Evaluation of flip-chip bonding electrical connectivity for ultra-large array infrared detector.

Flip-chip bonding is a key technology for infrared focal plane array (IRFPA) detectors. Due to the high cost of device preparation, the ultra-large array infrared detector cannot be directly used for the flip-chip bonding experiment, and the connectivity rate cannot be measured. To evaluate the flip-chip bonding process, a test device which has the same interconnecting structure as current IRFPA detectors is proposed. Indium bumps are electrically extracted to test electrodes. Electrical measurements were performed to characterize the connection and adhesion of the indium bumps and to calculate the connectivity rate. The electrical connectivity characteristics of the test devices correspond to the observation results of the indium bump extrusion, effectively detecting the interconnecting anomalies such as disconnection, adhesion, overall misalignment, etc., and verifying the feasibility of the test method. The test device has similar multi-layer components and thermal properties as HgCdTe infrared detector for process evaluation and post-processing experiment. The connectivity rate of the test device is up to 100%, and remains above 99% after thermal recycle experiment. The contact resistance of the interconnecting structure is calculated to be about 31.84 Ω based on the test results.

Systematic prediction method for flip-chip bonding connectivity of ultra-large array infrared detector.

The flip-chip bonding technique utilized in ultra-large array infrared detectors has a substantial impact on connectivity rates. The electrical connectivity of the flip-chip bonding process exhibits randomness due to the difficulties in the surface control of large-scale devices. This restriction hinders the development of ultra-large array devices. In this work, the surface shape matching calculation is performed based on the surface shape distributions measured from infrared detector chips and readout circuits. The multi combinations and multi rotation angles are employed to calculate the distribution of combined surface distances, and the combined PV (peak-to-valley) value is applied to describe the severity of surface mismatch. Test devices with combined PV values ranging from 7.460 µm to 4.265 µm are prepared and tested, and the connectivity rate achieves an improvement from 74.57% to 99.75% between mismatched devices and matching devices. The electrical test results of test devices indicate that disconnections tend to cluster in areas where surface distance is over 5 µm, which is determined by extracting and analyzing the surface distance correlated to electrical test results. A standard based on the combined PV value is established to select matching combinations and ensure a high connectivity rate of 99% or 97% for infrared detectors, while the connectivity rates of randomly selected devices are no higher than 91%. This work presents a systematic method to predict and improve the connectivity rate of flip-chip bonding process for ultra-large array infrared detector.

Molecular and cellular mechanisms of the first social relationship: A conserved role of 5-HT from mice to monkeys, upstream of oxytocin.

Maternal affiliation by infants is the first social behavior of mammalian animals. We report here that elimination of the Tph2 gene essential for serotonin synthesis in the brain reduced affiliation in mice, rats, and monkeys. Calcium imaging and c-fos immunostaining showed maternal odors activation of serotonergic neurons in the raphe nuclei (RNs) and oxytocinergic neurons in the paraventricular nucleus (PVN). Genetic elimination of oxytocin (OXT) or its receptor reduced maternal preference. OXT rescued maternal preference in mouse and monkey infants lacking serotonin. Tph2 elimination from RN serotonergic neurons innervating PVN reduced maternal preference. Reduced maternal preference after inhibiting serotonergic neurons was rescued by oxytocinergic neuronal activation. Our genetic studies reveal a role for serotonin in affiliation conserved from mice and rats to monkeys, while electrophysiological, pharmacological, chemogenetic, and optogenetic studies uncover OXT downstream of serotonin. We suggest serotonin as the master regulator upstream of neuropeptides in mammalian social behaviors.

The electrochemical immunosensor of the "signal on" strategy that activates MMoO4 (M = Co, Ni) peroxidase with Cu2+ to achieve ultrasensitive detection of CEA.

