of rneG in RhoKD conditions impaired the regulation not only of mRNA but also of RibM protein ( Figure 4B). This result re-  vealed the importance of ribM mRNA stability control by RNase E/G in the regulation of RibM protein expression. The control of transcription termination is one of the general mechanisms of many of the riboswitches discovered to date. A previous comparative genomics study predicted that ∼40% of riboswitches analyzed use liganddependent sequestration of an intrinsic transcription terminator to regulate downstream gene expression (28). In addition to these predictions, the experimental demonstration of ligand-dependent transcription termination has also been reported for many riboswitches since their discovery (29)(30)(31). These analyses were performed using an in vitro transcription assay, and they demonstrated that ligand binding facilitates premature transcription termination. In this study, we performed RNAP-ChIP-qPCR analysis instead of in vitro transcription analysis to examine if premature transcription termination occurs in ribM ( Figure 3B-D). Although this analysis cannot rule out the possibility that some factors other than the riboswitch or ligand are involved, this method has the advantage that the results obtained reflect the transcription efficiency in vivo. Using this analysis, we succeeded in demonstrating that the FMN riboswitch induces transcription termination in response to its ligand more directly than has been reported.
We also confirmed that Rho is involved in this termination regulation under +FMN conditions. The involvement of Rho-factor in the riboswitch function was suggested recently (5). Using in vitro transcription analysis, the authors clearly revealed that the magnesium riboswitch of S. enterica and the FMN riboswitch of E. coli control the Rhodependent termination independent of translation. They also tested the effect of Rho inhibition on reporter gene expression in vivo using transcriptional fusion construct. However, the physiological significance of Rho-dependent termination on target gene expression control remains obscure because it is possible that these riboswitches primar-ily control translation initiation to regulate downstream gene expression (15,32). In the present study, we demonstrated that disrupting the rho gene increased the transcription elongation efficiency under +FMN conditions ( Figure  3B and D). We also demonstrated that disruption of Rho function increased ribM mRNA level only under +FMN conditions ( Figures 3E and 4B). Together with the result that Rho expression was not increased in +FMN cells (Supplementary Figure S3), these results indicate that Rho monitors the FMN-dependent conformational change in the FMN riboswitch and selectively terminates transcription in the presence of FMN. Furthermore, our results show that disruption of Rho function significantly increases the RibM protein level in addition to the ribM mRNA level. These results strongly indicate that Rho-dependent termination practically participates in FMN riboswitch-based ribM gene expression control. Taken together, this is the first assessment of the physiological significance of Rhodependent termination in gene expression control by a riboswitch.
Although our results suggested that both RNase E/G and Rho monitor the FMN-dependent conformational change of FMN riboswitch, the precise locations of Rhodependent termination site(s) and RNase E/G cleavage site(s) were not determined. Probably these sites are located in expression platform and would be exposed in the presence of FMN as reported in other riboswitches previously (4,5). Structure-probing assay together with in vitro transcription/cleavage assay using various mutant DNA templates or RNAs is required to determine the accurate site(s).
A previous comparative genomics study predicted that most riboswitches of bacterial groups other than Firmicutes and Fusobacterium are controlled at the translational level (28). However, recent findings in ␥ -proteobacteria that suggested the participation of protein factors such as RNase or transcription termination factor Rho in these riboswitch functions raise the question about the generalization of these situations (4,5). In this study, we demonstrated that the FMN riboswitch of C. glutamicum ribM mainly controls ribM mRNA by using Rho and RNase E/G to regulate RibM protein expression. This observation not only revealed that the interplay between the riboswitch and protein factors is not confined to ␥ -proteobacteria but also raised a question about the control of many other riboswitches that have been thought to be controlled by cis-elements. In addition, recent studies in Sinorhizobium meliloti (␣proteobacteria), Treponema denticola (spirochaeta) and Streptomyces griseus (actinobacteria) have shown that riboswitches lacking an obvious intrinsic transcription terminator sequence seem to be controlled at the mRNA level (33)(34)(35). Our observations suggest that these riboswitches would utilize some protein factors as the FMN riboswitch of C. glutamicum ribM. Taken together, the participation of protein factors in riboswitch function would be a widely distributed mechanism.
One interesting observation is that the FMN riboswitch of C. glutamicum ribM utilizes both Rho and RNase E/G for its regulation. This is the first example of a riboswitch that employs two trans-acting protein factors acting in mRNA metabolism. Both Rho and RNase E/G act in mRNA metabolism but in different pathways. Although FMN is a key effector, the use of these protein factors would render the FMN riboswitch responsive to physiological states other than the FMN concentration. For example, in E. coli, it is speculated that Rho activity is unlocked from inhibition by the RNA chaperone Hfq in response to amino acid starvation via RelA protein-dependent Hfq oligomerization (36)(37)(38). Although these observations were made in E. coli and C. glutamicum does not have a homolog of hfq, we can speculate that some signals cause Rho activity to be inhibited, leading to an increased expression of RibM protein in C. glutamicum. On the other hand, our results of the rho disruptant showed FMN-dependent regulation of ribM expression probably owing to RNase E/G and translational regulation ( Figure 3E). Thus, redundancy in regulatory pathways might not only expand the diversity of effectors but also ensure the regulation under various growth conditions. Furthermore, it should be noted that the dynamic range of the ribM FMN riboswitch gene regulation is relatively large compared with other riboswitches (Supplementary Table S6). By using two factors that act in different pathways, this riboswitch has a relatively large dynamic range of regulation.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

FUNDING
Funding for open access charge: Scientific research fund of Research Institute of Innovative Technology for the Earth. Conflict of interest statement. None declared.


extfieldsofstudy: Chemistry Medicine

text:
Effects of Different Sizes of Glass Beads on the Release of Sporocysts from Eimeria tenella Oocysts

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.

