Function of the phosphatidylinositol synthase Pis1 in maintenance of endoplasmic reticulum function and pathogenicity in Candida albicans.

Candida albicans is a common conditional pathogenic fungus in the human body, and its infections have received widespread attention in recent years. Phosphatidylinositol and its derivatives have significant regulatory effects on many physiological processes, such as cell metabolism and growth. In this study, we identified and studied the function of the phosphatidylinositol synthase Pis1 in Candida albicans. The protein has a conserved CAPT motif and multiple transmembrane domains. GFP tagging revealed that Pis1 was located at the endoplasmic reticulum (ER). The PIS1 knockout mutant was constructed using an induction system regulated by the MET3 promoter. Growth assays showed that PIS1 is an essential gene for normal growth of Candida albicans. Overexpression of PIS1 led to high sensitivity to both ER stress and cell wall stress, and down-regulated expression of the genes involved in ER stress response and maintenance of cell wall integrity. Interestingly, PIS1 overexpression enhanced secretion of the extracellular hydrolases. Virulence assays further revealed that PIS1 overexpression increased the fungal virulence, leading to quicker death of the fungus-infected mice and more severe fungal burden in the mouse kidneys. In summary, Pis1 is involved in ER stress response, maintenance of cell wall integrity, and pathogenicity of Candida albicans.

Data assimilation of flow-acoustic resonance.

A data assimilation (DA) strategy was developed for accurate prediction of the flow-acoustic resonant fields within a channel-branch system. The challenges of numerical simulation of such internal aeroacoustic systems are primarily associated with determination of the transfer loss between the acoustic waves and the shear layer vortices. Thus, a data-assimilated momentum loss model that comprises a viscous loss item and an inertial loss item was established and embedded into the Navier-Stokes equations. During the DA, the acoustic pressure pulsations measured from a dynamic pressure array served as the observational data, the ensemble Kalman filter served as the optimization algorithm, and a three-dimensional transient computational fluid dynamics method comprising an explicit algebraic Reynolds stress model (EARSM) served as the predictive model system. EARSM was used because its ability to predict internal flow-acoustic resonances was superior to that of other eddy viscosity models and Reynolds stress models. The data-assimilated flow-acoustic resonant fields were then comprehensively validated in terms of their acoustic fields, time-averaged flow fields, and phase-dependent flow fields. The time-averaged flow fields were obtained from planar particle-image velocimetry (PIV) measurements, and the phase-dependent flow fields were obtained from field programmable gate array-based phase-locking PIV measurements. The results demonstrate that the use of DA afforded an optimal simulation that efficiently decreased the numerical errors in the frequencies and amplitudes of the acoustic pressure pulsations, thereby achieving better agreement between time-averaged flow distributions and fluctuations. In addition, the data-assimilated numerical simulation completely reproduced the spatiotemporal evolution of the shear layer vortices, that is, their formation, developing, transport, and collapsing regions.

Signal Enhancement of Pressure-Sensitive Film Based on Localized Surface Plasmon Resonance.

Pressure-sensitive films have been used for measurement in micro flow, but thin films have very limited intensity, resulting in poor signal-noise ratio (SNR). This paper presents a pressure-sensitive film whose emission signal is enhanced by silver nanoparticles (AgNPs) based on localized surface plasmon resonance (LSPR). Electronic beam evaporator and annealing furnace are used to fabricate silver nanotexture surface. PtTFPP and polystyrene are dissolved in toluene and then spin-coated on the silver nanotexture surface to prepare the pressure-sensitive films. Signal enhancement of film with AgNPs due to LSPR is analyzed and enhancement effect of samples with different particle sizes and spacer thickness are compared. Pressure and temperature calibrations are performed to assess the sensing performance of pressure-sensitive films. Pressure-sensitive films with AgNPs demonstrate signal enhancement due to LSPR and show promise for measurement in micro flow.

Function of Atg11 in non-selective autophagy and selective autophagy of Candida albicans.

