s2fieldsofstudy: Biology Medicine

text:
Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.

Introduction
Osteoarthritis (OA) is a most common degenerative joint disorder and is characterized by degradation of articular cartilage, thickening of subchondral bone, and synovial inflammation 1,2 . OA may occur in response to inappropriate mechanical stress and local or systemic inflammation associated with genetic predisposition, obesity, and metabolic syndrome 3,4 . Although it has been considered as a "wear and tear" disease for many decades, recently the roles of pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, have been implicated in the OA pathogenesis. The inflammatory mediators produced by abnormal chondrocytes and synovial cells can trigger the cartilage extracellular matrix (ECM) degradation and break the cartilage homeostasis 5 . Enzymes responsible for degrading extracellular include two groups: matrix metalloproteinases (MMPs) and aggrecanases 6 . MMP13 belongs to the former group and is a major enzyme hydrolyzing type-II collagen, a dominant protein component in cartilage ECM 7,8 . Compared with other MMPs, MMP13 is expressed more strictly in connective tissue and is usually produced by only cartilage and bone during development 9,10 . Under stimulation of cytokines from mechanical injury and inflammation, MMP13 overexpression is regulated by complicated signals containing mitogenactivated protein kinase (MAPK) and nuclear factor (NF)-κB 11,12 . Emerging evidence from OA patients and rodent experimental arthritis supports that MMP13 plays a crucial role in the disease development and is believed as an important biomarker to reflect arthritis progress and therapeutic effects [13][14][15] .
Heparan sulfate proteoglycans are one kind of important ECM proteins covalently linked by polysaccharide side chains that are polymerized by enzymes and further modified by sulfation, deacetylation, and epimerization 16 . Sulfotransferases and sulfatases catalyze to add or release sulfate group to or from heparan sulfate residues, respectively 17,18 . The expression of heparan sulfate 6-O endosulfatases including Sulf1 and Sulf2 (Sulfs) are increased in human OA cartilage, and Sulf-deficient mice show much severe OA pathology 19,20 , indicating that enzymes involved in the sulfation of proteoglycan is associated with OA development.
Heparan sulfate 6-O-sulfotransferases (HS6ST) catalyze the transfer of sulfate group to C-6 (an exocyclic carbon) of the glucosamine residue in heparan sulfate. HS6STs belong to Golgi-resident enzymes and include three isoforms named HS6ST1, HS6ST2, and HS6ST3 in humans 21 . HS6STs are involved in tumorigenicity 21 , angiogenesis 22 , and neuron development 23 . In previous study, we found that HS6ST2 expression was significantly reduced in the cartilage of patients with OA and Kashin-Beck disease 24 . In addition, HS6ST2 might modulate fibroblast growth factor-2 signaling to affect chondrocyte growth and differentiation 25 . But the exact role of HS6ST2 is still elusive and the gene expression regulation of HS6ST2 especially in OA cartilage remains unknown.
MiRNAs are short non-coding RNAs with~22 nucleotides that bind 3′-untranslated region (3′UTR) of mRNAs to regulate gene expression through either repressing translation or increasing mRNA degradation generally 26,27 . Recently, more studies have reported that miRNAs participate in chondrogenesis 28,29 , cartilage degradation, and OA development 30,31 . In previous study, we have identified miRNA repertoire in the different stages of articular development and also proved that miR-337 was associated with chondrogenesis through regulating TGFBR2 expression 32,33 . Considering the regulation of HS6ST2 expression in chondrocytes, we hypothesize that the downregulation of HS6ST2 could be mediated by a certain miRNA. Using miRNA target prediction algorithms, miR-23b-3p was assumed to bind mRNA sequence of HS6ST2. MiR-23b-3p is located in human chromosome 9q22.32 region with 900 base pairs. The region contains miR-23b/27b/24-1 cluster closed to encoded gene C9orf3 34 . Previous studies about miR-23b were focused on cancer 35,36 and metabolic diseases 37,38 . But there is a study showing that miR-23b influences the differentiation of mesenchymal stem cell (MSC) into chondrocytes by directly targeting protein kinase A (PKA) mRNA to inhibit PKA signaling 39 .
In the present study, we first observed lower HS6ST2 mRNA expression and higher expression of both miR-23b-3p and MMP13 mRNA in the damaged cartilage compared with matched smooth cartilage from the same OA patients. Then we elucidated the roles of miR-23b-3p by directly targeting HS6ST2 mRNA and intervened miR-23b-3p expression leading to downregulation of HS6ST2 in chondrocytes. The deregulation of HS6ST2 could result in increased expression of MMP13 by activating p38 MAPK pathway.

Result
There are relationships among the expression levels of HS6ST2, MMP13, and miR-23b-3p in damaged cartilage from OA patients The OA-damaged cartilage and smooth cartilage from the same patients undergoing knee arthroplasty surgery were separated (Fig. 1a, upper panel) and validated by Safranin O-fast green staining (Fig. 1a, lower panel). HS6ST2 mRNA expression (Fig. 1b, left panel) was attenuated and MMP13 mRNA expression (Fig. 1b, right panel) was enhanced in damaged cartilage from 18 OA patients. Furthermore, we detected HS6ST2 protein in 16 pairs of OA patients using immunohistochemistry (IHC) and determined that HS6ST2 expression was significantly lower in OA-damaged cartilage tissues than matched smooth cartilage tissues ( Fig. 1c and Supplementary Figure S1).
HS6ST2 mRNA is a target of miR-23b-3p To confirm whether HS6ST2 mRNA is the target of miR-23b-3p, dual luciferase reporter assay was performed. SW1353 cells were transfected with both mimic miR-23b-3p and pmirGLO vectors, and a significant reduction was shown in pmirGLO-HS6ST2 wild-type 3′UTR group relative to pmirGLO vector group but no significant reduction in pmirGLO-HS6ST2 mutant 3′UTR group (Supplementary Figure S2c and Fig. 2a). The significant enhancement was demonstrated in the pmirGLO-HS6ST2 wild-type 3′UTR group with anti-miR-23b-3p transfection rather than in the pmirGLO-HS6ST2 mutant 3′UTR group (Fig. 2b), suggesting that miR-23b-3p could bind to the 3′UTR of human HS6ST2 mRNA.
The expression of miR-23b-3p significantly increased under transfection of mimic miR-23b-3p (as much as 1500 times) compared with mimic negative control (NC) in SW1353 cells (Fig. 2c, left panel), and correspondingly the transfection of anti-miR-23b-3p sequences could suppress the level of miR-23b-3p dramatically (over 99%) were determined by RT-qPCR. c Left, representative immunohistochemical localization of HS6ST2 expression in OA smooth and damaged cartilage from same patient. Arrows stand for HS6ST2-positive cells. Right, statistical analysis of average optical density in immunohistochemical detection from OA patients (n = 16). Scale bar, 100 μm. d Relative expression of miR-23b-3p in damaged cartilage compared with smooth cartilage from the same OA patients (n = 18) were determined by RT-qPCR. e The correlation analysis between miR-23b-3p and MMP13 mRMA expression from OA patients was performed. f, g The correlation analysis between miR-23b-3p and HS6ST2 expression from OA patients was performed. U6 snRNA was used as an internal control in RT-qPCR for miRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen for mRNA detection. Paired t-test was used to identify differences statistically between OA smooth cartilage group and OA-damaged cartilage group. *P < 0.05 and **P < 0.01. The correlation analysis was performed by using Spearman correlation (Fig. 2c, right panel). The protein expression of HS6ST2 was downregulated after the administration of mimic miR-23b-3p, whereas transfection with the anti-miR-23b-3p sequences could upregulate the protein expression of HS6ST2 (Fig. 2c, lower panel). The results were confirmed in another human chondrocyte cell line C28/I2 40 (Fig. 2d). Collectively, the above-mentioned results suggested that miR-23b-3p could regulate HS6ST2 protein expression in chondrocytes by targeting HS6ST2 mRNA 3′UTR.

