Lan Xie

MicroRNAs‐372/373 promote the expression of hepatitis B virus through the targeting of nuclear factor I/B

MicroRNAs (miRNAs) play important roles in the posttranscriptional regulation of gene expression. Recent evidence has indicated the pathological relevance of miRNA dysregulation in hepatitis virus infection; however, the roles of microRNAs in the regulation of hepatitis B virus (HBV) expression are still largely unknown. In this study we identified that miR‐373 was up‐regulated in HBV‐infected liver tissues and that the members of the miRs‐371‐372‐373 (miRs‐371‐3) gene cluster were also significantly co‐up‐regulated in HBV‐producing HepG2.2.15 cells. A positive in vivo association was identified between hepatic HBV DNA levels and the copy number variation of the miRs‐371‐3 gene cluster. The enhanced expression of miRs‐372/373 stimulated the production of HBV proteins and HBV core‐associated DNA in HepG2 cells transfected with 1.3×HBV. Further, nuclear factor I/B (NFIB) was identified to be a direct functional target of miRs‐372/373 by in silico algorithms and this was subsequently confirmed by western blotting and luciferase reporter assays. Knockdown of NFIB by small interfering RNA (siRNA) promoted HBV expression, whereas rescue of NFIB attenuated the stimulation in the 1.3×HBV‐transfected HepG2 cells. Conclusion: Our study revealed that miRNA (miRs‐372/373) can promote HBV expression through a pathway involving the transcription factor (NFIB). This novel model provides new insights into the molecular basis in HBV and host interaction. (HEPATOLOGY 2011;)

Integration of Sperm Motility and Chemotaxis Screening with a Microchannel-Based Device

BACKGROUND Sperm screening is an essential step in in vitro fertilization (IVF) procedures. The swim-up method, an assay for sperm motility, is used clinically to select the ideal sperm for subsequent manipulation. However, additional parameters, including acrosome reaction capability, chemotaxis, and thermotaxis, are also important indicators of mammalian sperm health. To monitor both sperm motility and chemotaxis simultaneously during sperm screening, we designed and constructed a microdevice comprising a straight channel connected with a bibranch channel that mimics the mammalian female reproductive tract. METHODS The width and length of the straight channel were optimized to select the motile sperms. We selectively cultured cumulus cells in the bibranch channel to generate a chemoattractant-forming chemical gradient. Sperm chemotaxis was represented by the ratio of the sperm swimming toward different branches. RESULTS The percentage of motile sperms improved from 58.5% (3.8%) to 82.6% (2.9%) by a straight channel 7 mm in length and 1 mm in width. About 10% of sperms were found to be chemotactically responsive in our experiment, which is consistent with previous studies. CONCLUSIONS For the first time, we achieved the combined evaluation of both sperm motility and chemotaxis. The motile and chemotactically responsive sperms can easily be enriched on a lab-on-a-chip device to improve IVF outcome.

In vitro fertilization on a single-oocyte positioning system integrated with motile sperm selection and early embryo development.

In vitro fertilization (IVF) technology has been broadly applied to solve human infertility in recent years. However, the physical tools for IVF remain unchanged over several decades before microfluidic technology was introduced in this field. Here, we report a novel microdevice that integrates each step of IVF, including oocyte positioning, sperm screening, fertilization, medium replacement, and embryo culture. Oocytes can be singly positioned in a 4 × 4 array of octacolumn units. The four symmetrical straight channels, crossing at the oocyte positioning region, allowed efficient motile sperm selection and facilitated rapid medium replacement. The fertilization process and early embryonic development of the individual zygote was traced with microscopic recording and analyzed by in situ fluorescent staining. The murine sperm motility was increased from 60.8 ± 3.4% to 96.1 ± 1.9% through the screening channels. The embryo growth rate and blastocyst formation were similar between the routine Petri dish group and the microdevice group. The healthy blastocysts developed in the microdevice could be conveniently retrieved through a routine pipetting operation and used for further embryo transfer.

Hepatitis B Virus Encoded X Protein Suppresses Apoptosis by Inhibition of the Caspase-Independent Pathway

Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV.

Rapid fabrication of a microdevice with concave microwells and its application in embryoid body formation

Here, we report a novel method for the fabrication of polydimethylsiloxane microdevices with complicated 3-D structures, such as concave and crater shapes, using an easily machined polymethyl methacrylate mold combined with a one-step molding process. The procedure presented here enables rapid preparation of complex 3-D microstructures varying in shape and dimensions. To regulate embryoid body (EB) formation, we fabricated a microfluidic device with an array of concave microwells and found that EBs growing in microwells maintained their shape, viability, and a high degree of homogeneity. We believe that this novel method provides an alternative for rapid prototyping, especially in fabricating devices with curved 3-D microstructures.

