Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Elucidation of enzymes involved in the biosynthetic pathway of bioactive polyacetylenes in Bidens pilosa using integrated omics approaches.

Polyacetylene compounds from Bidens pilosa are known to have several pharmacological activities. In this study, we identified major genes encoding enzymes involved in the biosynthesis of polyacetylene in B. pilosa. Seven polyacetylene metabolites present in B. pilosa leaves were induced by methyl jasmonate (MeJA) treatment and physical wounding. Transcriptome analysis via high-throughput sequencing revealed 39 202 annotated gene fragment sequences. A DNA microarray established by the 39 202 annotated genes was used to profile gene expression in B. pilosa leaf and root tissues. As no polyacetylene compounds were found in roots, the gene expression pattern in root tissue was used as a negative control. By subtracting MeJA-induced genes in roots, we obtained 1216 genes in leaves showing an approximate three-fold increase in expression post-MeJA treatment. Nine genes encoding enzymes with desaturation function were selected for confirmation of expression by qRT-PCR. Among them, two genes, BPTC030748 and BPTC012564, were predicted to encode Δ12-oleate desaturase (OD) and Δ12-fatty acid acetylenase (FAA), respectively. In B. pilosa leaves, RNAi knock-down concomitantly decreased, while virus-mediated transient overexpression of either gene elevated polyacetylene content. In summary, we demonstrate that two important enzymes, Δ12-oleate desaturase and Δ12-fatty acid acetylenase, involved in desaturation of linear fatty acid precursors play a role in polyacetylene biosynthesis in an important medicinal plant, Bidens pilosa.

Transformation and Characterization of Δ12-Fatty Acid Acetylenase and Δ12-Oleate Desaturase Potentially Involved in the Polyacetylene Biosynthetic Pathway from Bidens pilosa.

Bidens pilosa is commonly used as an herbal tea component or traditional medicine for treating several diseases, including diabetes. Polyacetylenes have two or more carbon-carbon triple bonds or alkynyl functional groups and are mainly derived from fatty acid and polyketide precursors. Here, we report the cloning of full-length cDNAs that encode Δ12-fatty acid acetylenase (designated BPFAA) and Δ12-oleate desaturase (designated BPOD) from B. pilosa, which we predicted to play a role in the polyacetylene biosynthetic pathway. Subsequently, expression vectors carrying BPFAA or BPOD were constructed and transformed into B. pilosa via the Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of transgenes and selection marker genes in the obtained transgenic lines. The copy numbers of transgenes in transgenic lines were determined by Southern blot analysis. Furthermore, 4-5 FAA genes and 2-3 OD genes were detected in wild-type (WT) plants. Quantitative real time-PCR revealed that some transgenic lines had higher expression levels than WT. Western blot analysis revealed OD protein expression in the selected transformants. High-performance liquid chromatography profiling was used to analyze the seven index polyacetylenic compounds, and fluctuation patterns were found.

TESC Promotes TGF-α/EGFR-FOXM1-Mediated Tumor Progression in Cholangiocarcinoma.

Cholangiocarcinoma is a relatively uncommon but highly lethal malignancy. Improving outcomes in patients depends on earlier diagnosis and appropriate treatment; however, no satisfactory diagnostic biomarkers or targeted therapies are currently available. To address this shortcoming, we analyzed the transcriptomic datasets of cholangiocarcinoma from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and found that TESC is highly expressed in cholangiocarcinoma. Elevated cellular levels of TESC are correlated with larger tumor size and predict a poor survival outcome for patients. Knockdown of TESC via RNA interference suppresses tumor growth. RNA-sequencing analysis showed that silencing of TESC decreases the level of FOXM1, leading to cell cycle arrest. Correlation analysis revealed that the cellular level of TESC is correlated with that of FOXM1 in cholangiocarcinoma patients. We further observed that upon TGF-α induction, TESC is upregulated through the EGFR-STAT3 pathway and mediates TGF-α-induced tumor cell proliferation. In vivo experiments revealed that knockdown of TESC significantly attenuates tumor cell growth. Therefore, our data provide novel insight into TESC-mediated oncogenesis and reveal that TESC is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

OCT4B mediates hypoxia-induced cancer dissemination.

