The concentrations of Cd and Zn in the sediments of Taihu Lake exceeded Level I of the China National Quality Standards for Soil. Zn accumulation was identified to increase with the trophic level. The bioconcentration of the four heavy metals in aquatic organisms was evident, with the invertebrates showing the highest bioconcentration factor in the food webs. Several biomarkers were investigated, including metallothionein (MT), malondialdehyde, and Na+/K+-adenosine triphosphatase activity. A positive correlation relationship was found between the MT content and heavy metal accumulation in organism tissues.Water, sediments, and aquatic organism samples were collected from Taihu Lake in China. Four types of typical heavy metal  were analyzed to evaluate their concentrations and trophic transfer in food webs. The stable nitrogen isotope δ Samples were analyzed from Taihu Lake, the bioconcentration of metals was evident and MTs were used to monitor the heavy metal pollution. Increasing incidents of metal pollution have been reported. Thus, the Chinese government has placed great importance on this situation in recent years.4–6Concern about heavy metal contamination is increasing because human activities are continuously emitting heavy metals into the environment, especially in densely populated and industrialized areas.7 With the rapid urbanization around the Taihu region, it receives large amounts of wastes from municipal sewage, industrial wastewater, aquaculture, etc., causing serious heavy metal pollution and eutrophication. Accordingly, the Chinese scientific communities and government have paid increasing attention to the water quality safety and potential threat in this region.8 A comprehensive investigation on the major heavy metal elements in Taihu Lake is imperative. Most of researchers have reported on the heavy metal occurrence in Lake Taihu. However, these researchers either collected samples from limited locations of the lake, or focused only on metals in water and sediments.9,10Taihu Lake, located in the Yangtze Delta, is one of the most densely populated and most economically developed areas in China. It is important for limnology, regional hydrology, and for fish production. It is also an irreplaceable drinking water source for many local cities.et al. reported in newly-formed wetlands of the Yellow River Delta, China,11 Quinn et al. researched in two streams within the New World Mining District near Cooke City, Montana, USA,12 and Wang studied among different marine food webs.13 Dietary exposure is widely recognized as a major route for the transfer of metals in aquatic food webs.14 Currently, stable isotopes are used to investigate the trophic relationships and potential biomagnification of contaminants in food webs. δ15N is effective in quantifying the trophic position because the enrichment of nitrogen isotope incrementally occurs across trophic levels (TLs) with a constant rate (3–4‰).15,16 Furthermore, biomarkers can reflect exposure and provide a reliable indication of toxic effects. Among them, metallothioneins (MTs) in aquatic organisms has become a promising tool for detecting metal contaminations.17,18Many studies have performed the transfer of metals through the aquatic food web, and substantial progress has been made over the past decades, such as Cui The present study aimed to (1) determine the levels of heavy metals  in food webs in Taihu Lake, (2) evaluate and quantify the biomagnification of heavy metals by calculating the bioaccumulation factor (BAF) and biomagnification factor (BMF), and (3) determine several biomarkers in organisms and assess the relationship between these biomarkers and the heavy metal content.2.2.1.2. It lies on the hinterland of the Yangtze Delta, spanning from 30°55′42′′ N to 31°33′50′′ N, and from 119°53′45′′ E to 126°36′15′′ E , silver carp , whitebait , and bighead carp (Aristichthys nobilis) are the main fish species in Taihu Lake.20Taihu Lake is the third largest freshwater lake in east China, with an area of 2428 km6′15′′ E . The lak2.2.Samples, including water, sediments, plankton, invertebrate, and fish, were collected from 9 sites in different representative parts of the lake from April to May 2010 . Water sExopalaemon modestus) and freshwater shrimp (Macrobrachium nipponense); and six fish species, namely, anchovy (Coilia ectenes taihuensis), whitebait , white semiknife-carp (Hemicculter leuciclus), silver carp , bighead carp (Aristichthys nobilis), and common carp (Cyprinus carpio). All organism samples were stored at −20 °C before analysis.Towing of the plankton conical net was performed to obtain phytoplankton (30 μm mesh) and zooplankton (270 μm mesh) samples. Shrimps and fish were collected from the area near the sampling sites using local commercial gill nets. The samples, which have been showed in 2.3.3 (Aristar grade) at 110 °C. The Cu, Zn, Cr, Cd and other HMs were then measured by ICP-MS (VG PQ2 TURBO), and the metals speciation of them was calculated with Visual MINTEQ (ver. 3.1). The sediment and organism samples were pooled and homogenized. All sediment samples were then digested using the National Standard Method (GB/T17140-1997) with HCl–HNO3–HF–HClO4 acid. The extracted solution from each sediment sample was analyzed for the total content of the four metals using a flame atomic absorbance spectrophotometer (Hitachi Z-2000). Organism samples were digested by a microwave system with HNO3 under controlled pressure. The concentrations of metals were determined by ICP-MS (VG PQ2 TURBO). Each sample had three replicates. The metal concentrations were calculated based on the dry weight of the sediments and organism samples. For quality assurance and quality control, standard reference materials and process blanks were digested and analyzed with each batch of samples.Before the digestion, we used natural withering to dry up the sediments. And for those animals and plants, they were dehydrated at the temperature of 80 °C for one night to test their dry weight. Filtered water samples were digested with 68% HNO2.4.16 were expressed in δ notation according to the following:The whole body of phytoplankton and zooplankton were adopted for stable isotope analyze. While for those invertebrates, we separated them with their crusts and tested all those tissues left. As for fishes, the muscles were used to realize isotope analyze. After drying at 50 °C (48 h), the samples were ground into a homogeneous powder and treated with a 2 : 1 chloroform : methanol solution to remove lipids. Before isotope analysis, the samples were further dried at 80 °C for 4 h, and then 0.5 mg of samples were set in 8 mm × 5 mm Sn capsules and combusted from 1000 °C to 1050 °C. Nitrogen was transported through the interface and analyzed using a mass spectrometer. Stable isotope ratiosRstandard can be determined based on atmospheric nitrogen.The values of consumer is the TL and the TL of phytoplankton is assumed to be 1.5.21The trophic level (TL) for each aquatic organism is given as2.5.22 Organism samples were weighed and homogenized by ultrasonication in 0.5 mL of sucrose buffer , and centrifuged at 16 000 × g in a refrigerated centrifuge for 20 min. The supernatant was diluted with the homogenate to 1.5 mL, and then mixed with Ag+ solution. After incubation at room temperature, 0.1 mL of red blood cell hemolysate was pipetted into the mixture. The newly homogenized solution was heated in a boiling water bath (100 °C) and subsequently centrifuged. The process was repeated twice prior to the collection of the final supernatant, which was digested with HNO3 and diluted. The Ag concentration was measured using ICP-MS. Three replicates of biological samples from T4 site were analyzed by the same process described above. The MT content in the samples was calculated as follows:c+Ag is the Ag+ concentration in the final mixture, V is the total volume of final mixture, and ww is the wet weight of each sample.Biological samples from Dapu sampling site (T4) were chosen for measuring the concentration of biomarkers as all the aquatic organisms could be collected there. And for invertebrates, the shrimp tissues after dissecting off their crusts  were used for measuring, while only the muscles were detected for fishes. MT measurement was performed using a modified silver saturation method.