A novel transgenic mouse model for corneal scar visualization.

Corneal opacities affect vision for millions of individuals worldwide. Fibrotic scar tissues accumulate in reaction to inflammatory responses and remain permanently in corneal stroma, and conventionally correctable only by donor corneal transplantation. Numerous studies have explored innovative approaches to reverse corneal scarring through non-surgical means; however, existing mouse models limit these studies, due to the lack of visibility of scar tissue in mouse corneas with steep curvature. Here, we reported that corneal scarring was modelled using a transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, in which enhanced green fluorescence protein (EGFP) reporter expression was driven by the promoter of collagen 3a1 (COL3a1), a stromal fibrosis gene. Similar to wildtype, Col3a1-EGFP transgenic corneas developed opacities after wounding by alkali burn and mechanical ablation, respectively, as examined under stereomicroscopy and Spectral Domain optical coherent tomography. The time course induction of EGFP was aligned with Col3a1 upregulation and matched with the elevated expression of other fibrosis genes (α-smooth muscle actin, fibronectin and tenascin C). Measured by flow cytometry and enzyme-linked immunosorbent assay, increased number of EGFP expressing cells and fluorescent intensities were correlated to corneal thickening and scar volume. After treatment with human corneal stromal stem cells or their exosomes, EGFP expression was downregulated together with the reduction of scar volume and fibrosis gene expression. These results have demonstrated that the transgenic mouse line, Tg(Col3a1-EGFP)DJ124Gsat, can be a valuable tool for the detection of corneal fibrosis and scarring in vivo, and will be useful in monitoring the changes of corneal fibrosis over time.

Association Between Preceding Viral Respiratory Infection and Subsequent Respiratory Illnesses Among Children: A Prospective Cohort Study in the Philippines.

Background: Acute respiratory infection (ARI) is of great concern in public health. It remains unclear whether viral infections can affect the host's susceptibility to subsequent ARIs. Methods: A prospective cohort study on ARIs of children below 5 years old was conducted in the Philippines from 2014 to 2016. The respiratory symptoms were recorded daily, and nasopharyngeal swabs were collected at both household and health facilities. The specimens were tested for respiratory viruses. We then determined whether viral etiology was associated with the severity of the present ARI and whether previous viral infections was associated with subsequent ARIs. Results: A total of 3851 children and 16337 ARI episodes were enrolled and recorded, respectively. Samples were collected from 24% of all ARI episodes; collection rate at the healthcare facilities was 95%. Enterovirus D68, rhinovirus species C, and respiratory syncytial virus were significantly associated with severe ARIs. The risk for subsequent ARIs was significantly enhanced after infections with adenovirus, influenza A virus, parainfluenza virus type 4, and rhinovirus species C. Conclusions: This study revealed that viral etiology plays a significant role in the severity of the present ARI and that viral infection affects the host's susceptibility to subsequent ARIs.

Deep learning based characterization of human organoids using optical coherence tomography.

Organoids, derived from human induced pluripotent stem cells (hiPSCs), are intricate three-dimensional in vitro structures that mimic many key aspects of the complex morphology and functions of in vivo organs such as the retina and heart. Traditional histological methods, while crucial, often fall short in analyzing these dynamic structures due to their inherently static and destructive nature. In this study, we leveraged the capabilities of optical coherence tomography (OCT) for rapid, non-invasive imaging of both retinal, cerebral, and cardiac organoids. Complementing this, we developed a sophisticated deep learning approach to automatically segment the organoid tissues and their internal structures, such as hollows and chambers. Utilizing this advanced imaging and analysis platform, we quantitatively assessed critical parameters, including size, area, volume, and cardiac beating, offering a comprehensive live characterization and classification of the organoids. These findings provide profound insights into the differentiation and developmental processes of organoids, positioning quantitative OCT imaging as a potentially transformative tool for future organoid research.

Molecular evolution of the hemagglutinin and neuraminidase genes of pandemic (H1N1) 2009 influenza viruses in Sendai, Japan, during 2009-2011.