A new type of ultrasensitive electrochemical immunosensor with "signal on" strategy was designed for quantitative detection of CEA. The sensing strategy design is based on the following principles: We use HMSNs-Cu2+@HA as the signal probe, the structure of HA is destroyed under acidic conditions, and the released Cu2+ activates the substrate material MMoO4 (M = Co, Ni) Peroxidase activity initiates the reaction of catalytic H2O2 and realizes the "signal on" condition of electrical signals. This strategy has the following advantages: (1) HA coating of HMSNs-Cu2+ can prevent Cu2+ leakage, has good biocompatibility and can be connected with more antibodies. (2) The prepared sensor has the characteristics of high sensitivity and a low detection limit. When the electrode substrate was CoMoO4, the detection range of the immunosensor was 0.01 pg/mL-40 ng/mL, and the detection limit was 0.0035 pg/mL (S/N = 3). This work innovatively applies the catalytic activity of metal ion-activated nanozymes in the detection of CEA, providing a new perspective for the monitoring and analysis of cancer markers.

Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair.

Homologous recombination (HR) is often used to achieve targeted gene integration because of its higher precision and operability compared with microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). It appears to be inefficient for gene integration in animal cells and embryos due to occurring only during cell division. Here we developed genome-wide high-throughput screening and a subsequently paired crRNA library screening to search for genes suppressing homology-directed repair (HDR). We found that, in the reporter system, HDR cells with knockdown of SHROOM1 were enriched as much as 4.7-fold than those with control. Down regulating SHROOM1 significantly promoted gene integration in human and mouse cells after cleavage by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), regardless of the donor types. The knock-in efficiency of mouse embryos could also be doubled by the application of SHROOM1 siRNA during micro-injection. The increased HDR efficiency of SHROOM1 deletion in HEK293T cells could be counteracted by YU238259, an HDR inhibitor, but not by an NHEJ inhibitor. These results indicated that SHROOM1 was an HDR-suppressed gene and that the SHROOM1 knockdown strategy may be useful for a variety of applications, including gene editing to generate cell lines and animal models for studying gene function and human diseases.

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

BACKGROUND: CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled. RESULTS: Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones. CONCLUSIONS: Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

Removal of Pharmaceutical and Personal Care Products (PPCPs) from Municipal Waste Water with Integrated Membrane Systems, MBR-RO/NF.

This study focuses on the application of combining membrane bioreactor (MBR) treatment with reverse osmosis (RO) or nanofiltration (NF) membrane treatment for removal of pharmaceuticals and personal care products (PPCPs) in municipal wastewater. Twenty-seven PPCPs were measured in real influent with lowest average concentration being trimethoprim (7.12 ng/L) and the highest being caffeine (18.4 ng/L). The results suggest that the MBR system effectively removes the PPCPs with an efficiency of between 41.08% and 95.41%, and that the integrated membrane systems, MBR-RO/NF, can achieve even higher removal rates of above 95% for most of them. The results also suggest that, due to the differences in removal mechanisms of NF/RO membrane, differences of removal rates exist. In this study, the combination of MBR-NF resulted in the removal of 13 compounds to below detection limits and MBR-RO achieved even better results with removal of 20 compounds to below detection limits.

Differential TGFß pathway targeting by miR-122 in humans and mice affects liver cancer metastasis.

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver function. Here we show a functional conservation of miR-122 in the TGFß pathway: miR-122 target site is present in the mouse but not human TGFßR1, whereas a noncanonical target site is present in the TGFß1 5'UTR in humans and other primates. Experimental switch of the miR-122 target between the receptor TGFßR1 and the ligand TGFß1 changes the metastatic properties of mouse and human liver cancer cells. High expression of TGFß1 in human primary liver tumours is associated with poor survival. We identify over 50 other miRNAs orthogonally targeting ligand/receptor pairs in humans and mice, suggesting that these are evolutionarily common events. These results reveal an evolutionary mechanism for miRNA-mediated gene regulation underlying species-specific physiological or pathological phenotype and provide a potentially valuable strategy for treating liver-associated diseases.

[Research progress on the application of IPAT model and its variants].

IPAT model and its variants could describe the quantitative relationships between human driving force and the environmental pressure, benefit fundamental understanding of the dynamics of coupled human and natural systems, and are regarded as effective tools in solving the environmental problems caused by the social and economic development. In this paper, the fundamental concepts of IPAT model and its variants were discussed including definitions, historical developments and the hot issues in their applications. The future research trends were put forward aiming to further expand the use of IPAT models in the sustainable development of environment and social decision-making.

