Sarcoptes scabiei, is one of the most severe ectoparasitic diseases in rabbits. Scabies seriously affects the commercial rabbit breeding, causing severe economic losses. Host resistance to S. scabiei is an important factor in further development of the rabbit industry. In the present study, we compared the host resistance to S. scabiei var. cuniculi of a new breed of domestic rabbit propagated by the Sichuan Animal Sciences Academy  compared with that of a traditional rabbit breed in the domestic rabbit industry .Scabies, caused by infestation of the mite S. scabiei var. cuniculi mites for 48 h. Then, during the course of four-week experimental infestation period, the body weight of rabbits was recorded every two weeks for calculating body-weight variations in comparison to the non-infested control rabbits. Skin lesions in the foot area were assessed on weekly basis and serum samples were tested weekly for the estimation of changes in the total antibody levels . Moreover, DNA extracted from the blood samples was amplified for analysis of the genetic diversity in the major histocompatibility complex, class II, DQ Alpha (MHC-DQA) gene.Both QX and IRA rabbits were experimentally infested with live P < 0.05) relative body weight gain compared to the non-infested control rabbits and significantly lower (P < 0.05) scores for foot skin lesions and higher levels of IgG, IgE and IgM at weeks 1 to 4, week 2 and week 1 post-infestation, respectively. Furthermore, a polymorphism site at position 103 bp of exon two of MHC-DQA gene and a different gene frequency were found between two rabbit breeds, suggesting the genetic basis for the differential host resistance to the S. scabiei var. cuniculi between two rabbit breeds.Compared to the IRA rabbits, the QX rabbits showed a significantly higher (S. scabiei var. cuniculi compared to the IRA rabbits at the clinical, immunological and genetic levels. These results provide a reference for the breeding of rabbits with adequately improved and sustained host resistance to scabies in the domestic rabbit industry.The QX rabbits showed higher host resistance to  Sarcoptes scabiei. These mites can burrow into the skin, feed on epithelial cells and serum, leading to serious skin lesions  × 100%. The skin lesions were assessed by observing the inflammatory reaction in toe area of hind limb and measurements of the lesion areas were made using a caliper and the lesion scores were indexed as follows: score 0, no skin lesions; score 1, mild inflammatory reaction with reddening; score 2, severe inflammatory reaction with reddening; score 3, lesions  versus the corresponding concentration on the horizontal (X) axis. The concentration of serum total antibodies in each serum sample was then determined by comparing the OD450 nm of the samples to the standard curve.Serum samples of experimentally infested rabbits in the QX and IRA groups were separated from the blood and stored at − 20 °C to test serum total antibody levels. The total IgG, IgE and IgM levels were assayed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using the Rabbit IgG (Immunoglobulin G) ELISA Kit , Rabbit IgE (Immunoglobulin E) ELISA Kit (Elabscience) and Rabbit IgM (Immunoglobulin M) ELISA Kit (Elabscience), respectively. The procedures were performed as per the instructions of ELISA kits. Briefly, antibodies to rabbit IgG, IgM and IgE were pre-coated on ELISA plates, serum samples and calibration standard samples (used to construct a standard curve) were added to each well and detected using a detecting antibody linked to horseradish peroxidase (HRP), the color was developed with tetramethyl benzidine (TMB) after incubation and thorough washing. Color intensity was measured at 450 nm using a spectrophotometer and the OD450 nm of each sample was recorded. A standard curve of OD450 nm MHC-DQA gene. The blood samples were treated using an Animal Tissue Direct PCR Kit  for extracting the genomic DNA and perform the PCR reaction. The MHC-DQA gene was amplified by PCR using gene-specific primers: forward (5′-TCA TCA GCT GAC CAC GTT GG-3′) and reverse (5′-GCA GCA GTA GAG TTG GAG-3′), which were designed based on the published Oryctolagus cuniculus MHC-DQA gene (GenBank: EU686533.1). The PCR reaction system comprised a 20-μl mix containing 10 μl of 2× PCR Easy® Mix, 7 μl of ddH2O, 0.5 μl of each primer (20 mM) and 2 μl of DNA template. The reaction conditions were as follows: initial denaturation at 94 °C for 3 min; followed by 30 cycles of amplification at 94 °C for 10 s, 58 °C for 20 s and 72 °C for 20 s; and a final extension step at 72 °C for 5 min. PCR products (10 μl each) were electrophoresed using a 1% agarose gel, stained with ethidium bromide and visualized on a UV transilluminator. Remaining products were used for the analysis of genetic diversity in the second exon of the MHC-DQA gene. PCR products used for electrophoresis were extracted and purified using a QIAquick Gel Extraction Kit  and then cloned into vector pMD19-T . Transformants were selected on Luria-Bertani agar containing ampicillin (100 mg/ml) and then subjected to automated sequencing . The DNAMAN Software (version 8.0) was used to compare the obtained nucleotide sequences of MHC-DQA gene with the reference sequences (GenBank: EU686533.1).Blood samples of experimentally infested rabbits in the QX and IRA groups were collected at week 4 PI and stored at − 20 °C for the extraction of genomic DNA and amplification of MHC-DQA gene between two rabbit breeds was analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with Mbo II enzyme . The 15-μl reaction mix for enzyme digestion was comprised of 8 μl of PCR products, 4 μl of ddH2O, 2 μl of 10 × l buffer, and 1 μl of Mbo II enzyme. The reaction conditions for enzyme digestion were 37 °C for 8 h. For confirming the size of the restriction fragments, enzyme-digested products (10 μl each) were electrophoresed using a 3% agarose gel, stained with ethidium bromide and visualized on a UV transilluminator.The genetic diversity on second exon of the P-value < 0.05 was considered to be statistically significant. All data are presented as the mean ± standard deviation (SD). Chi-square test was performed for comparing the genotype frequency, gene frequency of the MHC-DQA gene in each group and a P-values > 0.05 was considered to be reached the Hardy–Weinberg balance. The GraphPad Prism software (version 5.01) was used for producing graphs.All statistical analyses were performed using IBM SPSS statistics 20.0  software. One-way ANOVA analysis with Duncan’s multiple range test was applied to compare body weights, body weights variation, lesion score and antibody levels at different time points within the same group and at the same time point between different groups. A n = 18) was calculated  = 0.031, P = 0.862) and IRA  = 0.048, P = 0.828) groups. However, the initial average body weight of both experimentally infested  = 13.163, P = 0.001) and non-infested control  = 13.329, P = 0.001) rabbits in QX group was significantly lower compared to the IRA group. During the experimental infestation period (weeks 0–4 PI), all rabbits in QX  = 87.164, P < 0.0001; non-infested control: F = 101.691, P < 0.0001) and IRA  = 57.117, P < 0.0001; non-infested control: F = 108.363, P < 0.0001) groups showed a significant increase in average body weight. Meanwhile, body weight variation  = 150.413, P < 0.0001; weeks 0–4 PI: F = 161.605, P < 0.0001) and the relative body weight gain  = 81.782, P < 0.0001; weeks 0–4 PI: F = 26.530, P < 0.0001) in the non-infested control rabbits were significantly higher compared to the experimentally infested rabbits in both groups  = 3.567, P = 0.067; weeks 0–4 PI: F = 3.970, P = 0.082) and relative body weight gain  = 0.403, P = 0.501; weeks 0–4 PI: F = 2.060, P = 0.160) in the experimentally infested QX rabbits showed no significant difference compared to the IRA rabbits; however, values for body weight variation  = 107.071, P < 0.0001; weeks 0–4 PI: F = 163.155, P < 0.0001) and relative body weight gain  = 42.896, P < 0.0001; weeks 0–4 PI: F = 9.864, P = 0.003) were significantly lower in the QX non-infested control rabbits compared to the IRA  = 153.000, P < 0.0001) and IRA  = 185.809, P < 0.0001) groups developed progressive skin lesions caused by infestation of S. scabiei var. cuniculi, showing a significant increase in the average lesion score over the course of time , all the experimentally infested rabbits in the QX  = 11.439, P = 0.002; week 2 PI: F = 11.179, P = 0.002; week 3 PI: F = 11.490, P = 0.002; week 4 PI: F = 10.036, P = 0.003), together with less serious clinical signs compared to the IRA group. Meanwhile, the histogram in Fig. S. scabiei var. cuniculi compared to the QX rabbits.The average lesion score (mean ± SD) of experimentally infested rabbits in the two groups are shown in Table F = 2.355, P = 0.134; IgE: F = 3.653, P = 0.064; IgM: F = 2.535, P = 0.121). The variation in serum total antibody levels became more apparent as the infestation progressed and the comparison of antibody levels at each week PI showed differences between the experimentally infested rabbits in the two breed groups , the antibody-mediated humoral immune responses in the two groups of experimentally infested rabbits were elicited robustly. Analysis of the initial average total antibody levels at week 0 PI showed no significant difference in the experimentally infested rabbits of the two groups  = 14.055, P = 0.001; week 2 PI: F = 6.324, P = 0.017; week 3 PI: F = 12.299, P = 0.001; week 4 PI: F = 13.487, P = 0.001)  = 0.935, P = 0.340). However, at week 2 PI, the IgE levels in the QX experimentally infested rabbits were significantly higher compared to those in the IRA rabbits  = 19.307, P < 0.0001). From weeks 3–4 PI, the differences between the two groups of experimentally infested rabbits decreased and there were only slightly, but not significantly, higher IgE levels in the QX experimentally infested rabbits compared to the IRA rabbits  = 1.243, P = 0.273; week 4 PI: F = 2.218, P = 0.146)  = 7.348, P = 0.010). However, for the rest of the infestation period there were only slightly but not significantly, higher IgM levels were observed in the QX experimentally infested rabbits compared to the IRA rabbits  = 2.746, P = 0.107; week 3 PI: F = 1.592, P = 0.216; week 4 PI: F = 0.489, P = 0.489)  of the target gene fragment  and allele B (with the restriction enzyme cutting site for Mbo II at 103 bp). Besides, two genotypes were derived from two alleles, which were named as AA (without the restriction enzyme cutting site and only a 248-bp band was observed) and AB . From the PCR products in the QX and IRA groups, 10 out of 18 PCR products amplified from serum samples collected from experimentally infested rabbits in the QX group had the restriction enzyme cutting site for Mbo II at 103 bp; however, only 4 out of 18 PCR products in the IRA group had the restriction enzyme cutting site , the genotypes of MHC-DQA gene were 8 AA and 10 AB in QX and 14 AA and 4 AB in IRA  and experimentally infested IRA rabbits (0.889 gene frequency)  and IRA  groups.In terms of the second exon of F = 33.966, P < 0.0001; IRA: F = 15.279, P = 0.001) and significantly lower average lesion scores  = 12.937, P = 0.002; IRA: F = 12.903, P = 0.002) than AA individuals in the two breed groups. Meanwhile, both the AA and AB individuals between the two breed groups showed no significant difference in average relative body weight gain  = 0.070, P = 0.793; IRA: F = 0.499, P = 0.494) and lesion scores  = 3.219, P = 0.088; IRA: F = 1.407, P = 0.259).For further investigating the differences in the clinical condition of genotype AA and AB individuals in the two breeds, the average relative body