We, therefore, successively blocked NKCC1, KCC2, and GABAA  or activated GABAA (with isoguvacine) either during or after oxygen-glucose deprivation (OGD). Primary hippocampal cultures were exposed to a 2-h OGD or sham normoxia treatment, and viability was determined using the resazurin assay. Neuronal viability was significantly reduced after OGD, and was further decreased by DIOA treatment applied during OGD (p < 0.01) and by gabazine applied after OGD (p < 0.05). Bumetanide treatment during OGD increased viability (p < 0.05), while isoguvacine applied either during or after OGD did not influence viability. Our data suggests that NKCC1 and KCC2 function has an important impact on neuronal viability during the acute ischemic episode, while the GABAA receptor plays a role during the subsequent recovery period. These findings suggest that pharmacological modulation of transmembrane chloride transport could be a promising approach during stroke and highlight the importance of the timing of treatment application in relation to ischemia-reoxygenation.Neuronal ischemia results in chloride gradient alterations which impact the excitatory–inhibitory balance, volume regulation, and neuronal survival. Thus, the Na A) receptors or chloride transporters i, which in turn sets the strength and polarity of the GABAergic transmission in the brain. During development, KCC2 expression is upregulated, while NKCC1 levels are decreasing, leading to a lower [Cl−]i. KCC2 mRNA expression in the rat brain is minimal at birth and increases in early postnatal life, changes that are accompanied by a reduction in NKCC1 expression and are similar to those observed in the developing human brain [−]i. Thus, KCC2 mRNA and protein levels are significantly downregulated in rats after ischemia i could be associated with higher cellular viability. We have previously shown that chloride transport modulation in immature hippocampal cultures has a neuroprotective effect both in vitro during OGD i plays in both volume regulation and excitatory–inhibitory balance, we postulated that modulating [Cl−]i by blocking its transmembrane transport would influence cellular viability with an inversely proportional relation. Our results suggest a complex, timing-dependent effect of treatment on cellular viability.In the present study, we aimed to examine the effect of specific blocking of NKCC1 (bumetanide) , KCC2 (Rabazine)  as well Primary hippocampal cell cultures were obtained from Wistar rat pups at postnatal day zero (P0), following a protocol previously described . All anil-glutamine , and antibiotic-antimycotic (Invitrogen). The cell suspension was plated in 24-well plates coated with poly-d-lysine  at a density of 150,000 cells/well. The plates were placed in a humidified 5% CO2 incubator at 37 °C. After 5 days in vitro (DIV), the cultures were fed by removing half of the CM volume in each well (250 µL) and replacing it with 400 µL newly prepared CM.Briefly, the hippocampi were dissected, and the meninges and vascular plexuses were removed. The tissue was subjected to mechanical trituration and enzymatic digestion using papain , and the cell suspension obtained was kept in culture medium (CM) containing Neurobasal-A medium (Invitrogen) supplemented with B-27 (Invitrogen), A receptor activation from excitatory to inhibitory i either during OGD (T1) or post-exposure (T2). We also used the GABAA receptor antagonist gabazine  as well as the GABAA receptor agonist isoguvacine  to modulate the GABAA channel, both during OGD (T1) and during post-exposure (T2). All treatments were also applied to normoxic cultures. Experimental design and treatment times are illustrated in We blocked outward ClThe data was analyzed using Prism (GraphPad 7 Software). Data is presented as percentages normalized to the control values (normoxia + no treatment condition). The statistical unit was the number of wells tested per condition (n). The viability of cultures after different OGD treatments was compared to that of OGD without treatment from the same cultures. This results in slightly different OGD viabilities across treatment conditions. However, the degree of metabolic lesion was severe in all cultures, ranging from 42.5% to 59.6% (average 52.25%). All variables were tested for normality of distribution using the Shapiro–Wilk test and central tendencies are consequently reported as mean ± SD. Statistical significance was evaluated using ordinary two-way analysis of variance (ANOVA) with factors of oxygenation  and treatment  and Tukey’s multiple comparisons test, with α set at 0.05.n = 23, six cultures) as measured by the resazurin assay. Application of DIOA further decreased cellular viability when applied during OGD , but had no effect when applied post-exposure . Cellular viability of normoxia cultures treated with DIOA at T1 and T2 was 100.8% ± 7.2%  and 105.1% ± 4.8% , respectively (p < 0.0001) and DIOA treatment  = 3.283, p = 0.0407) as well as a significant OGD–DIOA interaction  = 5.851, p = 0.0037). After Tukey’s multiple comparisons, we found a significant difference between OGD and OGD + DIOA T1 , p = 0.0019), but no significant difference between OGD and OGD + DIOA T2 or between any of the normoxic treatment conditions.Our 2 h OGD protocol decreased neuronal viability to 57.3% ± 12.6%  to 49.6% ± 9.4%  when applied during OGD (Bumetanide T1). However, bumetanide administered post-exposure had no effect on cellular viability   and 104.7% ± 2.6% , respectively. There was a significant main effect of OGD  = 935.7, p < 0.0001) and bumetanide treatment  = 3.635, p = 0.0306) but no significant OGD–bumetanide interaction  = 0.4370, p = 0.6474). After Tukey’s multiple comparisons, we found a significant difference between OGD and OGD + Bumetanide T1 , p = 0.0251), but no significant difference between OGD and OGD + Bumetanide T2 or between any of the normoxic treatment conditions.Treatment with bumetanide, an NKCC1 blocker, resulted in an increase of cellular viability from 42.5% ± 8.9% (nide T2) . ViabiliA blocker, did not affect cellular viability when administered during OGD . However, gabazine treatment post-exposure decreased cellular viability from 49.6% ± 20.7%  to 35.5% ± 12.1%   and 100.1% ± 1.8% , respectively (p < 0.0001), but no significant effect of gabazine  = 2.423, p = 0.092) and no significant OGD–gabazine interaction  = 2.016, p = 0.136). However, Tukey’s multiple comparison test reports a significant difference between both OGD and OGD + Gabazine T2 , p = 0.0078) and OGD + Gabazine T1 and OGD + Gabazine T2 , p = 0.0281). There was no significant difference between OGD and OGD + Gabazine T1 or between any of the normoxic treatment conditions.Gabazine, a GABAzine T2) . Viabiliectively . After tn = 11, two cultures) and slightly lower at T1 and T2 after isoguvacine treatment . Viability of sham wells, which were kept for 2 h in normoxia and treated with isoguvacine at T1 and T2, was 100.6% ± 2.2  and 102.4% ± 4.7, , respectively (p < 0.0001), but no significant effect of isoguvacine  = 0.4831, p = 0.6209) and no significant OGD–isoguvacine interaction  = 1.579, p = 0.2204). After Tukey’s multiple comparisons, we report no significant difference between OGD and OGD with administration of isoguvacine treatment at T1 or T2 or between any of the normoxic treatment conditions.Cellular viability after OGD exposure with no treatment was 59.6%  in several in vitro models: cultured cortical neurons [−]i has been directly linked to neuronal death pertaining to ischemic episodes i to allow Cl− influx via ligand-gated Cl− channels, and buffering of external K+ concentration i, which causes the inactivation of GABAA receptors and subsequently prevents Cl− influx during OGD i following OGD [− outflow, as the Cl− equilibrium potential (ECl) is markedly shifted towards positive values [Cl exceeds the membrane potential for spiking, which only occurs in severe ischemic insults [in vitro models of ischemia report neuroprotective effects of GABAergic drugs [Additionally, our findings indicate that blocking GABAwing OGD ,22,35,36e values ,56,57,58 insults . Thus, mic drugs ,60,61. OA receptor is quite common in stroke patients and it is only sensible to ask whether this pharmacological manipulation could potentially have any detrimental effects on neuronal viability in this setting, especially since literature data are controversial regarding this matter [A receptor activation with isoguvacine on cellular viability of neurons during either OGD or post-exposure.Inducing sedation by enhancing the activity of the GABAs matter ,63,64. IA receptor plays a key role in the recovery period that follows. Our findings highlight a temporal widow for pharmacological modulation of chloride membrane transport as a promising neuroprotective strategy.In conclusion, we showed that the timing of treatment application in relation to the moment of the ischemia-reoxygenation is of paramount importance: the function of chloride co-transporters NKCC1 and KCC2 has a decisive impact on neuronal viability during the acute ischemic episode, while the GABA


Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. −/−;Six5−/−Six4 fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of −/−;Six5−/−Six4 embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in −/−;Six5−/−Six4 embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in −/−;Six5−/−Six4 embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes  Summary: The homeobox genes Six4 and Six5 are involved in the regulation of cell proliferation and mesothelium formation in the primary body wall, and −/−;Six5−/−Six4 mice are a suitable animal model for human middle-type omphalocele. IGF2 by abnormal imprinting causes Beckwith-Wiedemann syndrome, which is characterized by a large body, large internal organs and severe omphalocele  defect theory and the secondary abdominal wall defect theory (see reviews by Tfap2α (previously known as AP-2α) and Pitx2 mutants show severe ventral body wall closure defects such as thoracoabdominoschisis and omphalocele  and cell repulsion . HoweverSix4/AREC3 and Six5, which are categorized as Six4/Six5 subfamily members  mice exhibited phenotypes that resembled human middle-type omphalocele, including both small and large omphaloceles. We analyzed the morphological defects, cell proliferation and apoptosis in the PAW, and properties of coelomic epithelial cells (CECs) in −/−;Six5−/−Six4 embryos. From the results, we propose that the regulation of cell proliferation and morphological change in the PAW at an early stage is a basis for omphalocele phenotype, and that −/−;Six5−/−Six4 mice are a suitable animal model for reproducing human middle-type omphalocele.In this study, we focus on  members ,b of Six members . We foun−/−Six4, −/−Six5 and +/−;Six5−/−Six4 mice are viable, and developmental abnormalities in these mice have not been reported 18.5 in the expected Mendelian ratio (Table S1). As we found various types of ventral body wall closure defects in fetuses analyzed at E18.5, we classified them into three types: large omphalocele with the protrusion of the liver and intestine, small omphalocele with the protrusion of the intestine, and enlarged umbilical ring with anteriorly elongated shape of the umbilical ring compared with normal fetuses (Table S2). A small number of −/−Six4 fetuses exhibited an enlarged umbilical ring at E18.5 (Table S2), but others showed no abnormalities in the ventral body wall . All of the −/−;Six5−/−Six4 fetuses exhibited small or large omphaloceles at E18.5  with complete penetrance. These results indicate that Six4 and Six5 together are indispensable for ventral body wall closure