Changes In Autofluorescence Research Articles

Changes in reactive oxygen species and autofluorescence under hypoxia at the hippocampal CA3 area: Role of calcium and zinc influxes

Reactive oxygen species (ROS) have an important role in cellular biology, being involved, in a way that depends on their levels, in cell signaling processes or in oxidative stress, probably associated with neurodegenerative and other diseases. Most of the studies about ROS formation were performed in ischemic conditions, and thus, there is limited knowledge about ROS formation in less severe hypoxic conditions. This study investigates neuronal ROS generation and autofluorescence changes in hypoxic conditions, focusing on the involvement of calcium and zinc. Using hippocampal slices from Wistar rats, ROS production was monitored by the permeant fluorescent indicator H2DCFDA under different oxygenation levels. Moderate hypoxia (40% O2) led to a small ROS increase, while severe hypoxia (0% O2) showed a more pronounced rise. KCl-induced depolarization significantly enhanced ROS formation, particularly under severe hypoxia. Inhibition of NMDA receptors reduced ROS generation without affecting autofluorescence, while chelation of zinc ions decreased ROS production and increased flavin adenine dinucleotide (FAD) autofluorescence. These findings suggest that, in hypoxic conditions, ROS formation is mediated by calcium entry through NMDA receptors and also by zinc influxes. Thus, these ions play a crucial role in oxidative stress, which may be related with neurodegenerative diseases associated with ROS dysregulation.

UV irradiation during fluorescence measurements affects the structures and fluorescence properties of isolated lignins in solution

There are no reports on the stability of autofluorescence, although lignins and their solutions are known to exhibit autofluorescence. An aim of this study is to investigate spectral change of lignin autofluorescence under prolonged excitation with ultraviolet (UV). Both fluorescence spectra of softwood milled wood lignin (MWL) and kraft lignin (SKL) in propylene glycol significantly changed upon an UV irradiation at 280 nm under O2 and Ar atmospheres even for 10 min. By the further irradiation, the autofluorescence intensities were remarkably changed together with the emission wavelengths. However, no tendency in the autofluorescence change was observed. The change varied between lignin preparations and gas atmosphere. These phenomena should be caused by autoxidation upon UV irradiation, and the reaction resulted in not only photodegradation but also photopolymerization.

Fundus Autofluorescence in Diabetic Retinopathy.

Diabetic retinopathy is a leading cause of visual morbidity worldwide. Fundus autofluorescence is a rapid, non-invasive imaging modality that has gained increased popularity in recent years in the multimodal evaluation of diabetic retinopathy and, in particular, of diabetic macular oedema. Acquired using either a fundus camera or the confocal scanning laser ophthalmoscope, short-wavelength and near-infrared autofluorescence are the most used techniques in diabetic retinopathy. In diabetic macular oedema, short-wavelength autofluorescence, in its cystoid pattern, is useful for detecting cystoid macular oedema. Increased spot hyperautofluorescence in short-wavelength and granular changes in near-infrared autofluorescence correlate well with other imaging findings, indicating photoreceptor and retinal pigment epithelium damage and being associated with decreased visual acuity. While also being a marker of oxidative stress, increased short-wavelength autofluorescence in the setting of diabetic macular oedema appears to be a prognostic factor for poor visual outcome, even after the resolution of the intraretinal fluid. Autofluorescence also helps in the assessment of diabetic retinal pigment epitheliopathy and choroidopathy. Fundus autofluorescence is an evolving technology that will assist in gaining further insight into the pathophysiology of diabetic retinopathy and allow for a more comprehensive evaluation of these patients.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. A repeatability analysis revealed change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

CALIX[4]ARENES С-715 AND С-772 AS INSTRUMENTS OF INFLUENCE ON Са2+-TRANSPORT AND BIOENERGETICS IN MYOMETRIAL MITOCHONDRIA