weight gain and lesion scores at week 4 PI were compared between genotype AA and AB individuals within each breed group and between the two breed groups Table . ResultsBoophilus microplus, Amblyomma hebraeum, Psoroptes ovis, and Ostertagia circumcincta [in vitro cellular immune responses between two different cattle breeds (Belgian Blue beef and Holstein-Friesians) during a natural infestation with Psoroptes ovis and revealed differences in the host resistance between the two cattle breeds at the immunological level. In the present study, we focused on one of the most common parasites infesting rabbits, i.e. S. scabiei var. cuniculi, and comprehensively investigated the differential host resistance to this parasite in two different rabbit breeds at the clinical, immunological and genetic levels.The difference in host resistance to infestation with parasites among different breeds of host animals is an attractive topic for investigation. Previous studies focusing on the host resistance in different breeds of domestic animals have mainly centered on cattle and sheep as models and involving the parasites (largely ectoparasites) such as umcincta –34. The S. scabiei var. cuniculi in rabbits causes serious scratching with skin lesions and considerable weight loss [S. scabiei var. cuniculi between different methods of infestation (by contact or by means of a dressing), it has been shown that these two clinical indices are important in evaluating the severity of the S. scabiei var. cuniculi infestation in rabbits [P = 0.002; week 2 PI: P = 0.002; week 3 PI: P = 0.002; week 4 PI: P = 0.003) together with more serious clinical signs during the infestation with S. scabiei var. cuniculi. These differences in clinical condition between the two rabbit breeds provide evidence that the QiXing breed displayed better clinical signs compared to the IRA breed following infestation with S. scabiei var. cuniculi and preliminarily indicated that differences in the host resistance to S. scabiei var. cuniculi existed between these two breeds of rabbit.It has been demonstrated that infestation with ght loss  and the  rabbits . IntriguS. scabiei var. cuniculi in rabbits [S. scabiei [P = 0.001; week 2 PI: P = 0.017; week 3 PI: P = 0.001; week 4 PI: P = 0.001), indicating that the QX rabbits developed a stronger IgG response to S. scabiei var. cuniculi infestation compared to the IRA breed.The gradual IgG response developed in both rabbit breeds with a progressive increase in the IgG levels. These findings corroborate similar trends reported in previous studies focusing on antibody responses to  rabbits , 12. Mea scabiei –37, indiP < 0.0001), indicating that the QX rabbits developed a stronger IgE response to the infestation compared to the IRA rabbits.IgE is an important component of the host defense against a variety of parasites and along with mast cells, basophils and eosinophils, serves as an essential element in allergic and parasitic inflammation . SimilarS. scabiei var. cuniculi and our results showed that the IgM levels in the two rabbit breeds during infestation were increased during the week 1 PI and significantly differed between the two groups only at week 1 PI (P = 0.010), but progressively decreased below the initial levels during the remaining infestation period. This was in agreement with the variation trend of the IgM response to infestation of S. scabiei reported previously [S. scabiei var. cuniculi infestation developed in QX rabbits (P = 0.010). Hence, the differences in IgG, IgE and IgM responses between these two rabbit breeds demonstrate that the host resistance to S. scabiei var. cuniculi differed between them being comparatively higher in the QX breed.IgM was the first antibody to appear in response to the antigen exposure from eviously , indicatMHC gene in vertebrates and closely associated with differential host resistance to parasitic infestation [MHC-DQA alleles have been reported showing high levels of polymorphism at the rabbit DQA locus [MHC class II DRB constitutions and the rabbits’ intestinal burden of nematodes and coccidia and demonstrated that rabbits with a particular allele of MHC gene showed a lower level of infestation with Eimeria stiedai. In our study, two alleles (A and B) for the second exon of MHC-DQA gene were identified using PCR-RFLP with Mbo II. Allele A was the dominant in both the QX rabbits (0.772 gene frequency) and the IRA rabbits (0.889 gene frequency). However, in terms of genotype frequency, the AB genotype was dominant in the QX breed (0.556 genotype frequency) and the AA genotype was dominant in the IRA breed (0.778 genotype frequency). Combined with the cloned and sequenced results of MHC-DQA gene PCR amplification products, all the AB genotype clones were identical in both QX and IRA breed individuals. Meanwhile, the comparation of average relative body weight gain and lesion scores between AA and AB individuals at week 4 PI within each breed group and between the two breed groups, showed that AB individuals had a significantly higher average relative body weight gain  and significantly lower average lesion scores  than AA individuals in the two breed groups. These findings indicate that the differences in host resistance to S. scabiei var. cuniculi between the two rabbit breeds were likely under the regulation and control of MHC-DQA allele B. In addition to the differences in host resistance to S. scabiei var. cuniculi at the clinical and immunological levels as discussed above, QX rabbits with the advantageous AB genotype  showed a higher resistance to S. scabiei var. cuniculi infestation compared to the IRA rabbits. These results also corroborate the findings of Oppelt et al. [High polymorphism and multi-base mutations are the prominent features in estation , 41. To QA locus . In rabbQA locus  evaluatet et al. , who shoMHC-DQA gene and appearance of the alleles are believed to be driven by selection for resistance to parasites and other pathogens, which are themselves driven to evolve the mechanisms for escaping the host immune responses [MHC-DQA gene in the host are likely because the polymorphism of each allele was driven by a long-term selection for the resistance to parasites and other pathogens and the differences in the host resistance of rabbits to the infestation of parasites are directly related to the genetic diversity of exon two of MHC-DQA gene.The existence of genetic diversity in esponses . Such seesponses . The IRAMHC-DQA gene, we observed sufficient differences in host resistance between two rabbit breeds following an experimental infestation with S. scabiei var. cuniculi. The findings of our study provide foundation for further research focusing on elucidation of the potential mechanism implicated in modulating the MHC-DQA alleles associated with S. scabiei var. cuniculi infestation. Further studies aimed at identifying other potential factors implicated in the differential host resistance to the S. scabiei var. cuniculi between these two rabbit breeds would be of a great value for understating the basis of differential breed susceptibility to S. scabiei var. cuniculi.In summary, using clinical condition assessment, humoral immune responses and analysis of the genetic diversity of the S. scabiei var. cuniculi between the QiXing and IRA rabbit breeds at the clinical, immunological and genetic levels, and revealed a differential host resistance to S. scabiei var. cuniculi between the two rabbit breeds. Intriguingly, the QiXing rabbit breed showed a relatively higher resistance to S. scabiei var. cuniculi infestation compared to the IRA rabbit breed. These results not only provide a reference for breeding of rabbits with adequately improved resistance to S. scabiei var. cuniculi in the domestic rabbit industry, but also provide reasonable foundation for further studies focusing on the differences in parasite resistance in other host species.In this study we compared the host resistance to


In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. Methods: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays. Results: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode’s solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5. Conclusions: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.Background: Previous studies demonstrated that calcium/calmodulin (Ca Reactive oxygen species (ROS) have classically been considered a harmful byproduct of oxidant metabolism, as excessive ROS leads to the progression of vascular senescence, atherosclerosis, plaque instability, and endothelial dysfunction in the vascular system ,2,3. Now2-), which is related to endothelial dysfunction and inflammatory activation i regulatory mechanism, characterized by the mobilization of Ca2+ from the endoplasmic reticulum (ER) and the subsequent Ca2+ influx from the extracellular space. Considering that sarcoplasmic reticulum Ca2+ release could activate the CaM/CaMKII pathway, as we previously demonstrated in o = 0 mM with 1 mM EGTA) or [Ca2+]o = 1 mM modified Tyrode’s solution, no difference in the effects of BK on NOX-derived ROS production was observed  (2+]o = 0 mM) for chelating of intracellular Ca2+, the effect of BK on NOX-derived ROS production was completely attenuated  . Taken together, these results indicated that ER Ca2+ contents and local Ca2+ mobilization from the ER were required to activate NOX, whereas SOCE was not involved in this phenomenon.Next, we investigated the involvement of intra- or extra-cellular Caificant) B. When B < 0.05) C,D. Furtoduction E,F  and KN-93 (a CaMKII inhibitor) on BK-induced NOX-derived ROS production. With W-7 pretreatment (100 mM), the effect of BK on NOX-derived ROS production was completely abolished    A,B. Howeificant) C,D. Thes2+/CaM-dependent protein kinases, such as CaMKI and CaMKIV, could phosphorylate NOX family members. We performed a phosphorylation assay using a Phos-tag™ SDS-PAGE Western blotting method. As shown in We finally examined whether other Ca2+]i participate in several types of cellular signaling. Changes in [Ca2+]i regulate the enzymatic activities of several sources of ROS, such as endothelial nitric oxide synthase, mitochondria, and NOX in vascular smooth muscle cells and neutrophils [2+]i, and SOCE plays a central role of this increase in [Ca2+]i in non-excitable cells. Indeed, several reports have demonstrated that an increase in [Ca2+]i via SOCE activates NOX and promotes O2- generation [2+ via Ca2+ release from the ER and investigated the role of [Ca2+]i elevation on the activation of NOX in PAECs. The major findings demonstrated that an increase in intracellular Ca2+ via ER Ca2+ mobilization (but not SOCE), which was introduced by BK stimuli, could activate NOX and it involved ROS generation. We also elucidated that the mechanism underlying this BK-induced NOX activation was through a Ca2+/CaM-dependent and phosphorylation-independent process. Our findings clarify the interplay between ER Ca2+ mobilization and regulation of NOX-derived ROS production in PAECs.Spatial and temporal increases in i  via the phospholipase-C-inositol 1,4,5-triphosphate signaling pathway [2+]i via ER Ca2+ release, followed by SOC-mediated extracellular Ca2+ influx in PAECs [2+]i in a dose-dependent manner [2+ entry for sustained activation [2+]i. Agonists of GPCR, including angiotensin II and endothelin-1, were shown to increase endothelial NOX activity i evoked by Ca2+ release from the ER.Intracellular Car spaces ,36. In etivators ,37,38,392+ signaling in the vascular system i activates the Ca2+/CaM pathway; (2) the Ca2+/CaM pathway directly activates NOX, leading to increased cytosolic ROS production; (3) Ca2+ mobilization from ER, but not store-operated Ca2+ entry, regulates CaM-mediated NOX-derived ROS production; and (4) we could not detect which NOX subtypes were activated by BK stimulation. We concluded that CaM could directly activate endothelial NOX independently from phosphorylation, and it accelerated ROS production in PAECs. This effect of CaM was strictly controlled by an elevation of [Ca2+]i evoked by ER Ca2+ mobilization. Further studies will be needed to elucidate which of the NOXs are engaged in BK-induced ROS production.The highlights of our study are: (1) BK increases Ca