in mice and that Six4 is the primary player between the two.reported . SIX4 hareported ,b, suggeand Six5 . Howeverody wall E,E′. Altral body I,I′, a shalocele J,J′. Remat E18.5 F-H′ . −/−Six5 and +/−;Six5−/−Six4 fetuses had normal skeletal structure . In −/−;Six5−/−Six4 fetuses, the numbers of ribs and sternums were normal, but formation of the ensiform cartilage was impaired . These results indicate that the defects in −/−;Six5−/−Six4 fetuses are mostly confined to the abdominal body wall.Patients with omphalocele with severe protrusion of the liver and intestine often show other malformations at the thoracic and lumbar levels . Therefo−/−;Six5−/−Six4 mice during abdominal wall formation.Previous studies have shown that abnormal formation of the secondary abdominal wall, including abdominal muscle differentiation, may be the cause of the large omphalocele phenotype in humans  and in m−/−;Six5−/−Six4 fetuses with large omphalocele and −/−Six5 or +/−;Six5+/−Six4 fetuses, which have no apparent defects in ventral body wall closure, at E15.5 and E18.5 . The abdominal wall was closed at the umbilical cord level and it completely covered internal organs at the level anterior to the umbilical cord in −/−Six5 fetuses . The herniated livers and intestines in −/−;Six5−/−Six4 fetuses were covered with the peritoneum and amnion at the umbilical cord level . The anterior abdominal wall was completely lost in −/−;Six5−/−Six4 fetuses .We compared ventral body wall formation in −/−;Six5−/−Six4 fetuses in a pattern similar to that observed in −/−Six5 fetuses at E18.5 . The epaxial extensor muscle  and panniculus carnosus muscle (PC) were normally positioned in −/−;Six5−/−Six4 fetuses and −/−Six5 fetuses . All abdominal muscles were separated and positioned in the ventral wall of −/−;Six5−/−Six4 embryos in a pattern similar to that in control +/−;Six5+/−Six4 embryos at E15.5 . Previous studies have shown that SIX family proteins are involved in skeletal muscle differentiation by activating the expression of muscle regulatory factors MYOG and MYOD1 . In addition, MYOG and MYOD1 were expressed in abdominal muscle cells in +/−;Six5+/−Six4 and −/−;Six5−/−Six4 embryos with similar patterns, except the downregulation in the PC at E13.5 . These results suggest that there are no severe defects in abdominal muscle differentiation in −/−;Six5−/−Six4 embryos. Our results indicate that omphalocele formation in −/−;Six5−/−Six4 mice is not attributable to impaired abdominal muscle differentiation and clearly suggest the other causes of large omphalocele.Myoblasts start to migrate into the PAW at E10.5 and differentiate into four types of hypaxial muscles, specifically the transversus abdominis muscle (TA), internal oblique muscle (IO), external oblique muscle (EO) and rectus abdominis muscle (RA), by E13.5 . These mnd MYOD1 . MYOG wa−/−;Six5−/−Six4 embryos, defects in PAW formation have been considered as the cause of omphalocele. Therefore, we next examined the effects of Six4 deficiency in a Six5-null background on PAW formation along the anterior-posterior axis in comparison with the phenotypes of control (−/−Six5) and −/−;Six5−/−Six4 embryos at E13.0  was formed in the region posterior to the thoracic diaphragm    but thicker than that of −/−Six5 embryos at the 50% position of the AnPAW   but thicker than that of −/−Six5 embryos at the 50% position of the LaPAW    but similar at the 50% position of the PoPAW  to that in −/−Six5 embryos  B-D in cost, n=3) A,C,D. Thst, n=3) E-F″,K,L.st, n=3) G-H″,M,N.s n=3/3, G,H. The st, n=3) G,H,M,O.  embryos I-J″,P,Q.−/−;Six5−/−Six4 embryos (n=3) with patterns similar to wild-type and −/−Six5 embryos at E12.5 . These results suggest that umbilical ring formation is impaired in −/−;Six5−/−Six4 embryos before the protrusion of the liver.To analyze the detailed three-dimensional structure of the umbilical region, we performed micro-CT scanning analysis, which is a powerful method to identify structural phenotypes in mutant mice even at the early stages  Fig. 3)Fig. 3). −/−;Six5−/−Six4 embryos at the stage before E12.5. Physiological herniation and looping of the midgut were observed in wild-type and −/−Six5 embryos , and −/−;Six5−/−Six4 embryos showed similar patterns to wild-type and −/−Six5 embryos at E11.5 . Remarkably, bending of the PAW was missing at the distal levels in −/−;Six5−/−Six4 embryos at E11.5 . The overlay images of SIX4 and SIX5 immunofluorescence indicated that individual PAW cells showed the differential expression levels of SIX4 and SIX5 . As SIX1, another member of the Six family protein, is known to compensate for the roles of SIX4 in other tissues, we examined possible contribution of SIX1 in PAW development. However, SIX1 was not detected in the PAW at E10.75 . Consistent with protein localization, Six4 mRNA was completely absent in the PAW  and Six5 mRNA was expressed in the PAW at E11.5 and E12.5 . These results suggest that the expressions of Six4 and Six5 are differentially regulated in the PAW and they cooperatively function in the PAW at a stage before E11.5.The expression of reported , but thetibodies  at E10.0tibodies . SIX4 anand SIX5 C,F. In cand SIX5 G-I. ExprAs formation of the umbilical ring was impaired before the migration of muscle progenitor cells into the PAW, we next hypothesized that impaired cell proliferation in the PAW may cause abnormal PAW morphogenesis. To examine the cell proliferation rate in the PAW at the anterior to middle levels, we labeled proliferating cells with 5-ethynyl-2′-deoxyuridine (EdU) at E10.5 and counted the numbers of EdU-positive cells after 30 min within a 250 μm area from the lateral somitic frontier of the PAW M.Fig. 5P=0.22, Student's t-test, n=3)      C,D,M,N, st, n=3) C-F,M,N. st, n=3) C,D,M,N. st, n=3) C,E,M,N. st, n=3) D,F,M,N, P=0.17, Student's t-test, n=3)  than those in wild-type embryos  , resulting in the disappearance of the left-right asymmetrical ratio   A,C,D,O.  embryos B,E,F,O. st, n=3) A,C,D,O. st, n=3) B,E,F,O. Six4 and Six5 enhances cell death frequency in the PAW      G,I,J,P. st, n=3) H,K,L,P. st, n=3) G,I,J,P. st, n=3) H,K,L,P c−/−;Six5−/−Six4 embryos at E11.5 Six4−/−;SFig. S5H,I) but not at E9.25 and E9.5 . Laminin was accumulated with a fragmented distribution at the right and left sides of the PAW in wild-type embryos at E10.5  and mesenchyme was observed at E10.25  or Six4-expression vector (pCAGIG-flag-Six4). In the control vector introduction, 64.9±3.1%, 17.9±2.1% and 17.2±2.4% of EGFP-positive cells were detected in the CE, in the mesenchymal region adjacent to the laminin-positive basement membranes and in the mesenchymal region, respectively  but increased the ratio in the mesenchymal region adjacent to the laminin-positive basement membranes  and mesenchymal region   Q. These Table S3). However, in most of these mutant mice, anomalies other than omphalocele, such as neural tube defects, cleft palate and limb malformation, were observed but defects in the PAW have not been examined (summarized in Table S3). In the present study, we found that −/−;Six5−/−Six4 mice show severe ventral body wall closure defects with few other anomalies, they have defects in PAW formation, and that omphalocele formation is in complete penetrance (Table S2). Therefore, we emphasize that −/−;Six5−/−Six4 mice are a suitable animal model for large and small middle-type omphalocele reproducing typical human omphalocele.Various gene mutations cause body wall closure defects in mice  and, to −/−;Six5−/−Six4 embryos than in −/−Six5 embryos at the 0% position at E13.0 when the liver was partially protruded , the thinner abdominal wall in −/−;Six5−/−Six4 embryos was thought to be attributable to defects in the PAW before E11.5. Indeed, we detected the early occurrence of structural anomalies in −/−;Six5−/−Six4 embryos through high-resolution micro-CT analysis. The umbilical ring of −/−;Six5−/−Six4 embryos was larger than that of wild-type embryos before myoblast migration, suggesting that the initial change in ventral body wall closure had already occurred at E11.5 , analyses of −/−;Six5−/−Six4 mice first revealed that the impaired morphogenesis of the PAW may lead to middle-type omphalocele.Notably, the phenotypes of previously identified mouse mutants with ventral body wall closure defects have not been fully compared with omphalocele in newborn humans. Our analyses revealed that the AnPAW, LaPAW and PoPAW were thinner in  the PAW , Fig. S7at E11.5 . The PAW−/−;Six5−/−Six4 embryos at E10.5 before the onset of morphological change of the PAW. We observed the differential cell proliferation rates between the left and right somatic mesoderm-derived cells in the PAW of wild-type embryos at E10.5 , genes which are involved in the regulation of EMT in cultured cell lines . PCR-based genotyping was performed as previously described . escribed . Six4+/−escribed . Midday For Hematoxylin staining, E18.5 fetuses were embedded in paraffin and cut into 8-μm sections using a microtome . For Hematoxylin or Hematoxylin & Eosin staining for fetuses, frozen sections were prepared using cryostats . For skeletal staining, fetuses were fixed in 4% paraformaldehyde (PFA)/phosphate buffered saline (PBS) at 4°C for a week, and then the skin, mesenchyme and internal organs were removed. Fetuses were dehydrated in ethanol and incubated in acetone for 1 day. After incubation in ethanol, fetuses were incubated in Alcian Blue solution  at 37°C overnight and hydrated. Fetuses were incubated in 0.5% trypsin diluted with sodium borate solution for 6 h at 37°C and subsequently incubated in Alizarin Red S (Sigma) diluted with 0.5% KOH at room temperature overnight. Stained fetuses were washed with 0.5% KOH and treated with glycerol.−/−Six5: AnPAW level, 13 sections; LaPAW level, 14-17 sections; PoPAW level, nine sections. −/−;Six5−/Six4−: AnPAW level, 12 sections; LaPAW level, 20 sections; PoPAW level, six sections) were stained with anti-laminin antibody and DAPI. The distance of the PAW between the midline or distal position of the PAW and the RA was measured using cellSens software (Olympus), then the midline , half and proximal positions were defined as the 0%, 50% and 100% positions, respectively.The immunostaining of frozen sections was performed as described previously . Mouse ewww.osirix-viewer.com) and 3D Slicer (www.slicer.org) software.Imaging procedures were performed as previously reported . EmbryosIn situ hybridization was performed on 12 μm frozen sections as described previously  were used for hybridization.eviously . DIG-laband Six5  and Pitx−/−;Six5−/−Six4 embryos were used to obtain counts of EdU-positive cells and TUNEL-positive cells in the left and right PAW regions.A cell proliferation assay using EdU was performed as previously reported . To labepCAGIG-flag-Six4 was generated by inserting Flag-tagged mouse Six4 cDNA into multi-cloning sites of pCAGIG, which contain the CAG promoter and IRES-EGFP  was injected into the coelomic cavity of the right side in E11.0 mouse embryos using a fine glass needle . For quantification, EGFP-positive cells were counted in the total of 20 or 21 sections obtained from three embryos in control and Six4 experiments, respectively. The embryos were electroporated in a 20 mm×20 mm electro-chamber using an electroporator   and further cultured for 16 h for analyses.Whole-embryo culture and electroporation were performed according to a protocol described previously for rat embryos . Embryo CAX-EGFP  or pCAGIImages were taken using microscopes  equipped with a CCD-camera , a confocal laser microscope , a virtual slide microscope  and a microscope  equipped with an HD-color camera  and a recorder .


