The Circular RNA Circ-ANAPC7 as a Biomarker for the Risk Stratification of Myelodysplastic Syndrome.

To assess the diagnostic value of circ-ANAPC7 expression levels in MDS and its risk stratification. This is a retrospective observational study. This study enrolled 125 patients diagnosed with MDS and divided them into five groups according to IPSS-R (very high group, 25; high group, 25; intermediate group, 25; low group, 25; and very low group, 25), and 25 patients with IDA were studied as control group from our bone marrow cell bank. Bone marrow cell were used as material in this study to measure the expression level of circ-ANAPC7 by qRT-PCR. An evaluation of diagnostic value was conducted using ROC curves. Circ-ANAPC7 expression levels were 5.623 ± 4.483, 28.396 ± 12.938, 91.867 ± 37.010, 202.525 ± 54.911, 337.633 ± 86.013, and 502.269 ± 98.410 from the control group to the very high group, respectively (p < 0.05). Circ-ANAPC7 expression was gradually upregulated with the risk stratification of MDS. The AUCs of circ-ANAPC7 were 0.973, 0.996, 0.951, 0.920, and 0.907 in the control group/very low group, very low group/low group, low group/intermediate group, intermediate group/high group, and high group/very high group, respectively. In this study, the expression level of circ-ANAPC7 was found to be a promising biomarker for MDS. It may be added to the scoring system to better identify risk groups.

Aberrant expression of splicing factors in newly diagnosed acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common type of blood cancer in adults. Emerging evidence is establishing a connection between AML and aberrant alternative splicing of pre-mRNA, which may result from aberrant expression of splicing factors, the mediators of splicing reactions. MATERIAL AND METHODS: Using quantitative real-time polymerase chain reaction, we measured mRNA expression of 7 splicing factors belonging to the serine/arginine-rich (SR) protein family, SRSF1 (SF2/ASF), SRSF2 (SC35), SRSF3 (SRp20), SRSF4 (SRp75), SRSF5 (SRp40), SRSF6 (SRp55), and SRSF7 (9G8), and 1 non-SR factor, heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), in peripheral blood mononuclear cells of 26 patients with newly diagnosed AML and 26 healthy controls. In addition, the relationship between splicing factors and the mRNA splicing patterns of the caspase-8 gene (CASP8) was investigated. RESULTS: Compared to healthy controls, the expression of splicing factors was obviously aberrant in newly diagnosed AML patients. The expression of SRSF1, SRSF3 and SRSF4 mRNAs was significantly decreased. Moreover, a significant correlation was observed between several splicing factors and caspase-8 pre-mRNA splicing in AML patients, but not in control subjects. CONCLUSION: These data suggest that aberrant expression of splicing factors in AML may potentially connect with abnormal expression of oncogenes and be useful for early diagnosis, prognosis, and therapy of AML.

Facile synthesis of chitosan-based acid-resistant composite films for efficient selective adsorption properties towards anionic dyes.

To effectively and selectively remove toxic anionic dyes which are heavily discharged and to promote them recovery, a sustainable cellulose nanofiber/chitosan (CNF/CS) composite film was elaborately designed through a facile procedure. Based on the strong supporting effect of CNF and excellent compatibility between CNF and CS, the composite film presents low swelling and acid-proof properties, which can prevent the adsorption process from the disintegration of adsorbent. Moreover, the positive electrical property of CNF/CS film increases the discrepancy in adsorption capacities for anionic and cationic dyes. The maximum adsorption capacity of anionic methyl orange (MO) on CNF/CS film reaches 655.23 mg/g with a desirable recyclability. The adsorption behavior attributed to a physico-chemical and monolayer adsorption process. This work opens a new route for the development of eco-friendly and highly efficient adsorbents on selective removal and recycling of anionic dyes from wastewater.

[Preparation of silymarin-loaded amphiphilic chitosan micelle and its in situ absorption in rat intestine].