Ca2+ concentration ([Ca2+]i) and changes in sarcoplasmic reticulum (SR) vesicle Ca2+ uptake and release rates.  A well-established rat model of high repetition high force (HRHF) work was used to investigate if such exposure leads to an increase in cytosolic Ca2+]i, the latter implied by around 50–100% increases in pCam, as well as in the Ca2+ handling proteins RyR1 and Casq1 accompanied by an ∼10% increased SR Ca2+ uptake rate in extensor and flexor muscles compared to those of control rats. This demonstrated a work-related altered myocellular Ca2+ regulation, SR Ca2+ handling, and SR protein expression.  Six weeks exposure of rats to HRHF increased indicators of fatigue, pain behaviors, and [Ca2+ into the SR may reflect an early adaptation to avoid a sustained detrimental increase in [Ca2+]i similar to the previous findings of deteriorated Ca2+ regulation and impaired function in fatigued human muscle. These disturbances may mirror intracellular changes in early stages of human work-related myalgic muscle. Increased uptake of Ca This is permitted through an instantaneous release of large amounts of Ca2+ through the sarcoplasmic reticulum (SR) Ca2+ release channel and ryanodine receptor (RyR), thereby increasing [Ca2+]i and a subsequent, almost simultaneous, reuptake of Ca2+ into the SR by the SR ATPases (SERCA) together with buffering of Ca2+ inside the SR by the protein calcequestrin (Casq1) [Work-related muscle pain is considered a public health problem and increases the incidence of sick leave absences in otherwise healthy individuals . Repetit (Casq1) –8.2+ regulation and SR Ca2+ release rates [2+ release rate [2+]i above normal for a prolonged time period i in HRHF rat muscles, compared to FRC rats, and adaptive compensatory changes in SR Ca2+ uptake and release rates. Since [Ca2+]i cannot be measured directly in an in vivo setting, a number of crucial and well-investigated proteins were chosen to elucidate possible Ca2+ flux rates, pCalmodulin kinase (pCam), a protein indicative of [Ca2+]i; RyR, a protein related to SR Ca2+ release; SERCA1, a protein regulating SR Ca2+ uptake; and finally Casq1, a protein buffering Ca2+ inside the SR. As pain is a perception and always self-reported, an animal model can only observe and test motor and sensory behaviors as indications of fatigue and pain. We analyzed for voluntary motor behaviors suggestive of fatigue and sensory behaviors suggestive of pain or discomfort, as well as inflammatory cytokines and Hsp72 as injury markers  was determined fluorometrically  using the fluorescent Ca2+ indicator indo-1 (1 μM). When [Ca2+] reached a plateau (Nadir Ca2+), SR Ca2+ uptake was blocked by adding Cyclopiazonic acid (40 μM) and Ca2+ release was initiated by adding 4-chloro-m-cresol (5 mM), and the fluorescence followed for at least 30 seconds. All raw data for Ca2+ release and uptake were imported into Matlab version 7.0.1  and mathematically analyzed  [2+ uptake was performed with data points between a free [Ca2+] of 800 nM and the free [Ca2+] 20 seconds prior to initiating Ca2+ release . The time for the free [Ca2+] to decrease by 63% of the initial free [Ca2+] (τ) was calculated as 1/b from the equation, y = aebt− + c, where y is the free [Ca2+], t is time and a, and b and c are constants assigned from Matlab. The onset of Ca2+ release was defined as the time when [Ca2+] increased above nadir [Ca2+]. The data points during the first 30 seconds of release were mathematically fitted to the equation, y = a [1 − eb (t−c)−]. The values obtained for SR Ca2+ release and uptake rates are expressed as arbitrary units of Ca2+ min−1 g−1 protein and the inverse rate constant τ, in seconds, respectively. Assays of uptake and release rates of Ca2+ were performed in triplicates (a few in duplicates due to limited tissue homogenate). Protein content in the muscle homogenate was measured in triplicates using a standard kit (Pierce BCA protein reagent no. 23225).Homogenates were analyzed for SR vesicle Caescribed , 46. Mus2+ uptake and release rates on region B samples from HRHF-RL and FRC-R muscles. Protein content in the tissue lysate was measured in triplicate using a standard kit . Laemmli buffer was added to lysates, the samples were heated at 90°C for three minutes, shortly vortexed, and spun in a microcentrifuge, and equal amounts (20 mg in 10 μl) were separated by SDS-PAGE  using precast 4–12% BIS TRIS NuPAGE gels  at 100 V for 55 minutes. Gels were blotted  onto polyvinylidene difluoride membranes  blocked for 30 minutes in 5% milk dissolved in TBS/0.05% Tween-20 (TBST) and incubated overnight at 4°C with primary antibodies diluted 1 : 1000 in 4% BSA in TBST with shaking: mouse anti-phospho-CaMKII (Thr286) ; mouse anti-ryanodine receptor 1 ; rabbit anti-calsequestrin-1 ; mouse anti-SERCA1 ATPase ; and rabbit anti-GAPDH . Membranes were washed in TBS/0.05% Tween  and then incubated for one hour with appropriate HRP-conjugated secondary antibodies diluted 1 : 3000. Membranes were visualized with Immun-star western kit . To conserve samples, prior to immunostaining as described above, membranes were cut into upper and lower halves and one-half stained for GAPDH (the loading control) and the other for the protein of interest, as appropriate for the molecular weight of the protein of interest. For example, the upper half of the membrane probed for Casq1 (∼60 kDa) was stripped and reprobed for SERCA (110 kDa). Images were obtained using ChemiDoc XRS  and subsequently analyzed for densitometry using Bio-Rad image lab software. All band densities were normalized to internal control samples loaded onto each gel . Normalized bands were compared to GAPDH, and these ratios were graphed for representative blots. Gels and blots were repeated until four to five different samples per group were assayed, for a minimum of 5 gels per protein of interest.Western blot analyses for pCam, RyR1, Casq1, and SERCA1 protein expressions were performed on tissue lysates also used for the analysis of SR vesicle Ca2+ vesicle uptake and release differed by group (HRHF versus FRC), location within the muscle (region A versus B), or muscle . Western blot results from the preferred reach limb muscles were assayed using two-way ANOVAs and the factors group (HRHF and FRC) and muscle. ANOVAs were followed by Tukey post hoc tests for multiple comparisons. Adjusted p values for the Tukey post hoc results of <0.05 were considered significant for all comparisons.Data are reported as mean and SEM, GraphPad PRISM v.7.0. One-way repeated measures ANOVAs were used to examine the number of reaches rewarded. Two-way repeated measures ANOVAs were used to analyze the reach rate and mean reach impulse across the three sessions, using the factors session and week. Grip strength, forelimb agility, and forepaw mechanical sensitivity results were assayed using two-way repeated measures ANOVAs, using the factors week and group, with the group data separated by limb . Spontaneous behavioral changes suggestive of discomfort tracked during task performance were compared using nonparametric Kruskal–Wallis tests, with data compared to week 1 HRHF results. ELISA cytokine results were analyzed using two-way ANOVAs and the factors group (HRHF and FRC) and limb (RL/R and SL/L) for the Flex muscles. Linear mixed models were conducted in Stata version 15.0 to determine if SR Cap=0.008), which progressively increased across weeks of task performance . There were also significant differences in mean reach impulse (grasp force × grasp time) by week (p < 0.0001) and session .We observed that HRHF rats had significant differences in the number of reaches rewarded (ANOVA p=0.006). Post hoc tests revealed that grip strength had declined in both the HRHF-RL and HRLF-SL, compared to week 0 levels  and group . Forepaw mechanical withdrawal responses showed a significant difference by week for the 0.4 g von Frey filament  and to the 0.4 g filament in week 6 in HRHF-RL forepaws, compared to FRC-R rats  compared to FRC-R and contralateral HRHF-SL , compared to FRC-L-Ext muscles. In summary, 6 weeks of muscle overuse increased the SR Ca2+ uptake rate, while the release rate was unaffected by overuse.In all SR vesicle measured in the FRC rats, there were no differences between the right and left limbs for each muscle examined. Therefore, we compared FRC-R limb results to HRHF-RL results and FRC-L to HRHF-SL results. Further, there were no differences between muscle regions A versus B for all analyses, and we therefore presented data from region B only. The SR vesicle CaFRC rats . Howeverp=0.004; . The incp=0.03), with post hoc analyses revealing higher levels in the HRHF-RL-Ext and HRHF-RL-Flex muscles compared to the same muscles in FRC-R , with higher levels in HRHF-RL-Ext and HRHF-RL-Flex muscles compared to the same muscles in FRC-R , with higher levels in the HRHF-RL-Ext muscles compared to the same muscle in FRC-R , while SERCA1 protein content only tended to be increased by HRHF (p=0.25). Further, there was a clear increase in Casq1, considered as the principal Ca2+ binding protein in the SR in Type II fibers. Thus, both the key SR Ca2+ binding and pump proteins were increased, clearly indicating an HRHF-related increased Ca2+ handling capacity.The obtained significant differences in SR vesicle Ca studied . In cont2+ homeostasis in all muscles examined was accompanied by adaptations in Ca2+ regulation shown as a faster uptake rate of [Ca2+]i in HRHF rats compared to FRC rats. The increased SR Ca2+ uptake rate may reflect an early adaptation to avoid a detrimental increase in [Ca2+]i leading to a sustained increased cytosolic [Ca2+]i above normal, similar to previous findings in fatigued muscle [2+ by the SR govern a crucial role in maintaining [Ca2+]i, and hence the SR properties are major determinants of muscle function and performance. Still, Ca2+ can leak out of the SR either through the RyRs or through the SERCA, under, e.g., metabolic stress or strenuous activity i. These disturbances may mirror intracellular changes in the early stage of musculoskeletal disorder development in the human myalgic muscle that may deteriorate the Ca2+ regulation and impair muscle function.Early-phase muscle adaptation to repetitive work, with altered Ca2+ homeostasis on a cellular basis may be due to a constant activation of the same muscle fibers i. This may be mediated by leaky RyR1 and/or uptake of extracellular Ca2+. Further, HRHF induces muscle metabolic stress as indicated by an increase in muscle Hsp72. The increased [Ca2+]i is accompanied by an increased SR vesicle Ca2+ uptake rate together with an increase in Casq1, each leading to an enhanced SR Ca2+ buffering capacity. The study offers an insight into the early events in muscle response to HRHF tasks. Such exposure induces an impaired muscle Ca2+ regulation interplay may be instrumental in early development of musculoskeletal disorders in human myalgic muscles. Implications for human work-related musculoskeletal disorders from this rat model include the importance of mechanical work exposure and the clear indication of a peripheral contribution to the pathomechanisms. Both aspects must be considered when designing effective treatment and prevention of musculoskeletal disorders.Previous results from human studies indicate that the proposed model of the Ca


