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The presence of disease-specific antigens and autoantibodies in the sera of patients with atherosclerosis-related diseases has been widely reported and is considered to result from inflammation of the arterial wall and the involvement of immune factors. The aim of this study was to identify a novel antibody in patients with ischemic stroke by serological identification of antigens using recombinant cDNA expression cloning from patients who had a transient ischemic attack (TIA). We identified the serpin peptidase inhibitor, clade E member 1 (SERPINE1), as a candidate antigen. The serum anti-SERPINE1 antibody levels quantified using amplified luminescent proximity homogeneous assay-linked immunosorbent assay were significantly higher in patients with ischemic stroke, including those with acute cerebral infarction (aCI), TIA, and chronic cerebral infarction, than in healthy donors. The antibody levels were strongly associated with old age, female sex, and presence of hypertension, diabetes mellitus, and cardiovascular disease. Age and intima-media thickness of the carotid artery were positively correlated with antibody levels, which suggests that SERPINE1 may reflect the progression of atherosclerosis. In a multivariate analysis, SERPINE1 antibody level was an independent predictor of aCI. Thus, the serum levels of anti-SERPINE1 antibody could potentially serve as a biomarker of atherothrombotic infarction.

To investigate the efficacy of 125I seed implantation in the treatment regimen of pelvic recurrence after early cervical cancer surgery and to analyse prognostic factors. To evaluate efficacy and analyse prognostic factors of 125I seed implantation for pelvic recurrence after early cervical cancer surgery. A prospective study was conducted on 62 patients who experienced pelvic recurrence after early cervical cancer surgery between August 2005 and September 2015. The 62 patients were treated and assessed in 2 groups (n = 30). All 62 patients were randomized into two groups that received two different treatment regimens: the treatment group (n = 30), which received 125I particle implantation therapy, and the control group (n = 32), which received whole-pelvic irradiation using the anteroposterior/posteroanterior field and cisplatin-based concurrent chemoradiation therapy. The efficacy/efficiency of 125I seed implantation and prognostic factors were analysed by logistic regression. Overall survival was determined by Kaplan–Meier analysis. Multivariate analysis results were obtained by the Cox proportional hazards regression model. The effective control rates at 1, 3, 6 and 12 months were 76.7%, 80.0%, 83.3%, and 86.7% in the 125I particle implantation group. The total effective control rates at 1, 3, 6 and 12 months were 65.6%, 65.5%, 62.5%, and 71.9% in the chemoradiotherapy group. Significant differences were observed between the two groups. The overall survival rates at 1, 2, 3, 4, and 5 years and the median overall were 96.7%, 93.3%, 86.7%, 71.9%, 65.6% and 4.34 years, respectively, in the 125I seed implantation group and 81.3%, 71.9%, 62.5%, 56.3%, 53.1% and 3.59 years, respectively, in the control group. There were statistically significant differences in survival rates depending on the diameter of the largest recurrent pelvic tumour (χ2 = 6.611, P = 0.010). The multivariate analysis showed that the survival rates were related to the diameter of the largest recurrent pelvic tumour (χ2 = 4.538, P = 0.033). 125I implantation is an effective, safe, and promising method for the treatment of pelvic recurrence after early cervical cancer surgery. The diameter of the recurrent pelvic tumour was identified as a significant independent prognostic factor in patients who received 125I implantation.

Increased heart rate (HR) is a reaction to head-to-toe gravito-inertial (G) force. The anti-G straining manoeuvre (AGSM) is the crucial technique for withstanding a high-G load. Previous studies reported the main effects of HR only or AGSM only on G tolerance. We assessed the combined effect of HR and AGSM on the outcome of 9G profile exposure. A total of 530 attempts for the 9G profile were extracted to clarify the association of interest. Subjects with an AGSM effectiveness of less than 2.5G had a 2.14-fold higher likelihood of failing in the 9G profile. Trainees with HR increases of less than 20% in the first five seconds also had higher odds of 9G profile intolerance (adjusted OR 1.83, 95% CI 1.09–3.07). The adjusted OR of 9G profile disqualification was 2.93 (95% CI 1.19–7.20) for participants with smaller HR increases and lower AGSM effectiveness. The negative effect of a smaller HR increase on the outcome was likely to be affected by improved AGSM effectiveness (adjusted OR 1.26, 95% CI 0.65–2.42). We speculate that low AGSM effectiveness and a small HR increase were separately associated with failure of high-G challenge. Nonetheless, good AGSM performance seemed to reduce the negative effect of weak HR responses on the dependent variable.

Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke, transient ischemic attack , diabetes mellitus (DM), cardiovascular disease , chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than that of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.