+/K+-ATPase activity were obtained by the methods discussed in the MT quantification. The MDA content in organism tissues were assayed using commercially available kits  according to the manufacturer's protocol. The MDA concentrations were expressed as nanomole MDA per milligram protein. MDA assays were conducted using the thiobarbituric acid colorimetric method.23 The Na+/K+-ATPase activity was measured by determining the amount of inorganic phosphate (Pi) liberated from the hydrolysis of the substrate ATP.24The homogenates for MDA content and Na2.6.predator and metalprey are the metal concentrations of the predator and prey species, respectively.25The BMF was calculated byCss is the heavy metal concentration (HMC) in organisms at a steady state (mg g−1 DW), and Cw is the HMC in water (mg mL−1).26The bioaccumulation factor (BAF) is defined as2.7.15N). When the p value was less than 0.05, the linear regression between the HMC and TL was considered significant. Half of the value of the limit of detection for each element was substituted for the values below the limit of detection, and then applied in the statistical analysis.Statistical analysis was performed using SPSS . Linear regression models were used to examine the associations among the HMCs and stable isotopic ratios (δ3.3.1.−1): Cd (0.005–0.033) < Cr (0.213–0.395) < Cu (1.170–3.013) < Zn (0.553–5.100). The total metal concentrations in sediments had minor variations among stations with the following ranges (μg g−1): Zn (68.12–158.92) > Cr (28.40–44.00) > Cu (16.75–23.20) > Cd (0.06–0.50). The range of metal concentrations in water from Taihu Lake was lower than Level I of the China National Environmental Quality Standards for Surface Water  . The Cu and Cr concentrations in sediments were in compliance with Level I of the China National Quality Standards for soil  , whereas Zn and Cd were in compliance with level II of the China National Quality Standards for soil  . The settlement of electroplating, printing, and dyeing wastewaters possibly led to a higher Cd concentration. Compared with previous reports, the HMCs in water and sediments were found to be declining in Taihu Lake. This finding indicated that the Chinese government and scientific community are achieving initial success in the pollution control of Taihu Lake over the past decades.27–29 The metal speciation of Cu, Zn and Cd in water samples showed that Cu mainly existed in Cu–DOM complexing form whereas Zn and Cd mainly existed as free ion  collected from a metal-impacted estuary for 68 days.35 In addition, De Schamphelaere et al. found that the growth and reproduction of Daphnia magna fed with contaminated algal for 21 days were obviously inhibited. However, the inhibit effects were not caused by the pathway of waterborne exposure or reduced nutritional quality of the algal.36 In this study, the concentration of heavy metals in water was relatively low . The δ15N values were converted to TLs, which increased from 1.5 to 3.8 when trophic level rose from phytoplankton to fish. However, it was found that no apparent difference occurred between phytoplankton and zooplankton as the primary consumer. It was speculated that zooplankton may also ingest particulate organic matter (POM) as main food, Wedchaparn et al. also reported a similar phenomenon.38 Besides, the planktivorous fishes exhibited rather high TL levels , which were even higher than omnivores. The difference of food habit may explain for that Mao et al. has reported that the Lake Taihu showed multiple trophic pathways, and organic matters as suspended particulate organic matter and sediment organic matter, could also serve as the bottom of the trophic chain.39 And they founded that the TL levels of planktivorous fish and invertebrates were higher than herbivorous fish, which was corresponding with what was observed in 40 It was also interesting to find that invertebrates from Taihu Lake had abnormal higher TL levels (3.4 or 3.0), which was consistent with previous report.41,42 Mao et al. reported that macrophytes also made sizeable contribution for several invertebrate and fish species,43 and it was observed that consumers feed on phytodetritus or macrophytes exhibited relative higher TL levels. Therefore, it was speculated that the abnormal high calculated TL levels of invertebrates might due to their food source.The δ3.3.e.g., Cd) in invertebrates with respect to higher TLs revealed a high availability of these metal ions or high uptake rates. Cu and Zn were also enriched, but the BAF for Zn in plankton was low, whereas the factor for Cu was lowest in fish. Cu and Zn were probably essential metals required by physiological processes, and may have different internal regulation ways in the cells of aquatic organisms.14The magnification of selected heavy metals in the lake food webs was not evident, except for Zn. However, the BAF of these metals was proven to differ among various organisms . The hig44BMF was determined based on the comparison of heavy metals in predators and preys . Cd and 3.4.+/K+-ATPase activities, as promising biomarkers for metal pollution, oxidative stress, and biomembrane damage, respectively, were investigated. The results are presented in 45,46 Hence, higher accumulations of Cd and Cr in shrimps led to higher levels of MT induction, and also confirmed the highest concentration ability of heavy metals in invertebrates. For the six species of fish, MT levels were significantly higher in the three high TL species. MDA is the final product of lipid peroxidation, and one of the most damaging effects of free radicals and oxidative injury in cells.47 The increased MDA contents revealed that the organisms were already under a status of oxidative stress induced by some xenobiotics that probably contained heavy metals. Na+/K+-ATPases play a significant role in ionic regulation of cellular components and maintenance of tissue osmolarity, and it is also related to membrane structural integrity.48 In the present study, specific differences among the Na+/K+-ATPase levels in fish from Taihu Lake were not evident. The observed levels demonstrated minimal membrane damage in aquatic