Asclepias linaria Cav. (Apocynaceae) is a shrubby plant endemic of Mexico which has been used in traditional medicine. However, the bioactive potential of this plant remains unexplored. In this study, the phenolic composition, antioxidant, and cytotoxic activities of A. linaria leaves were determined. In order to estimate the phenolic composition of the leaves, the total phenolic, flavonoid, and condensed tannins contents were determined. Furthermore, the antioxidant activity was measured by the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS•+) radicals and the total antioxidant capacity. The phenolic compounds identified in the A. linaria leaves by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) include phenolic acids, such as p-coumaric and ferulic acid, as well as flavonoids, such as rutin and quercetin. The leaves’ extracts of A. linaria showed a high scavenging activity of DPPH• and ABTS•+ radicals , high total antioxidant capacity values , and had a cytotoxic effect against K562 and HL60 hematologic neoplasia cells lines, but no toxicity towards the normal mononuclear cell line was observed. These results highlight the potential of A. linaria and could be considered as a possible alternative source of anticancer compounds. Asclepias (milkweeds) with about 150 species, distributed in North and South America  (ABTS•+), potassium persulfate, ammonium molybdate, ascorbic acid, sodium carbonate, aluminum chloride, formic acid, methanol, and water (HPLC grade) were purchased from Sigma-Aldrich . Nylon membranes  were used for ultra-performance liquid chromatography coupled to mass spectrometry (UPLC–MS) analysis. Roswell Park Memorial Institute culture medium (RPMI-1640), fetal bovine serum (FBS), streptomycin/penicillin, fluorescence-activated cell sorting (FACS) Flow cytometry solution, 4′,6′-diamino-2-phenylindole (DAPI), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), amphotericin B, and dimethylsulfoxide (DMSO) were purchased from Gibco . Ficoll-paque Plus was purchased from GE Healthcare Bio-Sciences . Endothelial basal medium-2 (EBM-2) was purchased from Cambrex . Parthenolide was purchased from Abcam . Turk solution and trypan blue were purchased from Hycel . Imatinib was purchased from SelleckChem .The Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl  . Standards were prepared as stock solutions in methanol. Working solutions of standards were obtained by diluting the stock solution in methanol to obtain concentrations ranging from 0.25–5 µg/mL. Working solutions of standards were stored in dark conditions at −18 °C.A. linaria specimens were collected during the months of September and October 2017 at Fray Dominguez, Pajuacarán, Mexican state of Michoacán . The specimens were authenticated by Dr. Monserrat Vázquez Sánchez, from Colegio de Postgraduados, Campus Montecillo, México. Voucher herbarium specimens were deposited at the herbarium of CIIDIR-IPN Unidad Michoacán, with herbarium number MVS29. Seven populations were collected. Leaves were separated from the plants and dried in an oven at 50 °C for 48 h. Then, the leaves were ground in a mortar until a fine powder was obtained. Subsequently, a sieve (number 60) was used in order to homogenize the particle size to 250 µm. Samples were stored at room temperature in dark conditions until they were used. v/v) by agitation at 100 rpm using a shaking table in the dark at room temperature for 24 h. Subsequently, the extracts were centrifuged at 2722× g for 10 min at room temperature. The supernatant was recovered and filtered through Whatman No. 1 filter paper (pore size 11 µm)  to form the crude extract. Aliquots of the extract were taken for the phenolic content and antioxidant determinations.The extraction of phenolic compounds was carried out using 1 g of dry milled tissue which was macerated in 20 mL of solvent  Na2CO3 solution was added and kept up in dark conditions for 2 h at room temperature. Absorbance was read at 760 nm using a PowerWave HT Microplate Spectrophotometer . Total phenolic contents were estimated using a gallic acid standard curve , obtained using seven known concentrations (0.04–0.46 mg/mL) of the compound. Total phenolic content was expressed as milligrams of gallic acid equivalents per gram of dry tissue (mg GAE/g DT).The determination of total phenolic contents of the samples was carried out using the Folin–Ciocalteu reagent method with modifications . Two hun3 method previously reported, with slight modifications  − 0.0036, 2R = 0.9845) was generated with seven concentrations of ascorbic acid (0.03–0.3 mg/mL). Total antioxidant capacity values were expressed as milligrams of ascorbic acid equivalents per gram of dry tissue (AAE/g DT).The total antioxidant capacity was evaluated using the phosphomolybdenum method . In thisg for 30 min at room temperature. NMC were isolated and washed twice with endothelial basal medium-2 (EBM-2) supplemented with 3% FBS, 10 ng/mL streptomycin/penicillin, and 0.25 μg/mL amphotericin B (complete EGM-2 medium). The number of viable nucleated cells was determined in a Neubauer chamber  using Turk solution as a diluent and trypan blue, respectively. All cell lines were grown in RPMI-1640 culture medium added with 10% FBS and 1% antibiotic (streptomycin/penicillin) in 5% CO2 atmosphere at 37 °C [K562 and HL60 hematologic neoplasia cells lines, purchased from American Type Culture Collection (ATCC), and normal mononuclear cells (NMC), obtained from a healthy individual were used. NMC were obtained according to the previously described methodology . One hunat 37 °C .A. linaria extracts, the cell lines (2 × 105) were plated in 24 well plates and exposed in vitro with different concentrations of dry extracts  diluted in 0.1% (v/v) DMSO for 48 h at 37 °C. Subsequently, the cells were washed using 1 mL of FACS Flow cytometry solution, and centrifuged at a speed of 423× g for 6 min at 19 °C. Then, the supernatant was decanted and 200 µL of FACS cytometry solution and 200 µL of DAPI fluorescent label (2 µL/mL) were added. After that, the cells were incubated for 15 min at the same conditions previously mentioned. Finally, the viability was evaluated in a BD FACSVerse flow cytometer . In all cases, a negative control (without extract) and a positive control (with 2.5 µM Imatinib) were used. The value of the median inhibitory concentration (IC50) was calculated using the FlowJo software version 10 .In order to determine the cytotoxic effect of the To measure the selective cytotoxicity of the extracts against cancerous cells and the safety of the extracts towards normal cells, the selectivity index (SI) was determined. Therefore, the extracts were tested for cytotoxicity in the NMC cell line. The SI was calculated using the following equation:50 value ≤ 30 µg/mL of cytotoxic activity. Consequently, 9 extracts that showed the highest potential were analyzed.The reactive oxygen species (ROS) assay was performed exclusively on the HL60 cell line. A discrimination filter was applied to select the extracts that were analyzed by the ROS assay. The filter consisted of identifying those extracts that had a SI ≥ 5, a moderate to high concentration of phenolic compounds (7 to 10 mg GAE/g DT), moderate to high concentration of flavonoid compounds (2.5 to 4 mg CE/g DT), and those with an IC50 value) of each of the selected extracts after discrimination, obtained in the cell viability and proliferation test against the HL60 cell line. First, the cells were stained with 2 µL of 20 µM DCFDA reagent which oxidizes and fluoresces when it reacts with ROS and incubated for 30 min at 37 °C. Then, the cells were exposed in the absence (negative control) or presence  to the extracts at two different evaluation times: 1 and 3 h. Parthenolide at 7.5 µM (dissolved in DMSO) was used as a positive control. Subsequently, the production of ROS was evaluated with a BD FACSVerse flow cytometry . Finally, the data obtained from the flow cytometry were analyzed with the FlowJo software version 10  and expressed as median fluorescence intensity (MFI).The ROS assay consisted of applying the extract concentration  to normalize the data. Dunnett’s test was used to assess the statistical significance of differences between test and control data (p < 0.05 and p < 0.01). Relationships between all determinations were tested using Pearson’s correlation. These analyses were carried out using SPSS software version 25.0 . All determinations were performed in triplicate. Results are reported as the mean ± standard deviation of three independent replicates. Results were subjected to an analysis of variance (ANOVA). Differences between values with a A. linaria leaves were determined. The phenolic composition of A. linaria leaves showed that the main compounds were flavonoids and phenolic acids. The recognized antioxidant flavonoids, rutin, and quercetin were identified in A. linaria leaves. In addition, the leaves extracts of A. linaria showed a high radical scavenging activity, high total antioxidant capacity values, and had cytotoxic effect against K562 and HL60 hematologic neoplasia cells lines, but no toxicity towards the normal mononuclear cell line was observed. It can be concluded that the A. linaria species has the potential to be a source of extraction of phenolic compounds with relevant antioxidant and cytotoxic activities, with applications in multiple research fields.The phenolic composition, antioxidant and cytotoxic activities of


