The Implementation of a Gesture Recognition System with a Millimeter Wave and Thermal Imager.

During the COVID-19 pandemic, the number of cases continued to rise. As a result, there was a growing demand for alternative control methods to traditional buttons or touch screens. However, most current gesture recognition technologies rely on machine vision methods. However, this method can lead to suboptimal recognition results, especially in situations where the camera is operating in low-light conditions or encounters complex backgrounds. This study introduces an innovative gesture recognition system for large movements that uses a combination of millimeter wave radar and a thermal imager, where the multi-color conversion algorithm is used to improve palm recognition on the thermal imager together with deep learning approaches to improve its accuracy. While the user performs gestures, the mmWave radar captures point cloud information, which is then analyzed through neural network model inference. It also integrates thermal imaging and palm recognition to effectively track and monitor hand movements on the screen. The results suggest that this combined method significantly improves accuracy, reaching a rate of over 80%.

Iterative Reconstruction of Micro Computed Tomography Scans Using Multiple Heterogeneous GPUs.

Graphics processing units (GPUs) facilitate massive parallelism and high-capacity storage, and thus are suitable for the iterative reconstruction of ultrahigh-resolution micro computed tomography (CT) scans by on-the-fly system matrix (OTFSM) calculation using ordered subsets expectation maximization (OSEM). We propose a finite state automaton (FSA) method that facilitates iterative reconstruction using a heterogeneous multi-GPU platform through parallelizing the matrix calculations derived from a ray tracing system of ordered subsets. The FSAs perform flow control for parallel threading of the heterogeneous GPUs, which minimizes the latency of launching ordered-subsets tasks, reduces the data transfer between the main system memory and local GPU memory, and solves the memory-bound of a single GPU. In the experiments, we compared the operation efficiency of OS-MLTR for three reconstruction environments. The heterogeneous multiple GPUs with job queues for high throughput calculation speed is up to five times faster than the single GPU environment, and that speed up is nine times faster than the heterogeneous multiple GPUs with the FIFO queues of the device scheduling control. Eventually, we proposed an event-triggered FSA method for iterative reconstruction using multiple heterogeneous GPUs that solves the memory-bound issue of a single GPU at ultrahigh resolutions, and the routines of the proposed method were successfully executed on each GPU simultaneously.

CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance.

Transmitted by ticks, the bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), the most common vector-borne disease in the Northern hemisphere. No effective vaccines are currently available. B. burgdorferi sensu lato produces the CspZ protein that binds to the complement inhibitor, factor H (FH), promoting evasion of the host complement system. We previously showed that while vaccination with CspZ did not protect mice from B. burgdorferi infection, mice can be protected after immunization with CspZ-Y207A/Y211A (CspZ-YA), a CspZ mutant protein without FH-binding activity. To further study the mechanism of this protection, herein we evaluated both poly- and monoclonal antibodies recognizing CspZ FH-binding or non-FH-binding sites. We found that the anti-CspZ antibodies that recognize the FH-binding sites (i.e., block FH-binding activity) eliminate B. burgdorferi sensu lato in vitro more efficiently than those that bind to the non-FH-binding sites, and passive inoculation with anti-FH-binding site antibodies eradicated B. burgdorferi sensu lato in vivo. Antibodies against non-FH-binding sites did not have the same effect. These results emphasize the importance of CspZ FH-binding sites in triggering a protective antibody response against B. burgdorferi sensu lato in future LD vaccines.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

Comparison of 18F-FDG, 18F-Fluoroacetate, and 18F-FEPPA for Imaging Liver Fibrosis in a Bile Duct-Ligated Rat Model.

