Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Automatic optical path configuration variation in off-axis mirror system design.

In current off-axis system design process, optical path configuration (OPC) of the system often remains unchanged due to the explicit physical constraints added in the optimization process, and this prevents designers from obtaining potential better results with other OPCs. In this paper, we present a method to change the OPC automatically by ray-quadrangle-based optimization. In our method, a vector is utilized to represent the OPC and a penalty function based on the difference between the current vector and its target value is added into the optimization error function. During OPC variation, obscuration can be avoided without human interference. Examples are given as validation of the proposed method.

Passively mode-locked Yb fiber laser with PbSe colloidal quantum dots as saturable absorber.

A passively mode-locked Yb fiber laser using PbSe colloidal quantum dots (CQDs) as saturable absorber (SA) is experimentally demonstrated. An all-fiber experimental scheme was designed to understand the SA property of PbSe CQDs. The non-saturable loss, modulation depth, and saturable intensity of SA measured were 23%, 7%, and 12 MW/cm2, respectively. The PbSe CQDs were sandwiched in a fiber connector, which was further inserted into the Yb fiber laser for mode-locking. As the pump power up to 110 mW, the self-starting mode-locking pulses were observed. Under the pump power of 285 mW, a maximum average laser power with fundamental mode-locking operation was obtained to be 21.3 mW. In this situation, the pulse full width at half maximum (FWHM), pulse repetition rate, and spectral FWHM were measured to be 70 ps, 8.3 MHz, and 4.5 nm, respectively.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

Experimentally exploiting the violation of the Lagrange invariant for resolution improvement.

It is experimentally demonstrated that even though the numerical aperture in the object space is fixed, the resolution of an imaging system still can be improved by adjusting the parameters in the image space. This strategy cannot be realized until the discovery of the violation of the Lagrange invariant in a kind of self-interference holography. With the violation, parameters in the image space can escape the constraint of the object space for resolution improvement. Experiments that directly confirm this new ability are implemented and results agree well with the theoretical prediction. Additionally, better performance on frequency recording and finer details beyond the diffraction limit have been recorded with this method.

Relatively low level of antigen-specific monocytes detected in blood from untreated tuberculosis patients using CD4+ T-cell receptor tetramers.

The in vivo kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target Mycobacterium tuberculosis (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4(+) T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14(+) monocytes ranged from 0.26-1.44% and 0.21-0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages in situ via immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3-6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14(+) cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the mechanisms of antigen presentation and its relationship with TB infection and latent TB infection.

Super-resolution differential interference contrast microscopy by structured illumination.

We propose a structured illumination differential interference contrast (SI-DIC) microscopy, breaking the diffraction resolution limit of differential interference contrast (DIC) microscopy. SI-DIC extends the bandwidth of coherent transfer function of the DIC imaging system, thus the resolution is improved. With 0.8 numerical aperture condenser and objective, the reconstructed SI-DIC image of 53 nm polystyrene beads reveals lateral resolution of approximately 190 nm, doubling that of the conventional DIC image. We also demonstrate biological observations of label-free cells with improved spatial resolution. The SI-DIC microscopy can provide sub-diffraction resolution and high contrast images with marker-free specimens, and has the potential for achieving sub-diffraction resolution quantitative phase imaging.

Violation of the Lagrange invariant in an optical imaging system.

The Lagrange invariant provides a basic description of an optical imaging system. Many important conclusions can be drawn from it. We discovered that the Lagrange invariant is violated in a self-interference holography system with particular characteristics. With a proof-of-principle system, we proved this violation both theoretically and experimentally. This finding enables future exciting possibilities in optical imaging.

Anti-mycobacterial activity of marine fungus-derived 4-deoxybostrycin and nigrosporin.

4-Deoxybostrycin is a natural anthraquinone compound isolated from the Mangrove endophytic fungus Nigrospora sp. collected from the South China Sea. Nigrosporin is the deoxy-derivative of 4-deoxybostrycin. They were tested against mycobacteria, especially Mycobacterium tuberculosis. In the Kirby-Bauer disk diffusion susceptibility test, they both had inhibition zone sizes of over 25 mm. The results of the absolute concentration susceptibility test suggested that they had inhibitory effects against mycobacteria. Moreover, 4-deoxybostrycin exhibited good inhibition which was even better than that of first line anti-tuberculosis (TB) drugs against some clinical multidrug-resistant (MDR) M. tuberculosis strains. The gene expression profile of M. tuberculosis H37Rv after treatment with 4-deoxybostrycin was compared with untreated bacteria. One hundred and nineteen out of 3,875 genes were significantly different in M. tuberculosis exposed to 4-deoxybostrycin from control. There were 46 functionally known genes which are involved in metabolism, information storage and processing and cellular processes. The differential expressions of six genes were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The present study provides a useful experiment basis for exploitation of correlative new drugs against TB and for finding out new targets of anti-mycobacterial therapy.