Intervention of miR-23b-3p affects specific gene expression in chondrocytes
To further confirm the roles of miR-23b-3p in chondrocytes, the mimic of miR-23b-3p was used to alter the expression of miR-23b-3p. MMP13 and ADAMTS4 significantly increased under treatment of mimic miR-23b-3p (Fig. 2e, f). The downregulation of HS6ST2 upon miR-23b-3p overexpression was observed and Aggrecan Core Protein or Cartilage-Specific Proteoglycan Core Protein (ACAN) also decreased at both protein and mRNA levels under overexpression of miR-23b-3p (Fig. 2e, f). Transfection with anti-miR-23b-3p led to significant downregulation of MMP13, MMP3, and ADAMTS4 in chondrocytes and upregulation of HS6ST2 (Fig. 2g, h). The findings provided evidence that miR-23b-3p could affect ECM metabolism in human chondrocytes.
To determine whether miR-23b-3p could influence matrix degradation induced by TNF-α, SW1353 cells were transfected with mimic miR-23b-3p under TNF-α stimulation, and the MMP13 expression was enhanced more severely under the stimulation of both miR-23b-3p and TNF-α compared with TNF-α alone (Fig. 3b), and anti-miR-23b-3p could inhibit the MMP13 expression induced by TNF-α (Fig. 3c). The matrix content of chondrocytes was determined by toluidine blue staining at 48 h after transfection. After image analysis, the mean optical density of the staining under miR-23b-3p transfection was significantly lower than that of the NC group with or without TNF-α (Fig. 3d), and the matrix staining was enhanced by anti-miR-23b-3p transfection (Fig. 3e), suggesting that miR-23b-3p might take part in the catabolism pathway induced by TNF-α.
To confirm the matrix degradation caused by mimic miR-23b-3p via the target relationship between miR-23b-3p and HS6ST2 mRNA, the plasmid for ectopic expression of HS6ST2 with FLAG tag was constructed. MMP13 protein level decreased and matrix staining increased under overexpression of HS6ST2 treated with TNF-α ( Fig. 4c, d). Although the protein level of MMP13 could be upregulated by miR-23b-3p, the upregulation of MMP13 from ectopic miR-23b-3p was attenuated by overexpression of HS6ST2 (Fig. 4e). Under TNF-α stimulation, the matrix staining was significantly reduced with ectopic miR-23b-3p, but the degradation was rescued by HS6ST2 overexpression (Fig. 4f), suggesting that miR-23b-3p participates in matrix degradation via regulating HS6ST2 expression.
Phosphorylation of p38 MAPK is dependent on HS6ST2 mediated by miR-23b-3p p38 MAPK, c-Jun N-terminal kinase (JNK) MAPK, and NF-κB signaling, regulated by extracellular stress factors, are crucial downstream pathways of TNF-α during (see figure on previous page) Fig. 2 MiR-23b-3p inhibits HS6ST2 expression by targeting HS6ST2 mRNA and effects specific gene expression in chondrocytes. a, b Dual luciferase reporter assay to validate target relationship between HS6ST2 and miR-23b-3p. SW1353 cells were transfected with mimic miR-23b-3p (a) or anti-miR-23b-3p (b) by control vector, pmirGLO-HS6ST2 wild-type 3′UTR vector or pmirGLO-HS6ST2 mutant 3′UTR vector, respectively, for 48 h. c, d Stem-loop RT-qPCR and western blotting results in SW1353 cells (c) or C28/I2 cells (d) transfected with 10 nM mimic miR-23b-3p (left panel) or 50 nM anti-miR-23b-3p sequence (right panel). e, f RT-qPCR (e) and western blotting (f) results of cartilage-specific gene expression in SW1353 cells transfected with mimic miR-23b-3p or negative control. g, h RT-qPCR (g) and western blotting (h) results of cartilage-specific gene expression in SW1353 cells transfected with anti-miR-23b-3p or negative control. RNA was harvested at 24 h, while protein was isolated at 48 h after transfection. U6 snRNA was used as internal controls in miRNA stem-loop RT-qPCR detection and GAPDH was used as internal controls in mRNA RT-qPCR and western blotting detection. Bars represent standard error of the mean (SEM) from three independent experiments. Mann-Whitney U test was used to identify statistical differences between two groups. *P value < 0.05 inflammation and matrix degradation 5 . Our data showed that, with or without TNF-α, p38 MAPK pathway was significantly regulated during gain or loss of miR-23b-3p function rather than JNK MAPK and NF-κB signaling (Supplementary Figure S5). The protein expression of MMP13 and p-p38 were both positively regulated by miR-23b-3p (Fig. 5a, b), suggesting that p38 MAPK signaling might participate in upregulation of matrix degradation from ectopic miR-23b-3p.
The p38 MAPK inhibitor SB203580 was used in chondrocytes transfected with miR-23b-3p under stimulation with TNF-α. The expression of MMP13 induced by TNFα could be inhibited by SB203580 ( Supplementary Figure S6a), and upregulation of MMP13 induced by miR-23b-3p could also be attenuated after treatment of SB203580 in SW1353 cells ( Fig. 5c and Supplementary Figure S6b) and C28/I2 cells (Supplementary Figure S7a), indicating that miR-23b-3p could regulate the expression of MMP13 depending on p38 MAPK. Furthermore, after treatment of si-p38 MAPK, the level of phosphorylation form of p38 MAPK and upregulation of MMP13 via miR-23b-3p were not attenuated (Fig. 5d, e), although total p38 MAPK was downregulated, suggesting that upregulation of MMP13 via miR-23b-3p could be influenced by the activity of p38 MAPK (phosphorylation form of p38 MAPK) rather than of total p38 MAPK.
Under the treatment of si-HS6ST2, p-p38 significantly increased ( Fig. 6a) with treatment of TNF-α and HS6ST2 overexpression could also attenuate the upregulation of p-p38 from ectopic miR-23b-3p (Fig. 6b). The results from C28/I2 cells also showed that phosphorylation of p38 MAPK could be influenced by ectopic miR-23b-3p or si-HS6ST2 (Supplementary Figure S4). Additionally, upregulation of MMP13 under si-HS6ST2 transfection was also blocked by SB203580 (Fig. 6c, Supplementary Figure S6c and Supplementary Figure S7b). Above findings provided evidence that HS6ST2 mediated by miR-23b-3p might regulate cartilage degradation depending on the activity of p38 MAPK.

Discussion
The present study provided evidence to support a novel critical homeostatic mechanism that HS6ST2 as a key regulator in cartilage degradation is mediated by miR-23b-3p in cartilage. Previous reports showed that HS6ST2 expression was significantly reduced in the cartilage of OA patients and the change of HS6ST2 could alter the expression of cartilage-related genes 24 , suggesting a pivotal role of HS6ST2 during chondrocyte differentiation and progression of OA. However, the molecular mechanisms for HS6ST2-induced cartilage-specific gene expression and possible regulators for HS6ST2 expression remain unclear.
Hence, in this study we first assessed the HS6ST2 expression levels in OA-damaged cartilage tissues and the matched smooth cartilage tissues. In OA-damaged cartilage tissues, HS6ST2 was reduced at RNA and protein levels, while MMP13 mRNA was enhanced, which are consistent with published reports about overexpression of MMP13 in OA-damaged cartilage 41 and our previous study 24 .
Second, we examined the posttranscriptional regulation of HS6ST2 expression by miRNAs in chondrocytes and assumed that its expression could be regulated by miR-23b-3p. Bioinformatics results showed the conservation of target relationship between HS6ST2 mRNA and miR-23b-3p in humans, mice, and rats (Supplementary Figure S2). There are other candidate miRNAs also targeting HS6ST2. miR-140, a well-known cartilage-specific miRNA, was predicted to bind 3′UTR of HS6ST2 mRNA, for example. There have been several reports for miR-140 verifying its targeting gene, expression regulation, and roles in cartilage development and arthritis progression [42][43][44] . miR-23b-3p was selected for further study as a regulator of HS6ST2 expression rather than other candidate miR-NAs based on the consistent results from several prediction algorithms, conservation in various species, and innovation in cartilage biology.
From OA patients, the increasing of miR-23b-3p in OAdamaged cartilage tissues related to matched smooth cartilage tissues was determined, which is in accord with previous large-scale microarray miRNA analysis 45 . Furthermore, the positive relationship between miR-23b-3p and MMP13 had been observed in the present study, indicating that overexpression of miR-23b-3p is an important factor in cartilage matrix degradation and OA (see figure on previous page) Fig. 3 TNF-α-treated chondrocytes increases matrix degradation regulated by miR-23b-3p. a Stem-loop RT-qPCR result of miR-23b-3p (left panel) and protein levels of HS6ST2 and MMP13 (right panel) were assayed in SW1353 cells under stimulation by 10 ng/ml TNF-α for 24 h. b, c Western blotting result of HS6ST2 and MMP13 protein in SW1353 cells transfected with 10 nM mimic miR-23b-3p (b) or 50 nM anti-miR-23b-3p sequence (c) and stimulated by 10 ng/ml TNF-α for 24 h. d, e Toluidine blue staining results of SW1353 cells transfected with 10 nM mimic miR-23b-3p (d) or 50 nM anti-miR-23b-3p sequence (e) and stimulated by 10 ng/ml TNF-α for 24 h. Scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Asterisk (*): compared with mimic NC or anti-NC group. U6 snRNA and GAPDH were used as internal controls in RT-qPCR for miRNA and western blotting detection, respectively. Bars represent standard error of the mean (SEM) from three independent experiments. One representative result and quantitative data from three independent western blotting and toluidine blue staining. Mann-Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value <0.05 (see figure on previous page) Fig. 4 MiR-23b-3p could enhance matrix degradation in human chondrocytes via regulating HS6ST2. a, b SW1353 cells transfected with 50 nM HS6ST2 siRNA mixture (containing three target sequences, si-HS6ST2) or negative control (si-NC) were stimulated by 10 ng/ml TNF-α for 24 h. Protein level of HS6ST2 and MMP13 were assayed by western blotting (a) and matrix content of chondrocytes was determined by toluidine blue staining (b). Asterisk (*): compared with the si-NC group. c, d SW1353 cells transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG-HS6ST2 vector (FLAG-HS6ST2) for 24 h were stimulated by TNF-α for another 24 h. The protein expression of MMP13 was assayed by western blotting (c) and matrix content of chondrocytes was determined by toluidine blue staining (d). Asterisk (*): compared with the FLAG-Ctrl group. e, f Under stimulation with TNF-α, SW1353 cells were treated with mimic NC or mimic miR-23b-3p for 24 h and then transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG-HS6ST2 vector (FLAG-HS6ST2) for another 24 h to observe the rescuing effect of mimic miR-23b-3p in MMP13 expression (e) and matrix content (f). Asterisk (*): compared with the mimic NC group. GAPDH was used as internal controls in western blotting detection. In toluidine blue staining results, scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Bars represent standard error of the mean (SEM) from three independent experiments. Mann-Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value <0.05. NS stands for not significant Fig. 5 MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a, b SW1353 cells were transfected by mimic miR-23b-3p (a) or anti-miR-23b-3p sequence (b) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC (a) or anti-NC group (b). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si-p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann-Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value <0.05. NS stands for not significant pathogenesis. Dual-luciferase assay and protein detection after transfection indicated that miR-23b-3p could bind to the 3′UTR of human HS6ST2 mRNA and regulates its expression in chondrocytes. The observation that HS6ST2 in damaged cartilage tissue manifested lower expression and was downregulated by miR-23b-3p in chondrocytes implicated that relationship between HS6ST2 and miR-23b-3p might be an important regulatory mechanism in OA progression.
Pro-inflammatory cytokines, such as TNF-α and IL-1β, play crucial roles in OA pathogenesis, which could promote catabolism in arthritic cartilage and enhance the expression of matrix-degradation genes 5 . In present study, TNF-α was selected in inducing matrix degradation of Fig. 6 HS6ST2 could regulate the matrix degradation depending on the activity of p38 MAPK. a SW1353 cells were transfected by si-HS6ST2 or negative control (si-NC) with or without TNF-α for 24 h, and the protein expression of p-p38 and p38 was detected by western blotting. Asterisk (*): compared with the si-NC group. b Under stimulation with TNF-α, SW1353 cells were transfected by empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG-HS6ST2 vector (FLAG-HS6ST2) under treatment with mimic miR-23b-3p, and p-p38 and total p38 were determined by western blotting. Asterisk (*): compared with the mimic NC group. c SW1353 cells were transfected with HS6ST2 siRNA with or without p38 MAPK inhibitor SB203580 (10 μM) after treatment of TNF-α, and the protein expression of MMP13 was determined by western blotting. Each relative expression of phosphorylation form was normalized by the total form. d Schematic representation of miR-23b-3p-HS6ST2 axis-mediated catabolic effects in human chondrocyte. GAPDH was used as internal controls in western blotting detection. Mann-Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value <0.05. NS stands for not significant chondrocyte. Under treatment of TNF-α, the protein level of HS6ST2 was reduced, while the miR-23b-3p level was elevated, suggesting that the downregulation of HS6ST2 was mediated probably by upregulating miR-23b-3p in catabolism of chondrocyte. NF-κB pathway known to play an important role in the pathogenesis of OA 5 has been proved as the upstream regulation of miR-23b-3p expression, which was reported in the metabolism and cancer development 37,46 . Hence, increasing of miR-23b-3p under TNF-α stimulation in our study might depend on the activation of NF-κB signal pathway.
MiR-23b has been found to induce chondrogenic differentiation in the differentiation of MSC into chondrocyte 39 . In this study, we intervened the expression of miR-23b-3p in mature cartilage cells with miR-23b-3p mimic or anti-miR-23b-3p, and we found that MMP13 expression and matrix content were affected by the change of miR-23b-3p in chondrocytes under TNF-α treatment. Even without TNF-α, both mimic miR-23b-3p and anti-miR-23b-3p could still influence the expression of cartilage-related catabolic genes and matrix content of chondrocytes. Together, our data support that miR-23b-3p could affect the catabolism of mature chondrocyte besides its role in the differentiation of MSC into chondrocytes.
In chondrocytes, downregulation of HS6ST2 could enhance the expression of MMP13 and aggravate matrix degradation similar to ectopic miR-23b-3p and HS6ST2 overexpression could rescue the detrimental effect of miR-23b-3p. Exogenous HS6ST2 with FLAG tag could improve the decline of HS6ST2 from miR-23b-3p and attenuate the expression of MMP13 and loss of matrix content in the activated phase of TNF-α treatment, and FLAG-HS6ST2 protein was not influenced by miR-23b-3p because of 3′UTR deleting. Thus we have confirmed that miR-23b-3p participates in matrix degradation via regulating HS6ST2 expression.
In order to verify the mechanism of MMP13 upregulation by HS6ST2 as well as miR-23b-3p, the downstream signal pathways of TNF-α including NF-κB, p38 MAPK and JNK MAPK 5 were detected. Activation of p38 MAPK pathway was altered by change of miR-23b-3p or HS6ST2, which is consistent with previous reports that activity of p38 MAPK signaling pathway induces ECM degradation [47][48][49] . Catalytic inhibitors of p38 MAPK SB203580 could block p38 MAP kinase activity 50 , and the expression of MMP13 induced by TNF-α could be attenuated by SB203580 but more seriously under miR-23b-3p or si-HS6ST2 treatment. Under treatment of si-p38, the phosphorylation of p38 MAPK was not attenuated dramatically though the total p38 MAPK was downregulated seriously, and increasing of MMP13 and p-p38 from ectopic miR-23b-3p could not be blocked, which might belong to feedback regulation. Above results suggested that enhancement of MMP13 regulated by miR-23b-3p and downregulation of HS6ST2 might depend on activity of p38 MAPK rather than total p38 MAPK.
Our results suggested that there was a close relationship between HS6ST2 and activation of p38 MAPK, and we assumed that HS6ST2 could interact with some proteins that influenced activity of p38 MAPK in posttranscriptional regulatory mechanism. We checked MEK3/6 (specific activators for p38 MAPK) 51 and DUSP10 (dualspecificity protein phosphatases 10) that dephosphorylates the stress-activated p38 MAPK 52,53 by coimmunoprecipitation in our system. But the results showed no interaction between HS6ST2 and abovementioned proteins (Supplementary Figure S8). The exact mechanism of relationship between HS6ST2 and activation of p38 MAPK needs further exploring.
In conclusion, we found that, compared with smooth cartilage, the expression of HS6ST2 is lower where miR-23b-3p and MMP13 mRNA are higher in damaged cartilage in OA patients, and miR-23b-3p binds with 3′UTR of HS6ST2 mRNA and inhibit its expression in human chondrocytes. HS6ST2 regulated by miR-23b-3p affects MMP13 expression via enhancing the phosphorylation of p38 MAPK rather than total p38 MAPK change (see Fig. 6d). The work is the first to demonstrate the regulation of HS6ST2 by a miRNA during cartilage matrix metabolism, and HS6ST2 might play a pivotal role in matrix degradation. Further studies such as administrating miR-23b-3p mimic/inhibitors (gain/loss function of its target HS6ST2, and other possible targets) on mouse OA models are needed to provide more solid evidence in future. The findings not only provide new insights into the role of sulfotransferase in matrix degradation regulation but also raise possibilities to intervene HS6ST2 as well as miR-23b-3p for developing a novel therapeutic strategy of OA.