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

TREM-1 as a potential therapeutic target in neonatal sepsis.

OBJECTIVE: Bacterial sepsis in neonates is associated with elevated morbidity and mortality. A role for the pro-inflammatory Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is suspected in the innate immune response to bacteria, but little is known about its activities in infants. To begin exploring the feasibility of treating neonatal sepsis by blocking leukocyte TREM-1, we compared TREM-1 membrane expression and mRNA in newborns without clinical or microbiological evidence of infection, to that of healthy adults. The functionality of pro-inflammatory reactions in leukocyte TREM-1 of newborns was also evaluated. METHODS: Twenty term newborns were enrolled in this study and cord blood samples were collected at birth. For comparison, peripheral blood specimens were collected from 20 healthy adults (control adult, CA). The expression of TREM-1 protein and mRNA in leukocytes was detected with flow cytometry and real-time qPCR, respectively. Whole cord blood was also stimulated by Escherichia coli or blocked by the TREM-1-specific peptide LP17 to identify changes in the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α, as well as soluble TREM-1 (sTREM-1) using enzyme linked immunosorbent assay (ELISA). RESULTS: Mean fluorescence intensity (MFI) of TREM-1 on leukocytes of newborns appeared comparable to healthy adults [monocytes: 37.5 ± 6.7 vs. 37.6 ± 8.7; polymorphonuclear cells (PMNs): 32.9 ± 6.6 vs. 33.6 ± 5.8]. However, the percentage of PMNs positive for TREM-1 was lower in newborns than in healthy adults (82.3 ± 7.1 vs. 98.6 ± 4.8; P < 0.01); the percentage of TREM-1-positive CD14-positive monocytes was comparable to that of healthy adults (97.1 ± 8.3 vs. 97.5 ± 7.4). Exposure of cord blood to E. coli resulted in increased secretion of IL-6, IL-8, TNF-α, and sTREM-1. In contrast, the concentrations of IL-6, IL-8, and TNF-α decreased by a minimum of 15% when TREM-1 was blocked by LP17 then exposed to E. coli, versus E. coli alone. In addition, the concentration of sTREM-1 was positively correlated with the levels of TNF-α (r = 0.519, P < 0.05), IL-6 (r = 0.507, P < 0.05), and IL-8 (r = 0.538, P < 0.05). CONCLUSIONS: Healthy newborns exhibit expression of TREM-1 on monocytes similar to that in healthy adults, and most PMNs express TREM-1 at the newborn stage. Detection of sTREM-1 in neonatal peripheral blood should be further investigated as a potential method for the diagnosis of neonatal infection. Finally, blocking the TREM-1 signal transduction pathway may reduce inflammatory responses of neonate leukocytes and thereby provide a new strategy for treatment of neonatal infection.

The synergistic regulation of VEGF-mediated angiogenesis through miR-190 and target genes.

VEGF is a major contributor to angiogenesis, a vital process in normal growth and development and tumor transition. However, the current clinical efficacy of VEGF inhibitors is limited, and the molecular mechanism underlying VEGF regulation remains to be elucidated. Here we show that miR-190 directly targets a group of angiogenic effectors besides VEGF per se. Noted, these effectors can transcriptionally regulate VEGF expression in an intracellular or intercellular manner, thus demonstrating that miR-190 modulates the VEGF-mediated tumor angiogenesis at three levels. The synergistic effect of miR-190 and its target genes demonstrates a complex but apparently more stable system, allowing for the tight control of the level of VEGF. Finally, we showed that miR-190 significantly suppresses tumor metastasis, especially angiogenesis. Together, these results indicate that miR-190 is a promising antitumor target in clinical applications.

Engineered TAL Effector modulators for the large-scale gain-of-function screening.

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE-DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE-TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation.

miR-19b promotes tumor growth and metastasis via targeting TP53.