Candida albicans is an important opportunistic pathogenic fungus in the human body. It is a common microbe inhabiting on the mucosa surfaces of healthy individuals, but may cause infections when the host immune system is weak. Autophagy is a "self-eating" process in eukaryotes, which can recover and utilize damaged organelles and misfolded proteins. Here we investigated the role of the autophagy-related protein Atg11 in C. albicans. Deletion of ATG11 led to the defect in growth under the nitrogen starvation condition. Western blotting and GFP localization further revealed that the transport and degradation of Atg8 was blocked in the atg11Δ/Δ mutant under both the nitrogen starvation and hypha-inducing conditions. Moreover, degradation of both Lap41 (the indicator of the cytoplasm-to-vacuole pathway) and Csp37 (the indicator of mitophagy) was also thoroughly suppressed in this mutant under nitrogen starvation. These results indicated that Atg11 plays an essential role in both non-selective and selective autophagy in C. albicans.

High-repetition-rate burst-mode-laser diagnostics of an unconfined lean premixed swirling flame under external acoustic excitation.

Lean premixed swirling flames are important in practical combustors, but a commonly encountered problem of practical swirl combustors is thermo-acoustic instability, which may cause internal structure damage to combustors. In this research, a high-repetition-rate burst-mode laser is used for simultaneous particle image velocimetry and planar laser-induced fluorescence measurement in an unconfined acoustically excited swirl burner. The time-resolved flow field and transient flame response to the acoustic perturbation are visualized at 20 kHz, offering insight into the heat release rate oscillation. The premixed mixture flow rate and acoustic modulation are varied to study the effects of Reynolds number, Strouhal number, and acoustic modulation amplitude on the swirling flame. The results suggest that the Strouhal number has a notable effect on the periodic movements of the inner recirculation zone and swirling flame configuration.

Phosphor-Doped Thermal Barrier Coatings Deposited by Air Plasma Spray for In-Depth Temperature Sensing.

Yttria-stabilized zirconia (YSZ)-based thermal barrier coating (TBC) has been integrated with thermographic phosphors through air plasma spray (APS) for in-depth; non-contact temperature sensing. This coating consisted of a thin layer of Dy-doped YSZ (about 40 µm) on the bottom and a regular YSZ layer with a thickness up to 300 µm on top. A measurement system has been established; which included a portable; low-cost diode laser (405 nm); a photo-multiplier tube (PMT) and the related optics. Coating samples with different topcoat thickness were calibrated in a high-temperature furnace from room temperature to around 900 °C. The results convincingly showed that the current sensor and the measurement system was capable of in-depth temperature sensing over 800 °C with a YSZ top layer up to 300 µm. The topcoat thickness was found to have a strong effect on the luminescent signal level. Therefore; the measurement accuracy at high temperatures was reduced for samples with thick topcoats due to strong light attenuation. However; it seemed that the light transmissivity of YSZ topcoat increased with temperature; which would improve the sensor's performance at high temperatures. The current sensor and the measurement technology have shown great potential in on-line monitoring of TBC interface temperature.

The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling.

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation.

CRISPR technology holds significant promise for biological studies and gene therapies because of its high flexibility and efficiency when applied in mammalian cells. But endonuclease (e.g., Cas9) potentially generates undesired edits; thus, there is an urgent need to comprehensively identify off-target sites so that the genotoxicities can be accurately assessed. To date, it is still challenging to streamline the entire process to specifically label and efficiently enrich the cleavage sites from unknown genomic locations. Here we develop PEAC-seq, in which we adopt the Prime Editor to insert a sequence-optimized tag to the editing sites and enrich the tagged regions with site-specific primers for high throughput sequencing. Moreover, we demonstrate that PEAC-seq could identify DNA translocations, which are more genotoxic but usually overlooked by other off-target detection methods. As PEAC-seq does not rely on exogenous oligodeoxynucleotides to label the editing site, we also conduct in vivo off-target identification as proof of concept. In summary, PEAC-seq provides a comprehensive and streamlined strategy to identify CRISPR off-targeting sites in vitro and in vivo, as well as DNA translocation events. This technique further diversified the toolkit to evaluate the genotoxicity of CRISPR applications in research and clinics.