Avian Eimeria spp. are recognized as the parasitic protozoans with the greatest economic impact on poultry production worldwide [1]. Protozoan parasites of the genus Eimeria multiply in the intestinal tract and cause tissue damage and increase susceptibility to other diseases [2]. Sporulation of Eimeria spp. oocysts is affected by moisture, temperature, and oxygen levels [3]. Oocysts are enclosed in a thick outer shell and consist of a single cell that undergoes the process of sporulation to enter the infective stage after approximately 48 hr. Four sporocysts, each containing 2 sporozoites, are generally discernible within the oocysts. The oocyst wall has 2 distinguishable layers: an inner and outer layers [4]. In vivo, the oocyst wall may be opened through mechanical injury, such as being crushed in the gizzard of a bird. A high percentage of sporocysts are quickly released from crushed oocysts within the gizzard [5], and the sporozoites then enter the intestine. Sporozoites of Eimeria tenella invade chick cecal folds ~4 hr after infection, followed by sporozoite migration into the crypt epithelium. The first asexual replication occurs 48 hr later [6]. At least 2 generations of asexual reproduction occur before the sexual phase, where motile microgametes seek out and unite with macrogametes. The zygote matures into oocysts, which are then released from the intestinal mucosa and expelled in the feces. The life cycle of E. tenella is typical of all Eimeria.
To break the wall of Eimeria spp. oocysts for PCR analysis, glass beads, a mortar and a pestle, or chemical agents may be used [7]. Eimeria antigens are obtained from suspensions of oocysts in PBS by disruption with glass beads followed by repeated freezing and thawing [8]. The excystation of sporozoites from Eimeria oocysts is an important step for in vitro studies of these parasites. The aim of the present study was to assess the degree of released sporocysts of E. tenella oocysts using different sizes of glass beads and treatment durations.
Oocysts were field-isolated samples (Namwon Farm, Namwon, Korea). The correct species assignment and purity of samples were confirmed by singular ITS-PCR. Genes of E. tenella were collected using a Nucleic Acid Purification Kit (Exprep TM Plasmid DNA, GeneAll, Seoul, Korea). Genes were sequenced at the JeonJu Biomaterials Institute and submitted to GenBank (accession no. FJ447468). Since 2009, our laboratory has maintained generations of E. tenella on chickens. Feces were collected from 5 to 7 days post inoculation. Oocysts were suspended at ~3.0× 10 5 per milliliter in 2% (w/v) potassium dichromate in a conical flask. Oocysts were sporulated under air supply within 5-8 days [9] and were checked for complete sporulation. A 200-µl sample of 3.0 × 10 5 oocysts per milliliter was added to 800 µl of saturated salt solution. The oocysts suspension was then mixed at a ratio of 1:1 with glass beads (0.5, 1, and 3 mm in diameter) [10] and vortexed for 5 sec, 30 sec, 1 min, or 5 min. For vortexing, Vortex-2 Genie (G 560, Scientific Industries, Springfield, Massachusetts, USA) was used. During vortexing, the conical tube was kept angular using a maximum speed (3,200 rpm). Same vortex speed was fixed in all groups. After oocysts were vortexed and added to 4 ml of saturated salt solution, the remaining oocysts were counted by the standard MacMaster method [11]. The data were subjected to ANOVA with SAS (Ver. 8x). Duncan's multiple range tests was used to determine significant differences among the various durations or bead sizes.
Treatment of the oocytes with 1 mm glass bead (Fig. 1A, B) for 5 sec caused approximately 20% sporocysts release (Fig.  1C), while treatment for 1 min caused the release of more than 90% of sporocysts (Fig. 1D). Release rates of sporocysts using 0.5 mm glass beads (56.8± 7.3%) were higher than those with other bead sizes after 5 sec of treatment (P < 0.01). Release rates of sporocysts with 0.5 mm and 1 mm glass beads were not significantly different after 30 sec of treatment, but release rates using 0.5 mm and 1 mm glass beads were higher than 3 mm after 1 min and 5 min of treatment (P < 0.01). Change of treatment times was highest at 5 min (P < 0.01). For all bead sizes, the release rates of sporocysts increased as the agitation time increased (Fig. 2).
During in vitro experiments, oocysts may be made to release 80% of viable sporocysts [16]. There are several methods that can be used to rupture the cell wall of Eimeria species; the most common includes sonication, microwave, heating, and pestle and mortar. The greatest advantage of using glass beads for wall rupture is that they are an efficient way to inexpensively process large quantities of oocysts. The concept of mechanical crushing with glass beads came from the in vivo mechanism by which oocysts are crushed in the gizzard [12]. Our study for oocyst disruption of Eimeria did not require enzymes. Landers [13] demonstrated that excystation of E. nieschulzi could not be achieved using pepsin or trypsin. It was also found that, for release of E. separta sporocysts, hydrochloric acid, pepsin, and trypsin were required [14]. Pre-treatment of E. nieschulzi oocysts with pepsin destabilizes the oocyst wall structure, ensuring that mechanical disruption by vortexing with glass beads is more efficient [15]. Chai et al. [16] described that the wall structure of E. tenella oocysts was ruptured using a teflon-coated tissue homogenizer. The high mechanical resistance of Eimeria spp. oocysts required a greater number of glass beads during vortexing. Dulski and Turner [17] described a protocol similar to ours for disrupting E. tenella oocysts using 3 mm glass beads. They found that roughly 60% of oocysts were broken by orbital shaking at 200 rpm. Sporocysts have also been released from oocysts by agitating oocyst suspensions with 4 mm glass beads [18]. E. tenella oocysts placed in an equal volume of 0.5 mm glass beads and exposed to the maximum speed of agitation resulted in invisible sporocysts [19]. If too many glass beads are used, sporocysts may be damaged; in order to ensure a high yield of undamaged sporocysts, the agita-  tion procedure must be stopped after a very specific duration. After the oocysts wall is crushed, the sporocysts and glass beads are collected separately. The use of smaller glass beads may result in greater difficulty of recovering sporocysts. Our results suggest that sporocyst release from oocysts was efficient after vortexing with 0.5 mm glass beads for 5 to 30 sec, with 1 mm glass beads for 30 sec to 1 min and with 3 mm glass beads for 1 to 5 min. However, the use of 0.5 mm glass beads presented greater difficulties in recovering the sporocysts compared to larger glass beads. Therefore, the present study recommended the use of 1 mm glass beads for 30 sec to 1 min in order to maximize sporocyst release and recovery. Oocyst walls of Eimeria spp. have various characteristics. Some Eimeria species are more susceptible to chemical agents while others are more susceptible to mechanical injury. Because each Eimeria species is unique, with each being lysed upon application of different degrees of either mechanical or chemical stresses, different methods should be tested to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.