Tumor suppressor TP53 (or p53) is one of the most important regulators in numerous physiological and pathological processes. Recently, the miRNA-mediated post-transcription regulation of p53 has been studied. However, systematic studies of miRNA targeting sites within the p53 gene are still a challenging task. Here, we developed a dual-color assay capable of identifying miRNA targeting sites in a certain gene, specifically p53, in a simple, direct, and robust manner. Results showed that p53 was a direct and critical target of miR-19b, but not miR-19a, regardless of sequence similarity. Overexpression of miR-19b observed in human cancer cells can diminish p53 protein levels and, subsequently, downstream components such as Bax and p21. This miR-19b-mediated p53 reduction was shown to promote cell cycle, cell migration or invasion, and repress senescence and apoptosis in vitro. Further investigation revealed that miR-19b controls tumor growth and metastasis in vivo. Therefore, it is possible that miR-19b antagomirs or sponges could be developed as therapeutic agents against tumor development.

An integrative approach for the large-scale identification of human genome kinases regulating cancer metastasis.

Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. FROM THE CLINICAL EDITOR: This team of authors have utilized a self-assembled cell microarray to screen genes that regulate cancer cell migration using a human genome siRNA library of kinases. They validated previously known genes and identified novel ones that may serve as therapeutic targets.

Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp(y11) mutant embryos or cardia bifida phenotypes in fau(tm236a) mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

[Association of genetic polymorphisms of angiotensin converting enzyme and matrix metallo proteinase-1 with idiopathic pulmonary fibrosis].

OBJECTIVE: To investigate the correlation between angiotensin converting enzyme (ACE) and matrix metallo proteinase (MMP)-1 gene polymorphisms and the risk of idiopathic pulmonary fibrosis (IPF) in a Han Chinese population from Hebei Province. METHODS: Eighty-four IPF patients and 100 controls were enrolled from the Second Hospital of Hebei Medical University. Polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect ACE gene insertion/deletion (I/D) polymorphism and MMP-1 polymorphism respectively. The MMP-1 polymorphism was genotyped by DNA sequence analysis. Radioimmunoassay and ELISA were used to analyzed AngII, MMP-1 and TIMP-1 levels in IPF patients and healthy controls. RESULTS: There was a significant difference between the 2 groups in allele and genotype frequency distribution of ACE Insertion/Deletion polymorphism; frequency distribution of DD genotype and D allele of IPF patients were higher than those of the healthy control group (χ(2) = 11.227, 4.318, P < 0.05). There was no difference from different genders and ages on allele and genotype frequency distribution of ACE Insertion/Deletion polymorphism. (χ(2) = 0.03 - 1.069, P > 0.05). There was no significant difference between the 2 groups in genotype and allele frequency distribution of MMP-1 1G/2G polymorphism (χ(2) = 0.94 and 0.001, P > 0.05). The AngII levels from DD genotype of both IPF patients and healthy controls were the highest, followed by the DI genotype and the II genotype. The AngII level of any genotype for ACE Insertion/Deletion polymorphism in the IPF group was higher than that in the healthy control group (all P < 0.05). The serum level of AngII, MMP-1 and TIMP-1, as well as MMP-1/TIMP-1 ratio in the IPF group were higher than those in the healthy control group (all P < 0.05). CONCLUSIONS: The ACE polymorphism might be associated with IPF, and the serum level of AngII was affected not only by the genetic background of ACE insertion/deletion polymorphism but also the environmental factors. The MMP-1 1G/2G polymorphism might be weakly associated with IPF.

An integrated chip for the high-throughput synthesis of transcription activator-like effectors.

A TALE of two assays: Transcription activator-like effectors (TALEs) are programmable proteins that can specifically recognize a DNA sequence. Previous strategies for the synthesis of TALEs were complicated and time-consuming. The solid-phase synthesis strategy demonstrated here allows quick and simple purification of the ligation product.

A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis.

miRNA globally deregulates human carcinoma. A critical open question is how many miRNAs functionally participate in cancer development, particularly in metastasis. We systematically evaluate the capability of all known human miRNAs to regulate certain metastasis-relevant cell behaviours. To perform the high-throughput screen of miRNAs, which regulate cell migration, we developed a novel self-assembled cell microarray. Here we show that over 20% of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. MiR-23b, which is downregulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including tumour growth, invasion and angiogenesis in vivo. It regulates a cohort of prometastatic targets, including FZD7 or MAP3k1. These findings provide new insight into the physiological and potential therapeutic importance of miRNAs as a new class of functional modulators.