Materials and Methods
Patients and articular cartilage collection OA patients were diagnosed according to the modified Outerbridge classification by Xi'an Honghui Hospital, Xian Jiaotong University. Articular cartilage samples were collected from 18 patients from Shaanxi province, China with knee OA undergoing knee arthroplasty surgery (14 women and 4 men; age (mean ± SEM) 66.6 ± 4.2 years). After washing with sterile phosphate-buffered saline (PBS) buffer, portions of cartilage with smooth articular surface and portions with damaged articular surface were used for histologic stain, RNA extraction, and IHC. This study was performed with the approval of the Ethical Committee of the Xi'an Jiaotong University Health Science Center, and all individuals provided full written informed consent before the operative procedure.

RNA extraction and quantitation analysis in cartilage specimens
For RNA extraction, cartilage tissues (n = 18) from smooth and damaged articular surface of the same patients were harvested and divided into small pieces (<2 × 2 mm 2 ). The pieces were frozen in liquid nitrogen immediately. Total RNA was isolated with Trizol reagent (Thermo Fisher, catalog 15596-026) according to the manufacturer's protocol. cDNA was synthesized from 2 μg of total RNA in 20 μl of reaction system using the Transcriptor cDNA Synthesize Kit (Roche, catalog 04897030001) with OligdT primer for detecting mRNA expression. Meanwhile, the same cDNA sample was synthesized from 1 μg of total RNA with the mir-X miRNA First-Strand Synthesis Kits (Clontech, catalog 638315). Gene expression levels were detected by quantitative PCR (qPCR) with the SYBR Green System (Roche, catalog 04913850001). The primers for measuring mRNA are shown in Supplementary Table S2. The forward primer for miR-23b-3p detection was designed by Tiangen Biotech (Beijing, China) and reverse primer for miRNA measure was from the mir-X miRNA First-Strand Synthesis Kits. Quantification of the relative expression levels was determined by ΔΔCt method.

Histology and IHC
Human cartilage tissues (n = 16) were fixed in 4% buffered paraformaldehyde for at least 48 h and subsequently decalcified with buffered EDTA (12.5% EDTA, pH 7.4). Dehydrated samples were embedded in paraffin, and 5 μm-thick sections were cut. Tissue sections were deparaffinized in xylenes and rehydrated through a graded series of alcohols. Representative sections from smooth cartilage and damaged cartilage of OA patients were stained with Safranin O and counterstained with fast green. For IHC, after 3% H 2 O 2 treatment and antigen retrieval, all tissue sections were blocked at room temperature with 5% bovine serum albumin (BSA) for 30 min and then incubated with primary antibody at the dilution stated: anti-HS6ST2 (1:50, Santa Cruz, catalog sc-98287). After sequential incubations with biotinylated secondary antibody (BOSTER, catalog SA1022, China) and horseradish peroxidase-conjugated avidin (BOSTER, catalog SA1022, China), protein staining was performed using the Diaminobenzidine Substrate Kit (BOSTER, catalog SA2022, China). Samples were counterstained with hematoxylin, and brown staining indicated the HS6ST2 immunoreactivity. The results of IHC were analyzed by photography under microscopy with 400× magnification and quantification with the Image-Pro ® Plus software.
Cell culture SW1353 cell line was obtained from American Tissue Culture Collection (ATCC), and chondrocyte cell line C28/I2 was kindly provided by Professor Junling Cao, from Institute of Endemic Diseases, Xi'an Jiaotong University Health Science Center. Both cell lines were cultured in RPMI-1640 medium (Hyclone, catalog SH30809.01) with 10% fetal bovine serum (FBS; ExCell, FCS500). Both cell lines were maintained at 37°C and in the presence of 5% CO 2 and cultured in monolayer and grown to confluence. The medium with 1% penicillin/ streptomycin (Thermo Fisher, catalog 15140122) was changed every 2 days. When the cultures reached 90% confluence, the cells were detached by treatment with 0.05% trypsin (ExCell, CB000-C022) and passaged in culture.

Target prediction and luciferase reporter assay
TargetScan (www.targetscan.org), miRDB (www.mirdb. org), and miRanda (www.microrna.org) were used to predict the target relationship between miR-23b-3p and HS6ST2 mRNA. HS6ST2 mRNA possesses a putative miR-23b-3p-binding site in its 3′UTR. Dual luciferase reporter assay was performed to validate the target relationship. The linker fragment (linker fragment primer sequences used for Dual-Luciferase vector are listed in supplementary Table S2) containing HS6ST2 wild-type or mutant 3′UTR-binding site was synthesized and inserted into the pmirGLO Dual-Luciferase vector (Promega, catalog E133A). SW1353 cells were co-transfected with 100 ng of pmirGLO vectors possessing the wild-type or mutant 3′UTR of HS6ST2 mRNA and 10 nM of mimic miR-23b-3p or 50 nM anti-miR-23b-3p with Lipofectamine 2000 (Thermo Fisher, catalog 11668-019) according to the manufacturer's instructions. After 48 h, SW1353 cells were harvested and luciferase activity was assayed using the Dual Luciferase Reporter Assay System (Promega, catalog E1910).
Chondrocyte treatment with TNF-α SW1353 cells were seeded in 12-well plates at 7 × 10 4 cells/well (C28/I2 seeded 1.2 × 10 5 cells/well). After 24 h, the chondrocytes should be FBS free for >10 h, and then TNF-α (10 ng/ml, Sino Biological lnc, catalog HG10602-M) was used to stimulate cells for appropriate time (SW1353 for 24 h and C28/I2 for 6 h). In the experiment of miRNA and TNF-α double stimulation, the TNFα treatment was for 24 h (SW1353 cells) or 6 h (C28/I2 cells) after transfection and FBS free for at least 10 h.

Transient transfection of siRNA and plasmids
For the knockdown of HS6ST2 or p38 MAPK expression, siRNA against HS6ST2 (si-HS6ST2), p38 MAPK (si-p38), and the negative control siRNA (si-NC) were prepared (Genepharma, Shanghai, China). SW1353 cells were seeded in 12-well plates at 7 × 10 4 cells/well. After 24 h, 50 nM siRNA mixture (containing three different target sequence) or si-NC were transiently transfected into the cells by Lipofectamine 2000 (Thermo Fisher, catalog 11668-019, 1.5 μl/well) and the efficiency of knockdown was determined by western blotting after transfection for 48 h. Different siRNA sequences against HS6ST2 or p38 MAPK are listed in Supplementary Table S1.
Toluidine blue staining SW1353 cells were seeded in 24-well plates at 3 × 10 4 for 24 h. After transfection of miRNAs or plasmids, the chondrocytes were incubated with TNF-α (10 ng/ml, Sino Biological lnc, catalog HG10602-M) for another 24 h. In order to determine the matrix content, the chondrocytes were fixed in 4% buffered paraformaldehyde for at least 20 min and stained with 1% toluidine blue for 10 min (miRNA or siRNA transfection) or 15 min (plasmid transfection). After washing with PBS buffer, the results of staining were analyzed by photography under microscope with 200× magnification and quantification with the Image-Pro ® Plus software.