Aim. The goal of work is to study the effects of calix[4]arenes C-715 and C-772 on Ca2+-transport, the electron transport chain activity, and the reactive oxygen species generation in the mitochondria of rat uterine smooth muscle. Methods. Measurement of changes in NADH autofluorescence, as well as the energy-dependent accumulation of Ca2+ and reactive oxygen species generation in the fraction of isolated mitochondria was carried out using the spectrofluorimetry method. The hydrodynamic diameter of mitochondria was measured using the laser correlation spectroscopy method. Results. It was found that calix[4]arenes C-715 and C-772 moderately inhibit both the NADH oxidation and the energy-dependent Ca2+ accumulation by isolated mitochondria. We also showed that the studied calix[4]arenes, depending on concentration and time, reduce the level of reactive oxygen species generation by mitochondria. Additionally, it was revealed that the studied compounds moderately increase mitochondria hydrodynamic diameter depending on concentration. Conclusions. Studied calix[4]arenes slow down the oxidation of NADH in isolated mitochondria, which represents an inhibitory effect on the electron transport chain functioning, in particular its complex I. Since this might lower the electrochemical potential of the inner mitochondrial membrane, we observed reduce of energy-dependent Ca2+ accumulation. Selected compounds decrease the level of reactive oxygen species generation by isolated mitochondria, which could be considered as protective effect on organelles. A moderate increase in mitochondria hydrodynamic diameter suggests that the studied compounds do not cause mitochondrial dysfunction. Researched calix[4]arenes can be used in experimental practice to influence the mitochondrial functional activity.

Prospective Study of Change in Skin Autofluorescence Over Time and Mortality in People Receiving Hemodialysis

Elevated skin autofluorescence (SAF), a measure of tissue accumulation of advanced glycation end products (AGEs), is a strong predictor of all-cause and cardiovascular mortality in the hemodialysis population. However, prospective studies investigating the association between changes in SAF over time and mortality are scarce. We therefore aimed to investigate the prognostic value of SAF trend for predicting mortality in a hemodialysis population. We enrolled 120 patients on hemodialysis in a 5-year observational, prospective study. SAF was measured at baseline, 3, 6, 9, 12, and 24 months. Rate of change in SAF (i.e., SAF trend) was calculated using linear regression. Time to event was the number of days from baseline to death, kidney transplantation, or March 31,2022. Mean age, mean baseline SAF, and median SAF trend were 65 ± 14 years, 3.4 ± 0.9 arbitrary units (AU), and an increase of 0.1 (-0.1 to 0.4) AU/yr, respectively. Median observation time was 42 months, during which 59 participants (49%) died. Univariable analysis identified age, history of smoking, lower serum albumin, higher baseline SAF, and increase in SAF as significant predictors of higher mortality. In multivariable analysis, higher baseline SAF (hazard ratio: 1.45; 95% confidence interval: 1.08-1.94; P= 0.01) and increasing SAF trend (2.37 [1.43-3.93]; P< 0.001) were independent predictors of increased mortality. An increasing SAF trend and higher baseline SAF were independent predictors of all-cause mortality in this hemodialysis population, suggesting that monitoring of SAF may have clinical utility. Strategies to improve outcomes by reducing or preventing the increase in SAF should now be investigated in prospective studies.

Macular pigment evaluation using dual-wavelength fundus auto-fluorescence imaging.

Macular pigment plays an important role in the reduction of oxidative stress and in preventing retinal diseases. Quick and easy measurements of the macular pigment are essential in both clinical and research settings. Dual wavelength fundus auto-fluorescence seems to be the optimal method. This study aims to investigate changes in fundus autofluorescence in patients taking daily lutein oral supplements and develop image processing methods for follow-up evaluations of the images. New devices allow us to examine fundus autofluorescence using both blue and green excitation wavelengths. This allows detection of the amount of macular pigment by subtracting these two images because the yellow pigment particles absorb blue wavelengths. We determined daily dose of 25 mg of lutein and 3 mg of zeaxanthin. Patients were followed up for 15 months at 3-month intervals. During our 15-month study, we observed a positive trend in pixel lightness values, suggesting an increase in macular pigments in the foveal area. In all patients taking daily lutein supplements, the foveal index significantly increased after six months, with a median change of 0.081. We did not observe a significant change after the first three months (0.006) and only a small change between the 6th and 12th-month visits (0.012). With appropriate patients and procedures for capturing autofluorescence images, this is a valuable technique for macular pigment evaluation in follow-up examinations using software image post-processing and analysis with commonly available hardware. To put this into everyday practice, developing tools to automate the assessment is necessary.