Analyzing the evolutionary pattern of the influenza A(H1N1)pdm09 strain in different regions is important for understanding its diversification. We therefore conducted this study to elucidate the genetic variability and molecular evolution of the influenza A(H1N1)pdm09 strains that circulated during the 2009-2010 and 2010-2011 influenza seasons in Sendai, Japan. Nasopharyngeal swab specimens were collected from patients with influenza-like illnesses who visited outpatient clinics in Sendai City, Japan, from September 2009 to April 2011. A total of 75 isolates were selected from September 2009 to April 2011 to analyze the genetic changes in the entire hemagglutinin 1 (HA1) segment of the HA gene and the neuraminidase (NA) gene based on sequence analysis. Bayesian coalescent Markov chain Monte Carlo analyses of HA1 and NA gene sequences were performed for further analysis. High sequence identities were observed for HA1 and NA in influenza A(H1N1)pdm09, displaying 99.06 and 99.33 % nucleotide identities, respectively, with the A(H1N1)pdm09 vaccine strain A/California/07/2009. The substitution rates of nucleotides for HA1 in the 2009-2010 and 2010-2011 were 1.5 × 10-3 and 1.6 × 10-3 substitutions per site per year, respectively. Phylogenetic tree analysis demonstrated that Sendai isolates were clustered into global clade 7, which is characterized by an S203T mutation in the HA1 gene. Moreover, two distinct circulation clusters were present in the 2010-2011 season. Mutations were present in antigenic or receptor-binding domains of the HA1 segment, including A141V, S143G, S183P, S185T, and S203T. The Bayesian skyline plot model illustrated a steady rate for the maintenance of genetic diversity, followed by a slight increase in the later part of the 2010-2011 season. Selection analysis revealed that the HA1 (position 197) and NA (position 46) sites were under positive selection; however, no known mutation conferring resistance to NA inhibitors such as H275Y was observed. The effect on control of the influenza A(H1N1)pdm09 virus, including vaccine strain selection, requires continuous monitoring of the strain by genetic surveillance.

Viruses that cross borders: factors responsible for global dissemination of viral infections.

OBJECTIVE: Pandemic viral infections as emerging infectious diseases are of a great global concern. However, for some viruses, particular strains are endemic to specific areas and can be genetically distinguished from strains in other regions. In contrast, for some other viruses, genetically similar strains can spread and circulate all over the world. This study addresses global dissemination of various viral infections. METHODS: We classified 34 viruses as per their ability to cross borders by review. We also described factors responsible for and the dynamics of global dissemination. We examined biological characteristics of viruses, manners or routes of transmission, host responses and epidemiological factors. RESULTS: Factors required for viruses to cross borders include 'non-blood infection', 'short incubation period', 'short infectious period', 'frequent re-infection', 'small basic reproductive number (R(0))' and 'high annual incidence'. CONCLUSION: Knowing the factors responsible for global dissemination of pathogens is useful for controlling and/or containing both classic and emerging infectious diseases.

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factor ß3.

BACKGROUND: Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect. METHODS: Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor ß3 (hTGFß3) and hTGFß1 were examined by immunofluorescence and qPCR. TGFß3 knockdown for > 70% in hCSSC [hCSSC-TGFß3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFß3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined. RESULTS: hTGFß3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFß3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFß3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring. CONCLUSION: This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFß3, inducing a scar-free tissue response.

Mesenchymal Stem Cells Reduce Corneal Fibrosis and Inflammation via Extracellular Vesicle-Mediated Delivery of miRNA.

Mesenchymal stem cells from corneal stromal stem cells (CSSC) prevent fibrotic scarring and stimulate regeneration of transparent stromal tissue after corneal wounding in mice. These effects rely on the ability of CSSC to block neutrophil infiltration into the damaged cornea. The current study investigated the hypothesis that tissue regeneration by CSSC is mediated by secreted extracellular vesicles (EVs). CSSC produced EVs 130-150 nm in diameter with surface proteins that include CD63, CD81, and CD9. EVs from CSSC reduced visual scarring in murine corneal wounds as effectively as did live cells, but EVs from human embryonic kidney (HEK)293T cells had no regenerative properties. CSSC EV treatment of wounds decreased expression of fibrotic genes Col3a1 and Acta2, blocked neutrophil infiltration, and restored normal tissue morphology. CSSC EVs labeled with carboxyfluorescein succinimidyl ester dye, rapidly fused with corneal epithelial and stromal cells in culture, transferring microRNA (miRNA) to the target cells. Knockdown of mRNA for Alix, a component of the endosomal sorting complex required for transport, using siRNA, resulted in an 85% reduction of miRNA in the secreted EVs. The EVs with reduced miRNA were ineffective at blocking corneal scarring. Furthermore, CSSC with reduced Alix expression also lost their regenerative function, suggesting EVs as an obligate component in the delivery of miRNA. The results of these studies support an essential role for extracellular vesicles in the process by which CSSC cells block scarring and initiate regeneration of transparent corneal tissue after wounding. EVs appear to serve as a delivery vehicle for miRNA, which affects the regenerative action. Stem Cells Translational Medicine 2019;8:1192-1201.

Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound-healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat-tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 µm by passive absorption. The CCG disks were dimensionally stable, easy to handle, and could be adhered securely to de-epithelialized mouse cornea with fibrin-based adhesive. CSSC in CCG maintained >80% viability for >1 week in culture media and could be cryopreserved in 20% fetal bovine serum-10%DMSO in liquid nitrogen. CCG containing as few as 500 CSSC effectively prevented visible scarring and suppressed expression of fibrotic Col3a1 mRNA. CSSC in CCG were more effective at blocking scarring on a per-cell basis than CSSC delivered directly in a fibrin gel as previously described. Collagen-embedded cells retained the ability to suppress corneal scarring after conventional cryopreservation. This study demonstrates use of a common biomaterial that can facilitate storage and handling of stem cells in a manner that may provide off-the-shelf delivery of stem cells as a therapy for corneal scarring. Stem Cells Translational Medicine 2018;7:487-494.

Evolutionary Dynamics of Tat in HIV-1 Subtypes B and C.

Evolutionary characteristics of HIV-1 have mostly studied focusing its structural genes, Gag, Pol and Env. However, regarding the process of HIV-1's evolution, few studies emphasize on genetic changes in regulatory proteins. Here we investigate the evolutionary dynamics of HIV-1, targeting one of its important regulatory proteins, Tat. We performed a phylogenetic analysis and employed a Bayesian coalescent-based approach using the BEAST package to investigate the evolutionary changes in Tat over time in the process of HIV-1 evolution. HIV-1 sequences of subtypes B and C from different parts of the world were obtained from the Los Alamos database. The mean estimated nucleotide substitution rates for Tat in HIV-1 subtypes B and C were 1.53 x 10(-3) (95% highest probability density- HPD Interval: 1.09 x10-3 to 2.08 x 10(-3)) and 2.14 x 10(-3) (95% HPD Interval: 1.35 x 10(-3) to 2.91 x 10(-3)) per site per year, respectively, which is relatively low compared to structural proteins. The median times of the most recent common ancestors (tMRCA) were estimated to be around 1933 (95% HPD, 1907-1952) and 1956 (95% HPD, 1934-1970) for subtypes B and C, respectively. Our analysis shows that subtype C appeared in the global population two decades after the introduction of subtype B. A Gaussian Markov random field (GMRF) skyride coalescent analysis demonstrates that the early expansion rate of subtype B was quite high, rapidly progressing during the 1960s and 1970s to the early 1990s, after which the rate increased up to the 2010s. In contrast, HIV-1 subtype C exhibited a relatively slow occurrence rate until the late 1980s when there was a sharp increase up to the end of 1990s; thereafter, the rate of occurrence gradually slowed. Our study highlights the importance of examining the internal/regulatory genes of HIV-1 to understand its complete evolutionary dynamics. The study results will therefore contribute to better understanding of HIV-1 evolution.

Intersubtype Genetic Variation of HIV-1 Tat Exon 1.

HIV-1 Tat is a regulatory protein that plays a pivotal role in viral transcription and replication. Our study aims to investigate the genetic variation of Tat exon 1 in all subtypes of HIV-1: A, B, C, D, F, G, H, J, and K. We performed phylogenetic, mutation, and selection pressure analyses on a total of 1,179 sequences of different subtypes of HIV-1 Tat obtained from the Los Alamos National Laboratory (LANL). The mean nucleotide divergences (%) among the analyzed sequences of subtypes A, B, C, D, F, G, H, J, and K were 88, 89, 90, 88, 86, 89, 88, 97, and 97, respectively. We revealed that subtype B evolved relatively faster than other subtypes. The second and fifth domains were found comparatively more variable among all subtypes. Site-by-site tests of positive selection revealed that several positions in all subtypes were under significant positive selection. Positively selected sites were found in the acidic domain at positions 3, 4, and 19, in the cysteine-rich domains at positions 24, 29, 32, and 36, in the core domain at position 40, and in the basic domain for the rest of the positions for all subtypes. Positions 58 and 68 in the basic domain were positively selected in subtypes A, B, C and B, C, F, respectively. We also observed high variability within positively selected sites in amino acid positions. Our study findings on HIV-1 Tat genetic variability may contribute to a better understanding of HIV-1 evolution as well as to the development of effective Tat-targeted therapeutics and vaccines.