organisms.The MT concentrations, MDA concentrations, and Naet al. indicated that cadmium accumulated by mussels of Mytilus galloprovincialis was primarily associated with MT.49 Damiens et al. also reported that the larvae of Oysters from a hatchery from Normandy (English Channel) were exposed to copper or cadmium for 24 h, larvae accumulated copper and cadmium with an increase in MT concentrations particularly with cadmium.50 Thus, MTs in aquatic organisms could be a tool for the biomonitoring of heavy metal pollutants in ecotoxicological studies. Regarding the MDA concentrations and Na+/K+-ATPase activities, no clear relationship was found with the molar concentration of heavy metals, which indicated that the other pollutants  also had affected lipid peroxidation and membrane injury.51,52 To establish a possible assessment method of the distribution and ecological risk of the heavy metals in Taihu Lake, more data on biomarkers  in different fish organs and integrated biomarker response analysis are required.The relationships between the biomarker levels and total molar concentrations of the four metals are presented in 4.15N stable isotope analysis was used to describe the transference of the four heavy metals in the food web in Taihu Lake. By analyzing the relations between trophic levels and the accumulations of heavy metals, Zn accumulation was identified to increase with the trophic level. Bioconcentration and biomagnification factors for the four metals in the food web also been calculated. The invertebrates showed the highest bioconcentration factor in the food webs. Finally, the relationships between three kinds of biomarkers  and the heavy metal accumulations was investigated. A positive correlation between MT levels and the accumulations gave us a possible tool in the passive monitoring of heavy metals pollutants in ecotoxicological studies. In summary, a comprehensive status of heavy metals is analyzed in aquatic organisms of different trophic levels from Taihu Lake, which would provide a useful theoretical basis of heavy metal pollution control and regulation for local governments.Nine sampling points from the entire Taihu Lake were selected for the survey of the heavy metals  in water, sediment, plankton, invertebrate and fish. The concentrations of Cd and Zn in sediments exceeded Level I of the China National Quality Standards for soil (GB 15618-1995). The δThis research did not involve human participants and the authors declare that any research involving human participants and/or animals in this study was carried out in strict accordance with the recommendations of the approval of Institutional Authority for Laboratory Animal Care of Beihang University. All efforts were made to minimize suffering of the animals.The authors declare that they have no conflict of interest.RA-008-C7RA11677B-s001


Osteoarthritis (OA) is the most prevalent degenerative joint disease, resulting in joint pain, impaired movement, and structural changes. As the ability of joint tissue to resist stress is mainly imparted by fibrillar collagens in the extracellular matrix, changes in the composition and structure of collagen fibers contribute to the pathological remodeling observed in OA joints that includes cartilage degeneration, subchondral bone (SCB) sclerosis, and meniscal damage. Using the established OA model of destabilization of the medial meniscus (DMM) in C57BL/6J mice, we performed a comprehensive analysis of the content and structure of collagen fibers in the articular cartilage, subchondral bone, and menisci using complementary techniques, which included second harmonic generation microscopy and immunofluorescence staining. We found that regions exposed to increased mechanical stress in OA mice, typically closest to the site of injury, had increased collagen fiber thickness, dysregulated fiber formation, and tissue specific changes in collagen I and II (Col I and Col II) expression. In cartilage, OA was associated with decreased Col II expression in all regions, and increased Col I expression in the anterior and posterior regions. Col I fiber thickness was increased in all regions with disorganization in the center region. In the superficial SCB, all regions exhibited increased Col I expression and fiber thickness in OA mice; no changes were detected in the deeper regions of the subchondral bone except for increased Col I fiber thickness. In the menisci, OA led to increased Col I and Col II expression in the vascular and avascular regions of the anterior meniscus with increased Col I fiber thickness in these regions. Similar changes were observed only in the vascular region of the posterior meniscus. Our findings provide, for the first time, comprehensive insights into the microarchitectural changes of extracellular matrix in OA and serve as guidelines for studies investigating therapies that target collagenous changes as means to impede the progression of osteoarthritis. Although OA has been initially classified as a disease of articular cartilage degeneration, recent studies have demonstrated the impact of OA on other joint tissues. Accordingly, subchondral bone sclerosis, osteophyte formation, synovitis, and meniscal tears have emerged as hallmarks for OA development and progression6. Crucially, altered composition of the extracellular matrix (ECM) in any of the joint tissues is expected to impact the functionality and structural integrity of the whole joint. Type I and Type II collagen are the most abundant proteins in the ECM of bone and cartilage, respectively7, while menisci are fibrocartilaginous, with an ECM that is also composed of both type I and II collagen9. Type I and II collagens are both fibrillar collagens, meaning their collagen molecules are cross-linked into fibrils, which self-assemble into fibers10. Collagen fibers preserve the structure of the tissues by resisting deformation11, allowing the tissue to withstand stress. Examination of the collagen network in the ECM during OA progression is therefore crucial, as it provides valuable insights into the causes that underpin OA-associated loss of joint structural integrity. To characterize the collagenous changes of the ECM in OA joint tissues, we used second harmonic generation (SHG) microscopy to image anterior, center, and posterior regions of the medial knee compartment, defining alterations in not only articular cartilage, but subchondral bone and menisci as well.Osteoarthritis (OA) is the most prevalent degenerative joint disease and is characterized by limited mobility and impaired joint function. The ability of joints to withstand stress is affected by factors such as age, injury, obesity, and genetics, which can predispose individuals to develop OA13. During the SHG process, samples are exposed to two photons of equal wavelength, which combine and are emitted as either forward (FSHG) or backward (BSHG) signal if the wavelength passes through the structure or is reflected off the structure. Type I collagen typically produces a strong FSHG signal, while Type II collagen typically produces a strong BSHG signal. While other fibrillar collagens  are present in the joint tissues, they generate weak SHG signals15. FSHG and BSHG signals can then be analyzed to determine fiber