Hypoxia, the reduction of oxygen levels in cells or tissues, elicits a set of genes to adjust physiological and pathological demands during normal development and cancer progression. OCT4, a homeobox transcription factor, is essential for self-renewal of embryonic stem cells, but little is known about the role of OCT4 in non-germ-cell tumorigenesis. Here, we report that hypoxia stimulates a short isoform of OCT4, called OCT4B, via a HIF2α-dependent pathway to induce the epithelial-mesenchymal transition (EMT) and facilitate cancer dissemination. OCT4B overexpression decreased epithelial barrier properties, which led to an increase in cell migration and invasion in lung cancer cells. OCT4B knockdown attenuated HIF2α-induced EMT and inhibited cancer dissemination in cell-line and animal models. We observed that OCT4B bound the SLUG promoter and enhanced its expression, and SLUG silencing inhibited OCT4B-mediated EMT, accompanied with decreased cell migration and invasion. Correlation analysis revealed that OCT4B expression was significantly associated with the SLUG level in lung tumors. These results provide novel insights into OCT4B-mediated oncogenesis in cancer dissemination.

Establishment of a mouse model for the complete mosquito-mediated transmission cycle of Zika virus.

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host.

Experience of Surgical Treatment for Occipital Migraine in Taiwan.

PURPOSE: Refractory migraine surgery developed since 2003 has excellent results over the past 10 years. According to the pioneer of migraine surgery, Dr. Bahman Guyuron, 5 major surgical classifications of migraines are described in the field of plastic surgery, namely, frontal migraine, temporal migraine, rhinogenic migraine, occipital migraine, and auriculotemporal migraine. In this study, we present the preliminary surgical results of the occipital migraine surgery. MATERIALS AND METHODS: A total of 22 patients with simple occipital migraines came to our outpatient clinic for help from June 2014 to February 2015. Thirteen cases were excluded owing to ineligibility for operation or other reasons. The patients who concurrently experienced other types of migraines were precluded even if they received combined migraine surgery. Therefore, 9 simple occipital migraine cases were enrolled in this study. Migraine severity was evaluated by uniform questionnaires to identify the source of migraine. Neurolysis was performed under general anesthesia, with the patient in a prone position. Postoperative conditions were evaluated at the second, fourth, sixth, and eighth weeks by posttreatment questionnaires. RESULTS: Of all the 9 patients, 5 experienced single-sided migraines of greater occipital nerve origin (2 left-sided and 3 right-sided cases). Two patients had bilateral migraines of greater occipital nerve origin, and unilateral right lesser occipital nerve origin was noted in one patient. The last patient had right-sided migraines of greater and lesser occipital nerve origin. As a result in the follow-up, a response rate greater than 90% was documented, and complete resolution was observed in 2 patients. Drug doses were reduced more than 50% in the remaining patients. The overall efficacy of occipital migraine surgery in this study was 88.8% (8/9 cases). CONCLUSION: Some patients with migraine are good candidates for surgical resolution with appropriate and meticulous selection. Similar to what is observed in Western countries, the migraine surgery is promising and could provide a better quality of life to selected refractory migraine patients in Taiwan.

Low-power wireless ECG acquisition and classification system for body sensor networks.

A low-power biosignal acquisition and classification system for body sensor networks is proposed. The proposed system consists of three main parts: 1) a high-pass sigma delta modulator-based biosignal processor (BSP) for signal acquisition and digitization, 2) a low-power, super-regenerative on-off keying transceiver for short-range wireless transmission, and 3) a digital signal processor (DSP) for electrocardiogram (ECG) classification. The BSP and transmitter circuits, which are the body-end circuits, can be operated for over 80 days using two 605 mAH zinc-air batteries as the power supply; the power consumption is 586.5 µW. As for the radio frequency receiver and DSP, which are the receiving-end circuits that can be integrated in smartphones or personal computers, power consumption is less than 1 mW. With a wavelet transform-based digital signal processing circuit and a diagnosis control by cardiologists, the accuracy of beat detection and ECG classification are close to 99.44% and 97.25%, respectively. All chips are fabricated in TSMC 0.18-µm standard CMOS process.