Chromosomal abnormalities are diagnostic and prognostic key factors in acute myeloid leukemia (AML) patients, as they play a central role for risk stratification algorithms. High hyperdiploidy (HH), a rare cytogenetic abnormality seen commonly in elder male AML patients, is normally categorized under AML with complex karyotype (CK). Accordingly, patients with HH generally are associated with low remission rates and a short overall survival.EVI1/PRDM16 gene fusion, and monoallelic loss of tumor suppressor gene TP53. After successful chemotherapeutic treatment the patient experienced a relapse to AML-M1, and she developed secondary AML-M6 with tetraploidy and HH. Unfortunately, the young woman died 8.5 months after initial diagnosis.Here we report a case of 21-year-old female, diagnosed with a de novo AML-M1 according to WHO classification and a CK at diagnosis. Cytogenetic, molecular cytogenetic approaches (standard fluorescence in situ hybridization (FISH), array-proven multicolor banding (aMCB)) and high resolution array comparative genomic hybridization (aCGH) analyses revealed a unique complex but still near diploid karyotype involving eleven chromosomes was identified. It included pentasomy 4, three yet unreported chromosomal aberrations t, t, and t, and a combination of two cytogenetic events, yet unreported to appear in together, i.e. a reciprocal translocation t leading to TP53 at diagnosis, and HH in secondary AML were not previously reported. Thus, the combination of the here seen chromosomal aberrations in adult primary AML seems to indicate for an adverse prognosis.To the best of our knowledge, a comparable adult AML associated with such a CK, coexistence of 3q rearrangements with loss of  It is well established, that acquired chromosomal rearrangements play a central role in risk stratification of the disease –4. AccorApproximately 10 to 15% of AML patients had a CK , 4, 6, 7MLL translocations) can confidently be assigned to the adverse risk group, whereas those with numerical changes only, should be classified into the intermediate risk group , anemia (Hgb 9.6 g/dl) and thrombocytopenia (Plt 17 × 109/l). Now the patient stopped the treatment on her own request for 1 month. Afterwards she suffered from fever (more than 40 °C for more than 3 days), menorrhagia and blurred vision in the right eye. Approximately 8.5 months after initial diagnosis she died in her house and no autopsy was performed. Her husband agreed with scientific evaluation of her case and the study was approved by the ethical committee of the Atomic Energy Commission, Damascus, Syria.Ten days later, the patient relapsed; her PB shows cytopenia [WBC 1.5 × 10Conventional cytogenetics analysis on unstimulated BM sample according to standard procedures was performed  prior anETV6 break apart probe and a specific probe for 17p13 (TP53)  were applied according to manufacturer’s instructions. Array-proven multicolor banding (aMCB) probes sets for chromosomes 1, 2, 3, 8, 10, 11, 12 and 17 were used [48–50,X,- X,der(1)t,der(1)t1;3),+ 4,+ 4,+ 4,+ 6,t,t10;12),dic× 2  ,+ 4,dic48–50,X,-X,der(1)t,der(1)t,der(2)(:1p36.21- > 1p35::2p22- > 2qter),+ 4,+ 4,+ 4,+ 6,der(8)t,der(10)t,del(10)(q21q21),dic,del(15)q14q14),del(15)(q21.1q21.1),del(15)(q22.32q24)del(17)(q12q12) [q14,del; CNAs involving variable numbers of genes (e.g. deletion on 17q21.3); CNAs involving large parts of chromosomal p or q arms (e.g. duplication of 3q26.1q29) and CNAs of whole chromosomes  was positive for CD45To the best of our knowledge we report here the first case of a patient with an AML-M1 relapsing with a secondary AML-M6. In AML-M1 the patient presented a CK involving eleven chromosomes and yet unreported acquired chromosomal aberrations, while in AML-M6 a completely different, two-clonal karyotype with tetraploidy and HH was observed.According to the literature, HH (≥49 chromosomes) and tetraploidy (4n = 92 chromosomes) has been reported to date in 15 and 99, respectively, of 18,334 AML cases listed in Mitelman database [Gains of chromosomes, in particular tetrasomies 4, 8, 13, 14, 20 and 21, as well as pentasomies 13, 21 and 22, have been observed in AML rarely. However, there was no influence on survival observed according to the number or types of trisomies or tetrasomies . Also, tIn general, HH and tetraploidy appears infrequently in AML; it seen primarily in de novo disease in older male patients > 60 years) with low remission rates and short overall survival (OS) , 10. Unf0 years wTP53, ETV6, BRCA1 genes and or gain of copy numbers for EVI1  gene. TP53 gene mutation is observed in approximately 5–10% of all AML cases, occurring frequently in elderly subjects and cases with FAB classification M6, as well as in cases with CK; it is associated with unfavorable prognosis [EVI1 gene occurs in approximately 6–8% of AML cases and has been associated with poor treatment outcome [EVI1 gene maps to chromosomal band 3q26.2 and was first identified to be aberrantly upregulated in almost all AML cases with t [EVI1 locus at 3q26 is translocated to PRDM16  at 1p36, being highly homologous to EVI1 (PRDM3) [Concerning aberrations observed in the present case there was specifically in AML-M1 monoallelic losses for rognosis . Aberran outcome , 23. The1;q26.2)  or inv. , 25. In TP53 deletion and translocation t involving EVI1 gene, but also by HH. ICE therapy might have been helpful here, however, due to interrupted treatment this cannot be finally assessed.According to the literature the here observed, we report the first AML-M1 case relapsing to a completely independent biclonal secondary AML-M6 case. Adverse outcome of the case may be partially caused by adverse mutations in AML-M1 like


To introduce the technique and investigate the clinical efficacy of transcatheter arterial chemoembolization (TACE) in combination with simultaneous DynaCT-guided radiofrequency ablation (RFA) in the treatment of solitary large hepatocellular carcinomas (HCCs) .Forty-six patients who received TACE combined with simultaneous DynaCT-guided RFA for solitary large HCCs between January 2012 and August 2016 were reviewed, and the success rate, safety, local tumor progression (LTP), and overall survival (OS) were retrospectively investigated. OS and time to progression were analyzed with the Kaplan–Meier method.Technical success rate was 100%, average operative time for DynaCT-guided RFA was 45.3 ± 4.8 min, average radiation dose was 730.5 ± 78.8 mGy, and no life-threatening complications were observed. At 1-month follow-up enhanced MRI, complete remission was achieved in 82.6% of patients (38/46), and partial remission in 17.4% (8/46). The median follow-up period was 29.5 months (interquartile range 4.0–69.0 months). At 1, 2, and 3 years after surgery, the LTP rates were 4.3, 13.1, and 30.4%, respectively, and the OS rates were 89.1, 71.7, and 56.5%, respectively.DynaCT-guided TACE + RFA is safe and feasible for the treatment of solitary large HCCS. TACE combined with simultaneous RFA provides a new treatment option for solitary large HCCs in which DynaCT has important clinical value. Compared with other countries, large HCCs  have a higher incidence in Asia, especially in China. This is primarily due to the large Chinese population, poor economic conditions, and limited awareness of proper treatment. Large HCCs are often identified at initial diagnosis . The spaFor unresectable large HCCs, especially solitary large HCCs, transcatheter arterial chemoembolization (TACE) has become the primary treatments –6. Of noAll patients were diagnosed with HCC by imaging or pathological examination and received TACE with simultaneous DynaCT-guided RFA. A total of 46 patients received treatment for solitary large HCCs in our hospital between January 2012 and August 2016. There were 32 males and 14 females  (Table The inclusion criteria were as follows: (1) Imaging or pathological examination confirmed unresectable solitary HCCs and the maximal tumor diameter was > 5 cm; (2) the tumor selected for ablation was ≥ 1 cm away from the important organs and tissues ; (3) the Child–Pugh grade of liver function was A and B or BCLC grade was A and B, and the Karnofsky score was  >  70; and (4) patients received initial treatment.The exclusion criteria were as follows: (1) patients were unsuitable for interventional treatment due to other serious diseases ; (2) patients were sensitive to iodine and femoral puncture was infeasible; (3) patients had large arteriovenous fistula in the liver, involvement of the bile duct, tumor thrombus within the portal vein, and extrahepatic metastasis; (4) the expected survival time was shorter than 3 months; and (5) patients had poor lung function and the duration of breath holding was shorter than 8 s even after breathless training.Artis zee DSA , Syngo Workplace workstation , and radiofrequency therapeutic apparatus  were used in this study. A radio frequency of 460 kHz was chosen, and a RITA multipolar radiofrequency ablation electrode needle (outer tube diameter: 14G) with nine hook-shaped bundle electrodes (umbrella-like opening with the diameter of 5 cm) was used.After routine skin sterilization, local anesthesia with lidocaine was administered. Modified Seldinger catheter was used for femoral puncture, and a Simon I 4F catheter was employed for celiac artery and superior mesenteric artery angiography. If necessary, selective hepatic artery angiography was performed.The artery supplying the tumor was determined by DSA. Then, a 2.6 F microcatheter was used for superselective catheterization of the supplying artery, followed by chemoembolization Fig. a. The enA DR scanning interval of 8 s was used for DynaCT, and the angle of the