content, width, and orientation. SHG has been utilized to examine collagenous changes in articular cartilage of early stage OA16. SHG is also emerging as a promising in-vivo-imaging application for diagnostic evaluation of collagenous changes in articular cartilage and menisci18.SHG imaging is a powerful, label-free method for visualizing fibrillar collagens19, but they either explored structural changes to the collagenous network in one tissue type22, or described singular parameters of the collagen network22. Kinematic studies of OA knees have shown that changes in distribution of joint loading result in regional changes in the tibial plateau, leading to degenerative changes in the medial compartment and varus deformities24. To our knowledge, no previous studies have provided a detailed account of the regional changes in collagenous structures in different tissues of late-stage OA knee joints. Here, we provide a comprehensive analysis of the pathological changes in the structural organization and composition of collagen fibers in the different tissues of knee joints in late-stage OA.Previous studies have examined the relationship of joint tissue degeneration between cartilage, subchondral bone, and menisci1. For a preliminary evaluation of the collagen microarchitecture, we stained knee joints of both sham and DMM-operated mice with Picrosirius red (PSR)3, in which samples were imaged with a polarized light microscope to selectively image collagen fibers  staining of Col I and Col II in sham and DMM mice and analyzed the area % and average intensity of the expression of both proteins in the anterior, center, and posterior regions of the cartilage Fig. A–D. BecaPSR indicated increased collagen fiber thickness illustrated by an increase in red-staining fibers in sclerotic SCB Fig. E and red29. Therefore, we assessed changes in collagen composition in the vascular (outer 10–30% of the meniscus) and avascular (inner 70–90% of the meniscus) regions30 of both the anterior and posterior menisci. In the anterior meniscus of DMM mice, the expression area of both Col I and Col II was significantly increased in both the vascular and avascular compartments . All animal procedures were performed according to the National Institute of Health (NIH) Guide for the care and use of laboratory animals and approved by the Animal Care and Use Committee of Pennsylvania State University.53. Briefly, mice were prepped for surgery with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg), and the skin of the medial knee was cleaned with betadine. A 5 mm incision was made through the skin to reveal joint structures and the patellar tendon was identified. The MMTL was transected with a #11 scalpel allowing for increased movement of the medial meniscus within the joint. To close the incision site, 4–0 silk sutures in an interrupted pattern were placed and the site cleaned with betadine. Sham surgeries were performed on the left knees, in which the same procedure was performed without disruption of the MMTL. Analgesia was provided by intraperitoneal injection of buprenorphine (0.5 mg/kg) every 12 h for 3 days. Sutures were removed after 7 days. After 12 weeks post-DMM, mice were sacrificed by whole animal perfusion (with 10% NBF). Mouse knee joints were harvested, fixed with 10% neutral buffered formalin (NBF) for one week, then decalcified in two changes of decalcification buffer  over one week. Samples were then embedded in paraffin wax and serial sections of DMM and sham knee joint samples were obtained at 5 µm thickness with a rotational microtome. Sections were then used for Safranin-O/Fast Green, Picrosirius red (PSR), and immunofluorescence staining and histological analysis. Safranin-O/Fast Green images were acquired with OsteoMeasure Software (https://www.osteometrics.com/).DMM surgeries were performed on right knees of twelve-week old C57BL/6J male mice, through transection of the meniscotibial ligament (MMTL) as previously described3. Slides were imaged at 10 × magnification with polarized light microscopy and were quantified using ImageJ. Images were split into red and green channels , then areas were selected and area percentage and average intensity were quantified.5 µm sections of the medial knee joint were deparaffinized and stained with picrosirius red solution for 1 h at RT. Slides were then rinsed 2 × in 0.5% acetic acid solution, then dehydrated, cleared in xylene, and mounted with resinous medium15. Briefly, we utilized a InSight DS + mode-locked single-box laser system, set to acousto-optic tunable filters (AOTF) to protect samples from damage. Care was taken to ensure that power setting was consistent between sample imaging. Samples were imaged with a Nikon A1 MP + Multi-Photon upright Microscope system (Nikon Instruments) with water immersion objective lens (CFI75 Apo Water 25X/1.1 LWD 2.0-mm WD) for FSHG collection, and 440/20 nm band pass filters for BSHG detection.We have previously described the general SHG microscopy setupSafranin-O/Fast Green stained knee joint sections were used for SHG microscopy. Tibial cartilage, subchondral bone, and anterior and posterior horns of the medial meniscus from DMM and sham knee joints were imaged using SHG microscopy, in order to compare and quantify collagenous changes within specific regions of interest. Images of the tibia were acquired at anterior, middle, and posterior regions in reference to the anterior and posterior meniscal horns, for an even representation of the cartilage , superficial subchondral bone , and deep subchondral bone, immediately superior to the growth plate. Because the superficial and transitional zones in our DMM samples were too degraded to measure, we imaged the remaining uncalcified cartilage in the radial zone. The anterior and posterior horns of the medial menisci were analyzed by comparing vascular  and avascular  regions for collagenous changes due to differences in blood supply. Three ROIs were captured from each area for a representative measure of the entire sample.Each ROI was captured at a lower laser power to measure collagen content and at a higher laser power to measure fiber orientation and width. Using lower laser power prevented oversaturation of pixels between ROIs of any samples, while the higher laser power images visualized smaller fibers with lower expression during later analysis. Notably, care was taken to avoid oversaturation in each imaging session by utilizing the saturation map during image capture, as the width of discrete fibers are impossible to define if excess signal merges the fibers together.15 using Volocity software , to analyze collagen content, fiber width, and orientation index.Utilizing the 3D reconstructions created from Z-stacked SHG images, analyses were performed as previously describedTo determine the collagen content in the ROIs, volume fraction (VF) for both BSHG and FSHG signal was calculated and confirmed with immunofluorescence staining. For each ROI, volume fraction was calculated by dividing the volume in the ROI producing BSHG or FSHG signal by the total volume of the image in order to quantify the amount of signal within the ROI. Thicker fibers allow SHG signal to traverse the structure to the FSHG detector, while thinner fibers