Ultrasound Assessment of the Gastrocnemius Muscle as a Potential Tool for Identifying Sarcopenia in Patients with Type 2 Diabetes.

Objective: This study aims to examine the clinical significance of ultrasound evaluation of the gastrocnemius muscle (GM) in identifying sarcopenia in patients with type 2 diabetes (T2D). Methods: One hundred and fifty-three patients with T2D were included in this study. We measured the appendicular skeletal muscle mass index (ASMI), handgrip strength, and 6-meter walking speed. The US-derived muscle thickness (MT), cross-sectional area (CSA), and shear wave ultrasound elastography (SWE) of GM were also measured. We assessed the correlations between clinical indicators and US features. The model for screening sarcopenia was established using stepwise logistic regression. Stepwise linear regression was used to identify a set of variables that jointly estimated ASMI. The model's ability to identify sarcopenia and low muscle mass was assessed by receiver operating characteristic (ROC) curve analysis. Results: The prevalence of sarcopenia in this study was 24.2%. The CSA, MT and SWE values of the patients with sarcopenia were lower than those of patients without sarcopenia (all p < 0.05). ASMI was positively correlated with CSA (r = 0.56, p < 0.001) and MT (r = 0.39, p < 0.001). Handgrip strength was positively correlated with CSA (r = 0.45, p < 0.001), MT (r = 0.25, p < 0.001), and SWE (r = 0.26, p = 0.002). A diagnostic model for sarcopenia was established with a sensitivity of 81.1%, specificity of 75.0%, and an area under the curve (AUC) of 0.800. The estimated ASMI equation was developed and found to have a positive correlation with actual ASMI (r = 0.70, p < 0.001). It was also effective in diagnosing low muscle mass, with an AUC of 0.787 for males and 0.783 for females. Conclusion: Ultrasonographic assessment of the gastrocnemius muscle was found to be a useful and convenient method for detecting sarcopenia in patients with T2D.

Fluorescence holography with improved signal-to-noise ratio by near image plane recording.

Holographic fluorescence imaging is very promising, as it can obtain three-dimensional fluorescence imaging without scanning. However, the current method usually records holograms far from the image plane, with the fluorescence decaying when spreading broadly. Here we show that the signal-to-noise ratio (SNR) of fluorescence holography can be improved by recording the high-contrast interferogram near the image plane. We found that this can be achieved by setting the focal length of the lens for the reference wave (f(2)) close to that for the object wave (f(1)). With experiments, we demonstrate an example of an increase of about 21 times in SNR by changing f(2) from infinity to 226 mm, which is close to f(1) (323 mm).

Aberration-free digital holographic phase imaging using the derivative-based principal component analysis.

SIGNIFICANCE: Digital holographic microscopy is widely used to get the quantitative phase information of transparent cells. AIM: However, the sample phase is superimposed with aberrations. To quantify the phase information, aberrations need to be fully compensated. APPROACH: We propose a technique to obtain aberration-free phase imaging, using the derivative-based principal component analysis (dPCA). RESULTS: With dPCA, almost all aberrations can be extracted and compensated without requirements on background segmentation, making it efficient and convenient. CONCLUSIONS: It solves the problem that the conventional principal component analysis (PCA) algorithm cannot compensate the common but intricate higher order cross-term aberrations, such as astigmatism and coma. Moreover, the dPCA strategy proposed here is not only suitable for aberration compensation but also applicable for other cases where there exist cross-terms that cannot be analyzed with the PCA algorithm.

Hierarchical CNN-based occlusal surface morphology analysis for classifying posterior tooth type using augmented images from 3D dental surface models.

OBJECTIVE: 3D Digitization of dental model is growing in popularity for dental application. Classification of tooth type from single 3D point cloud model without assist of relative position among teeth is still a challenging task. METHODS: In this paper, 8-class posterior tooth type classification (first premolar, second premolar, first molar, second molar in maxilla and mandible respectively) was investigated by convolutional neural network (CNN)-based occlusal surface morphology analysis. 3D occlusal surface was transformed to depth image for basic CNN-based classification. Considering the logical hierarchy of tooth categories, a hierarchical classification structure was proposed to decompose 8-class classification task into two-stage cascaded classification subtasks. Image augmentations including traditional geometrical transformation and deep convolutional generative adversarial networks (DCGANs) were applied for each subnetworks and cascaded network. RESULTS: Results indicate that combing traditional and DCGAN-based augmented images to train CNN models can improve classification performance. In the paper, we achieve overall accuracy 91.35%, macro precision 91.49%, macro-recall 91.29%, and macro-F1 0.9139 for the 8-class posterior tooth type classification, which outperform other deep learning models. Meanwhile, Grad-cam results demonstrate that CNN model trained by our augmented images will focus on smaller important region for better generality. And anatomic landmarks of cusp, fossa, and groove work as important regions for cascaded classification model. CONCLUSION: The reported work has proved that using basic CNN to construct two-stage hierarchical structure can achieve the best classification performance of posterior tooth type in 3D model without assistance of relative position information. The proposed method has advantages of easy training, great ability to learn discriminative features from small image region.