C-arm and location of the treatment bed were adjusted for the calibration of anterior–posterior and lateral X-rays. DynaCT of the abdomen was performed to include the area of lesion. The following parameters were used: rotation angle of C-arm: 200°; rate of image capture: 50 frames/s; increment in each frame: 0.5°; X-ray dose: 0.36 μGy/frame; total time: 8 s; and total number of frames: 396. Patients were asked to hold their breath during image acquisition, and the images were sent to the Syngo Workplace workstation for reconstruction of the three-dimensional volumetric images as well as axial, sagittal, and coronal images.1) Using the DynaCT workstation interface, the width and location of the windows were adjusted to clearly display the lesion. iGuide mode was selected, and a cross was used to localize the lesion in axial, sagittal, and coronal planes. The inner puncture target spot was selected, and the surface puncture was marked, according to the puncture routine automatically selected by the iGuide software  developed by the American Association for the Study of Liver Diseases (AASLD) : Completx ± s). OS rates and LTP rates were estimated using the Kaplan–Meier method. A P value < 0.05(two-tailed) was considered statistically significant.Statistical analysis was performed using the SPSS version 24.0 . Quantitative data were expressed as mean ± standard deviation , lidocaine in normal saline was injected to separate the lesion from surrounding tissues, followed by puncture. The technical success rate was 100%. The average duration of DynaCT-guided RFA was 45.3 ± 4.8 min, and the average radiation dose was 730.5 ± 78.8 mGy Table .Table 2OForty-six patients were followed up from the initial treatment to October 2017. The average duration of follow-up was 29.8 ± 17.3 months (range 4–69 months). At 1 month after surgery, the enhanced MRI was performed, and the therapeutic efficacy was assessed using m-RECIST criteria. The results showed a CR in 82.6% (38/46) of patients, and a PR in 17.4% (8/46) with a clinical efficacy rate of 100%. After initial treatment, ten patients received a second TACE or RFA during follow-up, of whom CR was achieved in seven patients . During the follow-up period, the LTP was observed in 25 of 46 patients (54.3%). LTP was found in 14 patients (30.4%). At 1, 2, and 3 years after treatment, the LTP rates were 4.3% (2/46), 13.1% (6/46), and 30.4% (14/46), respectively. During the follow-up period, 17 patients died from tumor progression, two died from gastrointestinal bleeding, and one died from hepatic encephalopathy. One, 2, and 3 years OS rates were 89.1% (41/46), 71.7% (33/46), and 56.5% (26/46), respectively , sand diaphragmatic damage with pleural effusion (n = 1).Complications were evaluated according to the Cardiovascular and Interventional Radiological Society of Europe (CIRSE) classification system . No gradIn recent years, RFA has become a common nonsurgical treatment besides TACE and its effectiveness has been confirmed in small HCCs, comparable to that of radical treatments . However, RFA cannot assure peripheral necrosis for large or irregular HCCs, which increases the risk for residual tumor, recurrence, and metastasis . With thDynaCT employs CTCB using the Artis zee DSA system  and can realize synchronized acquisition with flat-panel detector during C-arm rotation. After image reconstruction using the workstation, three-dimensional images of the target lesion and CT images of soft tissues can be obtained, which overcomes the limitations of traditional instruments. Thus, spiral CT and DSA can be performed on the same treatment bed. Using this technique, DynaCT-guided RFA was employed for the treatment of HCCs in this study. In the present study, DynaCT-guided RFA was conducted immediately after DSA-guided TACE with a technical success rate of 100%. At 1, 2, and 3 years after surgery, the survival rate was 89.1% (41/46), 71.7% (33/46), and 56.5% (26/46), respectively, which is consistent with the survival rates reported by Takaki et al. . These rBased on our findings, DynaCT-guided TACE with simultaneous RFA appears safe and reliable for treatment of solitary large HCCs. The advantages of this technique include simultaneous combination of TACE and RFA which reduces the time interval between two treatments, thus avoiding the clearance of lipiodol and chemotherapeutics and the formation of new collateral vessels and vascular recanalization after embolization in this time interval . In addiOf note, DynaCT has several limitations including: (1) the puncture should be continuously monitored by DynaCT, which increases the radiation exposure to clinicians; (2) the images obtained from DynaCT should be subjected to MPR reconstruction in the workstation, and the reconstructed images have poor resolution and poor intensity differences between tissues as compared with those from routine CT . BesidesOur results indicated that DynaCT-guided TACE with simultaneous RFA is a safe and efficacious treatment for solitary large HCCs. TACE combined with simultaneous RFA provides a new treatment option for solitary large HCCs in which DynaCT has important clinical value. When CT-guided interventional therapy is unavailable or the conditions of the surgical unit are less advanced, DynaCT is recommended for RFA or TACE with simultaneous RFA in the treatment of large HCCs with relatively complete capsules and favorable locations.


Weak magnetic fields affect reactive oxygen species levels, stem cell proliferation/differentiation, and new tissue growth. Biological systems are constantly exposed to electromagnetic fields (EMFs) in the form of natural geomagnetic fields and EMFs emitted from technology. While strong magnetic fields are known to change chemical reaction rates and free radical concentrations, the debate remains about whether static weak magnetic fields  also produce biological effects. Using the planarian regeneration model, we show that WMFs altered stem cell proliferation and subsequent differentiation via changes in reactive oxygen species (ROS) accumulation and downstream heat shock protein 70 (Hsp70) expression. These data reveal that on the basis of field strength, WMF exposure can increase or decrease new tissue formation in vivo, suggesting WMFs as a potential therapeutic tool to manipulate mitotic activity. Exposure to electromagnetic fields (EMFs) occurs both from modern technology and Earth’s natural geomagnetic field, which averages 25 to 65 μT  using the robust planarian regeneration model. Planaria are free-living flatworms that are capable of regenerating all tissues, including the central nervous system and brain, owing to a large adult stem cell (ASC) population that comprises ~25% of all cells  and examined blastema outgrowth at 3 days postamputation (dpa)  followinn = 12; P < 0.01 by Student’s t test).We hypothesized that WMF effects were due to altered ROS levels, which peak at the wound site by 1 hpa and are required for planarian blastema formation , phenocoTo investigate genetic mechanisms by which ROS levels (and thus WMFs) regulate regenerative outgrowth, we examined their effects on heat shock protein 70 (Hsp70) expression. Hsp70 is a stress response protein that acts as a chaperone for protein folding during repair, promoting both normal cell survival and cancer cell growth , similarTo determine whether the observed changes in blastema size were due to changes in proliferation, we examined mitotic activity via phospho–histone H3 (pH3) staining at the wound site at 4 hpa. Our data revealed that 200 μT WMF exposure, direct ROS inhibition, and direct Hsp70 inhibition all resulted in significantly reduced mitotic activity as compared to control conditions . In planOur data confirm that WMFs affect biological systems and establish a nascent mechanistic pathway by which this occurs. Currently, the main hypothesis for how magnetic fields interact with biological systems is through radical pair recombination   was maintained at 18°C in the dark. All planarians were kept in worm water; worm water consists of Instant Ocean salts (0.5 g/liter) in ultrapure water of Type 1. Animals were fed no more than once a week with “natural” (no antibiotics or hormones) liver paste made from whole calf liver (Creekstone Farms). Liver was frozen and thawed only once before feeding animals. Worms 5 to 7 mm in length were starved at least 1 week before experimentation. S. mediterranea were amputated into trunk fragments via scalpel cuts made just above and below the pharynx. Amputations were done under a dissecting microscope on a custom-made cooling Peltier plate, as previously described  and was fed through the x and y coils in order to create a uniform static WMF. Before each experiment, Helmholtz coils were characterized using a Gauss meter (AlphaLab models GM1-ST or GM1-HS), which was also used to verify the magnetic field at the end of each experiment.A magnetic field enclosure (MagShield box) was constructed of μ-metal (which blocks magnetic fields) with a vertical μ-metal partition for running parallel experiments . Two custom-built triaxial Helmholtz coils were positioned in the exact center of each partition (via a stack of plastic well plates) to ensure that experiments were uniformly exposed to each specific magnetic field. Each coil was composed of a Plexiglas skeleton around which a ceramic-insulated copper wire was wrapped multiple times running in two parallel strands on each of the n = 24; 45 μT, n = 29; and 200 μT, n = 25. For the temporal trials, planarians were exposed beginning at 0 hpa , 30 