A tactile vision substitution system for the study of active sensing.

This paper presents a tactile vision substitution system (TVSS) for the study of active sensing. Two algorithms, namely image processing and trajectory tracking, were developed to enhance the capability of conventional TVSS. Image processing techniques were applied to reduce the artifacts and extract important features from the active camera and effectively converted the information into tactile stimuli with much lower resolution. A fixed camera was used to record the movement of the active camera. A trajectory tracking algorithm was developed to analyze the active sensing strategy of the TVSS users to explore the environment. The image processing subsystem showed advantageous improvement in extracting object's features for superior recognition. The trajectory tracking subsystem, on the other hand, enabled accurately locating the portion of the scene pointed by the active camera and providing profound information for the study of active sensing strategy applied by TVSS users.

A wireless ECG acquisition and classification system for body sensor networks.

This paper demonstrates a wireless ECG acquisition and classification system with a bio-signal processor (BSP), a super regenerative transceiver, and a digital signal processor (DSP). The BSP, which is implemented with low complexity architecture, includes only a low noise amplifier with chopping techniques and a high-pass sigma-delta modulator (HPSDM). The super-regenerative on-off keying (OOK) transceiver is applied for the low power, short range transmission and low date rate wireless communication. For the signal processing and analyzing, the DSP circuit is adopted in the receiver. The whole system is implemented in a TSMC 0.18 µm 1P6M CMOS process under the supply voltage of 1.2 V. In the near body node, the power consumption including a BSP and a transmitter is 587 µW only. With two PR44 zinc-air batteries of 605 mAh, the near body node circuit can be operated about 100 days. In the receiving node, the power consumption with a receiver and a DSP is 926 µW.

A low-power 13.56 MHz RF front-end circuit for implantable biomedical devices.

A low-power fully-integrated CMOS RF front-end circuit for a passive 13.56 MHz biomedical implant is presented. A 13.56 MHz binary phase shift keying (BPSK) signal is received by an internal coil. This front-end circuit is composed of a full-wave bridge rectifier, a linear regulator, a BPSK demodulator, and a clock/data recovery (CDR). A full-wave bridge rectifier converts the carrier waveform with the BPSK signal to an unregulated DC voltage. A linear regulator stabilizes the unregulated DC voltage to 1.8 V that serves as the DC source for the implant. A BPSK demodulator detects the incoming BPSK signal from the internal coil and translates the demodulated data to the CDR which can successfully recover the clock and data for the system controller. This chip with a core area of 0.45 mm(2) has been fabricated in a TSMC 0.18 µm 1P6M CMOS technology. The total power consumed is only 632 µW.

Wireless front-end with power management for an implantable cardiac microstimulator.

Inductive coupling is presented with the help of a high-efficiency Class-E power amplifier for an implantable cardiac microstimulator. The external coil inductively transmits power and data with a carrier frequency of 256 kHz into the internal coil of electronic devices inside the body. The detected cardiac signal is fed back to the external device with the same pair of coils to save on space in the telemetry device. To maintain the power reliability of the microstimulator for long-term use, two small rechargeable batteries are employed to supply voltage to the internal circuits. The power management unit, which includes radio frequency front-end circuits with battery charging and detection functions, is used for the supply control. For cardiac stimulation, a high-efficiency charge pump is also proposed in the present paper to generate a stimulated voltage of 3.2 V under a 1 V supply voltage. A phase-locked-loop (PLL)-based phase shift keying demodulator is implemented to efficiently extract the data and clock from an inductive AC signal. The circuits, with an area of 0.45 mm², are implemented in a TSMC 0.35 µm 2P4M standard CMOS process. Measurement results reveal that power can be extracted from the inductive coupling and stored in rechargeable batteries, which are controlled by the power management unit, when one of the batteries is drained. Moreover, the data and clock can be precisely recovered from the coil coupling, and a stimulated voltage of 3.2 V can be readily generated by the proposed charge-pump circuits to stimulate cardiac tissues.

