An RF-Ultrasound Relay for Adaptive Wireless Powering Across Tissue Interfaces.

Single modality wireless power transfer has limited depth for mm-sized implants across air / tissue or skull / tissue interfaces because they either suffer from high loss in tissue (RF, Optical) or high reflection at the medium interface (Ultrasound (US)). This paper proposes an RF-US relay chip at the media interface avoiding the reflection at the boundary, and enabling efficient wireless powering to mm-sized deep implants across multiple media. The relay chip rectifies the incoming RF power through an 85.5% efficient RF inductive link (across air) using a multi-output regulating rectifier (MORR) with 81% power conversion efficiency (PCE) at 186 mW load, and transmits ultrasound using adiabatic power amplifiers (PAs) to the implant in order to minimize cascaded power loss. To adapt the US focus to implant movement or placement, beamforming was implemented using 6 channels of US PAs with 2-bit phase control (0, 90, 180, and 270°) and 3 different amplitudes (6-29, 4.5, and 1.8 V) from the MORR. The adiabatic PA contributes a 30-40% increase in efficiency over class-D and beamforming increases the efficiency by 251% at 2.5 cm over fixed focusing. The proof-of-concept powering system for a retinal implant, from an external PA on a pair of glasses to a hydrophone with 1.2 cm (air) + 2.9 cm (agar eyeball phantom in mineral oil) separation distance, had a power delivered to the load (PDL) of 946 µW. The 2.3 × 2 mm2 relay chip was fabricated in a 180 nm high-voltage (HV) BCD process.

The Rab-binding Profiles of Bacterial Virulence Factors during Infection.

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.

Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors.

The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.

Internet gambling among high school students in Hong Kong.

The study investigated Internet gambling involvement and pathological gambling among Hong Kong adolescents aged 12-19 years. The diagnostic and statistical manual (4th edition) multiple response format for juveniles (DSM-IV-MR-J) (Fisher in J Gambl Stud 16:253-273, 2000) was filled by 1,004 students (597 boys, 407 girls) recruited by random selection of classes. The response rate was 86.6 %. Results indicate that more respondents participated in land-based gambling than Internet gambling (63.5 vs. 3.5 %) but online gamblers are 1.5 and 3.2 times more likely to develop pathological and at-risk gambling than non-Internet gamblers. Using the DSM-IV-MR-J criteria, 5.7 and 22.9 % of the Internet gamblers could be classified as at-risk gamblers and pathological gamblers, respectively. Majority (94.3 %) wagered online at home, and 91.4 % made their first bet before 18 years. Many perceived Internet gambling as a trendy (71.4 %) and safe entertainment (54.3 %). Problematic Internet gambling was significantly associated with the male gender, school grades, online gambling frequency, amount wagered and a gambling family environment. Survey results have implications for gambling research and preventive programs.

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines.

Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification.

The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.

Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ.

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.IMPORTANCE Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. Citrobacter rodentium is a mouse pathogen used to model EPEC and EHEC pathogenesis in vivo Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors in vivo are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the in vivo processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type C. rodentium or an espJ catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response in vivo.

Macau parents' perceptions of underage children's gambling involvement.

OBJECTIVES: The study examined Macau parents' perceptions of underage children's gambling involvement, and parents' attitudes towards help seeking if their children had a gambling problem. The parents' gambling behavior in the past year was also investigated. METHODS: This is a parent survey using a self-administered questionnaire. A convenience sample of 311 Macau parents (106 fathers and 205 mothers) with underage children aged 3-17 years was recruited. The response rate is 77.8%. The participants were asked if they had ever approved or taught their underage children to gamble, and how did they award their children when they won in gambling games. The parents were also asked if they had gambled in the previous 12 months, and their gambling behavior was assessed by the Chinese Problem Gambling Severity Index (CPGSI). RESULTS: Half of the parents surveyed (52%) did not approve underage gambling but 81% taught their underage children to play different gambling games. Children were awarded with money (55%), praises (17.5%), toys (15%) and food (12.5%) when they won in games. One-fifth (20.6%) were distressed with their children's gambling problem. Many (68.8%) were willing to seek help to cope with children's gambling problems. Only 21.2% (n = 66) of the parents reported gambling in the past year. Using the CPGSI, 4.5% of these gamblers could be identified as problem gamblers, and 16.7% were moderate-risk gamblers. CONCLUSION: The study results indicate parent education should be included in prevention of underage gambling.