mpa, 1 hpa, 2.5 hpa, 24 hpa, and 48 hpa until the end of the 72-hour period. In addition, planarians were exposed beginning at 0 hpa and ending at 12, 24, or 48 hpa, at which time they were removed from the MagShield box and allowed to continue regenerating in standard incubators (as for untreated controls) until the end of the 72-hour period. Last, planarians were preexposed starting 24 hours before amputation and were either allowed to regenerate with no further exposure or returned to the MagShield box to be further exposed for either 24 or 48 hpa (after which time they continued regenerating in standard incubators until the end of the 72-hour period). Temporal experiments were performed once . Total biological replicates for each condition were as follows: 0 to 72 hours, n = 25; 30 min to 72 hours, n = 19; 1 to 72 hours, n = 12; 2.5 to 72 hours, n = 14; 24 to 72 hours, n = 17; 48 to 72 hours, n = 16; 0 to 12 hours, n = 18; 0 to 24 hours, n = 20; 0 to 48 hours, n = 17; −24 to 24 hours, n = 13; −24 to 48 hours, n = 20; and −24 to 0 hours (amputation), n = 15. For the field strength trials, experiments were performed once, except for 200 μT, which was repeated three times, and 45 μT, which was repeated eight times (as every field strength was run with concurrent controls). Total biological replicates for each condition were as follows: 45 μT, n = 119; 0 μT, n = 30; 100 μT, n = 28; 200 μT, n = 25; 300 μT, n = 18; 400 μT, n = 18; 500 μT, n = 17; and 600 μT, n = 16.The MagShield box was housed in a temperature-controlled room (20°C). The temperature inside the Helmholtz coils during magnetic field exposure assays was randomly tested twice a day over the 3-day assay, with an average temperature of 22°C (±1°C). Animals were placed in worm water in either 35 or 60 mm petri dishes at the indicated times into the MagShield box in the center of the partition (via a stack of larger plastic petri dishes). Animals were exposed to controlled static magnetic fields (in the dark) always in tandem: with one side of the MagShield partition set to Earth-normal 45 μT (for controls) and the other partition set to the indicated experimental WMF strength. Unless otherwise indicated, experimental planarians were exposed to 200 μT from 5 minutes postamputation (mpa) to 72 hpa. Experiments were repeated three times . Total biological replicates for each condition were as follows: untreated, n = 20 and DPI, n = 22.ROS accumulation was inhibited with diphenyleneiodonium chloride . Animals were presoaked in 10 μM DPI [made from a 3 mM dimethyl sulfoxide (DMSO) stock] for 5 hours before amputation. Newly amputated pharynx fragments were immediately returned to 10 μM DPI and were allowed to regenerate until 72 hpa, at which time planarians were imaged for blastema size analyses. Control experiments were placed in DMSO (vehicle control). Total biological replicates for each condition were as follows: DMSO, 2DCFDA; Molecular Probes C6827), was used to visualize ROS accumulation . One hour before imaging, worms were incubated in 25 μM CM-H2DCFDA made from 10 mM DMSO stock. For WMF experiments, following a 23-hour WMF preexposure period, whole worms were removed from the MagShield box, cut into pharynx fragments, placed into 25 μM CM-H2DCFDA, and returned to the MagShield box for the 1-hour incubation period. For ROS inhibition experiments, following a 5-hour pretreatment period, whole worms were removed from DPI, cut into pharynx fragments, and then placed into a combination of 10 μM DPI plus 25 μM CM-H2DCFDA for the 1-hour incubation period. After the 1-hour CM-H2DCFDA incubation period, all worms were rinsed three times in fresh worm water and then the ventral side was imaged using 35 mm FluoroDishes (WPI FD35-100) and 25 mm round no. 1.5 coverslips (WPI 503508). Signal intensity at the wound site was normalized to signal intensity of the central body to control for differences in dye loading between animals. Experiments were performed twice (except for DMSO and DPI). Total biological replicates for each condition were as follows: untreated, n = 37; 45 μT, n = 26; 200 μT, n = 24; DMSO, n = 19; and DPI, n = 15.The cell-permeant fluorescent general oxidative stress indicator dye, 5-(and-6)-chloromethyl-2′,7′-dicholorodihydrofluorescein diacetate (CM-HS. mediterranea SOD (SMU15011417) was used to generate SOD RNAi. The primers were 5′-ACTGGAGCCATCAATATCTGG and 3′-TAATCCGGCCTTACATTTTTG. A 552 bp region of S. mediterranea Hsp70 (SMU15039086) was used to generate Hsp70 RNAi. The region was from 5′-GGTTTTTGATTTGGGTGGTG to 3′-AGCTGTTGCTATGGGAGC. Worms were fed with RNAi three times over 8 days before being amputated on day 9, as indicated above. Control RNAi was double-stranded RNA to Venus-GFP, which is not present in the planarian genome. SOD RNAi rescue experiments were performed twice (except for 45 μT + SOD RNAi). Total biological replicates for each condition were as follows: 45 μT, n = 20; 200 μT, n = 20; 45 μT + SOD RNAi, n = 10; and 200 μT + SOD RNAi, n = 20. Total biological replicates for each condition for Hsp70 RNAi morphology experiments were as follows: control RNAi, n = 15 and Hsp70 RNAi, n = 15.RNAi was performed via feeding of in vitro–synthesized double-stranded RNAi, as previously described  was used to generate AGAT riboprobe. The primers were 5′-GGAGTTAAAGTGTCCATCCAG and 3′-GTTGCTAACCTGACTGACATGC. A 2461 bp region of S. mediterranea Piwi-1 (Q2Q5Y9.1) was used to generate the Piwi riboprobe. The region was from 5′-GATCCCAATTTAAGACCAAGAAGAG to 3′-TTTTTATGTATTCGATTAAAAAAAA. Total biological replicates for each condition for the Piwi probe were as follows: untreated, n = 7; Hsp70 RNAi, n = 9; 45 μT, n = 7; 200 μT, n = 7; DMSO, n = 6; and DPI, n = 6. Total biological replicates for each condition for the AGAT probe were as follows: untreated, n = 5; Hsp70 RNAi, n = 9; 45 μT, n = 6; 200 μT, n = 7; DMSO, n = 6; and DPI, n = 6.Colorimetric whole-mount in situ hybridization (WISH) was performed as previously described (Images were taken using a ZEISS V20 fluorescence stereomicroscope with AxioCam MRc or MRm camera and ZEN lite software (ZEISS). Regenerates were imaged while fully extended and moving to ensure the absence of any tissue bunching, which could affect analyses. Heat maps for visualizing intensity of ROS levels were generated using the standard rainbow lookup table (LUT) within the ZEN lite software. Adobe Photoshop was used to orient, scale, and improve clarity of images (but not for fluorescent images). Data were neither added nor subtracted; original images are available upon request.+ cells were counted with the RTCN tool in ImageJ; the whole regenerate was measured with the magnetic lasso tool in Adobe Photoshop, and the final counts were shown as cells per square millimeter. Significance was determined using either a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (using GraphPad Prism version 7.00 for Mac) or a two-tailed Student’s t test with unequal variance (using Microsoft Excel).For blastema size, the magnetic lasso tool in Adobe Photoshop was used to generate total pixel counts for both the anterior and posterior blastemas (as well as the entire regenerate). To control for worms of different sizes, blastema sizes were expressed as a ratio of blastema size/total regenerate size. For ROS indicator dye assay, the magnetic lasso tool was used to obtain mean gray intensity values for both the anterior and posterior blastema, as well as baseline mean pixel intensity values (from the center of the regenerate). Signal intensity was expressed as average blastema pixel intensity − average baseline mean pixel intensity. For the pH3 assay, images were masked to avoid background signal and pH3


Both ischemic and non-ischemic heart disease can cause disturbances in the myocardial blood volume (MBV), myocardial perfusion and the myocardial extracellular volume fraction (ECV). Recent studies suggest that native myocardial T1 mapping can detect changes in MBV during adenosine stress without the use of contrast agents. Furthermore, native T2 mapping could also potentially be used to quantify changes in myocardial perfusion and/or MBV. Therefore, the aim of this study was to explore the relative contributions of myocardial perfusion, MBV and ECV to native T1 and native T2 at rest and during adenosine stress in normal physiology.n = 41, 26 ± 5 years, 51% females) underwent 1.5 T cardiovascular magnetic resonance (CMR) scanning. Quantitative myocardial perfusion [ml/min/g] and MBV [%] maps were computed from first pass perfusion imaging at adenosine stress (140 microg/kg/min infusion) and rest following an intravenous contrast bolus . Native T1 and T2 maps were acquired before and during adenosine stress. T1 maps at rest and stress were also acquired following a 0.2 mmol/kg cumulative intravenous contrast dose, rendering rest and stress ECV maps [%]. Myocardial T1, T2, perfusion, MBV and ECV values were measured by delineating a region of interest in the midmural third of the myocardium.Healthy subjects . Myocardial perfusion, MBV and ECV all correlated with both native T1 and native T2, respectively .During adenosine stress, there was an increase in myocardial native T1, native T2, perfusion, MBV, and ECV , and native T1 .Multivariate linear regression revealed that ECV and perfusion together best explained the change in native T2 (ECV beta 0.21, Myocardial native T1, native T2, perfusion, MBV, and ECV all increase during adenosine stress. Changes in myocardial native T1 and T2 during adenosine stress in normal physiology can largely be explained by the