s2fieldsofstudy: Biology Medicine

text:
The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the "cuff model" of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Introduction
Neuropathic pain is usually chronic and arises as a consequence of a lesion or a disease affecting the somatosensory system. Maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system participate in this complex syndrome. Various models have been developed in rodents for studying neuropathic pain and its treatments [1][2][3] . Based on known etiologies, the models of neuropathic pain aim at mimicking the polyneuropathy observed in diabetes, the injuries to peripheral nerves, the central injuries, the trigeminal neuralgia, the neuropathies consecutive to chemotherapy, the post-herpetic neuralgia, etc. Different models of peripheral nerve injury in rodents focus on the sciatic nerve. These models depend on a compression and/or a section of this nerve. Indeed, the sciatic nerve affords relative easy surgery and allows for tests based on paw withdrawal reflexes. The models of chronic nerve compression include for example: the chronic constriction injury (CCI) 4,5 , the sciatic nerve cuffing [6][7][8][9] , the partial sciatic nerve ligation (PSL) 10 , the spinal nerve ligation (SNL) 11 , or the common peroneal nerve ligation 12 . Models referred to as "spared nerve injury" (SNI) are also widely used. They consist of a tight ligation and axotomy of two out of the three terminal branches of the sciatic nerve, while the third branch remains intact [13][14][15] . The various models of neuropathic pain, which target the sciatic nerve, result in a chronic mechanical allodynia (a nociceptive response to a normally non-nociceptive stimulus) on the injured hind paw.
Here, the detailed surgery procedure for the "cuff model" of neuropathic pain in mice is described. It consists in the implantation of a polyethylene cuff around the main branch of the sciatic nerve [6][7][8][9] . The use of von Frey filaments is also described. These filaments allow assessing the mechanical allodynia which is a long lasting nociceptive symptom present in this model.
1. Weigh the animal. Note: Mouse body weight should be over 20 g for the cuff insertion procedure described below. 2. Anesthetize the animal with an intraperitoneal injection of 4 ml/kg of a mixture of ketamine (17 mg/ml) and xylazine (2.5 mg/ml) in 0.9% NaCl, which provides around 45 min of anesthesia. 3. Check the absence of paw reflexes by pinching a hind paw with tweezers and check the absence of eye reflexes to make sure that the animal is fully anesthetized. 4. Shave the right leg from the knee to the hip using an electrical shaver. 5. Apply protective eye liquid gel to the eyes with a cotton-tipped swab. 6. Place the animal on its left side and place the right hind limb on a small pillow and maintain the right hind limb to the pillow with adhesive tape. 7. Disinfect the surgery field with chlorhexidine and 70% ethanol using gauze pad or cotton-tipped swab. 8. Find the femur using the forefinger and make an incision of approximately 0.5 cm, parallel to the femur and approximately 1.5 mm anterior to the femur. 9. Separate the muscles close to the femur with two autoclaved sticks. Notes: Never cut the muscle. Normally, the muscle layers separate easily without any bleeding and the sciatic nerve is then visible. In case of bleeding, use a sterile cotton-tipped swab to absorb the blood. 10. Insert two autoclaved sticks below the sciatic nerve to expose its main branch, and hydrate the nerve with a sterile physiological solution (0.9% NaCl). 11. Hold the pre-prepared sterile 2 mm section of split PE-20 polyethylene tubing (cuff), 0.38 mm ID / 1.09 mm OD, with the help of a pointed steel stick and a bulldog clamp. 1. Insert the pointed steel stick into the cuff, which will slightly open it. 2. Using the cuff lateral opening, insert the bulldog at one end of the cuff and parallel to the cuff. Rotate the bulldog (180°) so that it will hold the cuff by the side that is opposite to the lateral opening. Close the bulldog and remove the pointed steel stick. Note: The rotation is done to allow holding-on the cuff in an optimized position for the insertion, the bulldog clamp is also helping to maintain the cuff partly open. The model and the size of the bulldog clamp are critical for this step of the procedure.
12. Have a second experimenter hold the two sticks under the nerve and gently separate the sticks to facilitate the access to a section of sciatic nerve that is around 4 mm long. 13. Insert the 2 mm cuff around the main branch of the sciatic nerve, starting by inserting the part of the cuff that is distal to the bulldog around the part of the nerve that is proximal to the hip. 14. Close the cuff gently by exerting pressure on its two distal sides with pliers, without squeezing or changing the form of the tube. Turn the cuff to ensure that it is closed correctly. 15. Suture the shaved skin layer with surgical knots. 16. Place the mouse on its left side in a clean home cage. Keep it under the heat lamp until the mouse is awake. 17. Add extra water and place some chow directly in the home cage.

Surgery Procedure for Sham Controls
1. Apply the same surgery procedure as described above from step 2.1 to step 2.9, then follow with steps 2.15 to 2.17. For sham controls, omit the steps 2.10 to 2.14 that only concern the cuff insertion.

Representative Results
The data are expressed as mean ± SEM. Statistical analyses were performed using multi-factor analysis of variance (ANOVA) or unpaired t-tests in accordance with the experimental design. For these analyses, the Sham and Cuff surgery groups as well as the saline vs. drug treatments were considered as between-group factors. When appropriate, repeated measure analyses were used for the time course data. The post-hoc comparisons were performed using the Duncan test. Statistical significance was considered at p<0.05.
When using the procedures that are described above, the cuff implantation results in an ipsilateral allodynia as illustrated in Figure 1. Once the mouse is habituated to the testing procedure, the values for paw withdrawal thresholds in the von Frey test remain stable over time and are not affected by the surgical procedure per se, as illustrated in Sham animals. It should however be noted that a transitory post-surgical allodynia can usually be observed in Sham mice. When such allodynia is present, the paw withdrawal response returns to baseline after a few days postsurgery. In Cuff mice, the ipsilateral allodynia is already present on the first days post-surgery and is maintained for more than 2 months (see 9, and Figure 1; F8,344=29.5, p<0.001). The cuff-induced allodynia remains ipsilateral in C57BL/6J mice when it is measured by the von Frey test as described above, but in other conditions a presence of allodynia on the contralateral paw can also be observed 8 .
The absolute values for baseline are usually between 4 and 6 g in C57BL/6J mice, but the testing protocol may affect these values.
Tricyclic antidepressants are among clinical first-line treatments for neuropathic pain. In this model, the tricyclic antidepressant drug nortriptyline (5 mg/kg, intraperitoneal, twice a day) relieves the neuropathic allodynia after around 2 weeks of treatment, as illustrated in Figure 2 (F7,91=15.3, p<0.001; post-hoc: (CuffNor=Sham)>CuffSal at p<0.001 on days 29 -34). At this dose, no acute analgesic action of the antidepressant is observed 16,17 . To mimic the lasting pain relief that is present in patients taking such drugs, the mice can be tested before the morning drug administration rather than after. Such procedure allows assessment of a long-lasting effect primed by previous days of treatment. In this case, it requires 1 to 2 weeks of treatment to observe a lasting relief of the neuropathic allodynia. When the treatment is interrupted, a relapse is usually observed within 3 to 4 days 18 . Beside some antidepressants, gabapentinoids are the other first-choice treatments for neuropathic pain. Gabapentin has an acute and transitory analgesic action in this model 16 , but it also displays a delayed and longlasting antiallodynic action when testing the animal each day before the drug administration (Figure 3; p<0.001). This action is faster than with antidepressant drugs.