Blood vessels in the adult mammal exist in a highly organized and stable state. In the ischemic heart, limited expansion capacity of the myocardial vascular bed cannot satisfy demands for oxygen supply and the myocardium eventually undergoes irreversible damage. The predominant contribution of endogenous c-Kit+ cells is understood to be in the development and homeostasis of cardiac endothelial cells, which suggests potential for their targeting in treatments for cardiac ischemic injury. Quiescent cells in other tissues are known to contribute to the long-term maintenance of a cell pool, preserve proliferation capacity and, upon activation, facilitate tissue homeostasis and regeneration in response to tissue injury. Here, we present evidence of a Setd4-expressing quiescent c-Kit+ cell population in the adult mouse heart originating from embryonic stages. Conditional knock-out of Setd4 in c-Kit-CreERT2;Setd4f/f;Rosa26TdTomato mice induced an increase in vascular endothelial cells of capillaries in both neonatal and adult mice. We show that Setd4 regulates quiescence of c-Kit+ cells by the PI3K-Akt-mTOR signaling pathway via H4K20me3 catalysis. In myocardial infarction injured mice, Setd4 knock-out resulted in attenuated cardiomyocyte apoptosis, decreased infarction size and improved cardiac function. Lineage tracing in Setd4-Cre;Rosa26mT/mG mice showed that Setd4+ cells contribute to each cardiac lineage. Overall, Setd4 epigenetically controls c-Kit+ cell quiescence in the adult heart by facilitating heterochromatin formation via H4K20me3. Beyond activation, endogenous quiescent c-Kit+ cells were able to improve cardiac function in myocardial infarction injured mice via the neovascularization of capillaries.

To investigate the efficacy of 125I seed implantation in the treatment regimen of pelvic recurrence after early cervical cancer surgery and to analyse prognostic factors. To evaluate efficacy and analyse prognostic factors of 125I seed implantation for pelvic recurrence after early cervical cancer surgery. A prospective study was conducted on 62 patients who experienced pelvic recurrence after early cervical cancer surgery between August 2005 and September 2015. The 62 patients were treated and assessed in 2 groups (n = 30). All 62 patients were randomized into two groups that received two different treatment regimens: the treatment group (n = 30), which received 125I particle implantation therapy, and the control group (n = 32), which received whole-pelvic irradiation using the anteroposterior/posteroanterior field and cisplatin-based concurrent chemoradiation therapy. The efficacy/efficiency of 125I seed implantation and prognostic factors were analysed by logistic regression. Overall survival was determined by Kaplan–Meier analysis. Multivariate analysis results were obtained by the Cox proportional hazards regression model. The effective control rates at 1, 3, 6 and 12 months were 76.7%, 80.0%, 83.3%, and 86.7% in the 125I particle implantation group. The total effective control rates at 1, 3, 6 and 12 months were 65.6%, 65.5%, 62.5%, and 71.9% in the chemoradiotherapy group. Significant differences were observed between the two groups. The overall survival rates at 1, 2, 3, 4, and 5 years and the median overall were 96.7%, 93.3%, 86.7%, 71.9%, 65.6% and 4.34 years, respectively, in the 125I seed implantation group and 81.3%, 71.9%, 62.5%, 56.3%, 53.1% and 3.59 years, respectively, in the control group. There were statistically significant differences in survival rates depending on the diameter of the largest recurrent pelvic tumour (χ2 = 6.611, P = 0.010). The multivariate analysis showed that the survival rates were related to the diameter of the largest recurrent pelvic tumour (χ2 = 4.538, P = 0.033). 125I implantation is an effective, safe, and promising method for the treatment of pelvic recurrence after early cervical cancer surgery. The diameter of the recurrent pelvic tumour was identified as a significant independent prognostic factor in patients who received 125I implantation.

Dysarthria is universal in Parkinson’s disease (PD) during disease progression; however, the quality of vocalization changes is often ignored. Furthermore, the role of changes in the acoustic parameters of phonation in PD patients remains unclear. We recruited 35 PD patients and 26 healthy controls to perform single, double, and multiple syllable tests. A logistic regression was performed to differentiate between protective and risk factors among the acoustic parameters. The results indicated that the mean f0, max f0, min f0, jitter, duration of speech and median intensity of speaking for the PD patients were significantly different from those of the healthy controls. These results reveal some promising indicators of dysarthric symptoms consisting of acoustic parameters, and they strengthen our understanding about the significance of changes in phonation by PD patients, which may accelerate the discovery of novel PD biomarkers.