Reverse transcriptase-qPCR analysis in cells
Total RNA was extracted from the cells using the Trizol Reagent (Thermo Fisher, catalog 15596-026). cDNA was synthesized from 2 μg of total RNA in 20 μl of reaction volume using the Transcriptor cDNA Synthesize Kit (Roche, catalog 04897030001) with OligdT primer, and to detect the miR-23b-3p level the same cDNA sample was synthesized from 500 ng of total RNA with miR-23b-3pspecific stem-loop reverse transcription primer. Gene expression levels were measured by qPCR using the SYBR Green System (Roche, catalog 04913850001). Quantification of the relative expression levels was determined by ΔΔCt method. Triplicates were used in assay, and the cell experiments were repeated three times. All the primer sequences for mRNA detection are shown in Supplementary Table S2.

Protein sample preparation and western blotting
Whole-cell lysate was prepared in western blotting and immunoprecipitation lysis buffer (Beyotime, catalog P0013B) containing protease inhibitor mixture (Bimake, catalog B14002) and phosphatase inhibitor mixture (Biomake, catalog B15002). The cell lysate were incubated in ice for 30 min and then centrifuged at 12,000 × g at 4°C for 15 min, and the concentration of protein in cleared lysate was determined by BCA Protein Assay (TIANGEN, catalog PA115). Protein solution (about 15 μg) was first separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore, catalog IPVH00010). The PVDF membrane with the protein should been blocked in 5% no fat milk or 5% BSA at room temperature for 2 h, and then the membranes were incubated with primary antibodies, including anti-HS6ST2 (1:400, Abcam, catalog ab122220), anti-MMP13 Abcam, catalog ab140123). All the primary antibodies were diluted with 5% BSA/TBST buffer (with 0.1% Tween 20) and were incubated overnight at 4°C. After washing with TBST buffer (with 0.1% Tween 20) for three times, the membranes were incubated for 2 h with secondary horseradish peroxidase-conjugated anti-Mouse (1:10,000, Thermo Fisher, catalog A21202), anti-Rabbit (1:10,000, Thermo Fisher, catalog A11036), or anti-Goat IgG (1:10,000, Santa Cruz, catalog sc-2020). After washing with TBST and TBS, the target proteins were detected on membrane by ECL detection system (Millipore, catalog MA01821). Western blotting were scanned by GeneGnome XRQ System with the GeneTools analysis software (Syngene, MD, USA), and densitometry of specific western blotting bands was analyzed with the GeneTools analysis software.

Statistics
All of the cell experiments were performed with at least three independent biological replicates. Bars represent standard error of the mean (SEM) from three independent experiments. Mann-Whitney U test was used to identify statistically differences between groups. Paired t test was used to identify differences between OA smooth cartilage and OA-damaged cartilage from the same patients. The correlation analysis was performed by using Spearman correlation. P value <0.05 was considered statistically significant.


extfieldsofstudy: Chemistry Medicine

text:
Antitumor activity of Chlorella sorokiniana and Scenedesmus sp. microalgae native of Nuevo León State, México

Cancer cases result in 13% of all deaths worldwide. Unwanted side effects in patients under conventional treatments have led to the search for beneficial alternative therapies. Microalgae synthesize compounds with known in vitro and in vivo biological activity against different tumor cell lines. Therefore, native microalgae from the State of Nuevo Leon, Mexico may become a potential source of antitumor agents. The aim of the present study was to evaluate the in vitro cytotoxic effect of Nuevo Leon regional Chlorella sorokiniana (Chlorellales: Chlorellaceae) and Scenedesmus sp. (Chlorococcales: Scenedesmaceae). Native microalgae crude organic extracts cytotoxicity against murine L5178Y-R lymphoma cell line and normal lymphocyte proliferation were evaluated using the MTT reduction colorimetric assay. Cell death pathway was analyzed by acridine orange and ethidium bromide staining, DNA degradation in 2% agarose gel electrophoresis and caspases activity. Results indicated significant (p < 0.05) 61.89% ± 3.26% and 74.77% ± 1.84% tumor cytotoxicity by C. sorokiniana and Scenedesmus sp. methanol extracts, respectively, at 500 µg/mL, by the mechanism of apoptosis. This study contributes to Mexican microalgae biodiversity knowledge and their potential as antitumor agent sources.

INTRODUCTION
Most commercialized drugs are synthetic derivatives from natural products, or are the result of the systematic screening of terrestrial organisms, such as plants or microorganisms.
Analysis of molecules produced by aquatic organisms has shown that microalgae synthesize a large number of bioactive compounds, including pigments, sterols, polyphenols, fatty acids, proteins, vitamins, alkaloids, and sulfated polysaccharides. This group of microorganisms is extremely diverse and represents a number of unexploited natural sources for bioactive agents. Furthermore, the microalgae intake of polluting elements such as nitrogen, phosphorus, and sulphur for their own growing can be considered an advantage, since such elements can be also metabolized by harmful aquatic weeds to proliferate.
Common failure of conventional therapy against cancer indicates a critical need for beneficial alternative therapeutic agents (Rengarajan et al., 2013). Antitumor activity of microalgal compounds can be explained by their ability to cross the lipophilic membranes and interact with proteins involved in apoptosis. In addition, several microalgal compounds induce DNA-dependent DNA polymerases inhibition, cyclins expression alteration, or major transduction pathways interference. Microalgal compounds have been related to immune response stimulation (Baudelet et al., 2013), as well as cytotoxic against several cancer cell lines (Shanab et al., 2012;Lin et al., 2017). If any compound shows cytotoxic activity against cancer cells, it is important to discriminate if this compound does not represent a threat to normal cells and if its cellular toxicity mechanism is via necrosis or apoptosis. Both apoptosis and necrosis can occur independently, sequentially and/or simultaneously, where the stimuli degree and/or type determines either apoptotic or necrotic death cell (Elmore, 2007). In some cases, cancer chemotherapy treatments result in DNA damage, leading to apoptotic cell death (Elmore, 2007). Apoptosis involves DNA damage and caspases activation. Chlorella spp. extracts have resulted in cell death via DNA damage (Yusof et al., 2010) and caspases activation, demonstrating the apoptosis pathway (Lin et al., 2017).
The aim of the present study was to evaluate the potential of Nuevo Leon, Mexico native microalgae, C. sorokiniana and Scenedesmus sp. extracts, isolated from Nuevo Leon, Mexico, against murine L5178Y-R lymphoma cells. To our knowledge, this is the first report of antitumor activity of microalgae isolated from this geographical area.

Microalgae strains and culture
C. sorokiniana was isolated from San Juan River in the municipality of Cadereyta (26 • 21 55 N 98 • 51 15 O), whereas Scenedesmus was obtained from Pesquería River in the municipality of Apodaca (25 • 47 06 N 100 • 03 04 O) Nuevo Leon, Mexico. C. sorokiniana molecular identification using the P2F (5 -GGC TCA TTA AAT CAG TTA TAG-3 ) and P2R (5 -CCT TGT TAC GA(C/T) TTC TCC TTC-3 ) primers (Lee & Hur, 2009), which amplifies for a 1,700 bp fragment of the 18S gene, as previously reported by Cantú-Bernal (2017). Amplification conditions were an initial denaturation cycle at 95 • C for 5 min, 30-35 denaturation cycles at 95 • C for 30 s, alignment at 50-55 • C for 30 s, and an extension process at 72 • C for 105 s, followed by a final extension at 72 • C for 7 min. The PCR product was confirmed by electrophoresis on 1.5% agarose gel at 100 Volts for 35 min, where the expected 1,700 bp band was observed. Once the PCR product was confirmed, the band was purified, for which the Wizard SV Gel and PCR clean-up system kit (Promega, Invitrogen) was used. For the band sequencing, the product was sent to the synthesis and sequencing unit of the Institute of Biotechnology, Universidad Nacional Autónoma de México. The edition and analysis of the Chlorella sp. sequence similarity percentage was carried out using the program Bioedit Sequence Alignment Editor v. 7.1.9 by sequence identity matrix means, after being compared with sequences reported in GenBank.
For microalgae culture, water samples were taken on 50 mL sterile Falcon tubes and kept at 5 • C ± 2 • C on ice. Then, 5 mL were transferred to 250 mL Erlenmeyer flasks, containing 100 mL of LC culture medium, as developed and reported by López-Chuken, Young & Guzman-Mar (2010). Flasks were then incubated at room temperature (25 • C ± 3 • C) in a continuous shaker at 120× g and under light radiation using 100 Watt white fluorescent light bulb as a continuous artificial light source (1,000 lux approximately). Flasks were incubated for 14 d until green growth was observed, after which, 100 µL were transferred to Petri dishes containing the same culture medium, but solidified with 1.5% of bacteriological agar. Inoculated dishes were incubated at 30 • C ± 2 • C by using a 100 watt white fluorescent light bulb as a continuous artificial light until isolated green colonies were observed. Single colonies were collected using a bacteriological loop and placed in Erlenmeyer flasks containing 100 mL of algal LC liquid culture medium. Next, flasks were incubated under the same conditions described above. This process allowed us selecting a single microalgae genus by picking up a single colony; however, given that microalgae tend to grow in consortia with bacteria and yeasts, microalgal cultures were treated with an antibiotic and antimycotic solution containing 500 UI/mL penicillin, 500 µg/mL streptomycin, 50 µg/mL gentamicin, and 1.25 µg/mL fungizone. For this, 5 mL of LC liquid culture medium with antibiotics were placed in 15 mL conical tubes, after which 0.25 mL of the algal culture were added and tubes were incubated for 48 h, under same shaking and lighting conditions described above. After the incubation period, 500 µL of the cultures were transferred into 50 mL of sterilized LC liquid culture medium without antibiotics producing axenic cultures of C. sorokiniana and Scenedesmus sp. isolates. Each axenic culture was grown for 14 d in 1-L Erlenmeyer flasks containing 500 mL of LC liquid culture medium, until exponential growth phase was reached (based on growth curve, Fig. S1). Next, each complete culture was transferred to individual bioreactor tanks containing 14.5 L of LC culture medium. Photobioreactor tanks were designed by the López-Chuken work team, and consisted of circular acrylic tanks of 30 cm of diameter and height; aeration was supplemented by air pumps with an adapted 0.2 µm filter at 1-L/min flow rate, radiated by continuous artificial LED white lights at 1,500 lux of intensity, and agitation by rotary plastic pallets at 50 rpm (Fig. S2). Biomass production in bioreactors was monitored every 2 d (Tuesdays, Thursdays, and Saturdays) by taking a 10 mL sample with a sterile pipette and filtering through a previously weighed 0.7 µm-pore size microfiber paper. Then the paper was dried at 70 • C inside an oven and weighed again; this monitoring process was repeated until the biomass production showed no increase. Once the maximum biomass production was reached, bioreactor tanks were stored at 4 • C ± 2 • C, until most microalgae biomass precipitated, then, the supernatant was decanted (Fig. S3). The collected wet biomass was the centrifuged at 9,000 rpm for 10 min (ST16R model; Thermo Fisher Scientific, Waltham MA, USA) and frozen dried (Labconco, Kansas City, MO, USA).
Biomass dried samples of C. sorokiniana and Scenedesmus sp. were placed in separate Whatman cellulose extraction thimbles (33 × 80 mm, thickness 1.5 mm) (Sigma-Aldrich, St. Louis, MO, USA) and placed in a Soxhlet extraction apparatus (Reyna-Martínez et al., 2015), which is a continuous system consisting of a flat bottomed round flask, an extraction chamber with a siphon, and a condenser. This method was selected since this extraction is very practical and recommended by most of the methanol-soluble compounds for biological material recovering. A round flask filled with 600 mL of methanol was used and the extraction lasted 48 h for each microalgae. Methanol was selected based on preliminary results where methanol extracts showed the highest cytotoxic activity against L5178Y-R cell line; whereas chloroformic extracts did not show cytotoxic effects and hexane itself showed cytotoxicity against the tumor cell line tested. After the biological material compounds were extracted with methanol, the solutions were filtered using Whatman filter paper, and solvent was evaporated using a rotary evaporator, leaving approximately 10 to 15 mL of liquid material. Remaining solvent was further removed by a vacuum desiccator. Extracts were dissolved in RPMI medium at a concentration of 1 mg/mL and kept frozen until use. From this stock, serial 1:1 dilutions from 500 to 7.8 µg/mL were prepared.
The tumor cell line L5178Y-R (mouse DBA/2 lymphoma) was purchased from the American Type Culture Collection (LY-R, ATCC R CRL-1722 TM ; ATCC, Rockville, MD, USA), maintained in culture flasks with RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamine, and 0.5% penicillin-streptomycin solution (referred as complete RPMI medium) at 37 • C, in a humidified atmosphere of 5% CO 2 in air. Cellular density was kept between 10 5 and 10 6 cells/mL.