Figure 1. Mechanical paw withdrawal thresholds in the cuff model of neuropathic pain in mice.
Adult male C57BL/6J mice were habituated to the von Frey procedure until a stable baseline was obtained (the baseline is represented at point 0 on the graph). Both paws were tested. The Cuff mice display ipsilateral mechanical allodynia as showed by the lowered paw withdrawal thresholds (n=10 per group).

Discussion
The "cuff" model was initially developed in rats to obtain a standardized and reproducible chronic constriction injury with the implantation of multiple cuffs around the sciatic nerve 6 . It was then modified to implant a single cuff 7,8 , even though some research groups still use multiple cuff insertion [19][20][21][22] . It was then adapted to mice 9,23 , which opened the possibility to use transgenic animals. The cuff is usually 2 mm long, but other lengths have also been used in rats 22 . The polyethylene tubing depends on the species: PE-20 in mice 9 , and PE-60 24,25 or PE-90 7,8,26,27 in rats.
The mechanical allodynia is measured with von Frey hairs. In this test, the absolute values for paw withdrawal thresholds may depend upon the surface on which the animal stands 28 or upon the duration of filament bending 3 , but these factors do not affect the detection of the neuropathic allodynia.
Another interest of the model is its response to current treatments of neuropathic pain, i.e., gabapentinoids and antidepressants. Similar to clinical observations: gabapentinoids display both an acute short-lasting analgesic action at high dose and a delayed sustained relieving action that is observed after a few days of treatment, tricyclic antidepressants and selective serotonin and noradrenaline reuptake inhibitors have no acute analgesic effect at relevant dose but they display a delayed sustained relieving action that requires 1 to 2 weeks of treatment, and the selective serotonin reuptake inhibitor fluoxetine is ineffective 16 . The model is thus appropriate to study the molecular mechanism underlying these treatments [16][17][18]44,45,47 , which may reveal new therapeutic targets to test in patients [48][49][50][51] . Lastly, the model also allows the study of the anxiodepressive consequences of neuropathic pain. Clinically, these consequences affect around a third of neuropathic pain patients but are preclinically less studied than the sensory aspects of pain. In this model, a time-dependent development of anxiety-like and depressive-like phenotypes is present 52 and the related mechanism can thus be addressed.
The standardized cuffs and procedures in this mouse model of neuropathic pain result in low interindividual variability for the mechanical allodynia. The possibility to use genetically modified animals 17,18,[44][45][46][47]52 , the long-lasting allodynia, the response to clinically used treatments and the time-dependent development of anxiodepressive symptoms make this model appropriate for the study of the various aspects and consequences of neuropathic pain and its treatments, which have already brought valuable information to this field of research.

Disclosures
The authors declare that they have no competing financial interests.


ANTI-HYPER LIPIDEMIC ACTIVITY OF METHANOLIC EXTRACT OF SMILAX WIGHTII A.DC. IN STREPTOZOTOCIN INDUCED MALE WISTAR ALBINO RATS

Smilax wightii, an endemic medicinal plant is found in the shola forests at high altitudes in Nilgiri Biosphere Reserve, the Western Ghats, Southern India . The present study, was undertaken to find out the effect of methanolic extract of this plant on the body weight, fasting blood glucose levels and lipid profiles in all the streptozotocin (STZ) induced rats. The extract exerted a significant (P<0.05) effect in the body weight of the experimental animals when compared to the control group. Treatment with the extract and glibenclamide resulted in a significant (P<0.01) reduction in the fasting blood glucose levels in diabetic rats when compared to the normal. The lipid profile such as TC, TG, LDL, and VLDL contents in the serum registered a significant (P<0.01) hike and a decline in the HDL contents in diabetic control group, which were retrieved to near normalcy in the plant extract treated groups. The effect produced by this plant extract was comparable with that of glibenclamide. The decreased fasting blood glucose levels and lipid lowering properties clearly showed the anti-hyperlipidemic effect of S.wightii.

INTRODUCTION
Traditional medicinal herbs have served as a potential alternative source of medicine and different healthcare systems (Kamboj, 2000). Diabetes mellitus is a metabolic disorder, characterized by hyperglycemia together with impaired metabolism of glucose and other energyyielding fuels such as lipids and proteins (Scheen, 1997). In modern medicine, no satisfactory effective therapy is still available to cure the diabetes mellitus (Piedrola et al., 2001). Herbal treatments are becoming popular as the herbal preparations have no or least side effects and also they are of relatively low cost (Rajasekaran et al., 2001).
The genus Smilax has more than 300 species, found on temperature zones, tropic and subtropics worldwide and belongs to Smilacaceae (Fnaec, 2000). The roots of Smilax wightii have been reported to cure dysentery, amoebiasis, veneral diseases, urinary complaints, fever, spermatorrhoea, antifertility, anaemia, rheumatic-arthritis, veterinary amoebiasis and gastric complaints (Adhikari et al., 2010). The species of Smilax have been reported to contain several phytoconstituents such as dioscin, steroids smilagenin and sarsapogenin (Coimbatore BSI, 1989). Several species of Smilax are used in Chinese traditional system of medicines as anticancer, anti-inflammatory and analgesic agents (Ozoy et al., 2008). Therefore, the present study was aimed to assess the anti-hyperlipidemic effects of methanolic extract of Smilax wightii in animal models.

Plant material
The plant materials were collected from Kodanadu, the Nilgiri Hills, Western Ghats, Southern India, Tamil Nadu. The plant was identified and authenticated by a plant taxonomist.

Preperation of extract
The whole plant materials were dried in shade after washing with cold water and then powdered using pulveriser and passed through sieve. About 100 g of dried plant powder was extracted with petroleum ether using soxhlet apparatus for 18 hours. The petroleum ether was evaporated from the extract and then the residue was re-extracted with methanol. This extract after evaporation of methanol, the filtered residue was stored at 4 0 C in refrigerator for further use.