combined changes in myocardial perfusion and ECV.Clinicaltrials.gov identifier NCT02723747. Registered March 16, 2016. Coronary artery disease (CAD) and several other non-ischemic heart diseases affect the myocardial microcirculation . AccuratParametric pixel-wise mapping has been developed to quantitatively measure and image the longitudinal relaxation time constant (T1) , and then = 43, 26 ± 5 years, 51% females) were included if they had no use of cardiovascular medication and had no previous history of cardiovascular symptoms or systemic disease, kidney disease, or asthma, were current non-smokers, and had a normal 12-lead electrocardiogram (ECG). The 12-lead ECG and a venous blood sample, used to determine blood hematocrit, were collected immediately prior to the CMR exam. Exclusion criteria included failed splenic switch off [Clinicaltrials.gov identifier NCT02723747.Healthy subjects  1.19 ms, repetition time(TR) 2.85 ms, matrix size =144 × 256, and field of view (FOV) 360 × 270 mm2.CMR was conducted at 1.5 T  with a phased-array eighteen-channel body matrix coil together with a spine matrix coil. All patients were examined in the supine position. Figure 2, slice thickness 8.0 mm and parallel acquisition technique factor 3.Three short-axis slices  were acquired during first pass perfusion imaging, both during adenosine stress  and at rest, during administration of an intravenous bolus of contrast agent . Adenosine was administered in one cannula, while the contrast agent was administered in a separate cannula in the opposite arm. All maps were automatically generated using inline perfusion software implemented in the Gadgetron online reconstruction framework  and the 2, slice thickness 8.0 mm, imaging duration 167 ms, TE/TR 1.12 ms/2.7 ms, matrix size =144 × 256, FOV 360 × 270 mm2 and breath hold 10.7 s. Three rest and stress ECV maps were generated offline from rest pre- and post-contrast T1 maps, and stress pre- and post-contrast T1 maps, respectively, and calibrated by blood hematocrit [Three short axis native myocardial T1 maps  were acquired using an ECG-gated modified Look-Locker inversion recovery (MOLLI) sequence  with a 5matocrit , employimatocrit .2, slice thickness 8.0 mm, acquisition window 700 ms, TD 483 ms, matrix size = 144 × 256, acceleration factor = 2. Inline motion registration was performed followed by a pixel-wise parametric fit to estimate the final T2 maps.Three myocardial native T2 maps  were acquired before and during adenosine stress (140 microg/kg/min infusion). T2 mapping was performed using the Siemens MyoMaps product sequence, which is a T2-prepared sequence. The T2 map was calculated from 3 single-shot acquisitions at different T2 preparation times . Typical imaging parameters included TE/TR = 1.06/2.49 ms, FA 70°, pixel size 1.4 × 1.4 mmThe subjects did not undergo late gadolinium enhancement (LGE) imaging as focal myocardial scar from silent myocardial infarction or myocarditis would be visible in rest ECV maps.Apical stress ECV maps were not of sufficient quality due to partial volume effects, and a sizable portion of the basal stress ECV maps were affected by the more prominent through-plane motion associated with imaging at a higher heart rate during stress. Thus, only the midventricular images were analyzed. The midventricular native T1, native T2, perfusion, MBV and ECV maps were analyzed with Segment software version 2.0 R5152 . All the respective average values were acquired by conservative manual delineation of a circumferential region of interest in the midmural third of the myocardium in the respective maps, both at rest and stress. To assess reproducibility, segmental analysis of the respective maps was performed by 3 independent observers.LV volumes, ejection fraction and myocardial mass were quantified using the SyngoVia Software, VA30 , including manual corrections. Body surface area (BSA) was calculated with the Dubois formula . Volumet2. Significant univariate associations were entered into the multivariate linear regression analysis. Multivariate linear regression was assumed for native T1 and native T2, respectively, and was used to investigate the best explanatory parameters for native T1 and native T2, respectively, using manual stepwise linear regression modeling. By combining rest and stress absolute values in the same data set, the absolute changes in all parameters were investigated by linear regression. Mean values were compared by using the paired or unpaired t-test as appropriate in normally distributed data, as decided by the Kolmogorov-Smirnov test. Interobserver variability was assessed by intraclass correlation. Intraobserver variability was calculated as the average difference of two measurements divided by their mean, and expressed as a percentage. Statistical analysis was performed using Microsoft Excel®  and SPSS Statistics® , International Business Machines, Inc., Armonk, New York, USA). The significance level in all statistical analyses was defined as p < 0.05.Continuous variables were reported as means together with their standard deviation (SD). Ordinal variables were reported as percentages. The relationships between perfusion, MBV, ECV, heart rate, systolic blood pressure, rate pressure product and hematocrit, and native T1 and native T2, respectively, were assessed with Pearson’s linear correlation coefficient, and presented as Rn = 1, and low peak AIF, n = 1). Table n = 41). Stress ECV images could not be processed in 4 cases because pre and post T1 maps were mistakenly acquired with different FOV, and these cases were excluded from analysis of ECV. No subjects had any evidence of any focal myocardial scarring in any of the three short-axis maps. No subject was excluded due to poor image quality.In total, two of the healthy subjects were excluded , native T2 , MBV , myocardial perfusion , and ECV , see Fig. From rest to stress there was an increase in native T1 , Fig. p = 0.02; perfusion beta 0.66, p < 0.001, model R2 = 0.64, p < 0.001), and change in native T1 , Table The relationships between myocardial T1, T2, MBV, perfusion and ECV assessed individually at rest and stress, respectively, are presented in Additional file We demonstrate that native myocardial T1, native myocardial T2, myocardial perfusion, MBV, and ECV all increase in response to adenosine stress in healthy subjects. The changes in native myocardial T1 and T2 values are best explained by the combined changes in myocardial perfusion and ECV, thus supporting the notion that native myocardial T1 mapping, and T2 mapping indeed reflect the increase of blood in the myocardium during adenosine stress, and illustrating the mechanism behind the potential to use native T1 or T2 to detect CAD without the need for an intravenous contrast agent.99mTc-Red-Blood-Cell method [In the current study, we quantified myocardial perfusion and MBV using first-pass contrast-enhanced CMR. Several approaches have been proposed for quantifying myocardial perfusion and MBV. PET  and myocl method , 48. FurA study cohort of healthy subjects with low measurement variability may not fully elucidate the relationships between the ECV, MBV and perfusion with native T1 and native T2 at rest. Therefore, a multivariate linear regression analysis of the changes in the parameters was employed by investigating rest and stress in a combined fashion. This introduced a variability in native T1 and T2 necessary to evaluate the relevant relationships. The multivariate analysis showed that the combined changes in myocardial perfusion and ECV best explain the increase in native T1 and T2 mapping in response to adenosine stress. This is furthered supported by the finding in the univariate analysis for native T1 and native T2 at rest and stress, respectively. The analyses indicate that ECV was the main contributor to these measures, see Additional file In the current study the MOLLI 5s(3s)3s sequence was used due to the clinical routine at the time of the study. MOLLI sequences can be heart rate dependent due to two major factors; the time between inversion, and the SSFP readout influence on each inversion recovery. By setting the time between inversions in seconds instead of heart beats as in the MOLLI 5(3)3 protocol, heart rate dependency is mitigated , howeverA recent study suggests that stress native T1 mapping can be used to differentiate between obstructive epicardial disease and microvascular disease , the mecThis study was conducted in a cohort of healthy subjects free of known cardiovascular disease, and therefore there is a need for larger scale studies to confirm the findings in older subject and patients with CAD. Furthermore, some of the subjecs had a high level of fitness, which is reflected in the slightly higher LV volumes and slightly lower LV ejection fraction, which is in agreement with previously published values in athletes . The finThe T2 mapping technique used in this work was based on a T2 prepared sequence, which may introduce heart rate sensitivity, off-resonance, and T1 dependencies in the quantification.Myocardial native T1, native T2, perfusion, MBV, and ECV all increase during adenosine stress in healthy subjects. Changes in myocardial native T1 and T2 during adenosine stress in normal physiology can largely be explained by the combined changes in myocardial perfusion and ECV.Additional file 1. Reproducibility, segmental values and respective linear regression. Data include tables on intra- and interobserver variability, segmental values of the respetive maps, and linear regression at rest alone and stress alone.