Tumor cytotoxicity and apoptosis assays
To determine the cytotoxic effect of C. sorokiniana and Scenedesmus sp. methanol extracts against L5178Y-R tumor cells, cell cultures were collected and washed three times in RPMI medium, then suspended and adjusted to 5 × 10 4 cells/mL with complete RPMI medium. One hundred microliters of the cell suspensions were then added to flat-bottomed 96-well plates (Becton Dickinson, Cockeysville, MD, USA), containing 100 µL of complete RPMI, methanol microalgae extracts at various concentrations, vincristine (250 µg/mL) as positive control, and RPMI medium as negative control; all treatments were tested in triplicate. Microplates were incubated for 48 h at 37 • C with 5% CO 2 , then 15 µL of MTT were added (0.5 µg/mL, final concentration), and cultures were incubated for 3 additional hours. After this, supernatant was removed and 80 µL of DMSO were added to all wells. Optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (DTX 880 Multimode detector; Becton Dickinson, Schwechat, Austria) at 570 nm (Gomez-Flores et al., 2009). The percentage of cytotoxicity was calculated as follows: Apoptosis induction by C. sorokiniana and Scenedesmus sp. methanol extracts against L5178Y-R cell line was evaluated in vitro by acridine orange and ethidium bromide staining. For this, 1 × 10 6 L5178Y-R tumor cells were placed in 24-well plates in the presence of 500 µg/mL methanol extracts, and incubated for 24 h. Then, 500 µL of RPMI, plus 1-µL of acridine orange and 100 µg/mL ethidium bromide (1:1 ratio) were added to the wells. Next, cultured cells were incubated for 5 min, washed with 1-mL PBS, and suspended in 100 µL of RPMI medium; after incubation period, 10 µL of cell suspension were placed between a slide and a coverslip for fluorescence microscope visualization (Inverted Tissue Culture Fluorescence Microscope Olympus IX-70, Representaciones y Distribuciones FAL, S.A. de C.V., Naucalpan, State of México, Mexico). Acridine orange stains viable cells and dead cells (green cells), whereas ethidium bromide only stains those cells that have lost the integrity of their membrane (orange cells). Therefore, viable cells appear in a uniform green tone, cells found in apoptosis appear in a spotty green or granular in the center due to the condensation of chromatin and fragmentation of the nucleus, whereas cells in necrosis appear in a uniform orange hue (Coligan et al., 1995).
In addition, apoptosis induction was evaluated by DNA degradation (Orozco-Flores et al., 2017). For this, cells were incubated for 48 h with C. sorokiniana and Scenedesmus sp. methanol extracts at 500 µg/mL, testing their respective negative (culture medium) and positive (20 µg/mL Actinomycin D) controls. After the incubation period, cells were collected and centrifuged at 2,000 rpm for 10 min, then washed with PBS and extracted using the AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen, Tewksbury, MA, USA). In order to visualize the extracted DNA, the sample was separated by 2% agarose gel electrophoresis, using SB buffer for the electrophoretic shift at 70 V for 20 min and 110 V for 1 h. After this, gel was stained with 5 ng/mL ethidium bromide and photographs were documented under High Performance Ultraviolet Transilluminator (UVP, LLC, Upland, CA, USA) light. DNA like-ladder fragmentation indicates apoptotic activity, whereas DNA smear represents cell death by necrosis.
In early apoptosis stages caspase enzymes are activated. Caspase participate in the cleavage of protein substrates leading to cell disassembly. Cleavage of protein substrates leads to a fluorescent monoamide formation and finally to a rhodamine 110 conversion. For apoptotic pathway involving caspases, caspase can be monitored by measuring fluorescence intensity using microplate wells (Towhid et al., 2013). For this, L5178Y-R cells (5 × 10 5 cells/well) were seeded in a 48 wells plate, and treated with Actinomycin D (800 ng/mL) as positive control, or Chlorella and Scenedesmus methanolic extracts at 500 µg/mL. Cultures were then incubated for 24 h at 37 • C, after which, activated caspases were detected with the CaspGLOW TM red active caspase staining kit following manufacturer's instructions. Fluorescence intensity was measured at Ex/EM = 540/570 nm in a Varioskan Lux Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

Animals
Six-to eight-week old Balb/c female mice were purchased from Harlan Mexico S.A. de C.V. (D.F., Mexico). They were kept in a pathogen-and stress-free environment at 24 • C, under a light-dark cycle (light phase, 06:00-18:00 h) in a One Cage 2100 TM System (Lab Products, Inc., Seaford, DE, USA), and given water and food ad libitum. Animals were euthanized by asphyxiation in a 100% CO 2 chamber. Experiments involving the use of animals were reviewed and approved by our institutional animal care and use committee before being initiated, and were performed in accordance with the Guiding Principles in the Use of Animals in Toxicology, adopted by the Society of Toxicology in March 1999.

Murine thymus lymphocyte viability assay
Thymus was immediately removed after mouse death. Single-cell suspensions were prepared by disrupting the organ in RPMI 1640 medium. Cell suspensions were washed three times in this medium, and suspended and adjusted at 1 × 10 7 cells/mL in complete RPMI medium. Thymus lymphocyte viability was determined by a colorimetric technique using MTT (Gomez-Flores et al., 2009). Thymus suspensions (100 µL of 1 × 10 7 cells/mL) were added to flat-bottomed 96-well plates (Becton Dickinson, Cockeysville, MD, USA) containing triplicate cultures (100 µL/well) of complete RPMI medium (unstimulated control), or 100 µL of C. sorokiniana and Scenedesmus sp. methanol extracts at various concentrations, for 48 h at 37 • C in 95% air-5% CO 2 atmosphere. After incubation for 44 h, MTT (0.5 mg/mL final concentration) was added, and cultures were additionally incubated for 4 h. Cell cultures were then incubated for 16 h with extraction buffer (100 µL/well), and optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (Becton Dickinson, Cockeysville, MD, USA) at 570 nm (Gomez-Flores et al., 2009).
All experiments were repeated at least three times with similar results. The results were expressed as means ± SEM of triplicate determinations from a representative experiment. Statistical significance was assessed by one-way analysis of variance and by the Student's t test.

RESULTS
Microscopic evaluation revealed the presence of C. sorokiniana and Scenedesmus sp. (Table 1), whose isolated colony cultures were then produced under axenic conditions. Culture of both microalgae in photobioreactor tanks showed that the exponential growth phase started after 12 d by C. sorokiniana, whereas for Scenedesmus sp., that started after  19 d (Fig. 1). Once the biomass was dried, collected, and weighed separately for each bioreactor, the yield by C. sorokiniana was of 0.24 g/L (±0.01), whereas for Scenedesmus sp. was of 0.30 (±0.01) g/L. Microalgae methanol extracts tested in vitro against tumor cell resulted in concentrationdependent activity against the murine tumor cell line L5178Y-R. C. sorokiniana extract caused significant (p < 0.05) 17% and 61% tumor cell toxicity at concentrations of 250 and 500 µg/mL, respectively, whereas that of Scenedesmus sp. induced 15%, 25%, and 75% cytotoxicity at concentrations of 125, 250, and 500 µg/mL, respectively (Fig. 2). Collected data were used to determine the inhibitory concentration mean (IC 50 ) of C. sorokiniana and Scenedesmus sp., methanolic extracts. The observed IC 50 for C. sorokiniana, Scenedesmus sp., and vincristine were 460.0 ±21.5, 362.9 ± 13.5, and 76.83 ± 2.55 µg/mL, respectively.
C. sorokiniana and Scenedesmus sp. methanol extracts were shown to cause DNA fragmentation in L5178Y-R cells, with the typical ladder pattern, after 24 h of treatment, which was comparable with the results obtained with actinomycin D (Fig. 3A). Caspase activity assay showed that Scenedesmus sp. resulted in significantly higher (P < 0.05) apoptosis compared with the control (Fig. 3B). The AOPI staining analysis revealed that C. sorokiniana extract resulted in 74.4% tumor cell toxicity, 66% apoptosis, and 9% necrosis, whereas Scenedesmus sp. extract caused 54% tumor cell toxicity, 51% apoptosis, and 3% necrosis (Figs. 3B and 3D).