Experimental animals
Male Wistar Albino rats weighing180-250 g were obtained from Agricultural University, Animal house lab, Trissur, Kerala. The animals were fed on a standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. They were maintained in a controlled environment (12 h/12 h light/dark) and temperature (25 ± 2°C). The animals were acclimatized to the laboratory conditions for one week before starting the experiment. All the procedures performed on animals were approved and conducted in accordance with the Institution of Animal Ethics committee and by the Regulatory body of the government (659/02/a/CPCSEA).

Toxicity studies
The acute toxicity studies were performed in adult male albino rats weighing180-250g. The animals were fasted overnight and 100-1000 mg/kg of the test extract was provided to various groups containing six animals in each group. The treated animals were monitored for 14 days, for behaviour, general health and mortality.

Induction of diabetes
Diabetes was induced in overnight fasted Wistar albino rats by intraperitoneal (i.p injection of streptozotocin at a dose of 55 mg/kg b.wt. Forty eight hours after streptozotocin administration, blood samples were drawn from tail and glucose levels were determined to confirm diabetes. The rats with blood glucose level above 200mg/kg were considered diabetic and used for the study.

Experimental design
The rats were divided into six groups comprising of six animals in each group as follows:

Determination of body weight
The rats from all the groups were weighed prior to the experiment and their initial body weights were recorded. The final body weights of the rats were recorded on the last day of experiment.

Estimation of fasting blood glucose (FBG) levels
Fasting blood glucose levels were measured on 0, 7 and 14 days of treatment of methanolic extract of Smilax wightii (MESW) supplement from the animals of all the groups. Blood samples were obtained by nicking the tails with a sharp razor and glucose concentrations were determined using a one-touch glucometer (Johnson& Johnson medical Ltd., Mumbai). The glucose concentration was read and documented from the glucometer readings. The results were expressed in terms of (mg/dl) of blood.

Estimation of lipid profile
The experimental animals from all the groups were sacrificed under light ether anaesthesia at the end of the experimental period. The rats were sacrificed by decapitation and blood was collected with anti-coagulant and the serum was used to for the estimation of lipid profile-TC-Total cholesterol (Parekh and Jung,197), TG-Triglycerides (Rice, 1970), HDL-High Density Lipoprotein (Warnick et al., 1985), LDL-Low Density Lipoprotein and VLDL -Very Low Density Lipoprotein (Friedwald et al., 1972).

Statistical Analysis
All biochemical data are expressed as Mean ± SEM. Statistical analysis was performed using one-way ANOVA using SPSS statistical analysis programme. In all cases, p-value less than 0.05 was considered to be significant.

Toxicity studies
Acute toxicity studies revealed that the methanolic extract of Smilax wightii (MESW) was found safe to up to a dose of 1000 mg/kg body weight. No sign of toxicity was noticed on the general health of the animals, and no death was observed in the animals when exposed to the extract.
The activity of methanolic extract of Smilax wightii (MESW) on body weight in streptozotocin induced diabetic rats are indicated in Table 1. There was a significant weight loss in the final body weight in the diabetic control group (Group II). A significant improvement in the body weight was observed in the extract and glibenclamide treated groups when compared to that of the diabetic control group.   fasting blood glucose levels. The reducing effect of the plant extract at a dose of 400 mg/kg b.wt was found to be comparable to that of the reference drug Glibenclamide (600 mg/kg b.wt). The initial reduction in blood glucose was observed on the 7 th day after the administration of the Smilax wightii extract. Meanwhile, on the 14 th day, almost the FBG levels were reduced significantly at the dose of 400mg/kg b.wt in the extract and glibenclamide treated groups. Table 3 depicts the changes in the levels of TC (Total Cholesterol), TG (Triglycerides), HDL (High Density Lipoprotein), LDL (Low Density Lipoprotein) and VLDL (Very Low Density Lipoprotein) of the experimental animals. Diabetes induced by STZ in rats significantly (P<0.01) elevated the TC, TG, LDL, VLDL levels and decreased the HDL levels in diabetic control compared with normal control rats. In the present study, administration of MESW at dose of 100, 200 and 400 mg/kgb.wt to the diabetic rats showed significant (P<0.05, P<0.01) reduction in TC, TG, LDL, and VLDL levels and increase in HDL levels than diabetic control rats.

DISCUSSION
Diabetes is a major health problem affecting major populations worldwide. It is a chronic disorder in metabolism of carbohydrates, proteins, and fat due to absolute or relative deficiency of insulin secretion with/without varying degree of insulin resistance. There are more than 30 million people with diabetes mellitus in India and the incidence is increasing. Decreased physical activity, increasing obesity, stress and changes in food consumption have lead to the cause of diabetes (Jarald et al., 2008).
The present study was aimed to investigate the anti-hyperlipidemic activity of methanoic extract of S. wightii (MESW) in STZ-induced diabetic rats. The results of the study revealed that MESW at the doses of 400 mg/kg b.wt. significantly normalized elevated blood glucose level and restored serum lipid profiles towards normal values.
Streptozotocin STZ, a highly cytotoxic agent of pancreatic β-cells induces diabetes by damaging the cells and causes reduction in insulin secretion (Esmaeli and Yazdanparas, 2004). The increased glucose level in the diabetic control animals may be due to the destruction of the pancreatic cells caused by the STZ induction. The increased levels of plasma glucose in STZ-induced diabetic rats were lowered by the administration of Smilax wightii extract.
Diabetes affects both glucose and lipid metabolism (Sperling and Saunders, 2000).
Hyperlipidemia was reported as common in adults with diabetes and it is characterized most often by increased triglyceride and reduced HDL cholesterol levels. This is generally observed in both type 1 and type 2 diabetes, representing the defect of insulin action in each, either due to inadequate secretion or resistance (Garg and Grundy, 1990). Increased fatty acid concentrations also increased the β-oxidation of fatty acids, producing more acetyl-CoA and cholesterol in diabetics (Yokogoshi andOda, 2000, Mard et al., 2010). Alterations in plasma lipoprotein metabolism are common in diabetes, which tend to exaggerate any pre-existing tendencies towards elevated lipid levels (Merzouk et al., 2004). The high density lipoprotein (HDL) was significantly reduced in the diabetic rats which indicate a positive risk factor for atherosclerosis (Bopanna et al., 1997). Previous studies in Smilax chinensis has reported similar results in the fasting blood glucose levels and lipid profile in Streptozotocin induced diabetic rats (Venkidesh et al., 2010). On the basis of the present study, it could be concluded that the methanolic extract of S.wightii exerted a significant antidiabetic and antihyperlipidemic effect. This could be due to different types of active principles present in the extract which serve as a good adjuvant in the present armamentarium of antidiabetic drug and has therapeutic value.