reflect SHG signals to the BSHG detector. Thus, the amount of FSHG and BSHG signal within a ROI is indicative of the amount of thick (Col I) and thin (Col II) fibers respectively. FSHG/BSHG VF ratio was then calculated as a measure of expression changes in Col I relative to Col II. Ratios from each ROI were averaged by sample, then plotted with GraphPad Prism. The FSHG/BSHG VF ratio was only calculated for cartilage because other joint tissues experienced a simultaneous increase in both Col I and II expression.16, this calculation does not distinguish between collagen fibers that produce both FSHG and BSHG signal, and can only be used as a measure in locations with only one type of collagen if statements are to be made about the thickness of Col I v. Col II fibers. It therefore does not provide a concrete measurement of the fibers themselves, but rather provides a relative measure of the amount of thick and thin fibers present in a ROI.Fiber width was determined by measuring four discrete fibers per ROI by using the line tool in Volocity to draw a line perpendicular to a fiber, measuring FSHG and BSHG signal intensity against distance across the line. FSHG signal intensity v. distance was plotted using Origin software, yielding peaks of width equal to that of the fiber. For each ROI, 3–5 fibers were measured, and the resulting widths plotted in Graph Pad by region. Fiber widths were averaged by sample. Although FSHG/BSHG ratio has been commonly used as a parameter to evaluate the thickness of collagen fibersFiber orientation was determined using a MatLab Fast Fourier Transform (FFT) script, in order to quantify the organization of collagen fibers in the ECM. This calculation determines how aligned the fibers are within the sample, providing a numerical value for the angle at which the fibers are placed in relationship to each other .https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html).Immunofluorescence staining was utilized to determine localization of collagen types I and II. 5 µm sections were deparaffinized. Antigen retrieval was achieved by incubation in 0.5% Pepsin/0.5 N HCl solution at 37 °C for 30 min, followed by incubation in hyaluronidase solution (1.5 mg/1 mL 1xTBS) at 37 °C for 5 min. The slides were then permeabilized by incubation in 0.03% Triton X in 1xTBS for 30 min. Samples were blocked in 10% normal goat serum at RT for two hours, then incubated overnight at 4 °C with rabbit anti-mouse Col II antibody . Slides were then washed 3 times with 1X TBS, incubated with goat-anti rabbit AF568 antibody (1:100) for 1 h at RT. Then, samples were incubated in goat biotinylated Col I antibody  overnight at 4C, washed in 1X TBS, and incubated with Streptavidin AF647 Conjugate  for 1 h at RT. Negative control slides for Col I and II were stained as described, but incubated O/N at 4 °C with IgGs instead of primary antibodies. All samples were mounted with DAPI Prolong Gold, cover-slipped, and imaged with a Zeiss Axio Observer Inverted Microscope at 40 × magnification. Images were acquired with Zeiss Zen Blue 2.6 edition  were selected for measurement of average fluorescence intensity (pixel intensity) and area percentage (number of pixels within an image containing fluorescence) of each collagen of interest. Threshold was set specifically for each collagen in each tissue type  to quantify fluorescence in the matrix, and was consistent throughout analysis of each sample.Data for each SHG and IF parameter studied was averaged per sample for n = 5 for both sham and DMM mice. Data from sham and DMM samplers were compared using unpaired Welch’s t-test. Comparison between the three regions of the cartilage  was performed using one-way ANOVA. All calculations were performed using the GraphPad Prism 9.1.0 program. Graphed values are expressed as mean ± SEM.Supplementary Figures.


This study aimed to examine the associations with epidemiological, behavioral and clinical parameters of IgG antibody responses against the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after immunization with two doses of the BNT162b2 vaccine in a cohort of healthcare workers  in Greece. We used a mixed effects model to investigate the potential associations of antibody levels one and three months after vaccination and examined by bootstrapping t-tests the putative effects of gender and age for each period. We also employed exact tests of independence in R × C contingency tables to explore associations between behavioral and gender variables with vaccinations side effects. We found significant differences between males and females as well as between subjects in the youngest (21–30 years) and the older age groups in both study periods. We also detected a decrease in titers with age and time. Males had steeper elimination rates across the age span in both periods, in contrast to females who exhibited a softer elimination titer rate with age in the first period and almost constant titers in the second. Concerning side effects, we found a significant association between pain at the injection site and female sex. Hence, our real-world data analyses revealed potentially important clues into the associations of antibody responses to SARS-CoV-2 spike. We discuss the importance of these findings in view of current mass vaccination perspectives and provide useful clues for the design and optimal timing of booster doses for COVID-19. The swift development of several highly effective vaccines that mitigate the devastating consequences of the coronavirus disease 2019 (COVID-19) pandemic just over a year after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), restored humanity’s optimism for an accelerated return to a new normalcy. Pfizer-BioNTech’s BNT162b2 , which is based on the novel, readily adaptable platform of messenger RNA (mRNA), was the first vaccine that was granted emergency use authorization in late December 2020, full approval recently in individuals 16 years of age and older and emergency use authorization in children aged 12–15 .The first large-scale study to evaluate the antibody response to the BNT162b2 vaccine in HCWs in Israel across different ages, sexes, and comorbidities found that almost all study participants developed IgG and neutralizing antibodies that were highly correlated, rapidly after two vaccine doses . Severalet al., who studied SARS-CoV-2 anti-spike IgG kinetics in 116 Israeli HCWs receiving BNT162b2, found a statistically significant difference in IgG levels between subjects younger than 50 years and older individuals .The analysis of emerging serological data following vaccination enhances our understanding of the immunological response to the novel COVID-19 vaccines and the factors that may influence them, thereby informing public health policy decisions . This stSera were collected between February and May, 2021 on a voluntary basis from 458 HCWs of the General Hospital of Arta, a public hospital in northwestern Greece providing primary and secondary health services. Of those, twelve had been previously infected with SARS-CoV-2 as determined by a confirmed positive RT-PCR result and were thus excluded from vaccination (and the study), whereas seven subjects who had not been previously infected or vaccinated served as