Molecular characterization of full-length Tat in HIV-1 subtypes B and C.

HIV-1Tat (trans-acting activator of transcription) plays essential roles in the replication through viral mRNA and genome transcription from the HIV-1 LTR promoter. However, Tat undergoes continuous amino acid substitutions. As a consequence, the virus escapes from host immunity indicating that genetic diversity of Tat protein in major HIV-1 subtypes is required to be continuously monitored. We analyzed available full-length HIV-1 sequences of subtypes B (n=493) and C (n=280) strains circulating worldwide. We observed 81% and 84% nucleotide sequence identities of HIV-1 Tat for subtypes B and C, respectively. Based on phylogenetic and mutation analyses, global diversity of subtype B was apparently higher compared to that of subtype C. Positively selected sites, such as positions Ser68 and Ser70 in both subtypes, were located in the Tat-transactivation responsive RNA (TAR) interaction domain. We also found positively selected sites in exon 2, such as positions Ser75, Pro77, Asp80, Pro81 and Ser87 for both subtypes. Our study provides useful information on the full-length HIV-1 Tat sequences in globally circulating strains.

Comparison of selection pressures on the HA gene of pandemic (2009) and seasonal human and swine influenza A H1 subtype viruses.

The 2009 human pandemic influenza (H1N1) virus possesses the HA gene of the H1 subtype. The evolutionary process of the 2009 H1N1 virus remains to be defined. We performed genetic analyses of the HA gene by comparing the 2009 H1N1 virus with seasonal human and swine viruses. We analyzed sequences of 116 2009 H1N1 viruses, and obtained 1457 seasonal H1N1, 365 swine H1, and 1332 2009 H1N1 viruses from the database. Selection pressure for the 2009 H1N1 virus was higher than that for the swine virus and equivalent to that for the seasonal virus. Positions 206 and 264 were found to be positively selected sites. We also identified sites under different selection pressures from the seasonal or swine virus that may be involved in imparting significant biological characteristics. The evolutionary characteristics of the H1 gene of the 2009 H1N1 virus differed from those of seasonal and swine viruses.

Differentiation of human influenza A viruses including the pandemic subtype H1N1/2009 by conventional multiplex PCR.

April 2009 witnessed the emergence of a novel H1N1 influenza A virus infecting the human population. Currently, pandemic and seasonal influenza viruses are co-circulating in human populations. Understanding the course of the emerging pandemic virus is important. It is still unknown how the novel virus co-circulates with or outcompetes seasonal viruses. Sustainable and detailed influenza surveillance is required throughout the world including developing countries. In the present study, a multiplex PCR using four primers was developed, which was designed to differentiate the pandemic H1N1 virus from the seasonal H1N1 and H3N2 viruses, to obtain amplicons of different sizes. Multiplex PCR analysis could clearly differentiate the three subtypes of human influenza A virus. This assay was performed using 206 clinical samples collected in 2009 in Japan. Between February and April, four samples were subtyped as seasonal H1N1 and four as seasonal H3N2. All samples collected after July were subtyped as pandemic H1N1. Currently, pandemic viruses seem to have replaced seasonal viruses almost completely in Japan. This is a highly sensitive method and its cost is low. Influenza surveillance using this assay would provide significant information on the epidemiology of both pandemic and seasonal influenza.

Simplified screening method for detecting oseltamivir resistant pandemic influenza A (H1N1) 2009 virus by a RT-PCR/restriction fragment length polymorphism assay.

The sudden emergence of the pandemic influenza A (H1N1) 2009 virus in early 2009 has resulted in a rapid transmission of this virus worldwide. Within a short time span, sporadic cases infected with this virus that shows oseltamivir resistance have also been reported. These resistant viruses have an amino acid change from histidine to tyrosine at position 275 (H275Y) of the neuraminidase gene. In this study, a reverse transcriptase PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay was developed to detect the H275Y mutation. Resistant and sensitive viruses could be differentiated using the RFLP patterns. This RT-PCR/RFLP assay is a simple method and also very specific and sensitive for detecting the H275Y mutation of pandemic influenza A (H1N1) 2009 viruses, and can be used in resource-limited settings.