A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated CD4 downregulation.

Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery-it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of dimethylsulfoxide and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

Analysis of Channel Current Noise in Small Nanoscale Metal Oxide Semiconductor Field Effect Transistors.

The shrinkage of metal oxide semiconductor field effect transistor (MOSFET) to the small size of the nanoscale results in changes in their channel current noise composition. This paper determines the channel current noise composition of 90 nm MOSFET through experiments, and according to the device material and noise characteristics analysis, the channel current noise of 90 nm and below is obtained, which not only contains thermal noise and suppressed channel shot noise, but also adds suppressed gate tunneling shot noise and cross-correlation noise. Then, Monte Carlo simulation of 10 nm MOSFET noise is further used to determine the channel current composition of small size nanoscale devices. Subsequently, based on the device structure and fundamental characteristics of channel current noise, the channel current noise model is established. Finally, this model is employed to analyze the relationship between thermal noise, suppressed shot noise, cross-correlation noise, and channel current noise in relation to bias parameters and device characteristics. The theoretical results are basically consistent with the experimental and the simulated results, and the channel noise increases with the increase of bias voltage. This achievement holds promise for enhancing the operational efficiency, reliability, and lifetime of nanoscale small-sized MOSFET devices.

Fluorescent Noncovalent Organic Framework for Supporting Gold Nanoparticles as Heterogeneous Catalyst with Merits of Easy Detection and Recycle.

A porous noncovalent organic framework with AIE effect is designed and synthesized as the support for gold nanoparticles (AuNPs). The framework is fabricated through the electrostatic complexation between carboxymethyl cellulose and tetraphenylethene-containing ammonium surfactant, which can complex AuNPs via the noncovalent interactions to offer a heterogeneous catalyst. Compared to the covalent modification on cellulose, this noncovalent framework gains superiorities in the catalyst synthesis and the size control of AuNPs. The AIE property and water-insolubility allow such heterogeneous catalysts to be easily detected, separated, and recycled, opening a new pathway for the reduction of nitrobenzene compounds and some dye compounds in aqueous conditions, which present the features of green chemistry. The use of cellulose for developing new heterogeneous metal catalysts, especially in a noncovalent way, would promote the value-added utilization of cellulose. This work provides a design strategy for gaining heterogeneous metal catalysts by taking advantage of natural bioresources.

Effect of Short-term Deep Breathing Exercises on Perioperative Anxiety and Pain in Pediatric Orthopedic Patients: A Randomized Controlled Trial.

PURPOSE: There are currently no pediatric studies examining the effects of deep breathing on perioperative pain and anxiety. This study sought to determine the effect of short-term deep breathing exercises on perioperative anxiety and pain in pediatric patients and their parents. DESIGN: A randomized controlled trial was conducted in the Department of Orthopaedic Surgery where pediatric patients about to undergo surgery were allocated to a control group or a deep breathing group. In the intervention group, patients and their main guardian were guided to practice 10 minutes of deep breathing exercises twice a day for 3 to 4 days prior to surgery. Perioperative anxiety and pain were measured for both the children and parents as outcome indicators. METHODS: Perioperative anxiety was measured using the modified Yale Preoperative Anxiety Scale-Short Form (mYPAS-SF) and state anxiety was measured using the State-Trait Anxiety Inventory (STAI). Patients reported their pain levels daily using the Wong-Baker FACES Pain Rating Scale. The following cutoffs were determined as high levels of anxiety: STAI (adult) > 44, STAI (child) > 36, and mYPAS-SF ≥ 30. FINDINGS: No significant differences were found in the STAI, mYPAS-SF, and Wong-Baker FACES Pain Rating Scale scores of the patients between the intervention and control group. Overall statistics showed that parents had significantly higher postoperative state anxiety levels toward female children (44.93 ± 9.01) compared to male children (40.18 ± 9.89). Preoperative and postoperative parental state anxiety levels were correlated with the child's postoperative anxiety. Furthermore, children's postoperative state anxiety was slightly correlated with postoperative pain. CONCLUSIONS: Short-term use of our deep breathing exercises was ineffective in reducing incidences of perioperative pain and anxiety in pediatric orthopedic patients. A longer period of deep breathing administration may be required for the intervention to be effective. Parental anxiety may have an effect on anxiety levels in children, and postoperative parental anxiety may be affected by the gender of the child.