Developing sensitive diagnostic methods for a longitudinal evaluation of the status of liver fibrosis is a priority. This study is aimed at assessing the significance of longitudinal positron emission tomography (PET) imaging with 18F-labeling tracers for assessing liver fibrosis in a rat model with bile duct ligation (BDL). Twenty-one 6-week-old Sprague-Dawley male rats were used in this study. Longitudinal PET images using [18F]N-2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([18F]FEPPA) (n = 3), [18F]fluoroacetate ([18F]FAc) (n = 3), and 18F-fluoro-2-deoxy-D-glucose ([18F]FDG) (n = 3) were obtained at 0, 1, and 2 weeks after BDL. Biochemical assays, histological assays, immunohistochemical staining assays, and next generation sequencing analyses were also performed at 0 (n = 3), 1 (n = 3), 2 (n = 3), and 3 (n = 3) weeks after BDL, which demonstrated the severe damage in rat livers after BDL. Regarding [18F]FEPPA and [18F]FDG, there was a significantly higher uptake in the liver after BDL (both P < 0.05), which lasted until week 2. However, the uptake of [18F]FAc in the liver was not significantly different before and after BDL (P = 0.28). Collectively, both [18F]FEPPA and [18F]FDG can serve as sensitive probes for detecting the liver fibrosis. However, [18F]FAc is not recommended to diagnose liver fibrosis.

A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain-based vaccine candidate, RBD219-N1C1.

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost. KEY POINTS: • Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined. • Three purification protocols for a COVID-19 vaccine antigen were compared. • Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown. Graphical abstract.

The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease.

The spirochete Borrelia burgdorferisensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

Tamponade in surgery for retinal detachment associated with proliferative vitreoretinopathy.