EARLY STAGES OF COLORECTAL CANCER CHARACTERIZATION BY AUTOFLUORESCENCE 3D MICROSCOPY: A PRELIMINARY STUDY.

Colorectal cancer is one of the most prevalent pathologies worldwide whose prognosis is linked to early detection. Colonoscopy is the gold standard for screening, and diagnosis is usually made histologically from biopsies. Aiming to reduce the inspection and diagnostic time as well as the biopsies and resources involved, other techniques are being promoted to conduct accurate in vivo colonoscopy assessments. Optical biopsy aims to detect normal and neoplastic tissues analysing the autofluorescence spectrum based on the changes in the distribution and concentration of autofluorescent molecules caused by colorectal cancer. Therefore, the autofluorescence contribution analysed by image processing techniques could be an approach to a faster characterization of the target tissue. Quantify intensity parameters through digital processing of two data sets of three-dimensional widefield autofluorescence microscopy images, acquired by fresh colon tissue samples from a colorectal cancer murine model. Additionally, analyse the autofluorescence data to provide a characterization over a volume of approximately 50 µm of the colon mucosa for each image, at second (2nd), fourth (4th) and eighth (8th) weeks after colorectal cancer induction. Development of a colorectal cancer murine model using azoxymethane/dextran sodium sulphate induction, and data sets acquisition of Z-stack images by widefield autofluorescence microscopy, from control and colorectal cancer induced animals. Pre-processing steps of intensity value adjustments followed by quantification and characterization procedures using image processing workflow automation by Fiji's macros, and statistical data analysis. The effectiveness of the colorectal cancer induction model was corroborated by a histological assessment to correlate and validate the link between histological and autofluorescence changes. The image digital processing methodology proposed was then performed on the three-dimensional images from control mice and from the 2nd, 4th, and 8th weeks after colorectal cancer chemical induction, for each data set. Statistical analyses found significant differences in the mean, standard deviation, and minimum parameters between control samples and those of the 2nd week after induction with respect to the 4th week of the first experimental study. This suggests that the characteristics of colorectal cancer can be detected after the 2nd week post-induction. The use of autofluorescence still exhibits levels of variability that prevent greater systematization of the data obtained during the progression of colorectal cancer. However, these preliminary outcomes could be considered an approach to the three-dimensional characterization of the autofluorescence of colorectal tissue, describing the autofluorescence features of samples coming from dysplasia to colorectal cancer. • A new digital image processing method was developed to measure intensity in 3D autofluorescence images of colorectal samples using a CRC mouse model. • This method showed that autofluorescence intensity in colon mucosa is similar in healthy tissue but changes significantly in tumor development. • Statistical analysis revealed CRC traits detectable from the second week post-induction, aiding in early CRC detection. • The study provides a basis for 3D autofluorescence characterization in colorectal tissue from dysplasia to cancer, although variability in autofluorescence limits data systematization during cancer progression.

Live-Cell Imaging Quantifies Changes in Function and Metabolic NADH Autofluorescence During Macrophage-Mediated Phagocytosis of Tumor Cells