extfieldsofstudy: Biology Medicine

text:
Anti-onchocerca Metabolites from Cyperus articulatus: Isolation, In Vitro Activity and In Silico ‘Drug-Likeness’

The aims of this investigation were to isolate active ingredients from the roots/rhizomes of Cyperus articulatus used as herbal medicine in Cameroon for the treatment of human onchocerciasis and to assess the efficacy of the metabolites on the Onchocerca worm. The antifilarial activity was evaluated in vitro on microfilariae (Mfs) and adult worms of the bovine derived Onchocerca ochengi, a close relative of Onchocerca volvulus. Cytotoxicity was assessed in vitro on monkey kidney epithelial cells. The structures of the active compounds were determined using spectroscopic methods and their drug-likeness evaluated using Lipinski parameters. Two secondary metabolites, AMJ1 [containing mustakone (1) as the major component] and linoleic acid or (9Z,12Z)-octadeca-9,12-dienoic acid (2) were isolated. Both compounds were found to kill both the microfilariae and adult worms of O. ochengi in a dose dependent manner. The IC50s for AMJ1 were 15.7 µg/mL for Mfs, 17.4 µg/mL for adult males and 21.9 µg/mL for adult female worms while for linoleic acid the values were, 15.7 µg/mL for Mfs, 31.0 µg/mL for adult males and 44.2 µg/mL for adult females. The present report provides the first ever evidence of the anti-Onchocerca efficacy of AMJ1 and linoleic acid. Thus, these secondary metabolites may provide a lead for design and development of new antifilarial agents. Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0023-5) contains supplementary material, which is available to authorized users.

Introduction
Human onchocerciasis or subcutaneous filariasis is a parasitic disease caused by the filarial worm Onchocerca volvulus. It is transmitted through the bites of infected blackflies of Simulium species, which carry immature larval forms of the parasite from human to human. In the human body, the larvae form nodules in the subcutaneous tissue, where they mature to adult worms. After mating, the female adult worm can release up to 1000 microfilariae a day. These move through the body, and when they die they cause a variety of conditions, including blindness, skin rashes, lesions, intense itching and skin depigmentation [1]. About 37 million persons are infected with O. volvulus, of whom 270000 are blind and 500000 visually impaired with about 90 million vulnerable to get the disease [2,3]. The burden of onchocerciasis causes long term disability, social Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0023-5) contains supplementary material, which is available to authorized users. stigmatization and leads to a highly unproductive population and consequently to economic loss and slowdown of country development over the years since the affected population is forced to abandon the infested areas which usually have a high agricultural potential [4].
Ivermectin (IVM), the sole drug used in communitydirected treatment of onchocerciasis is microfilaricidal and with only limited activity against the adult worms. Parasite resistance to IVM and adverse reactions observed in patients co-infected with Loa loa limit the use of the drug [3]. To date, the only known macrofilaricide for onchocerciasis is suramin but it is toxic. There is need to develop safe and easily administered drugs that can kill the adult O. volvulus parasite to reduce the time needed for control programs to eliminate adult worms from an endemic area. Rational drug discovery approaches have made only limited advances in the discovery of a safe macrofilaricide against the Onchocerca worm. It has been suggested that plants used in folk medicine to treat parasitic diseases may provide an alternative source of macrofilaricides [5]. Previous studies in our laboratory showed that the hexane extract of the roots/rhizomes of Cyperus articulatus (used as herbal medicine in Cameroon to treat onchocerciasis) was active against the microfilariae and adult worms of the bovine parasite Onchocerca ochengi, a close relative of the medically important O. volvulus. C. articulatus (family: Cyperaceae) is a bushy grass mainly found along tropical rivers and streams. The present studies aimed at isolating pure compounds from the hexane extract (essential oil) of the roots/rhizomes of the plant and evaluate their anti-Onchocerca activity and drug-likeness.

Identification of Secondary Metabolites Isolated from the Hexane Extract of Cyperus articulatus
The roots/rhizomes of C. articulatus were extracted with hexane. Column chromatography of this extract as described in the Experimental section afforded two compounds ( Fig. 1) which were identified with the help of 1 H and 13 C NMR spectra as well as by comparison of these data with published literature values for mustakone [6,7] and linoleic acid [8].

Identification of AMJ1
AMJ1 was obtained as white oil. The ESI-MS (ESM Fig. S1) of AMJ1 revealed the presence of three compounds with a major compound with peak at 219.1. In fact, its 1 H NMR spectrum showed a peak at 5.71 ppm characteristic of an olefinic proton deshielded by inductive effect of a carbonyl group. Other protons resonated at d 1.50-1.70 ppm corresponding to axial and equatorial protons of a cyclohexane ring. The 13 C NMR spectrum (ESM Table S1) showed the signals of the 15 carbon atoms amongst others. Comparing the 1 H and 13 C NMR data of mustakone (1) [6,7] with those of AMJ1, further confirmed the structure, mustakone which was previously isolated from the rhizomes of C. articulatus [7] was the major compound. Linoleic acid or (9Z,12Z)-octadeca-9,12-dienoic acid (2) Mustakone (1)  For microfilariae (Mfs) and adult male worms IC 50 corresponds to the concentration of the phytochemical that inhibits worm motility by 50 % while for adult female worms, IC 50 is the concentration of the metabolite that inhibits formazan formation by 50 %. IC 50 for mammalian cells (monkey kidney cells) is the concentration of the metabolite that inhibits cell viability by 50 %. SI (Selectivity Index) = IC 50 on mammalian cells/IC 50 on parasite. IC 100 is the concentration of the bioactive metabolite that inhibits O. ochengi Mfs and adult male worm motility or formazan formation by adult female worms by 100 %.

In Silico ''Drug-likeness'' Profile of Plant-Derived
Compounds with Anti-onchocerca Activity Lipinski et al. [9] postulated the ''rule of five'', which states that a compound will likely not be orally bioavailable if its molecular weight (MW) is [500 Daltons (Da), its n-octanol/water partition coefficient (log P) [5, its number of H-bond acceptors (HBA) [10 and its number of H-bond donors (HBD) [5. An additional rule for the number of rotatable bonds (NRB) is often added to Lipinski's rule in order to take drug metabolism and pharmacokinetics into account, since this parameter is known to influence bioavailability in rats [10]. The above parameters were used to initially access the likely oral bioavailability of the isolated compounds. Lipinski evaluation showed that whereas linoleic acid shows only one violation of the Lipinski rule, mustakone had no violation (Table 3).