Highly sensitive detection of three protein toxins via SERS-lateral flow immunoassay based on SiO2@Au nanoparticles.

We developed surface-enhanced Raman scattering-lateral flow immunoassay (SERS-LFIA) biosensor strips based on SiO2@Au nanoparticles (NPs) for the specific and highly sensitive detection of ricin, staphylococcal enterotoxin B (SEB), and botulinum neurotoxin type A (BoNT/A). SiO2@Au NPs were used to prepare SERS tags with useful properties, such as light weight, uniform particle size, good dispersion, and high SERS performance. The detection limit of the SERS-LFIA strips developed herein for ricin, SEB, and BoNT/A was 0.1, 0.05, and 0.1 ng/mL. Their sensitivity was 100-fold higher than that of colloidal gold-LFIA strips, and the same batch of strips had good repeatability. Moreover, the test was completed within 15 min, indicating that the strips are suitable for the rapid and on-site detection of the said toxins. The SERS-LFIA strips based on SiO2@Au NPs developed herein for the detection of toxins are important to the prevention of bioterrorism attacks.

A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen "arms race" is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5 Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

Automatic and sensitive detection of West Nile virus non-structural protein 1 with a portable SERS-LFIA detector.

A portable surface-enhanced Raman scattering (SERS)-lateral flow immunoassay (LFIA) detector has been developed for the automatic and highly sensitive detection of West Nile virus (WNV) non-structural protein 1 (NS1) and actual WNV samples. Au@Ag nanoparticles (Au@Ag NPs) labeled with double-layer Raman molecules were used as SERS tags to prepare WNV-specific SERS-LFIA strips. On this platform, the WNV-specific antigen NS1 protein was quantitatively and sensitively detected. The detection limit for the WNV NS1 protein was 0.1 ng/mL, which was 100-fold more sensitive than visual signals. The detection limit for inactivated WNV virions was 0.2 × 102 copies/µL. The sensitivity of the SERS-LFIA detector was comparable to that of the fluorescence quantitative reverse transcription-polymerase chain reaction assay. The prepared SERS-LFIA strips exhibited high sensitivity and good specificity for WNV. Thus, the strips developed herein have clinical application value. Moreover, the portable SERS-LFIA detector enabled automatic and rapid detection of the SERS-LFIA strips. The platform established herein is expected to make a substantial contribution to the diagnosis and control of outbreaks of emerging infectious diseases, including WNV.

Base-Mediated Amination of Alcohols Using Amidines.

Novel and efficient base-mediated N-alkylation and amidation of amidines with alcohols have been developed, which can be carried out in one-pot reaction conditions, which allows for the synthesis of a wide range of N-alkyl amines and free amides in good to excellent yields with high atom economy. In contrast to borrowing hydrogen/hydrogen autotransfer or oxidative-type N-alkylation reactions, in which alcohols are activated by transition-metal-catalyzed or oxidative aerobic dehydrogenation, the use of amidines provides an effective surrogate of amines. This circumvents the inherent necessity in N-alkylation of an oxidant or a catalyst to be stabilized by ligands.

Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors.

HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α-chain. CD8-VpuCD was rapidly endocytosed in a clathrin- and AP-2-dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the ß-TrCP binding site and a leucine-based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV-dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP-2 and showed that this is enhanced by serine-phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (µ) subunits of AP-2 and AP-1; this interaction was dependent on serine-phosphorylation of Vpu and on basic residues in the µ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.

Phosphoserine acidic cluster motifs bind distinct basic regions on the µ subunits of clathrin adaptor protein complexes.

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

SERS-based lateral flow assay for quantitative detection of C-reactive protein as an early bio-indicator of a radiation-induced inflammatory response in nonhuman primates.