ABSTRACT Background The immune system has evolved to detect foreign antigens and deliver coordinated responses, while minimizing “friendly fire.” Until recently, studies investigating the behavior of immune cells were limited to static in vitro measurements. Although static measurements allow for real-time imaging, results are often difficult to translate to an in vivo setting. Multiphoton microscopy is an emerging method to capture spatial information on subcellular events and characterize the local microenvironment. Previous studies have shown that multiphoton microscopy can monitor changes in single-cell macrophage heterogeneity during differentiation. Therefore, there is a need to use multiphoton microscopy to monitor molecular interactions during immunological activities like phagocytosis. Here we investigate the correlation between phagocytic function and changes in endogenous optical reporters during phagocytosis. Methods In vitro autofluorescence imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was used to detect metabolic changes in macrophages during phagocytosis. More specifically, optical redox ratio, mean NADH fluorescence lifetime and ratio of free to protein-bound NADH were used to quantify changes in metabolism. Results Results show that IFN-γ (M1) macrophages showed decreased optical redox ratios and mean NADH lifetime while phagocytosing immunogenic cancer cells compared to metastatic cells. To validate phagocytic function, a fluorescence microscopy-based protocol using a pH-sensitive fluorescent probe was used. Results indicate that M0 and M1 macrophages show similar trends in phagocytic potential. Conclusion Overall, this work demonstrates that in vitro multiphoton imaging can be used to longitudinally track changes in phagocytosis and endogenous metabolic cofactors.

Four different gene-related cone-rod dystrophy: clinical and genetic findings in six Chinese families with diverse modes of inheritance.

Purpose: To investigate pathogenic variants in six families with cone-rod dystrophy (CORD) presenting various inheritance patterns by using whole-exome sequencing (WES) and analyzing phenotypic features. Methods: A total of six families with CORD were enrolled in Ningxia Eye Hospital for this study. The probands and their family members received comprehensive ophthalmic examinations, and DNA was abstracted from patients and family members. Whole-exome sequencing was performed on probands to screen the causative variants, and all suspected pathogenic variants were determined via Sanger sequencing. Furthermore, co-segregation analysis was performed on available family members. The pathogenicity of novel variants was predicted using in silico analysis and evaluated according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Of the six families, two families were assigned as X-linked recessive (XL), two families were assigned as autosomal recessive (AR), and two families were assigned as autosomal dominant (AD). Pathogenic variants were detected in CACNA1F in two X-linked recessive probands, among which family 1 had a hemizygous frameshift variant c.2201del (p.Val734Glyfs*17) and family 2 had a hemizygous missense variant c.245G>A (p.Arg82Gln). Both probands had high myopia, with fundus tessellation accompanied by abnormalities in the outer structure of the macular area. The homozygous splice variant c.2373 + 5G>T in PROM1 and the homozygous nonsense variant c.604C>T (p.Arg202Ter) in ADAM9 were detected in two autosomal recessive families of the probands. Both probands showed different degrees of atrophy in the macular area, and the lesions showed hypofluorescence changes in autofluorescence. The heterozygous variation in CRX c.682C>T (p.Gln228Ter) was detected in two autosomal dominant families. The onset age of the two probands was late, with better vision and severe macular atrophy. According to ACMG guidelines and the analysis of online in silico tools, all variations were labeled as potentially harmful or pathogenic. Conclusion: Pathogenic variants in CACNA1F, PROM1, ADAM9, and CRX genes were identified in six families affected by the diverse inheritance patterns of CORD. Furthermore, the potential impact of the nonsense-mediated decay (NMD) mechanism on the manifestation of CORD phenotypes was examined and addressed. Simultaneously, the spectrum of pathogenic variants and clinical phenotypes associated with the CORD gene was extended.

The Small Intestine in the Acute Period of Spinal Injury: Early Metabolic Disorders According to Fluorescence-Lifetime Imaging FLIM