The plasma metabolome is associated with multiple phenotypes and diseases. However, a systematic study investigating clinical determinants that control the metabolome has not yet been conducted. In the present study, therefore, we aimed to identify the major determinants of the plasma metabolite profile. We used ultra-high performance liquid chromatography (UHPLC) coupled to quadrupole time of flight mass spectrometry (QTOF-MS) to determine 106 metabolites in plasma samples from 2503 subjects in a cross-sectional study. We investigated the correlation structure of the metabolite profiles and generated uncorrelated metabolite factors using principal component analysis (PCA) and varimax rotation. Finally, we investigated associations between these factors and 34 clinical covariates. Our results suggest that liver function, followed by kidney function and insulin resistance show the strongest associations with the plasma metabolite profile. The association of specific phenotypes with several components may suggest multiple independent metabolic mechanisms, which is further supported by the composition of the associated factors. Metabolomics, as other “omics” such as genomics, transcriptomics and proteomics, aims for a holistic view of biology in health and disease. In contrast to the transcriptome and proteome, which change gradually, the metabolome reacts instantly upon environmental changes, or in response to disease development . Mass spThe aim of the present study was to identify the major determinants of the plasma metabolite profile. Using reversed phase ultra-high performance LC (RP-UHPLC) coupled to quadrupole time-of-flight MS (QTOF-MS), we measured plasma metabolite profiles of 2503 extensively phenotyped individuals. Our method covered a wide range of metabolite classes, including (phospho)lipids, amino acids, acylcarnitines, and bile acids. The metabolite profiles were converted into orthogonal metabolite factors and associated with 34 clinical covariates.We determined relative levels of 106 identified metabolites in plasma samples from 2503 individuals. These metabolites included amino acids, acylcarnitines, bile acids and (phospho)lipids. After removal of duplicates and metabolites that were detected in <85% of the individuals, 78 metabolites remained for further analysis. We first examined the composition of the metabolite profiles. Correlation analysis revealed several clusters of metabolites, which corresponded to distinct metabolite classes or pathways A. In parTo transform the correlated metabolites into uncorrelated factors, we analysed data using PCA. Thereby, the 78 correlated metabolites were reduced into 18 uncorrelated factors, which together explained 74.5% of the variation in the data B. Next, β).After reduction of the metabolite data into independent variables with metabolite class signatures, we investigated the association between the factors and clinical parameters. For this, we built linear models on the scaled data, and calculated standardized regression coefficients  . We usedβ = −0.056, q = 1.20e−27), triacylglycerides , and the apolipoprotein B/apolipoprotein A1 (ApoB/ApoA1)-ratio  , and additional adjustment for BMI   .β = 0.059, q = 1.60e−15) and plasma creatinine . In addition, blood pressure-related traits were associated with this component, including hypertension , and both systolic  and diastolic  blood pressure , plasma creatinine , and hypertension  remained significantly associated with component 2, as well as after additional adjustment for BMI  .Component 2 associated with measures of kidney function, including eGFR (1.26e−5) . Notablyβ = −0.065, q = 3.92e−2), fasting glucose , T2D , 120 min oral glucose tolerance test (OGTT) glucose level , and homeostasis model assessment of beta-cell function , as well as measures of obesity , β = −0.054, q = 3.17e−9) . Furthermore, HOMA-IR , fasting glucose , T2D , HOMA-B , and WHR  remained associated with component 3 after additional adjustment for BMI , FAI , and age , of which eGFR remained significantly associated with the component after adjustment for age and sex , and additional adjustment for BMI .Component 4 associated with eGFR , FAI , free estradiol , and age , and component 6 with age , FAI , and SHBG . After adjustment for sex and age, however, component 5 associated strongly with HOMA-IR , BMI , SHBG , high-density lipoprotein , apolipoprotein A1 , and WHR , and these associations remained significant after additional adjustment for BMI .Both component 5 and 6 associated with sex- and age-related parameters. Component 5 associated with sex hormone binding globulin , and this association remained significant after adjustment for sex and age , and additional adjustment for BMI .Component 7 associated strongest with TAG , which did not remain significant after adjustment for sex and age.Component 8 was only associated with eGFR , alcohol consumption , and eGFR . These associations remained after adjustment for sex and age . Both eGFR  and alcohol consumption  remained associated with this component after additional adjustment for BMI. Notably, the association of eGFR with component 9 was independent of alcohol intake .Component 9 was associated with measures of obesity  production, and also the major site of LDL catabolism . In addiMedium-chain acylcarnitines mainly contributed to component 2. This component is associated with hypertension and kidney function, as approximated by eGFR. Hypertension is a proven risk factor for CKD , and theN-phenylacetyl-l-glutamine, hippuric acid, and p-cresol. While the former have been reported to be elevated in dialysis patients and can be reduced by increased dialysis frequency [In addition to component 2, eGFR also associated strongly with components 4 and 9. Component 4 was mainly contributed by purines, suggesting an inverse association between kidney function and circulating levels of purines. Increased intake of purines has been linked to hyperuricemia and gout , which irequency , elevaterequency . AlthougInsulin is often considered the main determinant of the plasma metabolite profile . HoweverIn the unadjusted model, component 5 associated with sex, SHBG and FAI. However, after adjustment for age and sex, it was strongly associated with HOMA-IR. Notably, this component was composed of branched-chain and aromatic amino acids, which are known to be higher in males  and to aIn this study, we used RP-UHPLC/QTOF-MS, a technique that is widely applied in metabolomics and that allows the detection of metabolites from multiple pathways, which have previously been associated with the investigated phenotypes. However, other techniques, covering other parts of the metabolome, may yield different results. Hence, a limitation of the study is the bias that potentially has been introduced by the choice of technique. Consequently, the number of detected metabolites remain limited in comparison to comprehensive studies involving several orthogonal platforms. Nonetheless, we cover metabolites from multiple classes, including amino acids, lipids and acylcarnitines, all of which are involved in central metabolic processes. Another possible limitation may be the composition of the sample; as the examined sample was collected in a specific region of Sweden, the application of these results to other populations remains to be examined. Genotype, lifestyle factors, dietary habits such as caffeine consumption, and medication are likely to influence the metabolome. We were not able to account for a number of these factors, as the frequency of medication among the population was low, and as information for several of these factors were not available. Finally, we have not performed any functional studies to investigate which tissue is responsible for the metabolite profiles we determined. Studying the link between tissue function and metabolite profiles is an interesting subject for future studies.The rationale of and the methodology used in the Vara Skövde Cohort within the Skaraborg Project have been described in detail ,39,40. ILifestyle and personal history questionnaires and anthropometric data were collected at baseline. Blood samples were collected after overnight fasting and stored at −80 °C until analysis. In total, 114 clinical covariates were available. These parameters included blood pressure, measured twice in supine position, artery elasticity by pulse wave analysis, and glucose and insulin levels as determined during an oral glucose tolerance test (OGTT). Lipoprotein profiles, inflammatory markers, albumin and creatinine levels were determined at the Clinical Chemistry Laboratory, Lund University. Hypertension was defined in accordance with international expert guidelines and JNC 7 criteria  or as onAfter filtering for duplicates and variables with small variation , 44 clinical covariates remained. For duplicates, clinically determined parameters were favoured over patient-reported information.v/v) formic acid, and B, acetonitrile/isopropanol  with 0.1% (v/v) formic acid. The gradient was set to the following: 0–2 min, 2-20% B; 2–3 min, 20–40% B; 3–5 min, 40–95% B; 5–6 min, 95% B; 6–7.5 min, 95–2%; 7.5–10 min, 2% B. Analyses were performed in both positive (ESI+) and negative (ESI−) electrospray ionization mode. Metabolites were identified by MS/MS using in-house libraries and the MassHunter METLIN Metabolite PCDL . Leucine and isoleucine (leu/ile) were reported as a single metabolite, as these amino acids were not resolved with our method. All metabolites were manually integrated and confirmed in MassHunter Profinder B.06.00 Build 6.0625.0 . Only metabolites that were identified by MS/MS were used for data analysis; duplicates and metabolites with 100% missing values within a single batch were excluded. Within-batch variation was corrected for using the scores along the first principal component from PCA calculated in Simca P+ 12.0  on the un-centred and unit variance scaled internal standards [Metabolites were extracted from 40 μL blood plasma, spiked with internal standards, followed by analysis using RP-UHPLC/QTOF-MS as previously described in detail , with mitandards .β). For all analyses, significance was defined as q < 0.05 using multiple testing adjustments according to the false discovery rate method .All statistical analyses were conducted in R version 3.3.3 (2017-03-06). Metabolites were log2-transformed to approximate normal distributions. Samples were analysed over one year in batches of 150 samples. Hence, batch-to-batch variation was expected in the data. We used common variance compensation  to remove variation between batches , and useAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Skaraborg Project was approved by the local ethical review board at Gothenburg University (Ö199-01). Informed consent was obtained from all individual participants included in the study.The results in this study suggest that liver function is strongly associated with the differences in plasma metabolite profiles as determined by RP-UHPLC/QTOF-MS, followed by kidney function and glycaemic control. These results also indicate multiple independent components that are associated with the same phenotypic trait, which may suggest independent mechanisms determining the function of related organs. Whereas previous metabolomic studies have highlighted a vast number of biomarkers for various diseases, this study shows that a large number of these appear in a concerted manner. Moreover, several biomarkers previously detected in severe clinical conditions, e.g., in dialysis patients, could also be confirmed in the general population.


Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies.Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. This article has an associated First Person interview with the first author of the paper. Summary: The DMD KO rabbit engineered by CRISPR genome editing faithfully recapitulates the DMD pathologies, and could be a valuable tool for basic and translational studies to combat this disease. Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease characterized by progressive muscle weakening and wasting. DMD patients usually lose the ability to walk between 10 and 12 years of age and die of respiratory and/or cardiac failure by the age of 20-30 years . DMD affDMD gene, which encodes dystrophin protein  mice, a widely used animal model of DMD. However, the Mdx mice do not recapitulate human DMD patients in many aspects. For example, the phenotype of Mdx mice is much milder compared with that of DMD patients, with almost a normal life span . These clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout (KO) rabbits showed many features of muscular dystrophy including elevated serum creatine kinase (CK), muscle necrosis and regeneration, impaired mobility, and increased fibrosis in the heart and bladder. This novel DMD rabbit model could be a valuable resource for DMD research and preclinical studies.DMD in rabbits, we designed a pair of sgRNAs targeting exon 51, which is commonly mutated in human DMD patients  developed into the blastocyst stage, among which 78.0% carried mutations in DMD at the target sites. There were no significant differences in the developmental rate between the noninjected embryos and microinjected embryos (P>0.05). These results demonstrated that the dual sgRNA-directed CRISPR/Cas9 system is efficient for generation of mutations in the DMD gene in rabbit zygotes.In order to disrupt the open reading frame (ORF) of patients A,B. To tDMD KO rabbits, a total of 128 injected zygotes were transferred into the oviducts of four surrogate rabbits. All surrogates were pregnant to term and produced 33 live pups  newborn pups carried a DMD mutation, and 22 of them (84.6%) carried the biallelic DMD mutations. In order to examine the off-target effects in these DMD KO rabbits, the PCR products of the top ten potential off-target sites were subjected to Sanger sequencing and T7E1 cleavage assay. No off-target mutations were detected at these potential sites in the DMD KO rabbits .In order to generate ive pups . The genDMD gene in the KO rabbits, we performed quantitative reverse transcription PCR (RT-PCR) analysis of DMD expression in the gastrocnemius muscle of KO and wild-type (WT) rabbits using two sets of primers. As shown in Fig. S3, the expression of DMD was significantly reduced in the skeletal muscle of DMD KO rabbits compared with WT controls. To further examine the expression of dystrophin protein in the muscle of these rabbits, we performed immunofluorescence staining. Dystrophin was detected at the sarcolemma of skeletal muscle fibers of WT rabbits, and its expression was completely disrupted in the KO rabbits  died by 20 weeks of age  . The 6MWMovies 1 and 2).Muscle injury is a hallmark of muscular dystrophy, which releases muscle proteins such as CK into the circulation. We observed that serum CK was significantly elevated in the KO rabbits from 8 to 20 weeks of age compared with their age-matched littermate controls F. In addDMD KO rabbits, we performed Hematoxylin and Eosin (H&E) and Masson's trichrome staining of the gastrocnemius muscle sections from the rabbits at 16 and 20 weeks of age. As shown in DMD KO rabbits displayed typical muscular dystrophy signs, as evidenced by increased fiber size variation, centrally nucleated fibers, fibrosis and fatty replacement  and quadriceps  muscles. The diaphragm muscles, which are severely affected in DMD patients, mice and dogs .To further examine the histopathology of the lacement A,B. The lacement C,E, and lacement D,F. The lacement G showed  rabbits H. These and dogs , were alMdx mice, a widely used animal model for DMD, do not show obvious cardiac pathology until a year later  and fractional shortening (FS)  B,C. TherDMD rabbit model with close resemblance to its human counterpart. This new model could facilitate basic research to understand the pathogenesis of DMD, and translational studies to develop novel therapeutic strategies for this devastating disease.To the best of our knowledge, this is the first report of a The New Zealand rabbits used in this study were maintained at the Laboratory Animal Center of Jilin University. All experiments involving rabbits in this study were performed in accordance with the guide of the Animal Care and Use Committee of Jilin University.BbsI sites of pUC57-T7-sgRNA cloning vector (Addgene ID 51306) as described  and purified using a miRNeasy Mini Kit  according to the manufacturer’s instructions.The CRISPR/Cas9 sgRNA was designed and assembled as previously described . The annescribed . The vecStreptococcus pyogenes), was linearized with NotI and transcribed in vitro using a mMessage mMachine SP6 Kit (Ambion) and RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.The 3xFLAG-NLS-SpCas9-NLS vector (Addgene ID 48137) (containing Cas9 from in vitro-transcribed Cas9 mRNA (200 ng/µl) and sgRNA (50 ng/µl) was microinjected into the cytoplasm of zygotes. The injected embryos were transferred into Earle's balanced salt solution (EBSS) medium and cultured at 38.5°C in 5% CO2 for 20-30 min. Approximately 30-50 injected zygotes were then transferred into the oviducts of recipient rabbits.The microinjection procedure and embryo transfer were performed essentially the same as previously described . BrieflyDMD KO and WT rabbits was extracted from a small piece of ear tissue using a TIANamp Genomic DNA Kit  according to the manufacturer's instructions. The sgRNA target sites were amplified by PCR using primers . The PCR products were gel purified and cloned into pGM-T vector (Tiangen). A minimum of 14 positive clones were sequenced and analyzed using DNAman.The genomic DNA from The T7E1 cleavage assay was performed as described previously , 2013. Bhttp://crispr.mit.edu/). The PCR products for these potential off-target sites using the primers listed in Table S1 were subjected to T7E1 assay and Sanger sequencing.The top off-target sites were predicted using the online CRISPR Design tool developed by the Zhang group at Massachusett's Institute of Technology (DMD KO rabbits using TRNzol-A+ reagent (Tiangen), and treated with DNase I . The first-strand cDNA was synthesized using the cDNA first strand synthesis kit (Tiangen). The cDNA was used for regular RT-PCR and quantitative RT-PCR (qRT-PCR) analyses to examine the expression of DMD. The primers were used for regular RT-PCR  and qRT-PCR . qRT-PCR was performed using a BioEasy SYBR Green I Real Time PCR Kit , and the 2−ΔΔCT formula was used to analyze gene expression, Gapdh was used as a reference gene. All experiments were repeated three times for each gene. Data were expressed as the mean±s.e.m.Total RNA was isolated from the gastrocnemius muscle of WT and DMD KO rabbits were measured biweekly. All data were expressed as the mean±s.e.m., and a minimum of three individual animals of each genotype were used in all experiments.The body weight of age- and sex-matched WT and The blood samples were collected into heparinized tubes from the ear vein, and sera were prepared by precipitation and centrifugation. Serum CK, ALT and AST levels were measured using a CK test kit (N-acetyl-L-cysteine method), ALT test kit (continuous monitoring method) and AST test kit (continuous monitoring method), respectively .DMD KO and WT rabbits. The rabbits wearing the device on their right hind leg were placed in an ∼30 m2 room to allow free movement.A Millet Sports Bracelet Wearable Device was used to record movement steps within a 1-h period for DMD KO rabbits (n≥3 per group) by an SIUI all digital color doppler ultrasound diagnostic system . Rabbits were studied in right lateral recumbency from parasternal long and short axis views. The rabbits were held in the right position by restraining their limbs with people. A linear array probe and center frequency of 10.0 MHz were used. Cardiac dimensions  were determined and the percentage of FS and left ventricular EF calculated.Echocardiography recording was performed as described previously . BrieflyDMD KO and WT rabbits (euthanized at 5 and 12 months of age). The tissues were fixed in 4% paraformaldehyde at 4°C, dehydrated in increasing concentrations of ethanol , cleared in xylene and embedded in paraffin for histological examination. The 5-μm sections were cut for H&E , α-dystroglycan , α-sarcoglycan  and caveolin 3  were used. The slides were then extensively washed with PBS and incubated with secondary antibodies  for 1 h at room temperature. Finally, the glass slides were mounted using VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Then the slides were imaged with a Nikon Ti-E inverted fluorescence microscope equipped with an Andor Zyla sCMOS camera and a Nikon Super Fluor 20×/0.75 NA objective lens. Images were recorded using the NISElements Advanced Research software package (Nikon) and processed using Photoshop CS5 (Adobe).DMD KO and WT rabbits at 5 months of age were analyzed for fiber size, central nucleation and fibrosis. A minimum of three different regions were counted per section. The fiber size, percentage of central nucleated fibers and fibrotic area were calculated using ImageProPlus 6.0 software .The H&E-stained cross-sections of gastrocnemius, tibial anterior and quadriceps muscles from the t-test for two group comparisons, and one-way ANOVA with Bonferroni's post-tests for multiple group comparisons, using Prism 7.0 (GraphPad). P≤0.05 was considered significant.Data are expressed as mean±s.e.m. Statistical differences were determined by unpaired Student's