DISCUSSION
To our knowledge, this is the first report of Nuevo Leon, Mexico native microalgae, identified as C. sorokiniana and Scenedesmus sp., showing cytotoxicity against a murine lymphoma tumor cell line. Previous reports have shown microalgae potential for wastewater treatment and biodiesel production (Reyna-Martínez et al., 2015;Beltrán-Rocha et al., 2017). In the present study, microalga isolates were grown under artificial light, closed photobioreactors, and previously established physicochemical conditions. Biomass production was monitored every 2 d by taking a 10 mL sample, filtered, dried, and weighed during the fermentation time-course. Biomass calculated from time-course data indicated that Scenedesmus sp. resulted in higher (twice as much) biomass production compared with that produced by C. sorokiniana, since values were higher than 0.8 and up to 0.4, respectively (Fig. 1). Nonetheless, after collecting the final biomass produced by each microalgae, Scenedesmus sp. resulted in only 18% more biomass, compared with C. sorokiniana, for a total of 13.74 g and 11.21 g dried biomass, respectively. C. sorokiniana and Scenedesmus sp. production in the photobioreactors was stopped after 29 d because no additional biomass production was observed. Biomass production was lower compared with other reports using batch culture and phototrophic conditions (Brennan & Owende, 2010; Chen et al., 2011). However, sufficient biomass was produced to obtain an adequate amount of methanol extract to perform biological assays.
In vitro tumor cell toxicity assays resulted in concentration-dependent activity against the murine tumor cell line L5178Y-R (up to 61.9% and 74.8% cytotoxicity at 500 µg/mL C. sorokiniana and Scenedesmus sp. extracts, respectively). These results are comparable with other reports showing about 50% in vitro cytotoxicity by microalga extracts against cervical cancer (Yusof et al., 2010;Kyadari et al., 2013).
Apoptosis is the best known pathway for programmed cell death. Apoptosis and necrosis can occur independently, sequentially or simultaneously. The type and/or the stimuli degree may determine if cells die by apoptosis or necrosis. At low doses, a variety of injurious stimuli such as heat, radiation, hypoxia, and cytotoxic anticancer drugs can induce apoptosis, or lead to necrosis at higher doses (Elmore, 2007). After cells enter the apoptotic process, their DNA degrades, showing a ladder pattern of multiples of approximately 200 base pairs, which can be observed when extracting the DNA and making an agarose gel electrophoresis. Apoptosis involves the activation of caspases enzymes linked to the initiating stimuli. Caspase-3 is required for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse of the cell (Mantena, Sharma & Katiyar, 2006). After testing C. sorokiniana and Scenedesmus sp. methanolic extracts, using the caspase-3/7 microplate assay, results demonstrated that only the Scenedesmus sp. methanolic extract was significantly different compared with the untreated cells (negative control), whereas no differences were observed with either actinomycin D or C. sorokiniana methanolic extract. Microalgae-induced tumor cytotoxicity was observed to be mediated by apoptosis, as determined by the acridine orange and ethidium bromide staining, as well as DNA fragmentation (ladder pattern) (Nagata, 2000). In fact, microalga isolates methanol extracts resulted in similar effects against the cell line compared with actinomycin D, compound that resulted in cellular apoptosis (Quintanilla-Licea et al., 2012). After testing crude extracts of the cyanobacteria Nostoc sp., against human pancreatic tumor cells PaTu 8902, Voráčová et al. (2017) found that apoptosis was mostly mediated by caspases 3 and 7. In summary, DNA fragmentation, acridine orange/ethidium bromide staining, and caspases results support apoptosis as the cell-death pathway by the tested microalgae methanolic extracts (Towhid et al., 2013).
Tumor cancer cells may develop as a result of in situ formation of nitrosamines from secondary amines and nitrite in an acidic environment of the stomach. There are chemical agents known as chemopreventers, which help to reverse, suppress or prevent these nitrosamines formation. In fact, ascorbic acid or phenolic compounds are chemopreventers, since they prevent or reduced nitrosamines formation (Jahan et al., 2017). It has been shown that microalgae synthesize a number of bioactive compounds, including bioactive peptides, fucans, galactans, alginates, phenolic compounds, phycocyanins, phycobiliproteins, eicosapentanoic and arachidonic acids, carotenoids, tocopherols, sterols, and terpenoids (Lordan, Ross & Stanton, 2011). Some of these compounds may be responsible for the cytotoxicity induced by C. sorokiniana and Scenedesmus sp. methanol extracts, against the murine lymphoma cell line L5178Y-R.
In a recent report, C. sorokiniana water extracts were evaluated against two human non-small cell lung cancer (A549 and CL1-5 human lung adenocarcinoma cells) cell lines using a subcutaneous xenograft tumor model. Results demonstrated the tumors growth inhibition after extract oral intake in vivo, through mitochondrial-mediated apoptosis (Lin et al., 2017).
In the present study, no significant lymphocyte cytotoxicity was observed by C. sorokiniana and Scenedesmus sp. methanol extracts. Nevertheless, results were comparable with other reports, which show low than 20% lymphocyte cytotoxicity but around 50% cytotoxicity against cervical cancer cells by microalga extracts in vitro (Yusof et al., 2010;Kyadari et al., 2013).
The bio-guided fractionation of these extracts is ongoing, and further studies of the isolated pure compounds will be performed.

CONCLUSION
The native microalgae C. sorokiniana and Scenedesmus sp. isolates from Nuevo Leon, Mexico water bodies were produced under a semi-pilot level using closed photobioreactors, with artificial illumination and aeration. The produced microalgae methanol extracts were cytotoxic against the murine tumor cell line L5178Y-R in vitro, by the mechanism of apoptosis, without affecting normal murine lymphocytes.


extfieldsofstudy:
Computer Science
Medicine

text:
Bi-stability in cooperative transport by ants in the presence of obstacles

To cooperatively carry large food items to the nest, individual ants conform their efforts and coordinate their motion. Throughout this expedition, collective motion is driven both by internal interactions between the carrying ants and a response to newly arrived informed ants that orient the cargo towards the nest. During the transport process, the carrying group must overcome obstacles that block their path to the nest. Here, we investigate the dynamics of cooperative transport, when the motion of the ants is frustrated by a linear obstacle that obstructs the motion of the cargo. The obstacle contains a narrow opening that serves as the only available passage to the nest, and through which single ants can pass but not with the cargo. We provide an analytical model for the ant-cargo system in the constrained environment that predicts a bi-stable dynamic behavior between an oscillatory mode of motion along the obstacle and a convergent mode of motion near the opening. Using both experiments and simulations, we show how for small cargo sizes, the system exhibits spontaneous transitions between these two modes of motion due to fluctuations in the applied force on the cargo. The bi-stability provides two possible problem solving strategies for overcoming the obstacle, either by attempting to pass through the opening, or take large excursions to circumvent the obstacle.

and N ≡ ntot 2 we can describe the system as a one step jump process where at any given epoch, the system at state n can jump forward or backward (n → n + 1 or n → n − 1) with transition rates of α n and β n respectively within the sample space of n ∈ [0, N ].
The gain term α n is given by contributions from pullers at the back and lifters at the front: The probability of finding the system in state n at time t + dt is given by: P n (t + dt) = α n−1 P n−1 (t)dt + β n+1 P n+1 (t)dt + (1 − α n dt + β n dt)P n (t) (S4) which yields the master equation for the given jump process: By definition, the mean value of n can be written as: and therefore the rate of change in n(t) is given by: [(n + 1)α n P n (t) + (n − 1)β n P n (t) − n(α n + β n )P n (t)] (S10) Recognizing both terms on the r.h.s. of (S11) as the weighted averages of the gain and loss terms: and by neglecting fluctuations around the mean we obtain a deterministic equation for the rate: where by plugging in (S2,S3) into (S13) we obtain the rate equation: The equations of motion of the simplified model Starting by decomposing the force vector into an internal and external parts: The internal part will be the difference between the number of puller ants on both sides of the cargo lattice, and will also account the reduction of friction due to the lifter ants where where Θ is the Heaviside step function, f s is the static friction force due to the contact of the cargo with the surface, β is the magnitude of friction force reduced by a lifter ant, and n l is the number of lifter ants.
Eq.S17 implies that each lifter ant reduces a factor of β from the static frictional force f s , until enough lifters lifted the cargo above the surface (βn l > f s ), such that h(f s , n l ) → 0. For simplicity, we consider throughout the model that the cargo is fully lifted above the surface, and neglect the contribution of the lifter ants.
The external part of the force balance S55 is given by: In the limit of → 0 (tanh x → sign(x)), the informed ants act as a restoring force ∀x.
The c.o.m. velocity is given by normalizing f tot by the cargo mechanical response coefficient γ: and therefore the acceleration is given by: By plugging in the rate equation (S14) into the acceleration (S20) we obtain: For for a fully occupied lattice, the number of ants on both sides is given by: and by adding and subtracting (S19) from (S22,S23) we obtain: Therefore, by plugging in (S24,S25) into (S20) we obtain: where in (S27) we rescale the parameters with force units by γ:

COMPARISON TO THE TETHERED CARGO MODEL
In [1] the dynamics of a cargo that is constrained by a tether was investigated. It was found that the cargo is pulled in the direction of the nest until the tether is taught, and then begins to perform oscillations. Unlike our system, the informed ants pull the cargo in a direction that is mostly orthogonal to the allowed motion. The equations of motion for the both models are given by: The difference between the two models is the external force and its derivative (the emboldened terms).
While for the rigid obstacle case the restoring force is localized to x = 0, in the tethered cargo case it is dependent on the angle θ, and therefore effectively directed (in a non uniform manner) to a scent trail with width of 2Lsinθ max , where L is the tether length and θ max is the maximal aperture the tethered cargo can take with respect to the origin. Comparison between the solution of both models shows that when ≥ 0.5 both models coincide (Fig.A) at a parameter range where there is no bi-stability. Blue -tethered cargo. Red -rigid obstacle. Black -separatrix (linear obstacle solution).