Discussion
Onchocerciasis causes intense itching, blurred vision and sometimes irreversible blindness. Rational drug discovery approaches have made only limited advances in the discovery of a safe macrofilaricide against the Onchocerca worm [11]. It has been suggested that medicinal plants used in folk medicine to treat parasitic diseases may provide an alternative source of macrofilaricides [5]. We here report the efficacy of AMJ1 [containing mustakone (1) as major component] and linoleic acid [(9Z,12Z)-octadeca-9,12-dienoic acid] (2) isolated from the essential oil (hexane extract) of the roots/rhizomes of C. articulatus against microfilariae and adult O. ochengi worms, the closest relative and best model of the human parasite, O. volvulus [11]. Thus, it is feasible that compounds that are active against the bovine parasite will also affect the human parasite, O. volvulus. The effect of these metabolites on the Onchocerca worm seems to be specific since other compounds tested under similar conditions were ineffective (results not shown). AMJ1-containing mustakone was more active in vitro than linoleic acid on adult male and female worms. The combined effect of these two metabolites may explain in part the apparent anti-Onchocerca activity of decoctions of C. articulatus and justifies the use of this plant as traditional medicine for the treatment of human onchocerciasis. However due to the difficulty in obtaining very pure compounds from oils, it may be necessary to further evaluate the anti-onchocerca activity of these compounds by using commercially produced samples. Furthermore HPLC techniques should be used to separate AMJ1 to determine the active principle in it. Mustakone, a sesquiterpene previously isolated by Nyasse et al. [6,7] has been shown to exhibit anti-plasmodial activity [12]. Linoleic acid has also been reported to have anti-bacterial [13] and antifungal activity [14]. We here report for the first time their activity against the Onchocerca worm. Lipinski evaluation shows that these compounds are potential drug leads against onchocerciasis.

Extraction and Isolation of Metabolites
The powdered (roots/rhizome) sample (700 g) was macerated in hexane (3 9 3 days each), filtered and concentrated on a rotavapor to dryness. Four grams (4 g) of the crude hexane extract (oil) was dissolved in methylene chloride and impregnated with 4.0 g of Celite then concentrated to dryness over a rotary evaporator. The powder was chromatographed over 20 g, 60 mesh silica gel column of size 50 cm long 1.5 cm diameter. The column was eluted with hexane-EtOAc (starting from 5-70 %), then with methanol. Eighty (50 mL) fractions were collected, concentrated and grouped based on TLC profile. The fractions were combined based on comparative TLC profiles.

Treatment of Second Portion of Crude Hexane
Extract of C. articulatus Ten and half grams (10.5 g) of the crude hexane extract was treated similarly as mention above with methylene chloride then impregnation was done with 11.0 g of Celite and concentrated to dryness over a rotary evaporator. The powder was chromatographed over 53 g, 60 mesh silica gel column of size 50 cm long 4 cm diameter. The column was eluted successively with pure hexane while increasing the polarity with EtOAc. Sixty (50 mL) fractions were collected, concentrated and grouped based on TLC profile. Fractions 1-5 were combined and subjected to gel permeation via Sephadex LH-20 (CH 2 Cl 2 ) to afford a white oil designated 79 mg of AMJ2, R f = 0.54 TLC (Hex/EtOAc) 2:8).

Isolation of O. ochengi Adult Worms
The isolation of O. ochengi adult worms was done as described previously [15]. The duration from the slaughtering of a cow to the harvesting of parasites from the skin was always less than 2 h to avoid bacterial contamination. Briefly, fresh pieces of umbilical cattle skin with palpable nodules bought from local slaughterhouses were washed, drained and sterilized with 70 % ethanol. O. ochengi adult worms were carefully scraped out of the nodules as single masses and temporarily submerged in 1 mL complete culture medium, CCM [RPMI-1640 (SIGMA, USA) supplemented with 25 mM HEPES, 2 g/L sodium bicarbonate, 2 mM L-glutamine, 5 % new born calf serum (SIGMA, USA), 150 units/mL penicillin, 150 lg/mL streptomycin and 0.5 lg/mL amphotericin B (SIGMA, USA), pH 7.4)] using 24-well plates. The adult worms were allowed in the culture medium overnight in a CO 2 incubator, during which period the male worms migrated out of the nodular masses. Only wells containing viable worms received treatment with test metabolite. Damaged worms and worms from putrefied nodules were discarded. The viability of worms retained for the assay was ascertained by visual and microscopic examination of adult male worm motility using an inverted microscope.

Isolation of O. ochengi Microfilariae
The cattle skin was obtained as described for adult worms. About 5 skin snips were obtained from different locations of the skin and incubated separately in small amounts of CCM for 30 min. Emerged Mfs were qualified and quantified for O. ochengi species with the aid of an inverted microscope. A selected piece of skin, rich in O. ochengi Mfs was carefully shaved with a razor blade, and then rinsed with distilled water. It was dabbed with a clean tea cloth to eliminate excess moisture and covered entirely with 70 % ethanol. The latter was allowed to evaporate completely in a horizontal flow sterile hood. The ethanol treatment was repeated once. The sterilized skin was tautly attached onto an autoclaved, cylindrical piece of wood using autoclaved thumb nails and close (about 1 mm apart) criss-cross cuts were made into the epidermis and dermis. The assembly was incubated in the culture medium for 4-6 h. The emerged and highly motile O. ochengi microfilariae were concentrated by centrifugation at 4009g for 10 min and then quantified.

Preparation of Mammalian Cells
Monkey kidney epithelial cells (LLC-MK2) (ATCC, USA) were cultured at 37°C in humidified air with 5 % CO 2 in a HeraCell-150 incubator (Thermo Electron, Germany) until the cell layer is almost confluent. The cells were rinsed with a solution of 0.125 % trypsin and 0.5 mM EDTA in medium 199 (Sigma, USA) and kept in the same mixture for less than 1 h for them to be dislodged. The cell suspension was centrifuged at 5609g for 10 min, the supernatant discarded and the pellet re-suspended to 2 9 10 5 cells/mL in CCM. The cell suspension was dispensed into 96-well microtitre plates (200 lL/well) and kept in the incubator for 3-5 days for cells to grow and become fully confluent. These cells served as feeder layer for the Mfs assays and were also used for cytotoxicity studies.

Preparation of Stock Solutions of Isolated Metabolites
Twenty-five milligrams (25 mg) of each compound was weighed and dissolved in microtubes containing 1 mL of 99.9 % pure dimethyl sulfoxide (DMSO) (SIGMA, USA) to obtain stock solutions of 25 mg/mL. Complete dissolution was achieved by vortexing. The solutions were stored at -20°C before they were used in the assays.