In this work, we fabricate microfluidic probes (MFPs) in a single step by stereolithographic 3D printing and benchmark their performance with standard MFPs fabricated via glass or silicon micromachining. Two research teams join forces to introduce two independent designs and fabrication protocols, using different equipment. Both strategies adopted are inexpensive and simple (they only require a stereolithography printer) and are highly customizable. Flow characterization is performed by reproducing previously published microfluidic dipolar and microfluidic quadrupolar reagent delivery profiles which are compared to the expected results from numerical simulations and scaling laws. Results show that, for most MFP applications, printer resolution artifacts have negligible impact on probe operation, reagent pattern formation, and cell staining results. Thus, any research group with a moderate resolution (≤100 µm) stereolithography printer will be able to fabricate the MFPs and use them for processing cells, or generating microfluidic concentration gradients. MFP fabrication involved glass and/or silicon micromachining, or polymer micromolding, in every previously published article on the topic. We therefore believe that 3D printed MFPs is poised to democratize this technology. We contribute to initiate this trend by making our CAD files available for the readers to test our “print & probe” approach using their own stereolithographic 3D printers. It operates under the well-known Hele-Shaw cell approximation, whereby a quasi-2D stokes flow is generated between two parallel flat plates separated by an arbitrarily small gap, and has been previously demonstrated in the microfluidic dipole (MD) and microfluidic quadrupole (MQ) configurations  is a non-contact microfluidic system that combines the concepts of hydrodynamic flow confinement (HFC)see Fig. . The tec12, such as high shear stress and tiny patterning areas, while allowing localized delivery of reagents for biological applications14. Another major advantage of the MFP is its ability to pattern large, open planar surfaces using conventional lab equipment (such as cells on Petri dishes)15.The main advantage of the MFP is that it overcomes major limitations of conventional channel-based microfluidics17. One of this barrier is the fact that MFPs cannot be easily produced on demand due to their typically complex fabrication procedures. For example, the technology was originally designed with a silicon tip and Polydimethylsiloxane (PDMS) chip-to-world connector, which requires bulk micromachining of the silicon chip, fabrication of the PDMS block using soft lithography or micro-molding techniques, alignment and assembly of the different layers, and separate machining of the probe holder3. An attempt to standardize this fabrication procedure introduced the glass-silicon hybrid vertical MFP (vMFP) concept, with a gasket-integrated probe holder for a more compact world-to-chip interface4. This technique has been demonstrated to be very effective in fabricating MFPs with all fluidic apertures placed along a straight line, and is currently one of the most commonly adopted. However, its procedure requires costly microfabrication facilities, implies wafer bonding steps, is difficult to implement for arbitrary aperture arrangements, and results in long prototyping cycles. The 3D printing technology, which is already positively disrupting the development cycles of conventional microfluidic devices19, is poised to overcome these challenges. Not only does it afford a relatively seamless connectivity of several parts, it also offers a straightforward, simple, rapid, inexpensive, and yet robust methodology to manufacture these devices on demand , as shown in Fig. ing Fig. . The proThe second MFP  aims at maximum design simplicity with a commercial 27 µm resolution stereolithography printer . There is no built-in reservoir in the MFP, and the fluidic connections are simply made by plugging commonly available 1/32” tubes into the probe and gluing them in place. The result minimizes probe size within the limits imposed by the printer Fig. . SpecialIn this design, the MFP and its adapter form a slider Fig. . This alAkin to classical MFPs, these 3D printed MFPs are connected to micro-positioners and goniometers, that can be controlled in one or more axes, to control the MFP-to-substrate gap and angle of inclination respectively Fig. . The MFPThe MD and MQ are produced using the 3D printed MFPs from both designs. The profiles were investigated as functions of MFP’s aperture Spacing to Diameter ratio (S/D), aspiration to injection flow rates ratio . Close inspection reveals that the location of the stagnation point was matched with a relative error of approximately 2%. This might be due to experimental inaccuracies such as an imperfect tip surface or small angle between the MFP and the bottom substrate. The analytical model also underestimates the HFC size by approximately 25 µm on all borders. This relatively small error (<5%) is explainable by the fact that the analytical solution considers point source approximation (and thus a S/D ≫ 1). For a similar S/D ratio than the one used in this experiment (S/D = 5.5), error of the same magnitude have been previously reported21. Also, the analytical model doesn’t consider the diffusion at the HFC interfaces. However, diffusion affects our experimental measurement and the threshold we used to determine the experimental HFC envelope might slightly overestimate it.Comparison between numerical simulations and experiments for the MQ profiles Fig.  shows thα of 5, an increasing HFC footprint was obtained when S/D is varied from 1.5 to 4  of the culture with a solution of CellTracker® Green fluorescence dye (10 μM concentration), using a MFP-substrate gap of 60 µm. As expected, an increase in staining intensity is observed as the exposure duration of the cells to the HFC (of the CellTracker solution) is increased from 15–120 s , are relatively inexpensive via 3D mail-order printing services, and are potentially as reusable as Si-based MFPs. This approach allowed for the integration of new components like inbuilt reservoirs, convenient MFP probe holders, and using commercially available tubing connectors for a straight forward connectivity and minimization of dead volume–that would have been otherwise complicated to achieve with conventional MFP microfabrication techniques. Moreover, this work demonstrated how 3D printing brings scalability and flexibility to the MFP in terms of producing different designs, different numbers of apertures, different aperture configurations, and different fluidic connectors. Using two independent MFP designs, proof-of-concepts were presented in the form of developing MDs, MQs, and selective labeling of adherent live cells within their culture medium in a “writing” mode. The MD and MQ profiles produced by the 3D printed MFPs closely matched numerical and analytical calculations – the same way micromachined MFPs do – thus confirming similarities in their accuracies. Furthermore, cell staining applications were demonstrated, which validate the applicability of the 3D printed MFPs in performing bioassays.10. (2) The printed MFPs contained some unavoidable channel artifacts – one design more than the other – that had minor effect on the accuracy of the produced profiles. However, with 3D printing technology currently undergoing intense development to bring down costs and increase resolution, these limits are expected to be continuously pushed back as new generations of 3D printers come to the market. In the case an MFP is required for an application that requires a single cell resolution, the same technology discussed here can be adapted to more advanced 3D printers capable of printing 3D nanostructures, such as those involving direct laser writing and two-photon polymerization technologies25. (3) Biocompatibility of the polymers and resins used in SLA is still a concern for applications where cells are directly cultured on the device26. However, in the case of MFPs, cells are cultured in Petri dishes, or microtiter plates, and are never in contact with the 3D printed device, which limit the biocompatibility problem. Furthermore, research on new post-processing and sterilization techniques, and on biocompatible resin formulation, have been demonstrated to reduce protein adsorption and improve long-term cell viability after experiments with 3D printed materials29.The use of affordable 3D printing in this field, however, comes with some drawbacks: (1) the current resolution of the affordable 3D printers limits the minimum aperture sizes of MFPs. However, this didn’t affect the capability of presented MFPs to target cluster of tens of cells and generate concentration gradients at resolutions similar to the original MQThe HFC concept behind the MD and MQ have numerous potential applications, which could become even more accessible to the research community with this new fabrication strategy, lessons learned from our experiments, and the presented proof of its practical applicability. By demonstrating the simplicity and customizability of 3D printed MFPs, we believe that the technology will help spread the concept outside microfabrication laboratories and make its potential and versatility as an open reagent delivery tool directly available to life-science laboratories. To support this assertion, all CAD and stl files used to print the above designs are made available  for any stereolithographic 3D printer owner to test.: Designs were performed using a commercial CAD software (SolidWorks) and the MFPs were printed using a commercially available stereolithographic 3D printer . The printer has a labeled XY resolution of 82 µm, and Z resolution of 25 µm but our parametric study with this specific MFP design showed that the minimum diameter resolution for repeatable 3D channels is 250 µm using a polymeric resin . After printing, the chip was washed with ethanol, placed in a UV flood light curing system for 2 min and then washed again with DI water. The MFP holder was separately 3D printed .Design 1Design 2: Designs were made using Catia v5  while abiding by two rules to minimize the risk of aperture clogging: 1) the length of apertures was kept the smallest possible  and 2) aperture size was kept over 6 printer’s pixel size (160 µm). The MFPs were printed using an Asiga freeform Pico with a resolution of 27 µm . The resins used were PlasCLEAR V2 and PlasGRAY V2 (Asiga). MFPs were briefly soaked in an isopropanol bath to remove uncured resin. To thoroughly clean the microchannels, the MFPs were then put in an isopropanol bath in a sonicator  for 20 minutes. Thereafter, MFPs were post-cured with UV light for 15 minutes to fully harden . MFPs were designed to fit widely available 1/16″ O.D. tubing. Tygon tubes  were plugged in the MFP and glued with cyanoacrylate glue. To insure no glue would enter inside the MFP apertures and tubing, each tube was filled with water during this step. MFP tips were manually polished using fiber optic lapping sheets . Sheet with 5 µm, 3 µm, 1 µm and 0.3 µm grits were used consecutively. Water was injected with the MFP’s apertures after each polishing steps to make sure no polishing residues would clog the apertures. MFPs made with clear resin were coated with black ink  using an airbrush. A layer of aerosol clear coat  was then sprayed on the tip.20H10Na2O5) . For the MQ, a solution of Rhodamine B (C28H31ClN2O3) (Sigma Aldrich) was injected through the second injection aperture. Injection and aspiration were driven using the MFCS®-EZ microfluidic pressure pump  or by the neMESYS high precision syringe pump  depending on the design used. Images of the profiles created by fluorescein and rhodamine B solutions were captured using their respective microscope’s fluorescent filter cubes, and then combined using Image J (Design 1)  and MATLAB (Design 2). For Design 2 MQ, a background image was subtracted on both fluorescein and Rhodamine B channels before they were merged in a single image. The background used corresponds to an image of the MFP with injection or aspiration turned off. To deduct the diffusion region from the overall profile, we used the “Minimum Threshold” function in ImageJ, which was derived from the thresholding technique adapted in reference30. Profiles were then traced out using a plot digitizer (GNU Library). For the dipole characterization, the experiment was repeated with at least 3 different MFPs per profile to test reproducibility. The dimensions for each interval was averaged to produce the final plot with standard error estimates used as the error bars.To characterize the MD and MQ, the MFP was positioned above a Petri dish or a microscope slide with custom walls (depending on the design) located on the stage of an inverted microscope. Upon aligning the MFP with the objective of the microscope, the Z stage  is used to slowly approach the substrate. When the MFP contacts the substrate, it is then lifted by the required gap-height to produce the Hele-Shaw two parallel plates configuration. The Design 1 MFPs were visualized using the Epi-fluorescence inverted ECLIPSE Ti microscope , and images were captured using the DS-Qi2 camera (NIKON). Design 2 MFPs were visualized and imaged using the epi-fluorescence inverted