N-butyldeoxygalactonojirimycin (NB-DGJ) caused reversible infertility at 150 mg/kg/day when administered daily as single oral dose. NB-DGJ inhibited CD rat-derived testicular β-glucosidase 2 (GBA2) activity at 10 µM but did not inhibit CD rat-derived testicular ceramide-specific glucosyltransferase (CGT) at doses up to 1000 µM. Pharmacokinetic studies revealed that sufficient plasma levels of NB-DGJ (50 µM) were achieved to inhibit the enzyme. Fertility was blocked after 35 days of treatment and reversed one week after termination of treatment. The rapid return of fertility indicates that the major effect of NB-DGJ may be epididymal rather than testicular. Collectively, our in vitro and in vivo studies in rats suggest that iminosugars should continue to be pursued as potential lead compounds for development of oral, non-hormonal male contraceptives. The study also adds evidence that GBA2, and not CGT, is the major target for the contraceptive effect of iminosugars.This study shows for the first time that an iminosugar exerts anti-spermiogenic effect, inducing reversible infertility in a species that is not related to C57BL/6 male mice. In CD rats,  Access to orally bioavailable, safe, effective, and reversible male contraceptive agents would be an important advance in expanding current contraceptive options available to couples. While female oral hormonal contraceptive agents have been on the market since 1960, the development of male hormonal contraceptives has been slow ,3. SinceN-alkylated iminosugar N-butyldeoxynojirimycin  -NB-DNJ (160 mg/kg) and reported a t½ = 4.5 h, Cmax = 13,800 ng/mL (~63 µM) and tmax = 0.5 h [We noted that analysis  revealed = 0.5 h .Our study is the first to demonstrate that an iminosugar can induce reversible infertility in a species other than C57BL/6-related mice. The observed rapid reversibility of infertility in CD rats after cessation of drug treatment (one week), compared to mice (three weeks) suggests that other targets and/or species differences may be influencing the effects of the iminosugars. Thus, the results of our study provide new evidence that it might be possible to develop iminosugars as male contraceptive agents for human use. Our current study has limitations; however, the results are encouraging and lay a strong foundation for future investigations with higher dosage and/or modified analogs of iminosugars. We have already synthesized and determined the in vitro efficacy of various four- and eight-membered analogs of iminosugars as part of our structure–activity-relationship (SAR) approach to determine the best structure that can exhibit anti-fertility effects over a broad range of species . These iAll reagents, unless otherwise specified, were purchased from Sigma and were ACS grade. Thin layer chromatography (TLC) plates  were obtained from Whatman International Limited, England. Bouin’s Fixative was obtained from Fisher Diagnostics.2 chamber) following the blood collection at 48 h in accordance with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Animals used for harvesting testes tissues for microsome preparation were euthanized by CO2 asphyxiation followed by cervical dislocation, as per IACUC protocol approved by University of Kansas Medical Center.For all studies using animals, the minimum number of animals to achieve statistical robustness and reduction requirements of the IACUC-approved studies  were used. Animals for mating studies were euthanized . Body weight was noted every week for all treatment groups. Mice were euthanized at the end of a 35 days dosing regimen using COFor sperm retrieval, epididymis was removed from the animal and placed in Petri dish. One mL of capacitation buffer  at room temperature was added to the dish. Using forceps, the caput of the epididymis was held in place and the cauda sliced repeatedly with a razor blade. The spermatozoa were allowed to disperse for 15 min with gentle swirling intermittently. A 100 µL aliquot of the sperm suspension was dropped onto a standard glass microscope slide (Fisherbrand superfrost microscope slides 25 × 75 × 1.0 mm)) and smeared with side of pipette tip. The slides were air dried flat for 1 h at room temperature. The sperm smear was fixed in methanol for 2 min. The sperm smear was than stained using FITC-PSA  was diluted 1:7000 in PBS containing 1:10,000 DAPI dihydrochloride, FluoroPure™ grade, Invitrogen) (≥98% pure) to make the stock solution. To each slide, 100 µL of FITC-PSA was added and spread out with edge of pipette tip to cover the dried sperm smear. The slides were incubated at 37 °C for 10 min, and then washed with 5 changes of PBS for 5 min each. The slides were air-dried and mounted with 20 µL Vectashield mounting medium for fluorescence  and observed. Slides were viewed on a Nikon IX-81 microscope under a 40× objective using FITC and DAPI filter to visualize acrosome morphology. The phase contrast images were also collected at the same magnification to compare morphological details.NB-DGJ  via oral gavage at 150 mg/kg/day (in approximately 5 mL/kg of water) daily for 35 consecutive days. The third group (6 rats) was treated with 15 mg/kg NB-DNJ in distilled water containing 0.9% benzyl alcohol) via oral gavage (in approximately 5 mL/kg of water) daily for 35 consecutive days  via oral gavage (5 mL/kg/day), and the second group (6 rats) was treated with ive days .Rats were weighed on day 0 (first day of dosing) and weekly thereafter. Dosages were adjusted based on the most recent body weight. Males were cohabited with untreated females  weekly at weeks 1 through 9 ,39. FemaSperm were obtained from the left cauda epididymis and evaluated for sperm motility, as follows. The cauda epididymis was punctured with an 18-gauge needle and sperm expressed into the well of a culture slide. The sperm were diluted with capacitation buffer  containit-test was performed on body weights, paired testes weights, ventral prostrate weights, seminal vesicle weights, paired epididymal weights, spermatid head counts, percent motile and normal epididymal sperm. We used analysis of variance (ANOVA) for serum inhibin B, rFSH, and testosterone, since these measurements were repeated throughout the study period. The effect of NB-DNJ at 15 mg/kg/day dose in the rat model was studied following the same protocol as that for NB-DGJ. Since the mating trials did not show effect on fertility by week 6, the study was terminated at week 7 (data not presented).Statistical analysis using the Student’s NB-DGJ was formulated as described above to 33.75 mg/mL for in vivo administration. Rats were dosed orally by using a 15 G × 3” disposable gavage needle with a maximum volume of 5 mL/kg body weight. At various time points  following dosing, approximately 0.1 mL of blood was collected from each rat into BD Microtainer® with Lithium Heparin (Becton-Dickinson). Plasma was then isolated by allowing the samples to sit at room temperature for 30 min followed by microcentrifugation at 15,000× g for 5 min. Plasma samples were then stored at −70 °C until processing and analysis.Four male CD rats (225–235 g) were purchased from Harlan  with in-dwelling jugular cannulas and allowed to acclimate for 72 h in climate-regulated holding rooms in the University of Minnesota’s AAALAC-certified animal facilities. Standard rat chow and water were provided ad libitum throughout the study. N-hydroxyethyl-1-deoxynojirimycin) as an internal standard) and vortexed. The samples were then incubated on ice for 1 h and then microcentrifuged at 15,000× g for 5 min. The cleared supernatants were then transferred to 12 × 32 mm glass vials with inserts .Frozen plasma samples were thawed on ice and processed by adding 2 volumes of 100% acetonitrile . UPLC isocratic separation of analytes was performed using a Waters Atlantis Hilic 3 µm, 150 × 2.1 mm analytical column  maintained at 30 °C. The eluent was a mixture of acetonitrile/water/buffer  delivered at 0.25 mL/min. The buffer consisted of 100 mM ammonium acetate, pH 5. For the quantification of NB-DGJ and miglitol , the transitions m/z 220 (Q1) → m/z 158 (Q3) and m/z 208 (Q1) → m/z 146 (Q3) were monitored, respectively. Data reduction was performed with QuanLynx software. PK analysis was performed by WinNonLin using a non-compartmental model.Processed samples were analyzed by LC/MS/MS using a previously published method . A standv/v) reagent C, 10 mM DTT, 8 mM EDTA, 1 mM UDP-Glu and 1% CHAPSO) and dispersed by passage through a 25-gauge needle followed by an insulin needle.  The microsome suspension was stored as 100 μL aliquots in microcentrifuge tubes, flash frozen in liquid nitrogen for 1−2 min, stored at −80 °C, and used as needed.Testes in 5 g batches were placed in a 50 mL culture tube containing 25 mL of Reagent B , where reagent A is 1 μg/mL antipain, 1 μg/mL leupeptin, 10 μg/mL aprotinin, 13.2 μg/mL 4-amidinophenyl-methanesulfonyl fluoride hydrochloride (APMSF) and 250 mM KCl. The testes were minced with scissors and then blended by 10 s bursts repeated 2−3 times on Power Gen 700  at setting 5–6, while on ice. The homogenate was centrifuged at 7500 rpm for 10 min at 4 °C using a Beckman SW28 rotor (5660 g). The resulting supernatant was collected and centrifuged at 23,500 rpm for 1 h at 4 °C in a Beckman SW40 rotor. The supernatant was discarded and the pellet containing the microsomes was suspended in 600 μL of reagent D (66% (v/v) reagent A, 5 mM MnCl2, 0.1 mM phosphatidylcholine, 50 μM conduritol B epoxide, 2 mM EDTA, 5 mM UDP-Glu), 145 μL water, 50 μL iminosugar from serially diluted stock solutions prepared in water, and 100 μg testicular microsomes. Control tubes contained the same components, except microsomes. Reactions were initiated by the addition of 3 μL of bovine serum albumin–ceramide and incubated at 37 °C for 30 min, then terminated by addition of 1 mL of 2:1 (v:v) chloroform: methanol, vortexed, and incubated at room temperature for 30–60 min to allow phase separation. The upper phase and the mid-layer were removed and discarded, and 500 µL of chloroform:methanol: water (3:48:47) was added to the bottom layer, vortexed, and allowed to sit for 15 min at room temperature. The resulting upper phase was again removed and 100 µL of chloroform:methanol (2:1) was added, and then sample tubes were dried in a vortex evaporator overnight.The following solutions were added to each assay tube (Fisher 15 × 85 mm borosilicate glass): 295 μL of assay mix  were pre-treated by immersion in chloroform:methanol: water (50:50:15) for 5 min, air dried for 10 min, then immersed in 5% sodium borate (prepared in methanol) for 1 min, dried and heated at 120 °C for 1.5 h. The dried sample tubes were reconstituted with 100 µL chloroform:methanol (2:1) and vortexing, and 20 µL was then spotted onto the plates at the origin. The spotted plates were air-dried and placed in a sealed TLC chamber saturated with chloroform:methanol: water (60:30:5) and run approximately 1 h until the solvent reached <1 cm from the top of plate).50 values.The TLC plates were documented using a UV transilluminator (at 302 nm) and analyzed using AlphaEase  software. The integrated density value (IDV) was plotted against iminosugar concentration using Sigma Plot 10. A linear regression plot was used to determine ICd-glucoside  was added to each well using a multi-channel pipette, followed by 10 μL of iminosugar serial dilutions to yield 0, 5, 10, 50, 100, 500 and 1000 μM final concentrations, added from left to right, to provide increasing concentrations of the iminosugar from the top to the bottom of the plate. Then, 50 μL of testicular microsomes (1 μg/μL) was added to each well using a multi-channel pipette in the first column. Another multi-channel pipette was kept pre-loaded with terminator solution  and was added simultaneously to the first row as a 0-time point. Microsomes were then added to the remaining rows after setting the timer to 1 min. Every 1 min, the terminator solution was added to each row until the 12th row. Absorbance was then detected at 360/460 nm using Synergy HT Multi-Mode Microplate Reader (BioTek). The absorbance values were subtracted from background (MUG only). A linear regression plot was created using Prism software (Graph Pad Prism 5) to determine the IC50 values.The assay was carried out in 96-well plates: 50 μL of 3 mg/mL 4-methyl umbelliferyl-beta-NB-DGJ, administered orally, induced reversible infertility in male CD rats. Thus, unlike, previously reported, iminosugars exert anti-fertility effects in rodent species other than C57Bl6J, indicating that iminosugars and their derivatives should continue to be explored as potential non-hormonal male contraceptive compounds. They are attractive candidates due to their safety, efficacy and excellent bioavailability.