To improve the oral bioavailability of silymarin, the silymarin-loaded amphiphilic chitosan micelles (SM-OGC) were prepared. The absorption of SM-OGC in rat intestine was investigated. SM-OGC was prepared by dialysis method. The size and zeta potential of SM-OGC were investigated. Compared to silymarin suspension, the absorption of SM-OGC was investigated using in situ single pass perfusion model. The diameters and zeta potential SM-OGC were (162.4 +/- 3.0) nm and (+32.6 +/- 0.98) mV, respectively. The encapsulation efficiency was (39.17 +/- 0.98)% and the drug loading of SM-OGC was (28.15 +/- 0.43)%. The absorption of SM-OGC at different segments of intestine was significantly higher than that of silymarin suspension (P < 0.05). The apparent absorption rate (K(a)) and effective permeation coefficient (P(eff)) at the duodenum were the largest. K(a) and P(eff) had no significant difference between jejunum, ileum and colon. OGC micelles might significantly promote the absorption of silymarin in the intestine tract.

Association of SERPINC1 Gene Polymorphism (rs2227589) With Pulmonary Embolism Risk in a Chinese Population.

Background and Aims: Genetic variants in the gene SERPINC1 have been shown to be associated with antithrombin deficiency, which subsequently contributes to the susceptibility to venous thrombosis. However, several other studies have shown conflicting results regarding the association of SERPINC1 gene polymorphisms (rs2227589) with the risk of thrombosis. Hence, in the present study, we conducted a case-control study to further evaluate the association between the variant rs2227589 with antithrombin deficiency in pulmonary embolism (PTE). A pooled systematic analysis was also conducted to evaluate the risk of rs2227589 in venous thromboembolism (VTE) among multiple populations. Methods: This case-control study involved 101 patients and 199 healthy controls. The allele frequency of SERPINC1 variant rs2227589 was analyzed by Sequenom assay. Antithrombin anticoagulant activity was detected using an automatic coagulation analyzer. In addition, a pooled systematic analysis on 10 cohorts consisting of 5,518 patients with VTE and 8,935 controls was performed. Results: In total, 27 (26.7%) PTE subjects were diagnosed as having antithrombin deficiency. Our results showed that antithrombin plasma activity was slightly lower in T allele carriers than that in C allele carriers. However, there was no significant correlation between rs2227589 genotype and antithrombin anticoagulant activity. The recessive model showed that rs2227589 was significantly associated (p = 0.026) with an increased risk {odds ratio [OR]: 2.31, 95% confidence interval [CI] (1.09-4.89)} of Chinese PTE. The pooled systematic analysis of all case-control study and meta-analysis showed that rs2227589 polymorphism was associated with an increased risk of VTE in the additive model [OR: 1.09, 95% CI (1.01-1.18), P = 0.029] and dominant model [OR: 1.10, 95% CI (1.01-1.20), P = 0.034]. Conclusions: Our study demonstrated that variant rs2227589 is associated with an increased risk of PTE in a Chinese population but no correlation with antithrombin anticoagulant activity. However, pooled systematic analysis of multiple populations showed a significant association between rs2227589 and the risk of VTE in the additive and dominant genetic model.

[Solubilizing and sustained-releasing abilities and safety preliminary evaluation for paclitaxel based on N-octyl-O, N-carboxymethyl chitosan polymeric micelles].

A series of novel self-assembled polymeric micelles based on carboxymethyl chitosan bearing long chain alkyl chains (N-octyl-O, N-carboxymethyl chitosan, OCC) was synthesized. PTX loaded OCC polymeric micelles (PTX-OCC) were prepared by dialysis method. The effects of the degree of substitutions (DS) of octyl groups on the solubilizing abilities of OCC for paclitaxel were studied. The PTX-OCC were characterized using drug loading content, drug encapsulation efficiency, dynamic light scattering, zeta potential and transmission electron microscopy (TEM). Take PTX injection (PTX-INJ) as control, the safety of PTX-OCC including hemolysis, hypersensitiveness in guinea pigs and acute toxicity in mice were also evaluated. OCC showed excellent loading capacities for paclitaxel with the DS of octyl groups in the range of 37.9% - 58.6%. Drug loading contents were up to 24.9% - 34.4% with drug encapsulation efficiency 56.3% - 89.3%, which both increased with the increasing of DS of octyl groups. The mean size of PTX-OCC was 186.4 - 201.1 nm which decreased with the increasing of DS of octyl groups. The zeta potential was -47.5 to -50.9 mV, which had no obvious relation with the DS of octyl groups. The TEM images showed a spherical shape. No burst release phenomena were observed and drug cumulative release was in the range of 60% -95% in 15 days. PTX-OCC with higher DS of octyl groups showed stronger sustained releasing ability. In terms of the induction of membrane damage and hypersensitiveness, PTX-OCC was superior to PTX-INJ. The LD50 and its 95% confidence interval of PTX-OCC were 134.4 (125.0 - 144.6) mg x kg(-1), which was 2.7 fold of PTX-INJ. The present PTX-OCC could be potentially useful as safety carriers for intravenous delivery.