Oral administration of indigo naturalis (IN) can induce remission in ulcerative colitis (UC); however, the underlying mechanism remains unknown. The main active components and targets of IN were obtained by searching three traditional Chinese medicine network databases such as TCMSP and five Targets fishing databases such as PharmMapper. UC disease targets were obtained from three disease databases such as DrugBank,combined with four GEO gene chips. IN-UC targets were identified by matching the two. A protein–protein interaction network was constructed, and the core targets were screened according to the topological structure. GO and KEGG enrichment analysis and bioGPS localization were performed,and an Herbs-Components-Targets network, a Compound Targets-Organs location network, and a Core Targets-Signal Pathways network were established. Molecular docking technology was used to verify the main compounds-targets. Ten core active components and 184 compound targets of IN-UC, of which 43 were core targets, were enriched and analyzed by bioGPS, GO, and KEGG. The therapeutic effect of IN on UC may involve activation of systemic immunity, which is involved in the regulation of nuclear transcription, protein phosphorylation, cytokine activity, reactive oxygen metabolism, epithelial cell proliferation, and cell apoptosis through Th17 cell differentiation, the Jak-STAT and IL-17 signaling pathways, toll-like and NOD-like receptors, and other cellular and innate immune signaling pathways. The molecular mechanism underlying the effect of IN on inducing UC remission was predicted using a network pharmacology method, thereby providing a theoretical basis for further study of the effective components and mechanism of IN in the treatment of UC.

Relationships among clinical characteristics, iron metabolism and neurotransmitters in Parkinson disease (PD) patients with restless legs syndrome (RLS) remains unclear. We divided 218 patients into PD with and with no RLS (PD-RLS and PD-NRLS) groups by RLS-rating scale (RLS-RS) score. Motor and non-motor symptoms were rated by related scales. Iron and related proteins, and neurotransmitters in cerebrospinal fluid (CSF) and serum were measured. PD-RLS frequency was 40.37%. PD-RLS group had longer duration, higher stage and scores of motor symptoms, depression, anxiety, sleep disorders, fatigue and apathy, and increased transferrin and decreased iron, ferritin, dopamine (DA) and 5-hydroxytryptamine (5-HT) in CSF. In CSF of PD-RLS group, RLS-RS score was positively correlated with transferrin level and negatively correlated with iron and ferritin levels; RLS-RS score was negatively correlated with DA and 5-HT levels; transferrin level was negatively correlated with DA and 5-HT levels, and ferritin level was positively correlated with DA level. In serum, PD-RLS group had decreased iron and transferrin levels, which were negatively correlated with RLS-RS score. PD-RLS was common and severer in motor and some non-motor symptoms. Iron deficiency induced by its metabolism dysfunctions in peripheral and central systems might cause PD-RLS through decreasing brain DA and 5-HT.

Background B7-h6, a member of the B7 family molecules, participates in the clearance of tumor cells by binding to NKp30 on NK cells. The expression of B7-H6 in esophageal squamous cell carcinoma and the clinical significance is unknown. The goal of this study was to determine the expression of B7-H6 in esophageal squamous cell carcinoma and the clinical significance of B7-H6 expression. Patients and methods We retrospectively collected clinical data from 145 patients diagnosed with esophageal squamous cell carcinoma between January 2007 and December 2008. These patients had all previously undergone surgical treatment for esophageal cancer, were clearly diagnosed, and had not received chemotherapy or radiotherapy. In addition, pathological tissue samples from the 145 patients were collected to detect the expression of B7-H6 by immunohistochemistry. The chi-square (χ2) test was used to analyse the relationships between B7-H6 and clinicopathological characteristics. The prognosis of the patients were analysed by Cox proportional hazards regression analysis and Kaplan-Meier analysis. Results B7-H6 was present in 133/145 (91.72%) of the esophageal squamous cell carcinoma samples and all localized in the cytoplasm. The expression level of B7-H6 was correlated with T stage (P=0.036) and lymphatic metastasis status (P=0.044). According to the results of the ROC curve analysis, H-score =90 was selected as the cut-off value. The 145 patients were divided into two groups, the high B7-H6 expression (H-score>90) group and the low B7-H6 expression (H-score≤90) group. Cox proportional hazards regression analysis indicated that tumour size (P=0.021), B7-H6 expression (P=0.025) and lymphatic metastasis status (P=0.049) were independent prognostic factors for esophageal squamous cell carcinoma. Kaplan-Meier analysis with the log-rank test demonstrated that the patients with high B7-H6 expression (P = 0.003), lymphatic metastasis (P