Simultaneous 3D surface profile and pressure measurement using phase-shift profilometry and pressure-sensitive paint.

A simultaneous three-dimensional (3D) surface profile and pressure measurement method that integrates phase-shift profilometry and pressure-sensitive paint (PSP2) is proposed. The advantages of this novel technique over previous 3D pressure-sensitive paint (3D-PSP) techniques include a simplified system with low cost, no interference with PSP coatings, high spatial resolution, and high accuracy. A modified digital light-processing (DLP) projector-structured light generator is used to encode ultraviolet light and generate fringe projection to excite the pressure-sensitive paint. The 3D profile is reconstructed using four phase-shifting emission images. Meanwhile, the surface intensity ratio distribution is obtained. The PSP2 method is applied to a nitrogen jet impingement experiment onto a spherical model. The intensity ratio results obtained using the PSP2 method differ little from the conventional PSP results obtained using uniform excitation. The phase distortion due to the emission intensity fluctuation leads to errors in surface profile measurement, and the fringe projection with high contrast improves surface profile measurement accuracy. In most of the final results, the average total errors between the reconstructed 3D surface and the CAD geometry are less than 0.1 mm.

Vacuole and Mitochondria Patch (vCLAMP) Protein Vam6 Is Involved in Maintenance of Mitochondrial and Vacuolar Functions under Oxidative Stress in Candida albicans.

Candida albicans is one of the most common opportunistic fungal pathogens in human beings. When infecting host cells, C. albicans is often exposed to oxidative stress from the host immune defense system. Maintenance of mitochondrial and vacuolar functions is crucial for its resistance to oxidative stress. However, the role of vacuole and mitochondria patchs (vCLAMPs) in cellular oxidative stress resistance and in the maintenance of organelle functions remains to be elucidated. Herein, the function of the vCLAMP protein Vam6 in response to oxidative stress was explored. The results showed that the vam6∆/∆ mutant exhibited obvious mitochondrial swelling, mtDNA damage, reduced activity of antioxidant enzymes, and abnormal vacuolar morphology under H2O2 treatment, indicating its important role in maintaining the structures and functions of both mitochondria and vacuoles under oxidative stress. Further studies showed that deletion of VAM6 attenuated hyphal development under oxidative stress. Moreover, loss of Vam6 obviously affected host tissue invasion and virulence of C. albicans. Taken together, this paper reveals the critical role of vCLAMPs in response to oxidative stress in C. albicans.

Study on the Function of the Inositol Polyphosphate Kinases Kcs1 and Vip1 of Candida albicans in Energy Metabolism.

In Saccharomyces cerevisiae, inositol polyphosphate kinase KCS1 but not VIP1 knockout is of great significance for maintaining cell viability, promoting glycolysis metabolism, and inducing mitochondrial damage. The functions of Candida albicans inositol polyphosphate kinases Kcs1 and Vip1 have not yet been studied. In this study, we found that the growth rate of C. albicans vip1Δ/Δ strain in glucose medium was reduced and the upregulation of glycolysis was accompanied by a decrease in mitochondrial activity, resulting in a large accumulation of lipid droplets, along with an increase in cell wall chitin and cell membrane permeability, eventually leading to cell death. Relieving intracellular glycolysis rate or increasing mitochondrial metabolism can reduce lipid droplet accumulation, causing a reduction in chitin content and cell membrane permeability. The growth activity and energy metabolism of the vip1Δ/Δ strains in a non-fermentable carbon source glycerol medium were not different from those of the wild-type strains, indicating that knocking out VIP1 did not cause mitochondria damage. Moreover, C. albicans KCS1 knockout did not affect cell activity and energy metabolism. Thus, in C. albicans, Vip1 is more important than Kcs1 in regulating cell viability and energy metabolism.

Role of the inositol polyphosphate kinase Vip1 in autophagy and pathogenesis in Candida albicans.