[Preparation of doxorubicin-loaded chitosan polymeric micelle and study on its tissue biodistribution in mice].

To prepare doxorubicin-loaded N-octyl-N'-succinyl chitosan polymeric micelle (DOX-OSC) and study the biodistribution of DOX-OSC in mice, DOX-OSC was prepared by dialysis method. By using doxorubicin injection (DOX-INJ) as control, DOX-OSC and DOX-INJ were administered to mice through caudal vein at a dose of 5 mg x kg(-1) body weight. The RP-HPLC method was established to determine the DOX levels in the plasma and other tissues of mice. The tissues distribution and targeting efficiency were evaluated by pharmacokinetic parameters (AUC, MRT) and targeting parameters (Re, Ce and Te). The drug loading and entrapment efficiency of DOX-OSC were (35.8 +/- 0.4)% and (75.3 +/- 1.1)%, respectively. The diameter and zeta potential of DOX-OSC were (174 +/- 12) nm and (-37.1 +/- 3.0) mV, respectively. The transmission electron microscope result showed DOX-OSC with spherical shape. The biodistribution results showed that the concentration of DOX of both DOX-OSC and DOX-INJ decreased rapidly in blood after iv administration. While free DOX levels in blood at 12-96 h were not detectable for DOX-INJ, in contrast, DOX level in blood at 96 h was still found for DOX-OSC. In contrast to DOX-INJ group, DOX-OSC showed a higher targeting efficiency in the liver and spleen. The AUCs of DOX in the liver and spleen were 20.0 and 47.4 times and the MRT were 11.2 and 37.2 times, respectively. And the levels of DOX-OSC in the heart and kidney tissues were significantly reduced. And the drug distribution of DOX-OSC in the heart and kidney tissues were 17.0% and 11.4%, respectively. Hence, DOX-OSC shows an excellent drug loading capabilities and a higher targeting efficiency in the liver and spleen. That the levels of DOX-OSC in the heart and kidney tissues are significantly reduced, might improve the treatment efficacy of DOX and decrease the side effects.

Effects of miR-619-5p on proliferation, migration and invasion of human breast cancer cells / 中国药理学通报

Aim To investigate the involvement and mechanism of miR-619-5p in the proliferation, migration and invasion of human breast cancer cells. Methods The expression of miR-619-5p in breast cancer and normal breast tissue and cells was detected using bioinformatic analysis or qRT-PCR. After transfection with miR-619-5p mimics or inhibitors, the expression of miR-619-5p and EMT-related molecule mRNA was determined by qRT-PCR. Cell proliferation was detected using CCK-8 assay; cell migration and invasion capacity was estimated by the wound healing assay and Transwell assay. The protein levels of EMT-related molecules were analyzed by Western blot. The target genes of miR-619-5p were analyzed by bioinformatic a-nalysis, and a preliminary analysis of the potential target gene CREB1 was carried out. Results miR-619-5p was low expressed in breast cancer tissues and breast cancer cells. Compared with the control group, over-expression of miR-619-5p resulted in up-regula-tion of miR-619-5p expression levels and EMT epithelial markers, down-regulation of pro-EMT molecules and mesenchymal markers, impairment of cell proliferation, migration and invasion, and down-regulation of CREB1 expression. The results of the low miR-619-5p expression group were opposite to the above results. Conclusions In breast cancer tissue and cells, miR-619-5p expression is lower. miR-619-5p inhibits the proliferation, migration, invasion and EMT of breast cancer cells, and its possible mechanism of the effects may be targeting CREB1.

Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

The present study was performed to explore the role of galanin and galanin receptor 1 (GalR 1) in nociceptive modulation in the central nucleus of amygdala (CeA) in normal rats and rats with neuropathy, and the involvement of GalR 1 and PKC was also investigated. The hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations were increased in a dose-dependent manner after intra-CeA injection of galanin in both normal rats and rats with neuropathy. The increased HWLs were significantly attenuated by intra-CeA injection of galanin receptor antagonist M40, indicating an involvement of galanin receptor in nociceptive modulation in CeA. Furthermore, intra-CeA administration of the GalR 1 agonist M 617 induced increases in HWLs in normal rats, suggesting that GalR 1 may be involved in galanin-induce antinociception in CeA. Additionally, intra-CeA injection of the PKC inhibitor inhibited galanin-induced antinociception, showing an involvement of PKC in galanin-induced antinociception in CeA of normal rats. Moreover, there was a significant increase in GalR1 content in CeA in rats with neuropathy than that in normal rats. These results illustrated that galanin induced antinociception in CeA in normal rats and rats with neuropathy, and there is an up-regulation of GalR1 expression in rats with neuropathy.

[Preparation of paclitaxel-loaded chitosan polymeric micelles and influence of surface charges on their tissue biodistribution in mice].

AIM: To prepare paclitaxel-loaded cationic chitosan micelles (PTX-CCM) and paclitaxel-loaded anionic chitosan micelles (PTX-ACM) and study the influence of surface charges on the biodistribution of paclitaxel-loaded chitosan polymeric micelles in mice. METHODS: PTX-CCM and PTX-ACM were prepared by dialysis method and were administered to mice by caudal vein at a dose of 20 mg x kg(-1) body weight. The RP-HPLC method was established to determine the PTX concentrations in the plasma and other tissues of mice. The tissues distribution of PTX-CCM and PTX-ACM were evaluated by the pharmacokinetic parameters (AUC, MRT). RESULTS: The diameter and zeta potential of PTX-CCM were 164 nm and +23.7 mV, while those of PTX-ACM were 180 nm and -28.0 mV, respectively. The drug loading and drug encapsulation efficiency for PTX-CCM were 26.4% (w/w) and 76.2% , while those of PTX-ACM were 34.6% (w/w) and 89.9%, respectively. The highest uptake of PTX-CCM and PTX-ACM in liver were 64.72% and 91.84% of dose, respectively. Meanwhile, MRT of both were 5.50 h and 51.39 h prolonged. The highest uptake of PTX-CCM and PTX-ACM in spleen were 7.08% and 5.16% of dose, respectively. Meanwhile, MRT of both were 9.04 h and 26.82 h. For PTX-CCM group, the AUC and C(max) of PTX in the lung were 2.71 times and 5.87 times of those of PTX-ACM group respectively. While in both PTX-CCM and PTX-ACM groups, the highest uptake of PTX in the heart were only 0.36% and 0.24% of dose, respectively and PTX in the kidney were only 0.75% and 0.33% of dose respectively. CONCLUSION: PTX-CCM and PTX-ACM showed excellent drug loading capabilities with amount of cationic charges and anionic charges on their surface, respectively. Both PTX-CCM and PTX-ACM groups showed a higher targeting efficiency in the liver and spleen in vivo and accumulated in both tissues for relatively long time, especially in PTX-ACM group. In contrast to PTX-ACM, PTX-CCM showed a higher lung targeting efficiency in vivo while PTX-ACM had a stronger retention ability in the lung. Meanwhile in both groups the levels of PTX in the heart and kidney tissues were significantly lower which might decrease the side effects of PTX.

[Analysis of Cut-off Value in Screening of Thalassemia by Capillary Hemoglobin Electrophoresis for Pregnant Women from Shenzhen Region of China].