NaoXinTong Capsule (NXT), a Chinese medicine, is currently used to treat patients with cardiovascular and cerebrovascular diseases. Clinical observations indicate its anti-diabetic functions with unclear mechanisms. Herein, we report the effect of NXT on diabetic nephropathy (DN). Type 2 diabetic db/db mice were treated with NXT for 14 weeks. In the course of treatment, NXT reduced diabetes-increased glucose levels and improved renal functions. At the end of treatment, we found that NXT ameliorated serum lipid profiles and other biochemical parameters. In the kidney, NXT inhibited mesangial matrix expansion, expression of vascular endothelial growth factor A, fibronectin, advanced glycation end product and its receptor. Meanwhile, it reduced the diabetes-induced podocyte injury by increasing WT1 and nephrin expression. In addition, NXT inhibited accumulation of extracellular matrix proteins by increasing MMP2/9 expression through inactivation of TGFβ/Smad pathway and CTGF expression. Mechanically, NXT activated insulin signaling pathway by increasing expression of INSR, IRS and FGF21, phosphorylation of Akt and AMPKα in the liver, INSR phosphorylation in the kidney, and FGF21 and GLUT4 expression in adipose tissue and skeletal muscle. Taken together, our study demonstrates that NXT inhibits DN by ameliorating glucose/lipid metabolism, maintaining tissue structure integrity, and correcting diabetes-induced renal dysfunctions.

The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. However, thermostable proofreading DNA polymerases occasionally failed to amplify target fragment that could be amplified by Taq DNA polymerase. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. Here we showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. To the best of our knowledge, this is the first example of DNA sequence that could inhibit DNA polymerase in its single-stranded form.

Constipation is one of the most frequent non-motor symptoms of Parkinson disease (PD) and it may be ignored by PD patients, leading to this problem not to be reported in time. The relationships between constipation and demographic variables, motor symptoms and other non-motor symptoms of PD are still unknown. PD patients were evaluated by diagnostic criteria of functional constipation in Rome III and divided into PD with constipation (PD-C) and PD with no constipation (PD-NC) groups. PD patients were assessed by rating scales of motor symptoms and other non-motor symptoms, activity of daily living and quality of life. The frequency of constipation in PD patients was 61.4%, and 24.5% of PD patients had constipation before the onset of motor symptoms. PD-C group had older age and age of onset, longer disease duration, more advanced disease stage, and more severe motor symptoms and non-motor symptoms, including worse cognition and emotion, poorer sleep quality, severer autonomic symptoms, fatigue and apathy. Binary Logistic regression analysis showed that the age, H-Y stage, depression, anxiety and autonomic dysfunction increased the risk of constipation in PD patients. Constipation exerted serious impact on the activity of daily living and quality of life in PD patients.

Parkinson disease (PD) is associated with multiple factors, including iron, which is demonstrated to deposit excessively in PD brains. We detected iron deposition by susceptibility weighted image (SWI) and measured the levels of iron metabolism-related proteins and inflammatory factors in cerebrospinal fluid (CSF) and serum of PD patients and control subjects. Clinical symptoms of PD were evaluated by series of rating scales. Relationships among above factors were analyzed. Results showed that corrected phase (CP) value of substantia nigra (SN) was significantly decreased in PD group compared to control group, hence, SN was the main region with excessive iron deposition. In PD group, ferritin was significantly elevated in CSF and reduced in serum compared to control group, and levels of ferritin in CSF and serum were both significantly and positively correlated with CP value of SN, thus, abnormal iron metabolism in central and peripheral systems was associated with iron deposition. CP value of SN in PD group was significantly and negatively correlated with interleukin-1β level in CSF, so interleukin-1β might be a neuroinflammatory factor produced by excessive iron in SN. Iron deposition in SN was significantly correlated with motor symptoms and part of non-motor symptoms of PD.

Parkinson disease (PD) is identified as tremor-dominant (TD) and postural instability and gait difficulty (PIGD) phenotypes. The relationships between motor phenotypes and cognitive impairment and the underlying mechanisms relating pathological proteins and neurotransmitters in cerebrospinal fluid (CSF) are unknown. We evaluated the motor symptoms and cognitive function by scales, and detected the levels of pathological proteins and neurotransmitters in CSF. TD group and PIGD group had significantly higher levels of total tau, tau phosphorylated at the position of threonine 181(P-tau181t), threonine 231, serine 396, serine 199 and lower β amyloid (Aβ)1–42 level in CSF than those in control group; PIGD group had significantly higher P-tau181t level and lower Aβ1–42 level than those in TD group. In PD group, PIGD severity was negatively correlated with MoCA score and Aβ1–42 level in CSF, and positively correlated with Hoehn-Yahr stage and P-tau181t level in CSF. In PIGD group, PIGD severity was negatively correlated with homovanillic acid (HVA) level in CSF, and HVA level was positively correlated with Aβ1–42 level in CSF. PIGD was significantly correlated with cognitive impairment, which underlying mechanism might be involved in Aβ1–42 aggregation in brain and relevant neurochemical disturbance featured by the depletion of HVA in CSF.