STABILITY ANALYSIS DERIVATIONS
a. Linear stability The dynamical system is given by: Finding the fixed points:ẋ Linearizing (S30) and (S31) around the fixed point (x * , v * ) = (0, 0): The eigenvalues of (S34,S35) are given by: where: The fixed point will change stability when µ = 0, which corresponds to the point where Re[λ] = 0.
Therefore, the critical f ind for the Hopf bifurcation is given by:

. Estimation of the homoclinic bifurcation
The acceleration of system is given by: At the transition between phases (i) and (ii) two nullclines merge, and the free motion changes to relaxation oscillations [2] .The transition point is an extrema of (S40). By treating the system separately for each half space of x ( tanh x → sign(x)) we obtain: and by equating (S41) and (S42) we obtain the velocity at the transition: The approximation of the critical point of bifurcation is given by pluggin in (S43) into (S41):

RELATION BETWEEN CARGO SIZE AND INDIVIDUALITY
The number of ants N that can occupy a circular cargo with radius R is given by where L is the average width of an ant, and R is the radius of the circular cargo.
The number of pullers that occupy the cargo can be written as where b is a fraction. By Plugging Eq.S46 into Eq.S45, we obtain that the number of pullers that are attached to the cargo, is proportional to the cargo size The role switching rate is given by where f tot is the total applied force, p i is the body axis unit vector of an individual ant labeled by i, and F ind is the individuality parameter.
When the cargo is in a persistent motion, the total force f tot can be approximated by and the body axis unit vector of each ant can be approximated by where f 0 is the magnitude of force applied by a single puller. What Eqs. S49,S50 imply, is that the total force is given by the puller ants, that pull the cargo in a single persistent direction.
By plugging Eq.S49,S50 into Eq.S48 we obtain And by using the relation between the number of pullers and the cargo size (Eq.S47) we obtain Therefore an increase in R is equivalent to an effective decrease of F ind , and a decrease in R is equivalent to an effective increase of F ind . For the circular cargoes we use in the experiments, we have a linear relation between the mass and the radius. i.e m ∼ R (Fig.B).

FULL 2D MATHEMATICAL MODEL
For a full 2D description we model a ring-like cargo as a circular lattice with n max sites (Fig.Ca) [3].
Each site i can be occupied by a puller, a lifter, or remain empty and is denoted by: The total force and c.o.m. velocity are given by: where the force f 0 exerted by a single puller is considered equal for all the ants, and p i is the body axis unit vector of an ant on site i: where θ i is the angle of site i around the cargo with respect to the chosen frame of reference, and φ i is the local orientation of the ant with respect to θ i .
The static friction is parameterized by the coefficient is given by f s , which is reduced with respect to the lifter activity by:f where βf 0 is the reduction in friction force by a single lifter, and is equal for all sites of a cargo with radial symmetry. The angular velocity of the cargo is given by: where γ rot is the cargo rotation response coefficient and τ kin is the kinetic torque friction acting in the same manner as (S58).
The dynamics are stochastic and simulated using a Gillespie algorithm [4], where ants can attach, detach, role switch, reorient (change its angle φ i ) and forget (transition from informed to uninformed).
The general dynamic scheme of the 2D model is described in Fig

DISCUSSION OF THE INTERACTIONS OF SINGLE ANTS WITH THE OBSTACLE EDGES
In the experimental setup we used, the cargo did not reach the side edges. Although we attribute this fact to the informed ants that pull towards the hole [5], ants were free to forage everywhere inside the obstacle. Since according to our model single ants can hold information that can be valuable to the group, we ask if single ants that arrived to the side edges, can act as "informed" and take the cargo away from these edges. In Fig.D we provide an example from an analysis that counts the number of ants near the obstacle edges and inside the area of oscillations where the cargo moved.
The analysis shows that the number of ants near the edges is much smaller compared to the number of ants along the rest of the obstacle. Throughout all the experiments that we have conducted, we have not found any case where a single ant that reached the obstacle edges, changed its direction of motion towards the cargo and attached to it.


extfieldsofstudy: Medicine Biology

text:
Potential Coverage of the 4CMenB Vaccine against Invasive Serogroup B Neisseria meningitidis Isolated from 2009 to 2013 in the Republic of Ireland

The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland.