Primary Screens on Adult Worms
This was done to eliminate inactive metabolites. Adult worm assays were conducted in 24-well plates (NUNC, USA) at 37°C in humidified air containing 5 % CO 2 for 5 days (120 h) without change of medium. Nodular masses (each generally containing a few males and a female worm) were first put in the wells (with 1 mL CCM) without extract overnight to confirm their viability and for adult males to migrate from the nodule into the culture medium. 1 mL of CCM containing 1000 lg/mL of extract was then added into each of quadruplicate wells to give a single final concentration of 500 lg/mL. Four nodular masses each, were used in the negative control (2 % DMSO in CCM only) and in the positive control (10 lM NYBC01, a gold conjugated compound) wells in which each well also received only one nodular mass. After 5 days incubation adult male viability was assessed based on motility scores using an inverted microscope. Motility score was on a scale of 4 (vigorous or normal movement of whole worm, corresponding to 0 % inhibition of worm motility), 3 (near normal movement of whole worm or 25 % inhibition of worm motility), 2 (whole body of worm motile but sluggish i.e. 50 % inhibition of worm motility), 1 (only head or tail of worm moving i.e. 75 % inhibition of worm motility), 0 (completely immotile worm i.e. 100 % inhibition of worm motility). A metabolite was considered active if there was a 100 % inhibition of adult male worm motility; or moderately active for a motility inhibition of 50-99 %; and inactive if the inhibition was less than 50 %. Adult female worm viability was assessed by the MTT/ formazan assay in which each nodular mass was placed in a well of a 48-well microtitre plate containing 500 lL/well of 0.5 mg/mL MTT (Sigma, USA) in incomplete RPMI culture medium, and then incubated in the dark at 37°C for 30 min. Adult female worm viability was taken as mean % inhibition of formazan formation relative to negative control at 120 h post addition of plant extract. An extract was considered active on the adult female worm if there was a 90 % or greater inhibition of formazan formation compared to the negative controls; or moderately active if the inhibition was 50-89 %. It was considered inactive if the inhibition was less than 50 %. Adult worm death positively correlates with inhibition of formazan formation. Inhibition of formazan formation was assessed by visually observing the worms after incubation in MTT or by colorimetry. Each nodular mass was placed in 500 lL of DMSO in a 48-well plate to allow the colour to elute from the worm for 1 h. After shaking the plate, 200 lL of the coloured formazan solution from each well was pipetted into the wells of a microtitre plate (78 wells) and the optical density read at 490 nm. Adult female worm viability was taken as mean % inhibition of formazan formation relative to negative control at 120 h post addition of the test compound.

¼
OD of negative control À OD of treatment OD of negative control Â 100

Primary Screen on Microfilariae
The isolated metabolites were also tested on Mfs at a single concentration of 500 lg/mL, in triplicate wells. The Mfs assay was conducted in 96-well microtitre plates (15 mfs in 200 lL CCM per well) at 37°C in humidified air containing 5 % CO 2 for 5 days without any change of medium. Fully confluent monkey kidney epithelial cells, serving as feeder layer, were co-cultured with the Mfs. The medium used in preparing the feeder cell layer was removed by a swift decantation before fresh CCM containing plant test metabolite (100 lL) and worms (100 lL) were immediately added. Ivermectin (20 lg/mL) and 2 % DMSO served as the positive and negative controls respectively. Mfs motility scores (viability) were done on a scale of 0 (immotile), through 0.25 (only tail or head shaking occasionally), through 0.5 (whole body motile, but sluggishly or with difficulties), to 1 (almost vigorous to vigorous motility). Scores were made every 24 h, terminating at 120 h using an inverted microscope. Any culture with microbial contamination was not considered. Mfs viability was taken as the mean % reduction at 120 h (day 5) after addition of drug. A metabolite was considered active if there was a 100 % reduction in mfs motility; or moderately active for a motility reduction of 50-99 %; and inactive if the reduction was less than 50 %.

Secondary Screens on Microfilariae and Adult Worms
This was done to confirm the activity of the metabolites that showed filaricidal activity in the primary screen, and to determine their IC 50 , IC 100 and selectivity index (SI) values. The metabolites were retested as described under primary screens at serial dilutions from 500 to 7.81 lg/mL using 24-well plates for adult worms and 96-well plates for Mfs. All assays were repeated at least three times and the results obtained are the mean values at each concentration. The graphical analysis was done using Microsoft Excel 2010 (Microsoft Corporation, USA). IC 50 determination was done using GraphPad Prism software (version 6).

Cytotoxicity Studies
This was done as part of the Mfs assay on the active compounds through observations on the monkey kidney epithelial cells on day 5. An examination of the deformities and degree of detachment of the monkey kidney cells was done. Dead or deformed cells were usually detached from the bottom of the vessel and were rounded in shape. The IC 50 values for these mammalian cells were determined graphically using data from microscopy. The selectivity index (SI) values were calculated using the ratio: SI ¼ IC 50 of drug on mammalian cell= IC 50 of drug on parasite Mfs ð Þ

In Silico ''Drug-Likeness'' of Active Isolated Compounds
The ''drug-likeness'' of the isolated compounds was assessed using Lipinski criteria [9], from computed molecular properties of the geometry optimized structures. All 3D molecular structures of the compounds were generated using the graphical user interface (GUI) of the MOE software [16] running on a Linux workstation with a 3.5 GHz Intel Core2 Duo processor, and energy minimization was subsequently carried out using the AM1 semiempirical approach implemented in MOPAC24 [17] until a gradient of 0.001 kcal/mol was reached. The 3D structures generated were then saved as .mol2 files subsequently included into a MOE database (.mdb). The MW, NRB, lipophilicity parameter (log P), HBA, HBD and Lipinski violations were calculated using the molecular descriptor calculator included in the QuSAR module of the MOE package [16].

Conclusions
AMJ1 [containing mustakone (1) as major component] and linoleic acid (2) have been isolated from the roots/rhizomes of Cyperus articulatus. Our investigations show that both phytochemicals were able to kill both the microfilariae and adult worms of O. ochengi in a dose dependent manner. This study therefore provides the first ever evidence of the anti-onchcerca efficacy of AJM1-containing mustakone and linoleic acid. Efforts are being made to identify the actual compound imparting high activity to AMJ1. Thus these metabolites may provide a lead for design and development of new antifilarial agents.