Fatigue is a very common non-motor symptom in Parkinson disease (PD) patients. It included physical fatigue and mental fatigue. The potential mechanisms of mental fatigue involving serotonergic dysfunction and abnormal iron metabolism are still unknown. Therefore, we evaluated the fatigue symptoms, classified PD patients into fatigue group and non-fatigue group, and detected the levels of serotonin, iron and related proteins in CSF and serum. In CSF, 5-HT level is significantly decreased and the levels of iron and transferrin are dramatically increased in fatigue group. In fatigue group, mental fatigue score is negatively correlated with 5-HT level in CSF, and positively correlated with the scores of depression and excessive daytime sleepiness, and disease duration, also, mental fatigue is positively correlated with the levels of iron and transferrin in CSF. Transferrin level is negatively correlated with 5-HT level in CSF. In serum, the levels of 5-HT and transferrin are markedly decreased in fatigue group; mental fatigue score exhibits a negative correlation with 5-HT level. Thus serotonin dysfunction in both central and peripheral systems may be correlated with mental fatigue through abnormal iron metabolism. Depression, excessive daytime sleepiness and disease duration were the risk factors for mental fatigue of PD.

The PP2C family member Wild-type p53-induced phosphatase 1 (Wip1) critically regulates DNA damage response (DDR) under stressful situations. In the present study, we investigated whether Wip1 expression was involved in the regulation of DDR-induced and depression-related cellular senescence in mouse hippocampus. We found that Wip1 gene knockout (KO) mice showed aberrant elevation of hippocampal cellular senescence and of γ-H2AX activity, which is known as a biomarker of DDR and cellular senescence, indicating that the lack of Wip1-mediated γ-H2AX dephosphorylation facilitates cellular senescence in hippocampus. Administration of the antidepressant fluoxetine had no significant effects on the increased depression-like behaviors, enriched cellular senescence and aberrantly upregulated hippocampal γ-H2AX activity in Wip1 KO mice. After wildtype C57BL/6 mice were exposed to the procedure of chronic unpredictable mild stress (CUMS), cellular senescence and γ-H2AX activity in hippocampus were also elevated, accompanied by the suppression of Wip1 expression in hippocampus when compared to the control group without CUMS experience. These CUMS-induced symptoms were effectively prevented following fluoxetine administration in wildtype C57BL/6 mice, with the normalization of depression-like behaviors. Our data demonstrate that Wip1-mediated γ-H2AX dephosphorylation may play an important role in the occurrence of depression-related cellular senescence.

MK2 activation by p38 MAPK selectively induces inflammation in various diseases. We determined if a MK2 inhibitor (MK2i), improves cornea wound healing by inhibiting inflammation caused by burning rat corneas with alkali. Our study, for the first time, demonstrated that MK2i inhibited alkali burn-induced MK2 activation as well as rises in inflammation based on: a) blunting rises in inflammatory index, inflammatory cell infiltration, ED1+ macrophage and PMN+ neutrophil infiltration; b) suppressing IL-6 and IL-1β gene expression along with those of macrophage inflammatory protein-1α (MIP-1α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1); c) reducing angiogenic gene expression levels and neovascularization (NV) whereas anti-angiogenic PEDF levels increased. In addition, this study found that MK2i did not affect human corneal epithelial cell (HCEC) proliferation and migration and had no detectable side effects on ocular surface integrity. Taken together, MK2i selectively inhibited alkali burn-induced corneal inflammation by blocking MK2 activation, these effects have clinical relevance in the treatment of inflammation related ocular surface diseases.

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

Self-assembly of colloidal particles into colloidal films has many actual and potential applications. While various strategies have been developed to direct the assembly of colloidal particles, fabrication of crack-free and transferrable colloidal film with controllable crystal structures still remains a major challenge. Here we show a centrifugation-assisted assembly of colloidal silica spheres into free-standing colloidal film by using the liquid/liquid interfaces of three immiscible phases. Through independent control of centrifugal force and interparticle electrostatic repulsion, polycrystalline, single-crystalline and quasi-amorphous structures can be readily obtained. More importantly, by dehydration of silica particles during centrifugation, the spontaneous formation of capillary water bridges between particles enables the binding and pre-shrinkage of the assembled array at the fluid interface. Thus the assembled colloidal films are not only crack-free, but also robust and flexible enough to be easily transferred on various planar and curved substrates.