Aim: Inositol polyphosphate kinases are involved in regulation of many cellular processes in eukaryotic cells. In this study, we investigated the functions of the inositol polyphosphate kinase Vip1 in autophagy and pathogenicity of Candida albicans. Results: Loss of Vip1 caused significantly increased sensitivity to nitrogen source starvation, abnormal localization and degradation of autophagy protein, higher vacuolar pH and higher (rather than lower) intracellular ATP levels compared with control strains. Besides, the mutant showed attenuated hyphal development and virulence during systemic infection to mice. Conclusion: The results reveal that Vip1 is important to autophagy of C. albicans. The maintenance of vacuolar acidic pH contributed to the role of Vip1 in autophagy. Vip1 is also required for pathogenicity of C. albicans.

Direct-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen.

CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription, and epigenetic modifications in genetic screens. However, it remains challenging to assay the complex molecular readouts after perturbation at high resolution and at scale. By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the CRISPR perturbation and its transcriptional readouts can be profiled together in a streamlined workflow.

Influence of antegrade pulmonary blood flow on the hemodynamic performance of bidirectional cavopulmonary anastomosis: a numerical study.

The role and effect of preserved antegrade pulmonary blood flow (APBF) at the time of bidirectional cavopulmonary anastomosis (BCPA) in the management of single-ventricle physiology is controversial. We investigated the influence of APBF on the fluid dynamics of BCPA connection using computational fluid dynamics (CFD). Patient-specific, three-dimensional geometry of a BCPA connection with native pulmonary artery (PA) trunk was reconstructed and transient CFD simulations were done at four predetermined mean flow rates of PA trunk (0.5L/min, 1L/min, 1.5L/min and 2L/min). During a cardiac cycle, the flow ratio of left pulmonary artery (LPA)/right pulmonary artery (RPA) increased from 1.91 to 2.50, and average control volume power loss increased from 0.76 mW to 18.05 mW when the mean flow rate of PA trunk changed from 0.5L/min to 2L/min. The pulsatility of pressures in LPA, RPA and superior vena cava became more prominent as the amount of APBF increased. Local fluid structures in the connection area at four levels of APBF differed from each other.

Rapid tomographic reconstruction based on machine learning for time-resolved combustion diagnostics.

Optical tomography has attracted surged research efforts recently due to the progress in both the imaging concepts and the sensor and laser technologies. The high spatial and temporal resolutions achievable by these methods provide unprecedented opportunity for diagnosis of complicated turbulent combustion. However, due to the high data throughput and the inefficiency of the prevailing iterative methods, the tomographic reconstructions which are typically conducted off-line are computationally formidable. In this work, we propose an efficient inversion method based on a machine learning algorithm, which can extract useful information from the previous reconstructions and build efficient neural networks to serve as a surrogate model to rapidly predict the reconstructions. Extreme learning machine is cited here as an example for demonstrative purpose simply due to its ease of implementation, fast learning speed, and good generalization performance. Extensive numerical studies were performed, and the results show that the new method can dramatically reduce the computational time compared with the classical iterative methods. This technique is expected to be an alternative to existing methods when sufficient training data are available. Although this work is discussed under the context of tomographic absorption spectroscopy, we expect it to be useful also to other high speed tomographic modalities such as volumetric laser-induced fluorescence and tomographic laser-induced incandescence which have been demonstrated for combustion diagnostics.

Integration of pressure-sensitive paint with persistent phosphor: A light-charged pressure-sensing system.

A novel pressure-sensitive paint has been developed by mixing phosphor Sr4Al14O25:Eu,Dy with pressure-sensitive luminophore PtTFPP in a polyethylene binder. The phosphor particles served as hosts for PtTFPP molecules while providing excitation light via their bright blue/green luminescence. The unique long afterglow of Sr4Al14O25:Eu,Dy allows the coating to be charged by a light source (UV-LED or even sunlight) in advance, and then the luminescent signals can be collected by using a color camera for pressure measurement (without an external light source). The persistent luminescence and the pressure-sensitive signal correspond to the green and red channels, respectively. The errors due to time-varying persistent luminescence can be removed by taking an intensity ratio between the two channels. The current formulation of this light-charged pressure-sensitive paint (LC-PSP) has been optimized for the maximum signal level. It showed similar pressure sensitivity to typical pressure-sensitive paints (PSPs), and its capability was demonstrated through a nitrogen jet impingement experiment. This light-charged PSP can be truly free of light source during measurement, which greatly simplifies the optical system and avoids errors due to the inconsistent illumination field, and thus provides a solution for PSP applications in facilities with limited optical access.