negative controls in the serological assay. The remaining 439 individuals who received two doses of Pfizer-BioNTech’s BNT162b2  21 days apart according to the recommended schedule, were enrolled in this study. A form containing demographic and clinical data as well as adverse events (AEs) after each vaccine dose was completed by each participant. Data collected at baseline included gender, age, and basic medical history that entailed information on underlying conditions and received therapy as well as smoking and alcohol consumption. Post vaccination, potential AEs were recorded after each vaccine dose and anti-spike IgG responses were measured 3–4 weeks after the second dose (first period). To obtain an assessment of the durability of the antibody response, measurements were repeated three months after receipt of the second dose in 110 of 439 vaccine recipients (second period). Thus, this was a longitudinal (repeated measures) study.The study was approved by the Medical Ethics Committee of the General Hospital of Arta. Written informed consent was obtained from study participants. All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.® EXL™ SARS-CoV-2 IgG (CV2G) assay , according to the manufacturer’s instructions. Results were expressed in index units and samples with IgG (anti-S-RBD)>1000 index were deemed positive. Sera from not infected or vaccinated individuals were used for the estimation of the cut-off. The sensitivity and specificity of the test are both reported by the manufacturer to be 100%.Sera were stored at 2–8°C until testing for IgG antibodies against the RBD of the S1 region of SARS-CoV-2 using the DimensionData analyses were performed in R , except We cleaned and curated the data using the “tidyr” and “dplyr” libraries in R. After cleaning, the dataset was constructed in the long format by gathering the two sampling periods  into one variable named “Sampling”, and spreading each symptom  variable into the corresponding dichotomous (Yes or No) variable. We also constructed three dichotomous variables, named “Smoking”, “Alcohol” and “Drugs”, with the latter referring to received medication at baseline for at least one condition unrelated to COVID-19. We explored the data for structure and outliers using the “ggplot2” library in R. For flexibility in analysis, we constructed an age categorical variable by classifying individuals in five decade-spanning groups as follows: “21–30”, “31–40”, “41–50”, “51–60”, and “61–72”.Associations between antibody titers and age were examined separately for males and females in each sampling period by Spearman’s exact test  using a Monte Carlo sampling approach with a sample size of 10,000. Within each sampling period, we examined mean differences of variables of interest by t-tests, bootstrapping the corresponding pair variables 10,000 times, using the “boot.t.test” function of the “MKinfer” library in R. To prevent against inflation of the Type I error (rejecting the true null hypothesis purely by chance) as a result of multiple pair comparisons, we applied the Benjamini-Hochberg  correctivs. AGE was examined for the Gender and Smoking variables by using the “Simpsons” library in R. Power analysis was performed by simulation using the powerSim function of the simr R library, with 1000 bootstraps of a sample size of 220 and an α value of 0.05.To assess the potential effects of different factors on antibody titers, we used a mixed effects linear model appropriate for our experimental design which involved repeated measures (dependent observations). In this model, Titer was the dependent variable and Age, Gender, Smoking, Alcohol and Drugs prescription were the explanatory variables. Random intercepts for the sampling interval and subjects were also included in the model to control for random effects of the two variables. The potential interaction between the Gender and Age variables was also examined. Sampling interval was the three-month span between the two sampling periods. Age was the subjects’ age in years. Gender was the subjects’ sex , whereas Smoking and Alcohol represented the subjects’ smoking and alcohol consumption behavior (Yes or No), respectively. Only the subset of individuals who were sampled twice was considered in the model (n = 110). The selection of the subset of individuals was based on the coverage of the full range of antibody titers (after the second vaccine dose), from low to intermediate to high. Mixed effect models were analyzed by the “lmer” function of the “lmerTest” library in R. The model residuals were tested for normality, homoscedasticity, independence and outlier effects. As the random effects were independent to each other, a diagonal covariance matrix was assumed for variance calculation. The mathematical justification for the addition of random effects in the model was examined by comparing the log likelihoods of the AIC values between models containing the random effect of interest and models lacking it, in a step by step process . For modTo examine potential associations between local  or systemic AEs  after vaccination with the “Gender”, “Smoking” or “Alcohol” variables, we used exact tests of independence in contingency tables in which the rows depicted the levels of the “Gender”, “Smoking” or “Alcohol” categories and the columns depicted the levels of the vaccine-AE variable of interest . Exact tests were performed with the StatXact program of Cytel Studio . The application of exact tests was necessary to overcome the problems of sparse or unbalanced contingency tables for which asymptotic methods are inappropriate .The demographic features of the study participants are described in In total, 439 subjects , with a mean age (±SD) of 48.6 (±9.51) (range: 24.0–72.0) years who were vaccinated with two doses of the BNT162b2 vaccine, were included in the first period of the study analysis. The vaccinees’ cohort comprised healthcare professionals  staff, 13 laboratory technicians, four biochemists, three health visitors, and two pharmacists) and nonmedical personnel of the hospital  technicians). Of the 439 study participants, 34.6%  had a history of underlying diseases, such as arterial hypertension and diabetes, and were receiving therapy (unrelated to COVID-19 and not including immunosuppressive medications) at baseline . Most paNo solicitated AEs were reported post vaccination by almost half (~49%) of study participants (n = 439). The mostly mild, local and systemic AEs that were observed within the first day after each vaccine dose are presented in nd vaccine dose, again predominantly among middle-aged women; they most commonly included the following AEs after the 1st and 2nd dose, respectively: fatigue (4.1% and 9.6%), fever (1.8% and 9.3%), headache (5.5% and 7.7%), myalgias (1.1% and 7.5%)/arthralgias (0.9% and 4.6%), and chills (0.7% and 4.3%). No severe allergic reactions were observed.Systemic AEs were more pronounced following the 2We investigated likely associations between AEs and gender. After the first vaccine dose, 46 (30.7%) men and 133 (46.0%) women reported pain at the site of injection, while 104 (69.3%) men and 156 (54.0%) women did not report this AE. After the second vaccine dose, 23 (15.3%) men and 91 (31.5%) women reported regional