In accidental irradiation situations, rapid in-field evaluation of acute radiation syndrome is critical for effective triage and timely medical treatment of irradiated individuals. A surface-enhanced Raman scattering (SERS)-based lateral flow assay was developed for the quantitative detection of C-reactive protein (CRP) as an early bio-indicator of a radiation-induced inflammatory response in nonhuman primates. Raman reporter-embedded gold-core silver-shell nanoparticles with built-in hot spots were synthesized and conjugated with a CRP detection antibody to serve as SERS tags in the lateral flow assay. The proposed SERS-based lateral flow assay can rapidly detect CRP with a limit of detection of 0.01 ng mL-1 and quantitative analysis ability. Furthermore, the assay was applied to evaluate the CRP levels in plasma samples of irradiated nonhuman primates at 0 to 80 h after exposure to sublethal (4 Gy) and lethal (8 Gy) doses of total body irradiation (n = 3 animals per group). The plasma CRP levels increase rapidly within few hours after irradiation. The CRP level peaks are observed at 12 or 24 h after irradiation, with a concentration of 201.30, 386.06 and 475.18 µg mL-1 for the 4 Gy irradiated animals and 197.14, 69.52 and 358.03 µg mL-1 for the 8 Gy irradiated animals. The results indicate the potential application of the proposed SERS-based lateral flow assay to serve as a rapid and accurate point-of-care biodosimetry assay for the quantitative detection of bio-indicators to triage irradiated individuals in the field of a radiation accident.

Fast and non-invasive serum detection technology based on surface-enhanced Raman spectroscopy and multivariate statistical analysis for liver disease.

This study explored a rapid and nondestructive liver disease detection technique based on surface-enhanced Raman spectroscopy (SERS) to realize the early diagnosis, prevention, and treatment of liver disease. SERS signals of serum were obtained from 304 normal individuals, 333 patients with hepatopathy, and 99 patients with esophageal cancer. The Raman spectra of different diseases were compared and diagnostic models of liver disease were established using orthogonal partial least squares discriminant analysis (OPLS-DA). The classification efficiencies of the different models were comprehensively evaluated through the receiver operating characteristic (ROC) curve and ten-fold cross validation. Area under the ROC curve is of greater than 0.97, indicating excellent classification of the groups. The accuracy rate of the test set reached 95.33%, and the lowest was 81.76% using the ten-fold cross validation. Thus, OPLS-DA combined with serum SERS is a rapid and non-invasive technique for the diagnosis of liver disease.

Mutations in ASPH cause facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs, or Traboulsi syndrome.

We have previously described a syndrome characterized by facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs (FDLAB, or Traboulsi syndrome). In view of the consanguineous nature of the affected families and the likely autosomal-recessive inheritance pattern of this syndrome, we undertook autozygosity mapping and whole-exome sequencing to identify ASPH as the disease locus, in which we identified two homozygous mutations. ASPH encodes aspartyl/asparaginyl ß-hydroxylase (ASPH), which has been found to hydroxylate aspartic acid and asparagine residues on epidermal growth factor (EGF)-domain-containing proteins. The truncating and missense mutations we identified are predicted to severely impair the enzymatic function of ASPH, which suggests a possible link to other forms of ectopia lentis given that many of the genes implicated in this phenotype encode proteins that harbor EGF domains. Developmental analysis of Asph revealed an expression pattern consistent with the proposed link to the human syndrome. Indeed, Asph-knockout mice had a foreshortened snout, which corresponds to the facial abnormalities in individuals with Traboulsi syndrome. These data support a genetic basis for a syndromic form of ectopia lentis and the role of aspartyl hydroxylation in human development.

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2-11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

Analysis of 3D Channel Current Noise in Small Nanoscale MOSFETs Using Monte Carlo Simulation.