Gambling behavior among Macau college and university students.

This survey investigated gambling behavior among Chinese students studying in Macau colleges and universities. It also aimed to examine the relationship between problem gambling, affect states and sensation seeking propensity. A convenience sample of 999 students (370 men, 629 women) filled a self-administered questionnaire consisted of the Problem Gambling Severity Index (PGSI) (Ferris and Wynne in The Canadian problem gambling index: User manual. Canadian Centre on Substance Abuse, Toronto 2001a), the 8-item Brief Sensation Seeking Scale (BSSS-8) (Hoyle et al. Pers Individ Diff 32(3): 401-414, 2002), Bradburn's Affect Balance Scale (BABS) (Bradburn in The structure of psychological well-being. Aldine, Chicago 1969) and questions on gambling activities. The response rate is 65%. Results indicate 32.3% (n = 323) of the survey participants wagered on mahjong (61.8%), soccer matches (40.2%), Mark Six lottery (37.2%), card games (28.1%), land-based casino gambling (13.1%), slot machines (7.5%) and online casino games (2.0%). The average monthly stake was MOP $411. Seeking entertainment (18.7%), killing time (12.5%) and peer influence (11.1%) were the three main reasons for gambling. Using the PGSI, 3.6 and 5.3% of the students could be identified as moderate-risk and problem gamblers respectively. Men were significantly more vulnerable to gambling problems (X2(1) = 35.00, p < 0.01) than women. Most of the problematic gamblers (76%) made their first bet before 14 years. The PGSI scores are significantly correlated with the BSSS-8 scores (r = 0.23, p < 0.01) but not with the overall ABS scores (r = -0.06, p > 0.05). The study findings inform campus prevention programs and future research.

A Bidirectional Neural Interface IC With Chopper Stabilized BioADC Array and Charge Balanced Stimulator.

We present a bidirectional neural interface with a 4-channel biopotential analog-to-digital converter (bioADC) and a 4-channel current-mode stimulator in 180 nm CMOS. The bioADC directly transduces microvolt biopotentials into a digital representation without a voltage-amplification stage. Each bioADC channel comprises a continuous-time first-order ∆Σ modulator with a chopper-stabilized OTA input and current feedback, followed by a second-order comb-filter decimator with programmable oversampling ratio. Each stimulator channel contains two independent digital-to-analog converters for anodic and cathodic current generation. A shared calibration circuit matches the amplitude of the anodic and cathodic currents for charge balancing. Powered from a 1.5 V supply, the analog and digital circuits in each recording channel draw on average [Formula: see text] and [Formula: see text] of supply current, respectively. The bioADCs achieve an SNR of [Formula: see text] and a SFDR of [Formula: see text] , for better than 9-b ENOB. Intracranial EEG recordings from an anesthetized rat are shown and compared to simultaneous recordings from a commercial reference system to validate performance in-vivo . Additionally, we demonstrate bidirectional operation by recording cardiac modulation induced through vagus nerve stimulation, and closed-loop control of cardiac rhythm. The micropower operation, direct digital readout, and integration of electrical stimulation circuits make this interface ideally suited for closed-loop neuromodulation applications.

Prevalence estimates of problem and pathological gambling in Hong Kong.

OBJECTIVE: This government-commissioned survey gauged the prevalence of problem and pathological gambling in the general population of Hong Kong and advised the government on preventative and treatment services. METHOD: A modified Chinese version of the DSM-IV Gambling-Behavior Index was administered to 2,004 Hong Kong residents by means of telephone interviews. The respondents were recruited through random sampling. RESULTS: Results showed that 4.0% and 1.8% of the respondents could be classified as problem and pathological gamblers, respectively. Significant differences between the survey sample and the respondents classified as problem and pathological gamblers were found in sex and education. The predictors of problem and pathological gambling were sex, education level, family income, and three specific forms of gambling: betting on horse racing, soccer games, and casino games. CONCLUSIONS: Survey results promoted preventative and treatment services.

A new method to determine in vivo interactomes reveals binding of the Legionella pneumophila effector PieE to multiple rab GTPases.

UNLABELLED: Legionella pneumophila, the causative agent of Legionnaires' disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the Legionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the Escherichia coli biotin ligase BirA and infected the cells with L. pneumophila expressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni(2+) nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity. IMPORTANCE: The respiratory pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently used in vitro approaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of the Legionella infection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.