pain, whereas 127 (84.7%) and 198 (68.5) men and women, respectively, did not. In both cases, a strong association was found between gender and regional pain by three exact tests of independence , indicating a significant trend for women to develop this reaction compared to men .We further examined the available baseline characteristics of study subjects  for possible associations with any AEs. Only received therapy at baseline showed statistically significant associations with the following reported AEs: weakness, dizziness, and regional pain. The association with weakness was observed after both vaccine doses (p = 0.004 and p = 0.005), whereas the association with dizziness, and regional pain was observed after the second vaccine dose only .To control for the potential contribution of Gender and Age, we also examined the conditional associations of the above exploratory factors to the corresponding AEs. This analysis did not reveal an association with the “Weakness” variable after the first vaccine dose, a finding suggestive of gender or age effects. The association with “Regional pain” was also attenuated after the first vaccine dose (p = 0.049), suggesting a similar effect. In contrast, the “Weakness”, “Dizziness”, and “Regional pain” reactions had almost the same probability significances after the second vaccine dose , indicating a strong association between drug intake (prescription medication) at baseline and these AEs.The results of the serological testing during the two study periods are shown in vs. 58,697).Overall, lower titers were detected among participants in the second study period (~3 months after completion of the vaccination scheme) compared to the first , female participants, who were also younger than male participants , had higher antibody titers compared to males . In the 2nd period, females, who were only slightly younger than male participants , also had higher antibody titers compared to males, but these differences were not statistically significant  group in the 2nd period.Regarding age, the youngest group (21–30 years) exhibited the highest antibody titers in both study periods, with mean values of 76,987 and 40,924 in the 1To further explore the impact of age on gender differences in the two periods, we examined contrasts (differences within each sampling period and gender category) by paired t-tests of all age category combinations, bootstrapping the paired variables 10,000 times. The results of this analysis are summarized in vs. 63,466, Tables In the first period, the antibody titers of females declined with age up to 50 years from a maximum of 76,646 among the 21–30 years-old, and then increased again in the 51–60 and 61–72 age groups . Our const and 2nd period, respectively) and the remaining age categories where antibody titers were lower in both sampling periods .We also examined the impact of gender on differences among age groups within each sampling period by paired t-tests and bootstrapping .In the first period, apart from the 21–30 age group where men surpassed slightly (mean difference = 795), women had higher titers across age groups, with a minimum and a maximum titer difference of 3,002 in the 41–50 and 12,603 in the 31–40 group, respectively . HoweverIn the second period, men had significantly higher titers in the 21–30 age group (p = 0.021) and a higher rate of titer decrease up to the age of 60 compared to women . Thus, wvs. antibody titers created for each sampling period  of the test indicates no correlation between the examined variables, while the alternative hypothesis (H1:ρ≠0) indicates a correlation between them. The corresponding correlations (Spearman exact test) between age and titer of males and females for the first period were -0.21 and– 0.07, respectively, and they were statistically significant for males (p = 0.011), but not for females (p = 0.27). In the second period, titers were reduced for both sexes. Intriguingly, however, females’ antibody titers were constant across ages, while males still had a negative slope of antibody titers. Males exhibited statistically significantly higher titers than females in younger and middle ages, but lower titers in ages >50 years. The corresponding correlations of male and female age groups with titers for the second period was -0.36 and- 0.07975, respectively, and they were marginally statistically significant (p = 0.043) and not significant (0.48), correspondingly.Preliminary analysis by modelling using a mixed linear model included Titer as the dependent variable and demographic  and health status indicators  as the explanatory categorical variables. Alcohol consumption and received therapy at baseline had no significant effects on antibody titers (p>0.2) and they were thus removed from the model. A statistically significant interaction (p = 0.024) was found between Smoking and Gender. Hence, the selected model comprised of the antibody titer as the dependent variable, “Gender”, “Age” and “Smoking” as separate categorical explanatory variables with the Gender × Smoking interaction, and the individuals’ random effects for the two sampling periods. The reference in the model was the 21–30 age group of non-smoking females.The assumptions of collinearity, influential observations (outliers), normality of residuals and normality of random effects were met by the data. Slight deviations were observed for the linearity and homogeneity of variance assumptions. The model without random effects (glm model) had had a higher AIC value compared to the same model considering random effects which had the lowest of all combinations, indicating the best fit of data. The statistical power of our study, for our sample size of 220 and alpha = 0.05, was found to be 98.30%, 95% CI  using the simr package.Mixed effects model analysis showed that there were highly significant differences (p<0.001) between the 21–30 years group (youngest vaccinees) and the 31–40, 41–50, and 51–60 groups, as well as a marginally significant difference (p = 0.092) between the 21–30 and the 61–72 years group .In addition, the mixed effects model showed a higher antibody titer by 14,026 for females compared to males, which was statistically significant (p = 0.0066). However, since a weak interaction was found between Smoking and Gender , Fig 3, As determined by simple regression analysis using the same explanatory variables, the gender × smoking interaction was also significant in the first period in either the whole sample of study participants  or the subset of the sample used in the mixed effects model . In the second period, although non-smoking females had higher titers than smoking females and non-smoking males had lower titers than smoking males  the inteCompared to the reference group, the 31–40 years age category had the lowest antibody titers with mean differences of 29,601  for females and 15,575  for males. The 41–50 and 51–60 age categories had almost equally reduced titers, with estimated values of about 44,828  and 44,185  for females, and 30,802  and 30,159  for males, respectively. The minimum difference was observed between the eldest (61–72 years) and the youngest (21–30 years) age category. Because of the slight violation of the homoscedasticity and linearity, we