OBJECTIVE: To investigate the cut-off value in screening of thalassemia in pregnant women from Shenzhen region by capillary hemoglobin electrophoresis. METHODS: The data of capillary hemoglobin electrophoresis and genetic diagnosis of thalassemia from 2122 examined prenatal women were retrospectively analyzed. Capillary hemoglobin electrophoresis and α-, ß- genetic diagnosis of thalassemia were carried out for every woman. Hemoglobin electrophoresis was performed using Capillarys 2 full-automated electrophoresis instrument. Gap polymerase chain reaction and reverse dot blot were used for genetic diagnosis of thalassemia genotyping test. The cut-off value in screening of thalassemia was determined by receiver operating characteristic curve and next to analyze the value of HbA2 and HbF in screening of thalassemia using the decided cut-off value. RESULTS: The areas under the curve (AUC(Roc)) of HbA2 for diagnosis of α-, ß- thalassemia were 0.75 and 0.981 respectively, and the AUC(Roc) of HbF for diagnosis of ß-thalassemia was 0.787. When HbA2 ≤ 2.55 was taken as the cut-off value of HbA2 for diagnosis of α-thalassemia, the sensitivity, specificity, positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) were 89.5%, 54.8%, 1.98, 0.19 respectively. When HbA2 ≥3.9 was taken as the cut off value of HbA2 for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 96.1%, 99.8% 480.5, 0.04 respectively. When HbF ≥0.75 was taken as the cut off value of HbF for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 83.6%, 61.8% respectively. CONCLUSION: The cut-off value in screening of thalassemia by capillarys 2 full automated electrophoresis instrument is different from that of the traditional method of hemoglobin electrophoresis, such as cellulose acetate membrane electrophoresis and agarose gel electrophoresis. Each laboratory should establish their own respective cut off value.

Effects of Fluoride on DNA Damage and Caspase-Mediated Apoptosis in the Liver of Rats.

Fluoride compounds are abundant and widely distributed in the environment at a variety of concentrations. Further, fluoride induces toxic effects in target organs such as the liver. In this study, we investigated liver histopathology, DNA damage, apoptosis, and the mRNA and protein expressions of caspase-3 and -9 in the rat livers by administering varying concentrations of fluoride (0, 50, 100, 200 mg/L ) for 120 days. The results showed fluoride-induced morphological changes and significantly increased apoptosis and DNA damage in rats exposed to fluoride, especially in response to higher doses. The immunohistochemical and qRT-PCR results indicated that caspase-3, caspase-9 protein positive expression and mRNA relative expression enhanced with increasing NaF concentration. In summary, our findings suggest that chronic exposure to fluoride causes damages to liver histopathology and leads to liver apoptosis through caspase-mediated pathways.

[Experimental analysis and countermeasures for EDTA-dependent Pseudothrombocytopenia].

The purpose of this study was to investigate the incidence of clinically common EDTA-dependent pseudo-thrombocytopenia (EDTA-PTCP) and methols for treating this diseese. A total of 1326 cases of thrombocytopenia found at blood routine examination were amalyzed anong 71 535 patients hospitalized in our hospital from January 2010 to May 2013, and 87 cases of PTCP caused EDTA-K anticoagulant were analyzed again by using sodium citrate auticoagulant, at the same time the platelet formation distribution was observed by microscopy of smear with Wright-Giemsa staining. The results showed that the platelet count detected by using EDTA-K anticoagulant in 87 cases was (56 ± 27)×10(9)/L, while the platelet count detected by using sodium citrate was (185 ± 39)×10(9)/L (t = 1.83,P < 0.01). The pseudo-thrombocytopenia incidence cansed by EDTA-K was 0.12%, it was 6.56% for the total number of thrombocytopenia. It is concluded that the incidence of PTCP cansed by EDTA-K is 0.12%, the PTCP is easily misdiagnosed. Therefore, the specimens of platelet count <100 10(9)/L should be tested again. When the platelet aggregation is found, the specimens should be examined again by using sodium citrate in order to avoid misdiagnosis.

N-acetylglucosaminyltransferase V negatively regulates integrin α5ß1-mediated monocyte adhesion and transmigration through vascular endothelium.