IMPORTANCE The meningococcal antigen typing system (MATS) is an enzymelinked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n ϭ 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland. KEYWORDS 4CMenB, MATS, MenB, NHBA, NadA, Neisseria meningitidis, vaccine, fHbp, porA T he relatively low incidence of invasive meningococcal disease (IMD) currently experienced in Europe (1) is due in part to the routine use of capsular polysaccharide conjugate vaccines, which have reduced disease incidence from meningococci expressing capsular types C, W, and Y (2,3). In common with many other European countries, the population of the Republic of Ireland (RoI) predominantly experiences serogroup B IMD (1,4), with laboratory-confirmed incidence rates of 1.37 cases per 100,000 population in 2013 to 2014, 1.24 in 2014 to 2015, and 0.99 in 2015 to 2016 (http://www.hpsc.ie). Meningococcal disease incidence peaks within the first year of life and then declines until the middle to late teenage years, when a second, smaller peak is observed. Children under 5 years of age suffer the highest disease burden (http:// www.hpsc.ie). The majority of meningococcal disease observed is sporadic and is caused by meningococci with multiple distinct genotypes as defined by multilocus sequence typing (MLST) (5).
The MenB polysaccharide is poorly immunogenic, and possibly autoantigenic, and so a MenB polysaccharide vaccine is considered unfeasible (6,7). Consequently, subcapsular antigens have been exploited instead. Several outer membrane vesicle (OMV) vaccines have been demonstrated to be safe and effective, protecting against disease caused by homologous meningococci (8)(9)(10)(11). These vaccines elicit mostly strain-specific antibody responses; poor cross-reactive responses to heterologous strains are observed in adults, and none are observed in infants (12,13). OMV vaccines are therefore limited to combating occurrences of clonal expansion and are unsuitable in controlling heterologous endemic strains.
A four-component vaccine, 4CMenB (Bexsero) (14), is currently licensed for use in over 30 countries worldwide and was introduced into the schedule of routine immunization of infants (at 2, 4, and 12 months of age) in the RoI in December 2016 and in the United Kingdom in September 2015 (15,16). The major antigens include recombinant forms of neisserial adhesin A (NadA) peptide 3 (variant NadA-2/3), factor-H binding protein (fHbp) peptide 1 (variant 1), Neisseria heparin-binding antigen (NHBA) peptide 2, and New Zealand strain outer membrane vesicles (NZ OMV) containing the PorA protein with the variable-region 2 (VR2) peptide P1.4 (10,14). 4CMenB has demonstrated effective protection against diverse MenB in the United Kingdom, with 83% (95% confidence interval [CI 95% ], 24% to 95%) two-dose vaccine effectiveness against all MenB cases in the first 10 months of the infant national immunization program (17).
Vaccine effectiveness depends on host (immune response) factors and pathogen (strain coverage) factors. Polysaccharide and conjugate vaccine immunogenicity is assessed by the serum bactericidal antibody (SBA) assay, which measures complementmediated killing for representative strains expressing different serogroups (18)(19)(20)(21)(22). Strain coverage for vaccines which target the polysaccharide can be estimated by observing the serogroup frequencies of invasive meningococcal populations. This approach is not practical for subcapsular protein antigens, which are likely to vary both in their levels of expression and in their peptide sequences across the many different endemic disease-associated meningococcal strains (23,24). MLST-based lineage frequencies cannot be used either, due to the dynamic and highly recombining population structure of meningococcal species (25).
As an alternative, the meningococcal antigen typing system (MATS), which combines genotyping for PorA and a sandwich enzyme-linked immunosorbent assay (ELISA), was developed to measure the level of target antigen expression and ability of 4CMenB-induced antibodies to recognize the vaccine antigens on individual invasive MenB isolates (26). This capacity of vaccine-induced antibodies to recognize their respective vaccine targets is referred to as the relative potency (RP). Test isolates are considered covered by MATS if the RP level is greater than the positive bactericidal threshold (PBT) for at least one vaccine antigen (fHbp, NHBA, or NadA) or if they harbor PorA VR2 peptide P1.4 (27) or both. MATS-PBT values correlate vaccine-induced SBA titers with bactericidal killing and were established using postvaccination pooled sera from infants after a fourth dose of 4CMenB at 12 months (26). These experiments link MATS-PBTs and the meningococcal gold standard correlate of protection (SBA titer of Ն4), which is the accepted surrogate to assess vaccine effectiveness in the absence of protection studies (18,22,28). To ensure consistency of MATS data, interlaboratory standardization studies have been conducted in National Reference Laboratories for meningococcal disease (29), including Public Health England's meningococcal reference unit (PHE-MRU, Manchester, United Kingdom) and the U.S. Centers for Disease Control and Prevention (CDC, Atlanta, GE).
The aim of this study was to evaluate the potential coverage, as defined by MATS, of MenB IMD-associated isolates collected during the 2009-2010 to 2012-2013 epidemiological years in the RoI and from different age groups. We aimed to describe 4CMenB target peptide diversity and the relationship with meningococcal lineages, as defined by MLST, before the introduction of the vaccine.
Coverage estimates by age and epidemiological year. The overall coverage estimate for the 4CMenB vaccine was 69.5% (CI 95% , 64.8% to 84.8%) (Fig. 1A). Point estimates of predicted strain coverage by year were similar across the different age groups, with the highest anticipated for children 1 to 5 years of age (76%; CI 95% , 72% to 80%) and lowest for those under 1 year of age (61.9%; CI 95% , 57.1% to 90.5%) (Fig. 1A). A 2-sided chi-square test was used to test the differences across years in the predicted MATS coverage, and no statistical significant difference was observed (P value for the age groups overall, 0.376; one-to-one-comparison P values, 0.358, 0.376, and 1 for children under 1 year of age, 1 to 5 years of age, and more than 5 years of age, respectively). The 2-sided chi-square test was also used to test the differences across epidemiological years in the predicted MATS coverage from 2009 to 2013 (Fig. 1B). No statistical significant difference was observed (overall, P ϭ 0.76; one-to-one comparisons, P Ͼ 0.45 to P Յ 1). Point estimates varied from 73.7% (CI 95% , 71.1% to 86.8%) in 2009 to 2010 to 61.5% (CI 95% , 53.8% to 76.9%) in 2012 to 2013 (Fig. 1B).
Coverage by antigen and by antigen combinations. MATS results revealed that no isolate was predicted to be covered by all four vaccine antigens (Fig. 2). Thirty-two isolates (31%) were not expected to be covered. The proportion of isolates covered by a single antigen was 21% (n ϭ 22/105), and the proportion of isolates covered by two antigens was also 21% (n ϭ 22/105), while 28% of isolates were covered by three antigens (n ϭ 29/105). Strains responsible for invasive disease in subjects under 1 year of age were more likely to harbor 3 MATS-positive peptides than the strains from those over 1 year of age (P Ͻ 0.02).
Only cc32 isolates (n ϭ 5/5) harbored intact nadA genes potentially encoding functional proteins (peptide 1 [n ϭ 4] and peptide 100 [n ϭ 1]) but failed to generate  an RP value for NadA above the PBT. These RP values may be underestimated since NadA expression is repressed in vitro but not in vivo (31) and may contribute to in vivo killing. In any case, the overall coverage estimate would not change as these five isolates were anticipated to be covered by fHbp. All cc213 isolates possessed NadA-4/5 alleles, which were frameshifted at an intragenic homopolymer and, if in-frame, would not express cross-reactive peptides. Three further contained atypical nadA alleles not anticipated to express functional forms (alleles ml1157 [n ϭ 2] and ml103 [n ϭ 1] and a single unassigned ST [incomplete fumC sequence]). 4CMenB peptide prevalence among the most prevalent sequence types. The most prevalent sequence types, ST-154 (n ϭ 14) and ST-41 (n ϭ 7), were anticipated to be covered by fHbp. NHBA peptide 2 was expressed in 92.9% of ST-154 isolates (n ϭ 13/14) and 85.7% of ST-41 isolates (n ϭ 6/7). These two STs were more likely to be associated with IMD in those 5 years of age or younger (OR, 4.29; CI 95% , 1.17 to 15.68, P Ͻ 0.03) and were covered by either two antigens (n ϭ 3/21) or three antigens (n ϭ 18/21).
Coverage estimation by exact matches to the vaccine peptides-no crossprotection. The peptides used in the 4CMenB vaccine are PorA P1.4, NHBA peptide 2, fHbp peptide variant 1, and NadA peptide 3. Coverage was estimated using the more stringent criterion of 4CMenB exact vaccine peptide matches only. This estimate ignores the potential of vaccine components to stimulate cross-protective antibodies. This is an important consideration, as the level of cross-protection from vaccineinduced antibodies is lower in infants than in adults (25,26,32).
The levels of diversity of vaccine target peptides among the Irish MenB strains were consistent with previous descriptions of meningococcal diversity; a few variants accounted for the majority of observations, and a larger number of variants were observed at lower frequencies. The high MATS coverage for the cc41/44 isolates is also consistent with previous reports.
When the RP value determined for a particular antigen is greater than or equal to the corresponding PBT value (or if the isolate possesses a porA p1.4 peptide), an isolate is considered MATS positive. PBT values were established as a link to the correlate of protection in experiments using postvaccination sera taken from infants after the fourth dose at 12 months (26). This is important to consider when interpreting the present MATS coverage estimate of 61.9% (CI 95% , 57.1% to 90.5%) in infants under 1 year of age (the target population) and in regard to those immunized at 2, 4, and 12 months of age in the RoI.
Further, infants typically produce weaker immune responses that are of shorter duration and less likely to produce a cross-protective immune response than adult vaccinees (12,38). The difficulty in producing a cross-reactive immune response in infants has been demonstrated for fHbp (39,40). Half of the strains studied here expressed either no antigen (30.5%) or one antigen (21%). Single-antigen coverage was mostly dependent on the level of fHbp coverage (18.1%), with minor contributions from NHBA (1.9%) and porA (1%). MATS was deliberately designed with an element of conservatism. MATS RP values are based on individual antigen targets only, and any mutual cooperation between different vaccine antigens is ignored, as are any possible contributions from the OMV minor antigens, including any contribution of nonprotein antibodies. Also, NadA expression is repressed and suboptimally expressed under the in vitro experimental temperature condition of 37°C (31). One study which looked at a representative panel of United Kingdom MenB strains where MATS-predicted coverage was 70% (CI 95% , 55% to 85%) reported a coverage estimate of 88% (CI 95% , 72% to 95%) based on examination of pooled infant and adolescent using the human serum bactericidal antibody assay (hSBA) (41). The synergistic effect of antibodies binding to more than one target in vivo may result in enhanced bactericidal activity against a meningococcus expressing multiple vaccine targets (26).
A high proportion (27.6%, n ϭ 29/105) of Irish isolates were predicted to be covered by three vaccine antigens, PorA, fHbp, and NHBA, compared to the global triple-antigen coverage of 15.7% previously reported (43). Coverage estimates could be considered more robust for these strains than for those covered by a single antigen (26). A redundancy of targets is more likely to facilitate antibody binding, increasing the likelihood of protection. The high triple-antigen coverage in the most prevalent strains (ST-154 and ST-41) is reassuring, as is the association between invasive strains harboring 3 MATS-positive peptides and infants under 1 year of age. Donnelly and colleagues reported an association between the number of vaccine targets and the level of killing; strains harboring 2 or more antigens are more likely to induce bactericidal killing (96%) than strains harboring a single antigen only (80%).
Nonrandom associations between meningococcal clonal complexes and age have been previously demonstrated (44,45). The predominant disease-causing lineage during the studied period, cc41/44, was more likely to invade those less than 5 years of age, exhibited high exact peptide coverage (89%), and had a redundancy of MATSpositive targets.
Together, these data suggest that routine vaccination of infants during this period would have offered vaccinees protection against the most common disease-causing lineage and the two most frequently isolated disease-causing clones (ST-41 and ST-154). Nine deaths resulted from the 105 MenB IMD case episodes, six of which were predicted by MATS to be covered and might have been prevented by individual vaccination (http://www.HPSC.ie). Two of the three deaths which occurred in infants over the study period might have been prevented by 4CMenB vaccination. Those infants would have received one or two doses of 4CMenB, given their age at the time of death. There is some evidence to support the idea that partial vaccination may result in milder disease (37).
As well-engineered as the MATS in vitro assay is, it remains unlikely to be wholly representative of the more complex in vivo conditions of bacterial invasion. Individual immune responses among vaccinees exhibit variation. When Bexsero was used to control a MenB cc41/44 meningococcal outbreak that was caused by a clone expressing both fHbp and NHBA peptides which matched those in the vaccine, 33.9% of 499 adult vaccinees failed to generate a detectable protective antibody response against the outbreak strain 8 weeks after the second dose (46).
Bexsero has been licensed and deployed on the basis of serological and immunological criteria only. These methods are substitutes for measuring vaccine efficacy directly from protection studies, where a reduction in disease incidence in a sufficiently large population may be attributed to the vaccine. Data from the United Kingdom national immunization program have provided the first evidence of the effectiveness of Bexsero at the population level, where disease is caused by many genetically variable strains (17). Parikh et al. used the screening method to compare vaccine-eligible and vaccine-ineligible cohorts, 10 months following the introduction of the vaccine into the routine infant schedule, and observed an 83% level (CI 95% , 24% to 95%) of two-dose vaccine effectiveness against all MenB cases (17). A 50% (CI 95% , 0.36 to 0.71, P ϭ 0.001) incidence rate ratio (IRR) reduction was observed in the vaccine-eligible cohort compared to the estimated number of cases in infants of the same age for the 4 years prior to the vaccine's introduction. A reduction of 36% is reported following correction for a declining SgB incidence (CI 95% , 0.08 to 0.55, P ϭ 0.015). These data provide the first evidence of real-world vaccine effectiveness. Although the precise level of effectiveness is unclear as yet, these data are particularly comforting, as the small population and the incidence of MenB disease in the RoI will hinder similar measurements of the vaccine's effectiveness.
Predicted coverage of Bexsero varies throughout Europe as a consequence of the diversity of the meningococcal population, which contains elements that are stable and elements that are subject to temporal and geographic flux (4,47). Researchers in the United Kingdom have reported MATS data from two different periods and observed a coverage change from the 2007-2008 estimate of 73% (CI 95% , 57% to 87%) to the more recent estimate of 66% (CI 95% , 52% to 80%) for the 2014 -2016 period. The changing nature of meningococcal populations presents a challenge to predicting the impact of subcapsular vaccines both ahead of implementation and postdeployment. The validation of MATS estimations from national immunization programs such as those in the United Kingdom may enable the use of MATS to monitor the effectiveness of the vaccine in the RoI and elsewhere. While whole-genome sequencing (WGS) does not directly determine the extent of antibody immunological cross-reactivity, as the number of isolates that have generated MATS data and WGS data increases, it may be possible to refine a model to predict the MATS result from genotype data (48,49). This is important, since only a minority of IMD cases are culture positive.
Continuous invasive strain characterization, including 4CMenB vaccine peptide sequence data, will be essential to monitor changes in circulating meningococci and to recognize the emergence and expansion of new clones, especially those with novel 4CMenB peptides or strains exhibiting nonsusceptible vaccine peptide profiles, and to investigate potential vaccine failures.

MATERIALS AND METHODS
Isolates. Cultured invasive meningococcal isolates are sent to the Irish Meningitis and Sepsis Reference Laboratory (IMSRL) for characterization. A total of 326 real-time PCR-confirmed MenB IMD cases occurred during the 2009 -2010 to 2012-2013 epidemiological years. Of these, 105 (32.2%) were culture positive, and all 105 invasive isolates were included in the study for MATS testing.
Sequencing. The porA variable regions were determined by Sanger sequencing, and the data were then used to deduce the PorA VR peptides using minor adaptions of previously defined protocol (50) and were curated using the PubMLST Neisseria website (http://www.pubmlst.org/neisseria) (51). Purified genomic DNA for all isolates was prepared using a Wizard kit (Promega) in accordance with the manufacturer's instructions. DNA quantity and quality were assessed using a Qubit fluorometer (Life Technologies, Inc.) and standard 1% agarose gel electrophoresis, respectively. Genomic DNA was sequenced on an Illumina HiSeq sequencer at the Oxford Genomics Center, Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom. The short-read sequences were assembled de novo using the VelvetOptimiser algorithm (52) as part of an in-house-validated pipeline developed in Oxford and were then added to the Bacterial Isolate Genome Sequence Database (BIGSdb) on the PubMLST Neisseria website (51). Genomes were automatically scanned against alleles previously defined in the sequence definition database. MLST data and 4CMenB target peptides were extracted using the "MLST" and "antigen" options with the "scheme" tool in BIGSdb (53).
MATS. MATS assays were performed at PHE-MRU, Manchester, United Kingdom (29). MATS ELISA RP values were determined independently for each antigen by comparisons with reference strains expressing each vaccine antigen (26). An isolate was predicted to be covered if the PorA VR2 P1.4 peptide was present and/or if an RP value greater than its MATS-PBT (0.021 for fHbp, 0.294 for NHBA, and 0.009 for NadA) was obtained for at least one antigen, as described previously (26). The PBT is the minimum RP value that is predictive of whether a MenB isolate would be susceptible to killing in the human serum bactericidal antibody assay (hSBA) by 4CMenB-elicited antibodies. The 95% confidence intervals for the positive PBT thresholds were derived as described previously (29). The threshold values established are as follows: 0.014 to 0.031 for fHbp, 0.169 to 0.511 for NHBA, and 0.004 to 0.019 for NadA.