Quantitative stress measurement of elastic deformation using mechanoluminescent sensor: An intensity ratio model.

The mechanoluminescent (ML) sensor is a newly developed non-invasive technique for stress/strain measurement. However, its application has been mostly restricted to qualitative measurement due to the lack of a well-defined relationship between ML intensity and stress. To achieve accurate stress measurement, an intensity ratio model was proposed in this study to establish a quantitative relationship between the stress condition and its ML intensity in elastic deformation. To verify the proposed model, experiments were carried out on a ML measurement system using resin samples mixed with the sensor material SrAl2O4:Eu2+, Dy3+. The ML intensity ratio was found to be dependent on the applied stress and strain rate, and the relationship acquired from the experimental results agreed well with the proposed model. The current study provided a physical explanation for the relationship between ML intensity and its stress condition. The proposed model was applicable in various SrAl2O4:Eu2+, Dy3+-based ML measurement in elastic deformation, and could provide a useful reference for quantitative stress measurement using the ML sensor in general.

Extracardiac Fontan with direct cavopulmonary connections: midterm results.

OBJECTIVES: The aim of this study was to analyse the midterm results following superior and inferior direct cavopulmonary connections (DCPC) to create a Fontan circulation in patients with functionally univentricular hearts. METHODS: A retrospective review of patients operated on between January 2005 and December 2011 was carried out. RESULTS: Thirty consecutive patients who underwent this type of operation were retrospectively reviewed. There were 18 (60%) males and 12 (40%) females, with a median age of 69 months (range 16-150 months) and median weight of 23 kg (range 11-46 kg). Aortic cross-clamping was used in 10 patients, with a median cross-clamp time of 40 min (range 23-99) and a median cardiopulmonary bypass (CPB) time of 135 min (range 76-179 min). The remaining 20 patients were operated on without aortic cross-clamping. Their median CPB time was 104 min (range 78-139 min). Fenestration was performed in 16 patients. The associated intracardiac procedures were performed in 10 patients. The follow-up period ranged from 2 months to 6 years. Operative mortality and late mortality after discharge was zero. The major postoperative complications included supraventricular tachycardia in one patient, oliguria and peritoneal dialysis in one and chest drainage (>30 ml/day) persisting >7 days in five (20%). One patient developed sinus bradycardia in association with sinus pauses 2 months after discharge. One patient developed pericardial effusion 1 month after discharge. A computational fluid dynamic study was performed in one patient. The computational fluid dynamic study showed that DCPC may have a better power efficiency. CONCLUSIONS: Superior and inferior DCPCs to create a Fontan circulation in appropriately selected patients with functionally univentricular hearts can be performed with a low risk and a low rate of reinterventions. The midterm results are favourable.

Patient-specific computational fluid dynamic simulation of a bilateral bidirectional Glenn connection.

Computational fluid dynamics (CFD) have been used to investigate the hemodynamic performance in cavopulmonary anastomosis and resulted in improved operative design. In this study, CFD simulations were performed in a patient-specific bilateral bidirectional Glenn (BBDG) connection model and the power losses as well as flow features at different levels of predetermined pulmonary flow splits were calculated and compared. The control volume power loss varied between 0.64 and 1.02 mW when the flow ratio of left pulmonary artery/right pulmonary artery changed from 80:20 to 20:80. The flow patterns within the connection area and the static pressures in the four vessels differed from each other as the pulmonary flow split changed. Power loss and flow patterns of this BBDG connection were influenced by the pulmonary flow split.