RELEVANCE A special place in the development of enteral insufficiency is given to dysproteinemia, which is one of the leading causes of the development of decubital ulcers in patients with spinal cord injury. Early enteral nutrition partially solved this problem, but the incidence of bedsores still remains high and reaches 68%. The risk of metabolic disorders in the acute period of spinal injury is largely determined by non-occlusive intestinal ischemia against the background of spinal shock, neurohumoral dysregulation; intra-intestinal and intra-abdominal hypertension; change in intestinal microflora. Pathological changes in the intestinal wall occur during the first 20 days after injury and further exacerbate chronic maldigestion, malabsorption, intestinal dyskinesia in patients with traumatic spinal cord disease. New knowledge about the features of early enteral nutrition in patients in the acute period of traumatic spinal cord disease will reduce the risk of decubitus ulcerative defects.AIM OF THE STUDY To study the dynamics of metabolic processes in the tissues of the small intestine in the acute period of spinal injury.MATERIAL AND METHODS Wistar rats (n=22). Spinal injury was simulated by acute complete transection of the spinal cord at the level of Th5–Th6 vertebrae. The assessment of metabolic changes in the cells of the serous membrane of the intestine was performed immediately, 3 and 24 hours after injury. The metabolism was assessed in vivo using fluorescence time-resolved macroimaging technology FLIM by autofluorescence in the spectral channel of the metabolic cofactor nicotinamide adenine dinucleotide (phosphate).RESULTS The acute period of spinal cord injury is accompanied by a change in the endogenous autofluorescence of the serous membrane of the small intestine: a statistically significant decrease in the mean fluorescence lifetime (τm), the lifetime of the long component (τ2), and the relative contribution of the long component (а2) in 24 h after injury was recorded. The changes observed using FLIM confirm the catabolic type of metabolism in the tissues of the small intestine after spinal cord injury.CONCLUSION For the first time in the experiment in vivo it has been shown that the acute period of spinal injury is accompanied by a violation of metabolic processes in the tissues of the small intestine. This fact requires a more balanced approach in calculating the calorie content of nutrients used for early enteral nutrition in patients with spinal cord injury.

Клинический случай хороидеремии

Purpose. To present an overview of a choroideremia clinical case. Material and Methods. The article presents a clinical case of a 19-year-old patient with complaints of low distance vision, narrowing of the visual field and nictalopia since childhood. The patient underwent complex ophthalmological examination including visometry, autorefractometry, tonometry, biomicro-ophthalmoscopy with Goldman lens, computer perimetry, optical biometry, optical coherence tomography with angiography function (AngioOCT), fundus photoregistration with autofluorescence, electrophysiological examination (EP). The patient was consulted by a geneticist to confirm the basic diagnosis. Results. Visual acuity: OD = 0.1 Sph -2.0 = 1.0; OS = 0.15 Sph -1.5 = 1.0. The anterior-posterior eyeball size: OD – 24.98 mm, OS – 25.0 mm. Computer perimetry revealed narrowing of visual fields up to 10 degrees in both eyes, which correlated with changes in autofluorescence. According to EP, electrical sensitivity threshold: OD 198 µA, OS 202 µA, electrical lability: OD 37 Hz, OS 35 Hz. High visual acuity with correction in patient C. is caused by the macula condition, according to the results of AngioOCT. The retinal pigment epithelium and chorioid are thin. Chorioid outside the macular area lacks the Sattler's layer. Based on the results of a consultation with a geneticist, a pathogenic variant of the nucleotide sequence was detected in the CHM gene (chrX:85213886). Conclusion. Choroideremia is a rare genetic disease with a specific clinical picture and progressive development of visual disorder. Proper and early diagnosis allows the prevention of the disease, including prenatal diagnosis, and the development of new therapies. Keywords: choroideremia, genetic choroidal diseases, optical coherence tomography, autofluorescence, electrophysiological examination

Prolonged Lifetimes of Histologic Autofluorescence in Ectopic Retinal Pigment Epithelium in Age-Related Macular Degeneration.