microscope Axio Observer.Z1  and the LaVision imager sCMOS camera . MD images were taken by injecting a solution of dissolved green fluorescein sodium salt  was used on the numerical simulation result.The 3D numerical models of the MD and MQ were developed using finite element simulations . The solutions were obtained using a direct and stationary solver to compute the Navier-Stokes equation  coupled with the convection-diffusion equations. MFPs were modeled as circular surfaces with inlets and outlets that correspond to the apertures. Dimensions used were those of the designed MFPs. Water was considered incompressible with a density of 998.2 kg/m21:x/S and 2y/S respectively  diluted in PBS buffer with a final concentration of 10 μM/mL. The solution was then mixed with fluorescein sodium salt  for visualizing the HFC using the microscope. The solution was then loaded into the MFP’s reservoir – by suction through the aperture of the MFP – and the MFP was positioned above the cell culture with a MFP-substrate gap of 60 µm. Monitoring of the cells’ exposure time was performed using a stopwatch. The calligraphic labeling was performed by programming (C#) the XY stage movements in the Zaber scripting environment. The spacing between each letter was achieved by rapid movement of the stages (~29 mm/s), from the end of one letter to the beginning of the other letter .HeLa cells  were cultured in sterile Petri Dishes (Thermo Scientific) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 0.2 mM L-glutamine  at 37 °C and 5% COMFPs used with cells were sterilized by soaking in 99% ethanol for 24 h. Washing with DI water, and curing, were then repeated and followed by soaking in DMEM for 12 h. Before preparing cell dishes used for the cell viability experiment, marks were randomly made at the bottom of the culture plates to ensure traceability of the target region. Cell viability was estimated by applying NucGreen™ Dead Cell Indicator (2 drops/mL of cell medium) to the cell culture dish already exposed to the HFC from the MFP. As control, a cell dish prepared with the same cell confluence as the processed dish, was kept in the incubator for the duration of the experiment and also processed with the NucGreen™ Dead Cell Indicator (Thermo Scientific) after 24 h. Counting of cells to evaluate death rate , was done manually using the ImageJ “Multi-Point” feature.Supplementary Figures and MaterialsAutomated cell labeling 1Automated cell labeling2


Emerging evidence has shown that mitochondrial pathways are potential therapeutic targets for cancers. Mitochondria are the central organelles for energy production, but also play a pivotal role in lipid and amino acid metabolism, redox and calcium homeostasis, transcription regulation, as well as cell fate determination. Previously we and others have demonstrated that disruption of mitochondrial function selectively targets acute myeloid leukemia (AML) cells ,2. IdentIn our recent publication in Cell Stem Cell , we utilWe interrogated the function of TAZ in AML through a series of functional assays. Loss of TAZ reduced proliferation, supressed clonogenic growth and induced differentiation in AML cells. Of note, knocking down TAZ in mice did not affect normal hematopoiesis, suggesting a specific dependence of AML cells on TAZ.As TAZ is a key enzyme for phospholipid biosynthesis, we examined the phospholipid profile of AML cells upon TAZ knockdown. In addition to the expected decrease in mature cardiolipin, we noticed increased levels of phosphatidylserine (PS) and decreased phosphatidylethanolamine (PE). Further study showed that supplementing TAZ knockdown cells with PE did not rescue the loss in proliferation. In contrast, supplementation of PS mimicked the effects of TAZ knockdown and decreased AML growth and stemness.Furthermore, phosphatidylserine decarboxylase (PISD), a mitochondrial enzyme that converts PS to PE, was downregulated at post-transcriptional level following TAZ knockdown. By lipid-protein overlay assay, we found that PISD recombinant protein bound cardiolipin and the cardiolipin moiety phosphatidylglycerol. CRISPR/Cas9-mediated knockout and chemical inhibition of PISD recapitulated the effects of knockdown of TAZ. Taken together, we discovered that TAZ modulates PISD activity to control intracellular levels of PS, which in turn regulates AML stemness and differentiation.Mechanically, we discovered that TAZ knockdown or increasing levels of PS up-regulated genes in the Toll-Like Receptor (TLR) pathway. We also showed that activation of the TLR pathways was functionally important to induce AML differentiation. Finally we used preclinical animal models to demonstrate that PISD inhibitor, MMV007285, reduced AML disease burden and targeted AML stem cells without toxicity.in vitro and less leukemic engraftment in vivo. We also discovered that intracellular PS inhibited AML growth and stemness by activating the TLR pathway. This finding implicates a novel function of PS in regulating cell growth and stemness. It also drives further studies on how PS mediates the TLR activation and the roles of inflammatory pathways in AML differentiation.This work provides new insights into the roles of TAZ in AML, and highlights the important role of PS in maintaining AML stemness. PS is an essential phospholipid which is normally restricted to the inner leaflet of the cell membrane. Previous studies on PS focused on the translocation of PS to the cell surface. For example, the externalization of PS has been considered as a hallmark of apoptosis . ExposedAt the therapeutic level, this paper proposes a novel mechanism to target leukemia stem cells (LSCs), which are the subpopulation of cells responsible for AML progression and relapse. Previous studies have shown that LSCs display unique metabolic properties, including relatively low levels of reactive oxygen species , sensitiIn summary , we disc


This study examines the early signaling effects of [10]-gingerol on human colorectal cancer cells. It was found that this compound caused a slow and sustained rise of [Ca2+]i in a concentration-dependent manner. [10]-Gingerol also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 38%. In a Ca2+-free medium, the [10]-gingerol-induced [Ca2+]i rise was partially abolished by depleting stored Ca2+ with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). The elevation of [10]-gingerol-caused [Ca2+]i in a Ca2+-containing medium was not affected by modulation of protein kinase C activity. The [10]-gingerol-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers. At concentrations of 10-100 μM, [10]-gingerol killed cells in a concentration-dependent manner. These findings suggest that [10]-gingerol induces [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from non-L-type Ca2+ channels in SW480 cancer cells. The effect of [10]-gingerol on cytosol free Ca Zingiber officinale L. Zingiberaceae) is a common condiment for various foods and beverages. Ginger also has a long history of use in traditional medicine. The underground stems or rhizomes of this plant have been used as a medicine in East Asian, Indian, and Arabic herbal traditions since ancient times [in vitro and in vivo in experiments [Ginger -5-hydroxy-3- decanone], the major pungent principle of ginger, has potential anti-inflammatory, antioxidant, anticarcinogenic, and antimutagenic activities ,9,10. EvG1 phase . This coal cells . In the al cells  and the gingerol . However2+ signaling and cytotoxicity in human colon cancer SW480 cells has not been explored. Colorectal cancer is the third most frequent and second most lethal in the United States -gingerol induced a significant and prolonged [Ca2+]i increase and cytotoxicity in human colorectal cancer cells. The concentration-response relationship, the Ca2+ sources of the Ca2+ signal, and the role of protein kinase C in the signal have been investigated. The effect of [10]-gingerol on Cad States . Therefo2+]i in a concentration- dependent manner in the presence of extracellular Ca2+. 2+]i rise induced by 5-25 μM [10]-gingerol. [10]-Gingerol at concentrations between 5-25 μM increased [Ca2+]i . The [Ca2+]i rise induced by 5-25 μM [10]-gingerol comprised an immediate rise and a sustained phase in 250 sec. At a concentration of 25 μM, the [Ca2+]i rise had a net value of 752nM at 250 sec. At a concentration of 0.1 μM, [10]-gingerol had no effect on [Ca2+ entry and Ca2+ release from stores in the [10]-gingerol response. 2+ largely suppressed the [10]-gingerol-induced [Ca2+]i elevation. The concentration-response relationship of [10]-gingerol-induced [Ca2+]i rise in the presence and absence of extracellular Ca2+ is shown in 2+ removal inhibited the [Ca2+]i rise caused by 25 μM [10]-gingerol by 38% as the maximum value .Experiments were performed to evaluate the relative contribution of extracellular Ca2+ store in the majority of cells i rise that comprised an initial increase and a gradual decay toward baseline. The net maximum [Ca2+]i value was 96±4 nM (n = 5). After depleting the endoplasmic reticulum Ca2+ store with thapsigargin, addition of 25 μM [10]-gingerol did not induce a [Ca2+]i rise, as shown in 2+]i rise with a net value of 72±3 nM (n = 5), which was 25% (p < 0.05), smaller than the control thapsigargin response shown in Previous reports have shown that the endoplasmic reticulum is a major Caof cells . Figure 2+ entry, the effects of several L-type Ca2+ entry blockers on [10]-gingerol-induced [Ca2+]i rise were evaluated. 2+-containing medium, pretreatment with 1 μM nicardipine did not inhibited 25 μM [10]-gingerol-induced [Ca2+]i elevation . Similarly, the Ca2+ influx was not affected by 1 μM diltiazem, nifedipine, or verapamil (n = 5).To explore the pathways underlying [10]-gingerol-induced Ca2+ signal could be modulated by the activity of protein kinase C [2+]i elevation was investigated. 2+]i elevation was not altered by pretreatment with 10 nM phorbol myristate acetate  or 2 μM GF109203 X (a protein kinase C inhibitor) (n = 5).A Cakinase C ,18. The 2+)i response, and unregulated (Ca2+)i rises can be linked to cytotoxicity [Given the acute incubation with [10]-gingerol caused a substantial and lasting , 13C (100 MHz), DEPT, HETCOR, COSY, NOESY, and HMBC NMR spectra were obtained on a Unity Plus Varian NMR spectrometer. LRFABMS and LREIMS were obtained with a JEOL JMS-SX/SX 102A mass spectrometer or a Quattro GC-MS spectrometer with a direct inlet system. Silica gel 60  was used for column chromatography. Precoated silica gel plates  were used for analytical TLC, and precoated silica gel plates  were used for preparative TLC. Spots were detected by spraying with 50% H2SO4 and then heating on a hot plate. Optical rotations were measured with a JASCO DIP-370 digital polarimeter. UV spectra were obtained in MeCN using a JASCO V-530 spectrophotometer. The IR spectra were measured on a Hitachi 260-30 spectrophotometer. Zingiber officinale (ginger) were purchased from a local market of Kaohsiung in Taiwan in July 2006, which was identified by Dr. Yen-Ray Hsui of the Division of Silviculture, Taiwan Forestry Research Institute, Taipei, Taiwan. A voucher specimen (Hsui-Zo-1) was deposited at Fooyin University.The roots of Z. officinale were chipped and air-dried and extracted repeatedly with CHCl3 at room temperature. The combined CHCl3 extracts were then evaporated further separated into 20 fractions by column chromatography on silica gel with gradients of n-hexane/CHCl3. Fr. 9 eluted with CHCl3-MeOH (50:1) was next repeatedly subjected to silica gel CC and yielded [10]-gingerol (210 mg) i was calculated as described previously assuming a dK of 155 nM [2+, cells were bathed in Ca2+-free medium in which CaCl2 (2 mM) was substituted with 0.1 mM EGTA. Trypsinized cells (10Cell viability was determined by the trypan blue exclusion (TBE) method . Brieflyp value of less than 0.05 was considered as statistically significant.Data is presented as means ± standard deviation (SD) and analyzed using one-way ANOVA with Scheffe′s test. A