A free composite de-epithelialized anterolateral thigh and the vastus lateralis muscle flap for the reconstruction of a large defect of the anterior skull base: a case report.

Reconstruction of the anterior skull base is one of the greatest challenges for reconstructive surgeons. Sometimes, the defect is so large that a local flap is insufficient for the reconstruction. In this report, we present a case of malignant meningioma of the anterior skull base. The tumor was treated by surgical excision resulting in a large defect from the anterior skull base to the nasal cavity. The entire defect was within the cranial vault. The reconstruction was achieved using a free composite de-epithelialized anterolateral thigh and the vastus lateralis muscle flap. Postoperative monitoring included hand Doppler and daily endoscopic inspection. This patient was satisfied with the cosmetic result. After 10 months, magnetic resonance imaging (MRI), performed to assess the flap, demonstrated that the volume of the de-epithelialized skin paddle of the anterolateral thigh flap had not changed, and that there was no tissue atrophy between the patient's eyes that could have resulted in deformity.

Design and synthesis of iridium bis(carbene) complexes for efficient blue electrophosphorescence.

Five iridium bis(carbene) complexes, [Ir(pmi)(2)(pypz)] (1), [Ir(mpmi)(2)(pypz)] (2), [Ir(fpmi)(2)(pypz)] (3), [Ir(fpmi)(2)(pyim)] (4), and [Ir(fpmi)(2)(tfpypz)] (5) (pmi=1-phenyl-3-methylimdazolin-2-ylidene-C,C(2'); fpmi=1-(4-fluorophenyl)-3-methylimdazolin-2-ylidene-C,C(2'); mpmi=1-(4-methyl-phenyl)-3-methylimdazolin-2-ylidene-C,C(2'); pypz=2-(1H-pyrazol-5-yl)pyridinato; pyim=2-(1H-imidazol-2-yl)pyridinato; and tfpypz=2-(3-(trifluoromethyl)-1H-pyrazol-5-yl)pyridinato), were synthesized and their structures were characterized by NMR spectroscopy, mass spectroscopy and X-ray diffraction. These complexes showed phosphorescent emission with the emission maxima between 453 and 490 nm. Various spectrophotometric measurements, cyclic voltammetric studies, and density functional theory (DFT) calculations show that, unlike most of the phosphorescent cyclometalated iridium complexes, the lowest unoccupied molecular orbital (LUMO) energy and the emissive state of these iridium complexes are mainly controlled by the N,N'-heteroaromatic (N^N) ligand. Despite the fact that the LUMO levels of these complexes are mainly on the N^N ligands, the efficiencies of the electroluminescent (EL) devices are very high. For example, the EL devices using [Ir(mpmi)(2)(pypz)], [Ir(fpmi)(2)(pypz)], and [Ir(fpmi)(2)(tfpypz)] as the dopant emitters exhibited light- to deep-blue electrophosphorescence with external quantum efficiencies of 15.2, 14.1, and 7.6% and Commission Internationale d'Énclairage (x,y) coordinates (CIE(x,y)) of (0.14, 0.27), (0.14, 0.18) and (0.14, 0.10), respectively.

Salvage of the skin envelope in complex incomplete avulsion injury of thumb with venous arterializaiton: a case report.

Treatment of an avulsion or degloving injury of the hand is a difficult but not unusual operation for plastic reconstructive or hand surgeons. The avulsion may be salvaged by arteriovenous shunting technique. We present a patient with incomplete avulsion injury of the distal phalanx of thumb. Arteriovenous shunting was created and the wound reconstructed primarily under venous arterialization. The avulsed skin envelope was survived well and functional status was improved.

An easy-to-use tool for dressing finger injuries painlessly.

Finger injuries are among the most common injuries treated in the emergency department. After surgical management, surgeons find that patients with finger injuries will sometimes experience severe tenderness when dressings are being changed. To reduce patients' discomfort an easy-to-use tool for painless finger wound dressing has been developed.