The purpose of this study was to investigate histologic autofluorescence lifetimes and spectra of retinal pigment epithelium (RPE) on the transition from normal aging to RPE activation and migration in age-related macular degeneration (AMD). Autofluorescence lifetimes and spectra of 9 donor eyes were analyzed in cryosections by means of 2-photon excited fluorescence at 960 nm. Spectra were detected at 483 to 665 nm. Lifetimes were measured using time-correlated single photon counting in 2 spectral channels: 500 to 550 nm (short-wavelength spectral channel [SSC]) and 550 to 700 nm (long-wavelength spectral channel [LSC]). Fluorescence decays over time were approximated by a series of three exponential functions. The amplitude-weighted mean fluorescence lifetime was determined. Markers for retinoid activity (RPE65) and immune function (CD68) were immunolocalized in selected neighboring sections. We identified 9 RPE morphology phenotypes resulting in 399 regions of interest (ROIs) for spectral and 497 ROIs for lifetime measurements. RPE dysmorphia results in a shorter wavelength peak of spectral emission: normal aging versus RPE migrated into the retina (intraELM) = 601.7 (9.5) nm versus 581.6 (7.3) nm, P < 0.001, whereas autofluorescence lifetimes increase: normal aging versus intraELM: SSC 180 (44) picosecond (ps) versus 320 (86) ps, P < 0.001; and LSC 250 (55) ps versus 441 (76) ps, P < 0.001. Ectopic RPE within the neurosensory retina is strongly CD68 positive and RPE65 negative. In the process of RPE degeneration, comprising different steps of dysmorphia and migration, lengthening of autofluorescence lifetimes and a hypsochromic shift of emission spectra can be observed. These autofluorescence changes might provide early biomarkers for AMD progression and contribute to our understanding of RPE-driven pathology.

Implementation of a virtual screening service for hydroxychloroquine and chloroquine retinopathy: One‐year results

AbstractPurpose: To evaluate the results of a new virtual screening service for hydroxychloroquine (HQC) and chloroquine (CQ) retinopathy.Methods: Prospective study including patients under long‐term HCQ or CQ therapy referred for screening at a single ophthalmic centre in Portugal over a one‐year period. The virtual clinic consisted of a visit for functional and structural testing, in accordance with the guidelines on HCQ retinopathy screening1,2, followed by a later review by an experienced consultant. Individualized counselling for both patients and prescribing physicians was provided on retinal toxicity risk, safe dosing, and adequate screening intervals.Results: A total of 93 patients under treatment with HCQ attended the virtual clinic. Systemic lupus erythematosus (38.7%) and rheumatoid arthritis (18.3%) were the most prevalent diagnoses. Mean daily HCQ dosing was 5.2 ± 1.6 mg/Kg, with 46.2% patients using a daily dose greater than 5 mg/Kg. Median treatment duration was 48 months (range: 1–264) and 49.5% patients had been treated for longer than 5 years. Renal disease was present in 11 patients (11.8%) and 7 (7.5%) had pre‐existing retinal conditions. Structural changes in macular optical coherence tomography and fundus autofluorescence were rarely detected (3.3%). In contrast, 16 patients (17.2%) presented with abnormal 10‐2 visual field testing. Multifocal electroretinogram was performed in 14 patients (15.1%), detecting characteristic abnormalities of drug toxicity in one patient, with a definite diagnosis of severe HCQ retinopathy. Recommendation for dose adjustment was given in 47.3% of cases, including advice to discontinue HCQ use in 7 (7.5%) patients based on risk factors.Conclusions: Retinal toxicity is a rare adverse effect of HCQ, a therapy increasingly used in the management of autoimmune disorders. Virtual clinics could be a safe and sustainable care delivery system for adequate counselling, risk assessment and early retinopathy detection.References.1. Marmor MF, et al. Recommendations on screening for chloroquine and hydroxychloroquine retinopathy (2016 revision). Ophthalmology 2016; 123:1386–1394.2. Royal College of Ophthalmologists. Hydroxychloroquine and chloroquine retinopathy: recommendations on monitoring. London: Royal College of Ophthalmologists, 2020.

Depolymerization behaviors of naked oat stem during autohydrolysis in mild subcritical water Ⅱ: Correlative changes in tissue structure, cell morphology, and chemical composition.