BACKGROUND: Retinal detachment (RD) with proliferative vitreoretinopathy (PVR) often requires surgery to restore normal anatomy and to stabilize or improve vision. PVR usually occurs in association with recurrent RD (that is, after initial retinal re-attachment surgery), but occasionally may be associated with primary RD. Either way, for both circumstances a tamponade agent (gas or silicone oil) is needed during surgery to reduce the rate of postoperative recurrent RD. OBJECTIVES: The objective of this review was to assess the relative safety and effectiveness of various tamponade agents used with surgery for RD complicated by PVR. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (the Cochrane Library 2019, Issue 1), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to January 2019), Embase (January 1980 to January 2019), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to January 2019), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 2 January 2019. SELECTION CRITERIA: We included randomized controlled trials (RCTs) on participants undergoing surgery for RD associated with PVR that compared various tamponade agents. DATA COLLECTION AND ANALYSIS: Two review authors screened the search results independently. We used the standard methodological procedures expected by Cochrane. MAIN RESULTS: We identified four RCTs (601 participants) that provided data for the primary and secondary outcomes. Three RCTs provided data on visual acuity, two reported on macular attachment, one on retinal reattachment and another two on adverse events such as RD, worsening visual acuity and intraocular pressure. Study Characteristics Participants' characteristics varied across studies and across intervention groups, with an age range between 21 to 89 years, and were predominantly men. The Silicone Study was conducted in the USA and consisted of two RCTs: (silicone oil versus sulfur hexafluoride (SF6) gas tamponades; 151 participants) and (silicone oil versus perfluropropane (C3F8) gas tamponades; 271 participants). The third RCT compared heavy silicone oil (a mixture of perfluorohexyloctane (F6H8) and silicone oil) with standard silicone oil (either 1000 centistokes or 5000 centistokes; 94 participants). The fourth RCT compared 1000 centistokes with 5000 centistokes silicone oil in 85 participants. We assessed most RCTs at low or unclear risk of bias for most 'Risk of bias' domains. Findings Although SF6 gas was reported to be associated with worse anatomic and visual outcomes than was silicone oil at one year (quantitative data not reported), at two years, silicone oil compared to SF6 gas showed no evidence of a difference in visual acuity (33% versus 51%; risk ratio (RR) 1.57; 95% confidence interval (CI) 0.93 to 2.66; 1 RCT, 87 participants; low-certainty evidence). At one year, another RCT comparing silicone oil and C3F8 gas found no evidence of a difference in visual acuity between the two groups (41% versus 39%; RR 0.97; 95% CI 0.73 to 1.31; 1 RCT, 264 participants; low-certainty evidence). In a third RCT, participants treated with standard silicone oil compared to those receiving heavy silicone oil also showed no evidence of a difference in the change in visual acuity at one year, measured on logMAR scale ( mean difference -0.03 logMAR; 95% CI -0.35 to 0.29; 1 RCT; 93 participants; low-certainty evidence). The fourth RCT with 5000-centistoke and 1000-centistoke comparisons did not report data on visual acuity. For macular attachment, participants treated with silicone oil may probably experience more favorable outcomes than did participants who received SF6 at both one year (quantitative data not reported) and two years (58% versus 79%; RR 1.37; 95% CI 1.01 to 1.86; 1 RCT; 87 participants; low-certainty evidence). In another RCT, silicone oil compared to C3F8 at one year found no evidence of difference in macular attachment (RR 1.00; 95% CI 0.86 to 1.15; 1 RCT, 264 participants; low-certainty evidence). One RCT that compared 5000 centistokes to 1000 centistoke reported that retinal reattachment was successful in 67 participants (78.8%) with first surgery and 79 participants (92.9%) with the second surgery, and no evidence of between-group difference (1 RCT; 85 participants; low-certainty evidence). The fourth RCT that compared standard silicone oil with heavy silicone oil did not report on macular attachment. Adverse events In one RCT (86 participants), those receiving standard 1000 centistoke silicone oil compared with those of the 5000 centistoke silicone oil showed no evidence of a difference in intraocular pressure elevation at 18 months (24% versus 22%; RR 0.90; 95% CI 0.41 to 1.94; low-certainty evidence), visually significant cataract (49% versus 64%; RR 1.30; 95% CI 0.89 to 1.89; low-certainty evidence), and incidence of retina detachment after the removal of silicone oil (RR 0.36 95% CI 0.08 to 1.67; low-certainty evidence). Another RCT that compared standard silicone oil with heavy silicone oil suggests no difference in retinal detachment at one year (25% versus 22%; RR 0.89; 95% CI 0.54 to 1.48; 1 RCT; 186 participants; low-certainty evidence). Retinal detachment was not reported in the RCTs that compared silicone oil versus SF6 and silicone oil versus to C3F8. AUTHORS' CONCLUSIONS: There do not appear to be any major differences in outcomes between C3F8 and silicone oil. Silicone oil may be better than SF6 for macular attachment and other short-term outcomes. The choice of a tamponade agent should be individualized for each patient. The use of either C3F8 or standard silicone oil appears reasonable for most patients with RD associated with PVR. Heavy silicone oil, which is not available for routine clinical use in the USA, may not demonstrate evidence of superiority over standard silicone oil.

A method to probe protein structure from UV absorbance spectra.

Proteins primarily absorb UV light due to the presence of tryptophan, tyrosine, and phenylalanine residues, with absorbance maxima at 280, 275, and 258 nm, respectively. We now demonstrate that a simple value obtained by relating the absorbance at all three wavelengths, [A280/A275 + A280/A258], is a generally useful, robust, and sensitive probe of protein 'foldedness', and thus can be used to investigate unfolding, refolding, disulfide bonds, stability, buffer excipients, and even protein-protein and protein-ligand interactions.

Application of molecular cytogenetic techniques to characterize the aberrant Y chromosome arising de novo in a male fetus with mosaic 45,X and solve the discrepancy between karyotyping, chromosome microarray, and multiplex ligation dependent probe amplification.