Changes in the expression of glycosyltransferases that branch N-linked glycans are associated with many physiological and pathological events, such as cell adhesion, migration, proliferation and tumor cell malignancy. Here, the altered levels of N-acetylglucosaminyltransferase V (GnT-V) and its product ß(1,6)-linked GlcNAc in monocytes were observed during inflammation. The effects of GnT-V and aberrant N-linked ß(1,6) branching on monocyte adhesion through vascular endothelium and transmigration were investigated. During IFN-γ-induced inflammation, adhesion and transendothelial migration of THP-1 monocytes were enhanced, and the levels of GnT-V and ß(1,6)-linked GlcNAc in THP-1 monocytes were significantly decreased. Expression of the GnT-V shRNA vector in THP-1 cells reversed the abnormal IFN-γ-induced characteristics, indicating direct involvement of N-glycosylation in these biological effects. The enhanced adhesion and transendothelial migration were significantly inhibited by functional blockade with antibodies against integrin α5 or ß1 in IFN-γ-induced and GnT-V knockdown THP-1 cells, demonstrating the involvement of integrin α5ß1 in the monocyte-endothelium interaction. However, IFN-γ treatment and GnT-V knockdown in THP-1 cells lowered expression of N-linked ß(1,6) branching on integrin α5 and ß1, without affecting the total protein expression of the subunits. Decreased GnT-V expression caused marked enchancement of integrin-induced phosphorylation of focal adhesion kinase (FAK). The augmented FAK-mediated ERK phosphorylation and activation were observed in IFN-γ-induced THP-1 cells. Furthermore, ERK inhibitor pre-treatment nearly abrogated the highly elevated IFN-γ-induced monocyte adhesion and transmigration, concomitant with reversal of the decrease in GnT-V and ß(1,6) branching. Our results demonstrate for the first time that decreased GnT-V activity due to inflammatory cytokine induction in human monocytes resulted in enchancement of integrin α5ß1-dependent monocyte-vascular endothelium adhesion and transmigration. Consequently, the activation of integrin caused elevation of FAK phosphorylation. These effects promoted FAK-mediated downstream signaling, including the ERK pathway, and indicate that GnT-V may be a potential therapeutic target for vascular inflammatory conditions.

Alternative splicing of apoptosis-related genes in imatinib-treated K562 cells identified by exon array analysis.

Imatinib is the therapeutic standard for newly diagnosed patients with chronic myeloid leukemia (CML). In these patients, imatinib has been shown to induce an apoptotic response specifically in cells expressing the oncogenic fusion protein BCR-ABL. Previous studies in our lab revealed that imatinib-induced apoptosis in K562 cells involves a shift in production of Bcl-x splice isoforms towards the pro-apoptotic Bcl-xs splice variant. Here, we report the findings from our subsequent study to identify other apoptosis-related genes that are differentially spliced in response to imatinib treatment. Gene expression profiling of imatinib-treated K562 cells was performed by the Affymetrix GeneChip Human Exon 1.0 ST array, and differences in exon-level expression and alternative splicing were analyzed using the easyExon software. Detailed analysis by reverse transcription-PCR (RT-PCR) and sequencing of key genes confirmed the experimental results of the exon array. Our results suggest that imatinib treatment of K562 cells causes a transcriptional shift towards alternative splicing in a large number of apoptotic genes. The present study provides insight into the molecular character of apoptotic leukemia cells and may help to improve the mechanism of imatinib therapy in patients with CML.

Super-short solid silicon microneedles for transdermal drug delivery applications.

In this study, the super-short microneedles with a length of 70-80 microm were fabricated using silicon wet etching technology. As evident from the visual inspection of pierced human skin, appearance of blue spots array after Evans Blue (EB) application indicated that the super-short microneedles were able to pierce into skin by pressing and swaying against the microneedles backing layer continually with a finger. The micro-conduits created in skin were validated by histological examination. Transport studies revealed that: (i) skin pretreated with super-short microneedles resulted in a remarkable enhancement of the galanthamine (GAL) transport, and the permeated amount increased as the insertion force increased; (ii) the super-short microneedles with flat tips were better than that with sharp tips for enhancing skin permeability; (iii) the longer time of super-short microneedles detained in skin resulted in a higher increase of skin permeability; (iv) there was no linear correlation between the GAL permeated through skin and the number of microneedles. The EIIIA(+) (526 bp) segment, a very sensitive marker of tissue injury, did not expressed in the skin pierced by super-short microneedles. Meanwhile, the skin pretreated with super-short microneedles did not infected after incubation with the Staphylococcus aureus. These results suggest that super-short microneedles may be a safe and efficient alternative for transdermal drug delivery of hydrophilic molecules.