Green, efficient depolymerization of lignocellulose is a prerequisite and the key in high-value utilization of its components. Understanding of the changes in morphological chemistry of the cell wall, related to the biomass recalcitrance, is critical for optimizing the processing conditions of cell wall deconstruction. Thus, herein, based on our previous studies, subcritical water autohydrolysis of naked oat stems was conducted at 190 °C and 1.18 MPa for 0–60 min. The depolymerization behaviors of tissues and cell walls were monitored dynamically at 10 min intervals. The micro-and ultrastructural changes of autohydrolyzed residue sections were analyzed. Parenchyma tissue commenced dissociation after 10 min at 190 ℃, whereas mechanical tissue dissociation occurred after 40 min at 190 ℃, along with interlayer separation of the secondary walls of sclerenchyma fibers. The autofluorescence changes of lignin showed that the order of regional lignin removal in tissues was from sieve tubes and companion cells to parenchyma cells, and finally to sclerenchyma fibers and vessel elements. The turning point of tissue and cell wall structural depolymerization of naked oat stems under subcritical water autohydrolysis conditions occurred after 40 min at 190 ℃ and 1.18 MPa. Under this condition, the degree of tissue dissociation was as high as 80%, while the fiber cell morphology remained intact. Dynamic changes in the tissue and cell wall structures and cell morphologies of naked oat stems maintained at 190 ℃ for various durations were captured by differentiating the use of the optical microscope, fluorescence microscope, and scanning electron microscope. The extensive, detailed assay data yield novel insights into the dynamic depolymerization mechanism of lignocellulosic composite cell walls under subcritical water autohydrolysis conditions.

Association of Fundus Autofluorescence Abnormalities and Pachydrusen in Central Serous Chorioretinopathy and Polypoidal Choroidal Vasculopathy.

A specific form of drusen, known as pachydrusen, has been demonstrated to be associated with pachychoroid eye diseases, such as central serous chorioretinopathy (CSC) and polypoidal choroidal vasculopathy (PCV). These pachydrusen have been found in up to 50% of eyes with CSC and PCV and may affect the disease progression and treatment response. This study aims to investigate the association between pachydrusen and changes in fundus autofluorescence (FAF) in eyes with CSC and PCV. A total of 65 CSC patients and 32 PCV patients were evaluated. Pachydrusen were detected using both color fundus photography and spectral-domain optical coherence tomography. The relationships between pachydrusen and FAF changes were then investigated. The prevalence of pachydrusen in CSC and PCV eyes was 16.7% and 61.8%, respectively. The mean age of patients with pachydrusen was significantly older than those without pachydrusen (CSC: 56.3 vs. 45.0 years, p < 0.001; PCV: 68.8 vs. 59.5 years, p < 0.001). No significant difference was found in the mean subfoveal choroidal thickness between eyes with or without pachydrusen. Eyes with pachydrusen were significantly associated with more extensive FAF changes in both CSC and PCV (p < 0.001 and p = 0.037, respectively). The study demonstrated that pachydrusen are more prevalent in PCV than CSC. Increasing age and more extensive abnormalities in FAF are associated with the presence of pachydrusen, suggesting that dysfunction of retinal pigment epithelial cells is associated with pachydrusen.

Insights into the kinetic regulation of Zn bioaccumulation at trace levels: Lighting up Saccharomycescerevisiae

Zn displays a double-edged effect by acting both as a micronutrient and a toxic metal, and quantitative analysis of its kinetic flux under low environmental concentrations is critical to understand its intracellular regulation. In the present study, we employed a Zn sensitive model eukaryote, the yeast Saccharomyces cerevisiae, which responded to intracellular Zn levels by increasing its autofluorescence, to quantify Zn influx, transportation between labile and storage pools, and efflux under different Zn exposure levels (<1 μM). We demonstrated that the yeast regulated Zn uptake from the extracellular source by a gradually decreased accumulation following an initial high accumulation rate. The subsequent reduced accumulation rate resulted in a steady-state Zn accumulation at 0.061 and 0.073 μg Zn/μg P as the threshold values for the control yeast and Zn-depleted yeast, respectively, independently of the extracellular Zn concentration. Compared with the control yeast, the Zn-depleted yeast possessed a higher accumulation rate, but the difference of bioaccumulation was maintained at approximately 0.01 μg Zn/μg P under different concentrations of extracellular Zn. In contrast, transportation between labile Zn and storage Zn pools or Zn efflux to the extracellular environment was not obvious after Zn exposure, indicating that the Zn dose was below a basal requirement. Such stabilized Zn accumulation was only induced by controlling the Zn influx at the bio-interface. With the novel monitoring of the kinetic changes of autofluorescence, our study demonstrated a remarkably tight Zn regulation system in yeast, providing enlightenment for Zn homeostasis in eukaryotes under low Zn exposure in aqueous environments.

Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ

PurposeCertain types of repetitive sub-maximal knee loading cause microfatigue damage in the human anterior cruciate ligament (ACL) that can accumulate to produce macroscopic tissue failure. However, monitoring the progression of that ACL microfatigue damage as a function of loading cycles has not been reported. To explore the fatigue process, a confocal laser endomicroscope (CLEM) was employed to capture sub-micron resolution fluorescence images of the tissue in situ. The goal of this study was to quantify the in situ changes in ACL autofluorescence (AF) signal intensity and collagen microstructure as a function of the number of loading cycles.MethodsThree paired and four single cadaveric knees were subjected to a repeated 4 times bodyweight landing maneuver known to strain the ACL. The paired knees were used to compare the development of ACL microfatigue damage on the loaded knee after 100 consecutive loading cycles, relative to the contralateral unloaded control knee, through second harmonic generation (SHG) and AF imaging using confocal microscopy (CM). The four single knees were used for monitoring progressive ACL microfatigue damage development by AF imaging using CLEM.ResultsThe loaded knees from each pair exhibited a statistically significant increase in AF signal intensity and decrease in SHG signal intensity as compared to the contralateral control knees. Additionally, the anisotropy of the collagen fibers in the loaded knees increased as indicated by the reduced coherency coefficient. Two out of the four single knee ACLs failed during fatigue loading, and they exhibited an order of magnitude higher increase in autofluorescence intensity per loading cycle as compared to the intact knees. Of the three regions of the ACL - proximal, midsubstance and distal - the proximal region of ACL fibers exhibited the highest AF intensity change and anisotropy of fibers.ConclusionsCLEM can capture changes in ACL AF and collagen microstructures in situ during and after microfatigue damage development. Results suggest a large increase in AF may occur in the final few cycles immediately prior to or at failure, representing a greater plastic deformation of the tissue. This reinforces the argument that existing microfatigue damage can accumulate to induce bulk mechanical failure in ACL injuries. The variation in fiber organization changes in the ACL regions with application of load is consistent with the known differences in loading distribution at the ACL femoral enthesis.

Long-term development of lens fluorescence in a twin cohort: Heritability and effects of age and lifestyle.

The blue-green autofluorescence of the ocular lens increases with age, glycemia and smoking, as the irreplaceable structural proteins of the lens slowly accumulate damage from the encounter with reactive molecular species. We have conducted a prospective study of lens autofluorescence over two decades in a twin cohort. The study included 131 phakic, non-diabetic adult twins (median age at follow-up 58 years, range 41–66 years) who were examined twice at an interval of 21 years. Change in anterior lens peak autofluorescence was analyzed in relation to age, current and baseline glycemia, cumulative smoking and heritability. The level of lens autofluorescence in the study population increased as a function of age and smoking (p ≤.002), but not as a function of glycemia (p ≥.069). Lens autofluorescence remained a highly heritable trait (90.6% at baseline and 93.3% at follow-up), but whereas the combined effect of age and cumulative smoking explained 57.2% of the variance in lens autofluorescence at baseline in mid-life, it only accounted for 31.6% at follow-up 21 years later. From mid to late adulthood, the level of blue-green fluorescence remained overwhelmingly heritable, but became less predictable from age, smoking habits and glycemic status. Presumably, as the lens ages, its intrinsic characteristics come to dominate over environmental and systemic factors, perhaps in a prelude to the development of cataract.

Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec

Background/aimsVoretigene neparvovec (VN) is the first and only subretinal gene therapy approved by the Food and Drug Administration and European Medicines Agency. Real-world application has started in 2018 in patients...