As field effect transistors are reduced to nanometer dimensions, experimental and theoretical research has shown a gradual change in noise generation mechanisms. There are few studies on noise theory for small nanoscale transistors, and Monte Carlo (MC) simulations mainly focus on 2D devices with larger nanoscale dimensions. In this study, we employed MC simulation techniques to establish a 3D device simulation process. By setting device parameters and writing simulation programs, we simulated the raw data of channel current noise for a silicon-based metal-oxide-semiconductor field-effect transistor (MOSFET) with a 10 nm channel length and calculated the drain output current based on these data, thereby achieving static testing of the simulated device. Additionally, this study obtained a 3D potential distribution map of the device channel surface area. Based on the original data from the simulation analysis, this study further calculated the power spectral density of the channel current noise and analyzed how the channel current noise varies with gate voltage, source-drain voltage, temperature, and substrate doping density. The results indicate that under low-temperature conditions, the channel current noise of the 10 nm MOSFET is primarily composed of suppressed shot noise, with the proportion of thermal noise in the total noise slightly increasing as temperature rises. Under normal operating conditions, the channel current noise characteristics of the 10 nm MOSFET device are jointly characterized by suppressed shot noise, thermal noise, and cross-correlated noise. Among these noise components, shot noise is the main source of noise, and its suppression degree decreases as the bias voltage is reduced. These findings are consistent with experimental observations and theoretical analyses found in the existing literature.

Highly active and regioselective hydroaminomethylation of olefins catalyzed by Rh/sulfoxantphos with ZSM-5.

Rh-catalyzed hydroaminomethylation has been developed with acid sulfoxantphos and ZSM-5. Linear amines were obtained in good yields (71-95%) with high l/b ratios (up to 132.4) and excellent TON values (up to 23 760). The ZSM-5 and SO3H group of ligands improved the performances of hydroformylation and reductive amination.

Therapeutic Measures for Infections Originating from Scalp Incisions Following Deep Brain Stimulation in Patients with Parkinson's Disease.

BACKGROUND: Deep brain stimulation (DBS) is a well-established treatment for Parkinson's disease (PD). However, infection following DBS surgery is a serious complication that can lead to the recurrence and worsening of Parkinson's symptoms or related hardware re-implantation, causing considerable patient suffering and financial burden. OBJECTIVE: This study aims to compare the therapeutic efficiency of different treatment approaches for scalp incision infections after DBS surgery in PD patients. METHODS: We conducted a retrospective review of patients with Parkinson's disease who experienced scalp infections following deep brain stimulation at our hospital between January 2017 and December 2021. The patients were divided into two groups based on whether affected implants were removed or not. Fisher's exact test was applied to compare the reinfection rates between groups A and B. RESULTS: In group A, four patients underwent debridement only, and all of them experienced reinfection between 2-25 months after the initial treatment. In group B, nine patients underwent debridement and removal of potentially affected implants. Among them, eight patients underwent re-implantation of the DBS device within 3-6 months after the initial treatment, and no cases of reinfection occurred. However, one patient experienced reinfection in the postauricular incision and percutaneous tunnel 5 months after the initial treatment, resulting in the complete removal of the entire DBS system. The reinfection rate in group B (11.11%) was significantly lower than that in group A (100%, P=0.007). CONCLUSION: Scalp incision infections following DBS surgery can affect deep tissues, and the implementation of a comprehensive treatment strategy involving local debridement and removal of potentially affected implants can significantly reduce the risk of infection recurrence and its spread.

Enhanced identification and localization of metabolites in Scutellariae Radix using ion mobility enabled MALDI-Q-TOF/MS imaging.

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Structural insight into the mechanisms of enveloped virus tethering by tetherin.

Tetherin/BST2 is a type-II membrane protein that inhibits the release of a range of enveloped viruses, including HIV-1. Here we report three crystal structures of human tetherin, including the full-length ectodomain, a triple cysteine mutant and an ectodomain truncation. These structures show that tetherin forms a continuous alpha helix encompassing almost the entire ectodomain. Tetherin helices dimerize into parallel coiled coils via interactions throughout the C-terminal portion of the ectodomain. A comparison of the multiple structures of the tetherin dimer reveals inherent constrained flexibility at two hinges positioned at residues A88 and G109. In the crystals, two tetherin ectodomain dimers associate into a tetramer by forming an antiparallel four-helix bundle at their N termini. However, mutagenesis studies suggest that the tetrametric form of tetherin, although potentially contributing to, is not essential for its antiviral activity. Nonetheless, the structural and chemical properties of the N terminus of the ectodomain are important for optimal tethering function. This study provides detailed insight into the mechanisms by which this broad-spectrum antiviral restriction factor can function.