The construction of an interfacial valve-based microfluidic chip for thermotaxis evaluation of human sperm

Thermotaxis has been demonstrated to be an important criterion for sperm evaluation, yet clinical assessment of thermotaxis capacity is currently lacking. In this article, the on-chip thermotaxis evaluation of human sperm is presented for the first time using an interfacial valve-facilitated microfluidic device. The temperature gradient was established and accurately controlled by an external temperature gradient control system. The temperature gradient responsive sperm population was enriched into one of the branch channels with higher temperature setting and the non-responsive ones were evenly distributed into the two branch channels. We employed air-liquid interfacial valves to ensure stable isolation of the two branches, facilitating convenient manipulation of the entrapped sperm. With this device, thermotactic responses were observed in 5.7%-10.6% of the motile sperm moving through four temperature ranges (34.0-35.3 °C, 35.0-36.3 °C, 36.0-37.3 °C, and 37.0-38.3 °C, respectively). In conclusion, we have developed a new method for high throughput clinical evaluation of sperm thermotaxis and this method may allow other researchers to derive better IVF procedure.

Systematic profiling of mRNA and miRNA expression in the pancreatic islets of spontaneously diabetic Goto-Kakizaki rats.

Type 2 diabetes (T2DM) is a complex multifactorial metabolic disorder that affects >100 million individuals worldwide, yet the mechanisms involved in the development and progression of the disease have not yet been fully elucidated. The present study examined the mRNA and micro (mi)RNA expression profiles by microarray analysis in the pancreas islets of spontaneously diabetic Goto-Kakizaki rats with the aim to identify regulatory mechanisms underlying the pathogenesis of T2DM. A total of 9 upregulated and 10 downregulated miRNAs were identified, including miR-150, miR-497, miR-344-3p and let-7f, which were independently validated by quantitative polymerase chain reaction assays. In addition, differential expression of 670 genes was detected by mRNA microarray analysis, including 370 upregulated and 247 downregulated genes. The differentially expressed genes were statistically associated with major cellular pathways, including the immune response pathway and the extracellular matrix (ECM)-receptor interaction pathway. Finally, a reverse regulatory association of differentially expressed miRNAs and their predicted target genes was constructed, supported by analysis of their mRNA and miRNA expression profiles. A number of key pairs of miRNA-mRNA was proposed to have significant roles in the pathogenesis of T2DM rats based on bioinformatics analysis, one example being the let-7f/collagen, type II, alpha 1 pair that may regulate ECM-receptor interactions.

A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia

An air-insulated microfluidic chip was designed for the automatic centrifugal distribution of samples to 24-test cells, enabling the parallel identification of multiple clinical pneumonia-related pathogens in 1.45-μL reactions without cross-contamination in 45 min. A portable nucleic acid analyzer that integrates mechanical, confocal optical, electronic, and software functions was also developed to collect fluorescence data in a Ø3 mm imaging field near the optical diffraction limit for highly sensitive fluorescence detection of nucleic acid amplification in real time. This microfluidic chip-based portable nucleic acid analyzer could detect low abundance nucleic acids present at as few as 10 copies. In a blinded experiment, specific identification of Mycoplasma pneumoniae, Staphylococcus aureus, and methicillin-resistant S. aureus was achieved with 229 clinical patient sputum samples. The total coincidence rate of our system and traditional RT-PCR with an ABI 7500 was 99.56%. Four samples accounting for the 0.44% inconformity were retested by gene sequencing, revealing that our system reported the correct results. This novel microfluidic chip-based detection system is cost-effective, rapid, sensitive, specific, and has a relatively high throughput for parallel identification, which is especially suitable for resource-limited facilities/areas and point-of-care testing.

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis.

In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the devices microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting.

A microfluidic “treadmill” for sperm selective trapping according to motility classification

Sperm motility analysis is an indispensable test in male infertility diagnosis. The current microscopic method cannot select the sperm of specific velocity for further in vitro Fertilization (IVF) usage or research purposes. Here, we report a novel and simple microfluidic device capable of balancing sperms swimming speed with local flow velocity. The results suggested that sperm motility was quantitatively graded and sperm with different average straight line velocity (VSL) 40.5 ± 3.0, 27.2 ± 0.6 and 18.7 ± 0.6 µm/s respectively were automatically trapped in corresponding regions. Sperm motility classification (SMC) and sperm trapping may facilitate both the diagnosis of male infertility and further studies on sperm physiological behavior.