Cell adhesion is a process through which cells interact with and attach to neighboring cells or matrix using specialized surface cell adhesion molecules (AMs). Adhesion plays an important role in normal haematopoiesis and in acute myeloid leukaemia (AML). AML blasts express many of the AMs identified on normal haematopoietic precursors. Differential expression of AMs between normal haematopoietic cells and leukaemic blasts has been documented to a variable extent, likely reflecting the heterogeneity of the disease. AMs govern a variety of processes within the bone marrow (BM), such as migration, homing, and quiescence. AML blasts home to the BM, as the AM-mediated interaction with the niche protects them from chemotherapeutic agents. On the contrary, they detach from the niches and move from the BM into the peripheral blood to colonize other sites, i.e., the spleen and liver, possibly in a process that is reminiscent of epithelial-to-mesenchymal-transition in metastatic solid cancers. The expression of AMs has a prognostic impact and there are ongoing efforts to therapeutically target adhesion in the fight against leukaemia. In multicellular organisms, cells adhere to each other to form tissues, organs, and systems. For such a high degree of organization, it is essential to establish junctions between cells as well as between cells and extracellular matrix (ECM). Cell-to-cell and cell-to-matrix adhesions are the result of the interaction between ECM components , membrane-associated adhesion molecules (AMs) and the anchored cytoskeleton. These connections not only maintain a strict tissue structure, but also play a role in cell migration, differentiation, and communication. In normal tissues, AMs expression is tightly regulated. However, aberrant expression of AMs occurs during disease and in malignant transformation.Any of numerous specialized trans-membrane molecules on the cell surface that bring about adhesion by binding to molecules on the surface of other cells or to ECM are defined as AMs. There are four main families of AMs: integrins, selectins, cadherins, and the superfamily of immunoglobulins (IgSF). Integrins are heterodimeric trans-membrane glycoproteins consisting of an α and a β subunit, assembled in different ways to generate a wide range of dimers (reviewed in ). Each tDeregulation of adhesion is considered a hallmark of metastatic solid tumors that seemed to be less of an issue in acute myeloid leukaemia (AML). AML is a genetically-heterogeneous group of multi-cause malignancies  in whichNot all of the known AMs are discussed here. Instead, we zoom in on the molecules relevant to AML, as attested to by the available literature. Thus, we review the deregulation of adhesion in AML, focusing on the altered expression pattern and signaling from AMs, the functional consequences, prognosis and the possibilities of devising targeted therapies against aberrant interactions that may help to increase the cure rates for AML patients.HSCs express a host of AMs, as detailed in Other types of AMs expressed by HSCs include sialomucins such as CD34, CD45R, CD43, CD162 , and CD164 , as wellThe expression pattern of AMs present on normal HSCs and AML blasts, including LSCs, is largely similar. The differential expression includes the presence of VLA-1, -2, -3, and 6 that are usually absent on normal haematopoietic cells . LSCs alHSCs are not randomly spaced within the BM microenvironment, but are positioned in and interact with discrete units referred to as niches, which determine the fate of HSC. The niche, i.e., the coupling of haemopoietic, stromal cells, and ECM, is a functional element that permits HSCs to expand or adopt a quiescent phenotype protecting their integrity and properties . There aAdhesion to the niche is critical to AML pathogenesis and progression. LSCs require a disease-promoting BM microenvironment that plays a role in disease initiation as it may transform HSCs . SeveralOther than adhesion, AML cells are also regulated by soluble factors secreted by niche cells, such as SDF-1 or the CC ligand 2 (CCL2) chemokine. In normal haematopoiesis, the CXCR4-SDF1 system regulates leukocyte trafficking and homing . ChemothLSCs are capable of remodeling the niche to a self-reinforcing leukaemic niche ,52. NichMoreover, positive feedback loops mediated by disease can cause inflammatory overexpression of AMs on activated endothelial cells . AML celWhereas transplanted HSCs and progenitors injected into the bloodstream home to sites of haematopoiesis, they can be mobilized from these niches into the blood through either physiologically or pharmacologically-induced mechanisms A. SialomCytokines such as stem cell factor, chemokines such as SDF-1 and IL-8, and proteolytic enzymes such as the metalloprotease superfamily are also involved in migration and homing. HCELL is of particular relevance in these processes: It is the most potent ligand for E and L-selectins that regulate cell movement (cell rolling) and promote weak HSC adhesion to BM vessels . The expSimilarly to normal HSCs, LSCs home to the BM and they exit it B. LSC hoDislodging AML blasts from the niche is a way of increasing their chemosensitivity and improving the efficacy of anti-AML therapies and will be discussed in more detail in the section of targeted therapies.In addition to their structural roles in anchorage, AMs transmit signals into the cells in response to the adhesive state, cell’s location, and surrounding ECM. Such signaling is involved in tumor development and progression. Thus, accumulating evidence indicates that integrins are implicated in leukaemia signaling. The binding of the integrin tail to adaptor proteins such as SRC kinase, focal adhesion kinase (FAK), or integrin-linked kinase (ILK) leads to the activation of different signal transduction pathways including mitogen activated protein kinase cascades, phosphoinositide-3-kinase (PI3K)/AKT kinase and protein kinase C ,66. ILK Little is known about selectins’ signaling in AML, albeit Levesque and colleagues demonstrated that VE-selectin confers protection from chemotherapy by activation of pro-survival NF-κB signaling. Interestingly, this activation was not observed upon adhesion to P-selectin, CD31 or VCAM-1 . MoreoveCadherins’ cytoplasmic domain directly binds β-catenin, which in turn is attached to α-catenin that serves as a link to actin cytoskeleton. Some evidence has suggested that cadherins can influence Wnt signaling pathway by competing for the pool of β-catenin . MoreoveThe t translocation, which gives rise to the AML1/ETO oncogenic fusion, is among the most common rearrangements found in AML. We recently reported that AML1/ETO expression reduces adhesion and enhances migration of haemopoietic cells in vitro . Such a In such a context, EMT could be responsible for (or contribute to) not only the motility, but also for the differentiation block and stemness induction functions of AML-associated oncogenes. If such a hypothesis were true, EMT could be seen as a universal step needed for every cancer to develop, and thus a common hub to be targeted for cancer therapy.The serum levels of chemokine or adhesion receptors represent prognostic factors for AML patients with a clear impact on OS and relapse. In particular, expression levels of VLA-4 and CXCR4 have been associated with patients’ outcome. Several independent studies showed that a high level of expression of CXCR4 predicts low rates of OS and event-free survival, while a low level correlates with increased OS, relapse free survival and complete remission (CR) rate ,90,91,92High level of integrin β3 expression is associated with shorter OS of AML patients, especially in cases with FLT3-ITD mutations. The expression of integrin β3 was higher in the poor risk group than in the favorable and intermediate groups .A prognostic value of other AMs, e.g., FAK and protein tyrosine kinase-2 (PYK2), has been assessed . AML patInterestingly, important EMT markers seem to have prognostic relevance. In particular, a poor clinical outcome is linked to elevated mRNA expression of vimentin. Analysis of mRNA expression data from 173 AML patients from the cancer genome atlas dataset, suggested that patients older than 60 years, with a normal karyotype and high vimentin expression have poor clinical outcome . SimilarCurrently, no therapeutic strategies for AML patients perform better than conventional chemotherapy. Sadly, the outcome remains poor and the standard of cure represents an option that is not always applicable as many patients are unfit for intensive regimes ,103. ThuThe AM-ligand interactions harnessed in AML therapy so far include: VLA-4 with VCAM-1, VLA-4 and CD44 and E-selectin, E-selectin ligand-1 and E-selectin, as well as the integrin/CD44 interaction with osteopontin, a glycoprotein of the ECM.AS101 is an agent against VLA-4 targeting the fibronectin-bound form. Based upon in vivo studies, AS101 inhibits the PI3K/Akt signaling pathway and acts as chemosensitizer of chemoresistant cells . An ongoInhibition of E-selectin employing a specific small molecule, GMI-1271, augments the effect of chemotherapeutic agents and decreases tumor burden in xenograft mouse models . A curreGiven CXCR4/CXCL12 involvement in homing, quiescence and proliferation of AML blasts  it has lAn alternative approach for targeting the CXCR4/CXCR12 axis is via the inhibition of CXCR12. Recently, a study revealed that NOX-A12, a CXCR12 inhibitor, induces chemosensitisation and interferes with migration of chronic lymphocytic leukaemia (CLL) cells . It is bAn additional factor that impairs the effect of chemotherapy is the hypoxic BM microenvironment and the resulting hypoxia-inducible factor-1α transcription factor induction which in turn upregulates the expression of AMs  or CXCROther strategies aimed at increasing mobilization and response to drug treatment are under investigation. Indeed, targeting the kinases involved in the phosphorylation of CXCR4  with small-molecule inhibitors may decrease the retention of AML cells in the BM niche .Adhesion plays an important role in the physiology and pathology of tissue homeostasis. Several of the findings in the adhesion field are surprising and may be non-instinctive: AML is more “solid” than it seems, adhesion loss is not equal to cancer spreading and EMT plays a role in the progression of a non-epithelial tumor. For years, the notion that loss of adhesion leads to cancer metastasis has been considered a paradigm. An ever-growing body of evidence calls for paradigm shift by suggesting that the opposite may be true . Indeed,Current research explores new aspects, such as the mechanism by which physical forces and local pliability regulate adhesion and it seems that the signals they produce are of particular interest in tumor biology . The rolOverall, much progress has been made; however, some questions still await an answer and only a small number of therapeutic agents have emerged. It is certain that there is a great potential for devising novel adhesion-related diagnostic, prognostic, and therapeutic tools. For example, syndecan-1 that is expressed on AML cells, is a therapeutic target under investigation in multiple myeloma , while t