We present a rare male fetus with karyotype of mosaic 45,X that comprises two types of aberrant Y chromosomes arising de novo (Yq12 deletion and isodicentric Yq11.22). Both types of the aberrant Y chromosomes lack the AZFc region which are expected to result in oligospermia but unaffected male external genitalia. Genetic analyses by karyotyping, chromosome microarray (CMA), and multiplex ligation-dependent probe amplification (MLPA) for the fetus revealed conflicting results. Additional molecular cytogenetics tools including fluorescence in situ hybridization (FISH) and multicolor banding (mBAND) were performed, which help resolving the discrepancy and delineated the composition of the aberrant Y chromosomes. This report highlighted the importance of incorporating multiple genetic technologies for accurate characterization of complex chromosomal rearrangements, which aid in the prenatal diagnosis and genetic counseling.

Development of Limited-Angle Iterative Reconstruction Algorithms with Context Encoder-Based Sinogram Completion for Micro-CT Applications.

Limited-angle iterative reconstruction (LAIR) reduces the radiation dose required for computed tomography (CT) imaging by decreasing the range of the projection angle. We developed an image-quality-based stopping-criteria method with a flexible and innovative instrument design that, when combined with LAIR, provides the image quality of a conventional CT system. This study describes the construction of different scan acquisition protocols for micro-CT system applications. Fully-sampled Feldkamp (FDK)-reconstructed images were used as references for comparison to assess the image quality produced by these tested protocols. The insufficient portions of a sinogram were inpainted by applying a context encoder (CE), a type of generative adversarial network, to the LAIR process. The context image was passed through an encoder to identify features that were connected to the decoder using a channel-wise fully-connected layer. Our results evidence the excellent performance of this novel approach. Even when we reduce the radiation dose by 1/4, the iterative-based LAIR improved the full-width half-maximum, contrast-to-noise and signal-to-noise ratios by 20% to 40% compared to a fully-sampled FDK-based reconstruction. Our data support that this CE-based sinogram completion method enhances the efficacy and efficiency of LAIR and that would allow feasibility of limited angle reconstruction.

Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis.

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Low-molecular-weight-heparin can benefit women with recurrent pregnancy loss and sole protein S deficiency: a historical control cohort study from Taiwan.

BACKGROUND: Heritable thrombophilias are assumed important etiologies for recurrent pregnancy loss. Unlike in the Caucasian populations, protein S and protein C deficiencies, instead of Factor V Lieden and Prothrombin mutations, are relatively common in the Han Chinese population. In this study we aimed to investigate the therapeutic effect of low molecular weight heparin upon women with recurrent pregnancy loss and documented protein S deficiency. METHODS: During 2011-2016, 68 women with recurrent pregnancy loss (RPL) and protein S deficiency (both the free antigen and function of protein S were reduced) were initially enrolled. All the women must have experienced at least three recurrent miscarriages. After excluding those carrying balanced translocation, medical condition such as diabetes mellitus, chronic hypertension, and autoimmune disorders (including systemic lupus erythematosus and anti-phospholipid syndrome), coexisting thrombophilias other than persistent protein S deficiency (including transient low protein S level, protein C deficiency, and antithrombin III), only 51 women with RPL and sole protein S deficiency were enrolled. Initially they were prescribed low dose Aspirin (ASA: 100 mg/day) and unfortunately there were still 39 women ended up again with early pregnancy loss (12 livebirths were achieved though). Low-molecular-weight-heparin (LMWH) was given for the 39 women in a dose of 1 mg/Kg every 12 h from the day when the next clinical pregnancy was confirmed to the timing at least 24 h before delivery. The perinatal outcomes were assessed. RESULTS: Of 50 treatment subjects performed for the 39 women (i.e. 11 women enrolled twice for two pregnancies), 46 singletons and one twin achieved livebirths. The successful live-birth rate in the whole series was 94 % (47/50). Nineteen livebirths delivered vaginally whereas 28 delivered by cesarean section. The cesarean delivery rate is thus 59.57 %. Emergent deliveries occurred in 3 but no postpartum hemorrhage had been noted. CONCLUSIONS: Our pilot study in Taiwan, an East Asian population, indicated anti-coagulation therapy is of benefit to women with recurrent pregnancy loss who had documented sole protein S deficiency. TRIAL REGISTRATION: ISRCTN64574169. Retrospectively registered 29 Jun 2016.

Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.

Label-free multi-color superlocalization of plasmonic emission within metallic nano-interstice using femtosecond chirp-manipulated four wave mixing.

We demonstrate an as yet unused method to sieve, localize, and steer plasmonic hot spot within metallic nano-interstices close to percolation threshold. Multicolor superlocalization of plasmon mode within 60 nm was constantly achieved by chirp-manipulated superresolved four wave mixing (FWM) images. Since the percolated film is strongly plasmonic active and structurally multiscale invariant, the present method provides orders of magnitude enhanced light localization within single metallic nano-interstice, and can be universally applied to any region of the random film. The result, verified by the maximum likelihood estimation (MLE) and deconvolution stochastic optical reconstruction microscopy (deconSTORM) algorithm, may contribute to label-free multiplex superlocalized spectroscopy of single molecule and sub-cellular activity monitoring combining hot spot steering capability.

Study design, rationale and methods for a population-based study of myopia in schoolchildren: the Myopia Investigation study in Taipei.

BACKGROUND: To describe the study design, rationale and methodology of the Myopia Investigation Study in Taipei (MIT). DESIGN: The MIT was a city-wide, population-based cohort study. PARTICIPANTS: Participants were grade 2 students (Fall 2013) of all 153 elementary schools in Taipei City. METHODS: The baseline data on the risk factors for myopia development was collected by parent-administered questionnaire surveys covering demographics, medical history, parental myopia, time spent on near work and outdoor activities, reading habits and eye care-seeking behaviour. Ocular examinations focused on the measurement of visual acuity (unaided and best-corrected) and refractive status (before and after cycloplegia), which will be carried out for the eligible schoolchildren biannually for 3 years consecutively. Once myopic children are identified, case manager-led telecoaching for health-care instructions and reminders will be delivered to parents or caregivers. MAIN OUTCOME MEASURES: To build a comprehensive database for prevalence, incidence and risk factors of early childhood myopia over a 3-year follow-up period. RESULTS: Of all 19 374 eight-year-old schoolchildren (10 210 [52.7%] boys) eligible for the MIT, 16 486 (85.1%) responded to the questionnaire, 12 019 (62.0%) were examined during the third quarter of 2013 and 11 590 (59.8%) (6267 [52.9%] boys) completed cycloplegic autorefraction on both eyes and were enrolled for further data analysis. There was no significant difference in terms of demographics between the analysed participants and all grade 2 students in Taipei City. CONCLUSIONS: Data from the MIT will provide population-based information concerning the prevalence, incidence and risk factors for myopia development among young schoolchildren in a metropolitan area of Taiwan.

TREM-1 regulates macrophage polarization in ureteral obstruction.

Chronic kidney disease (CKD) is an emerging worldwide public health problem. Inflammatory cell infiltration and activation during the early stages in injured kidneys is a common pathologic feature of CKD. Here, we determined whether an important inflammatory regulator, triggering receptor expressed on myeloid cells (TREM)-1, is upregulated in renal tissues collected from mouse ureteral obstruction-induced nephritis. TREM-1 is crucial for modulating macrophage polarization, and has a pivotal role in mediating tubular injury and interstitial collagen deposition in obstructive nephritis. Lysates from nephritic kidneys triggered a TREM-1-dependent M1 polarization ex vivo, consistent with the observation that granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived M1 macrophages express higher levels of TREM-1 in comparison with M-CSF-derived cells. Moreover, agonistic TREM-1 cross-link significantly strengthens the inductions of iNOS and GM-CSF in M1 cells. These observations are validated by a strong clinical correlation between infiltrating TREM-1-expressing/iNOS-positive macrophages and renal injury in human obstructive nephropathy. Thus, TREM-1 may be a potential diagnostic and therapeutic target in human kidney disease.