Mapping and role of T cell response in SARS-CoV-2-infected mice.

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.

Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR.

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vgamma2Vdelta2 T cells, molecular mechanisms by which HMBPP interacts with Vgamma2Vdelta2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vgamma2Vdelta2 TCR of rhesus macaques to define HMBPP/APC interaction with Vgamma2Vdelta2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vgamma2Vdelta2 TCR tetramer. The Vgamma2Vdelta2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vgamma2Vdelta2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vgamma2Vdelta2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vgamma2Vdelta2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vgamma2Vdelta2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vgamma2Vdelta2 TCR.

Digital holographic phase imaging with aberrations totally compensated.

Digital holography is a well-accepted method for phase imaging. However, the phase of the object is always embedded in aberrations. Here, we present a digital holographic phase imaging with the aberrations fully compensated, including the high order aberrations. Instead of using pre-defined aberration models or 2D fitting, we used the simpler and more flexible 1D fitting. Although it is 1D fitting, data across the whole plane could be used. Theoretically, all types of aberrations can be compensated with this method. Experimental results show that the aberrations have been fully compensated and the pure object phase can be obtained for further studies.

Mycobacterium tuberculosis peptide E7/HLA-DRB1 tetramers with different HLA-DR alleles bound CD4+ T cells might share identical CDR3 region.

Human CD4+ T cells play an important role in the immune response to Mycobacterium tuberculosis (MTB). However, little is known about the spectratyping characteristics of the CD4+ T-cell receptor (TCR) α- and ß-chains CDR3 region in tuberculosis (TB) patients. We sorted MTB peptide E7-bound CD4+ T cells by using E7/HLA-DR tetramers constructed with different HLA-DRB1 alleles and extracted the CDR3 amino-acid sequences of TCR α- and ß-chains. The results showed that the CDR3 sequences of E7-bound CD4+ T cells were completely or partially identical in a single patient. The sequences of MTB peptide C5-bound CD4+ T cells shared another, and non-peptide bound CD4+ T cells, as well as unbound CD4+ T cells with tetramers were different from each other. Specifically, diverse CDR3 sequences of E7-bound CD4+ T cells displayed similar protein tertiary structure in one TB patient. In summary, the TCR α- and ß-chains of CDR3 lineage of CD4+ T cells in TB patients apparently drifted, and the predominant CDR3 sequences of TCR α- and ß-chains that recognized the MTB antigen exhibited peptide specificity, and certain HLA-DR restriction was also established. This study elucidates the possible causes and mechanisms of peptide-specific CD4+ T-cell-related presentation against MTB.

microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production.

Macrophages play a crucial role in host innate anti-mycobacterial defense, which is tightly regulated by multiple factors, including microRNAs. Our previous study showed that a panel of microRNAs was markedly up-regulated in macrophages upon mycobacterial infection. Here, we investigated the biological function of miR-146a during mycobacterial infection. miR-146a expression was induced both in vitro and in vivo after Mycobacterium bovis BCG infection. The inducible miR-146a could suppress the inducible nitric oxide (NO) synthase (iNOS) expression and NO generation, thus promoting mycobacterial survival in macrophages. Inhibition of endogenous miR-146a increased NO production and mycobacterial clearance. Moreover, miR-146a attenuated the activation of nuclear factor κB and mitogen-activated protein kinases signaling pathways during BCG infection, which in turn repressed iNOS expression. Mechanistically, miR-146a directly targeted tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) at post-transcriptional level. Silencing TRAF6 decreased iNOS expression and NO production in BCG-infected macrophages, while overexpression of TRAF6 reversed miR-146a-mediated inhibition of NO production and clearance of mycobacteria. Therefore, we demonstrated a novel role of miR-146a in the modulation of host defense against mycobacterial infection by repressing NO production via targeting TRAF6, which may provide a promising therapeutic target for tuberculosis.

Monitoring of peptide-specific and gamma interferon-productive T cells in patients with active and convalescent tuberculosis using an enzyme-linked immunosorbent spot assay.

To establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P > 0.05), but the median number of spot-forming cells/10(6) cells (SFP value) for positive results was higher by the former than by the latter assay (P < 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P < 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.