explored our model by using the “robustlmm” package. Slightly different estimates and almost the same probability values were obtained (data not shown). As the parameter estimation methods in this algorithm are robust as the name implies, the slight departure from the regression assumptions or other unobserved data contamination seems not to have had significant effects in our estimations. In support of our analysis, recent research showed no parameter bias in mixed effects models that violate the associated assumptions .In this work, we scrutinized the associations of IgG antibody responses against the RBD of the spike protein of SARS-CoV-2 after immunization with two doses of the BNT162b2 vaccine in a cohort of HCWs (n = 439) and in a subset of vaccinees (n = 110) three months post-vaccination, with epidemiological, behavioral and clinical parameters. In particular, we explored possible associations of vaccination side effects after each vaccine dose to gender and investigated the potential role of exploratory variables on these effects. We also probed the potential associations between generated antibody titers of vaccinees approximately one and three months following the second vaccine dose, and the demographic variables of gender and age, and clinical and behavioral variables at baseline that included received medication (unrelated to COVID-19), alcohol consumption and smoking. Using a mixed effects model with titer as the dependent variable and age groups, gender, smoking and smoking-gender interaction as exploratory variables, we found differences in antibody titers between age groups as well as between genders and we also detected an interaction between smoking and gender.The previously reported by many other studies [e.g. ], good gWe also found statistically significant associations between received therapy at baseline for underlying medical conditions (unrelated to COVID-19) and regional pain as well as dizziness and weakness. The associations with regional pain and dizziness held after the second vaccine dose only, whereas the systemic AE of weakness was evident after both vaccine doses. To the best of our knowledge, no detailed work has been done to study such associations. A more detailed approach is thus needed to exclude putative interacting or lurking variables to confirm this result. We cannot exclude the possibility of involvement of a particular drug or drug category, especially since in our study we examined prescribed medication as a pooled variable which included different types of drugs. A more detailed study is needed to possibly partition the observed association to different types of medication.Humoral immune responses following vaccination with BNT162b2 against COVID-19 have been reported to be age-and gender-dependent, and more robust in younger ages and female octogenarians . NeverthOne interesting observation is the disparity in the decreasing rate of titers with age between the two genders as evidenced by comparing the corresponding least squares trend lines for the two periods. Females exhibited a slower titer decrease rate across age one month after receipt of the second dose compared to males, ending up in lower, but almost constant levels three months after having the second dose. In contrast, males displayed a consistent decrease rate with age in both periods after vaccination, indicating a propensity of elderly males to have lower titers and vaccine responses compared to elderly females three months post vaccination. Although in most cases the differences between males and females were not statistically significant, these findings merit further investigation to understand sex-age differences in vaccine responses that could be important for guiding public health decisions.Influenza A virus [Influenza A virus [Our analysis also showed an interaction between gender and smoking in antibody titers, such that women who smoked had lower antibody titers than women who did not, whereas men who smoked had higher antibody titers than nonsmoking men. Smoking, in general, is known to be associated with lower titers after vaccination against some viruses such as  A virus . However A virus ,41. Rece A virus . In our Limitations of our study include the unavailability of serological testing of all study participants in the second period, the overrepresentation of females and the imbalanced distribution of subjects across age groups, as well as the lack of assessment of additional antibody classes  or other components of immune responses, such as neutralizing antibodies and cell-mediated immunity. However, issues that could have distorted our results have been avoided through our meticulous analysis of available data. The next rounds of results of this prospective study, six- and nine months post vaccination, will shed additional light to the value of identified associations of antibody titers with epidemiological, behavioral and clinical parameters.S1 FigA. Received therapy. B. Smoking. C. Alcohol consumption. Of the 439 participants, 287 had no underlying disease, while the distribution of conditions among the remaining 152 subjects (25 of whom reported more than one conditions) was as follows: Arterial hypertension 41, thyroid abnormalities 33, diabetes 25, cardiovascular diseases 22, autoimmune diseases 19, asthma 11, cancer 5, arthritis 5 and other diseases  16.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableMean (SD) values are shown.(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Low tidal volume combined with an appropriate PEEP is the best option from the match between ventilation and pulmonary blood flow. Increasing PEEP can solve the problem of excessive shunt at high CO, and the V/Q ratio tends to match. At low CO, the increased dead space can reach as high as 64.64 ± 7.13% when PEEP = 20 cmH2O. With increasing PEEP, the microcirculation index deteriorates, including total vessel density (TVD), proportion of perfused vessel (PPV), perfused vessel density (PVD), and microcirculatory flow index (MFI). The periodic collapse of pulmonary capillaries or interruption of blood flow obviously occurred with high PEEP. The hemodynamic parameters indicated that the transpulmonary capillary wall pressure (Pcap) of the low CO group was negative at PEEP = 5 cmH2O, which determines the opening and closing of the pulmonary microcirculation and controls lung perfusion and the production of extravascular lung water. Therefore, it is essential to couple macrocirculation and pulmonary microcirculation during mechanical ventilation by improving shunting and optimizing Pcap.This study was performed to visualize the hemodynamic effects of pulmonary microcirculation and ventilation/perfusion (V/Q) matching after mechanical ventilation under different cardiac outputs and positive end-expiratory pressures (PEEPs). Ten experimental pigs were randomly divided into high and low tidal volume groups, and ventilation/perfusion were measured by electrical impedance tomography (EIT) at different PEEPs. Then, all the pigs were redivided into high cardiac output (CO) and low CO groups and measured by EIT at different PEEP levels with a low tidal volume. Additionally, sidestream dark field (SDF) was used to measure pulmonary microcirculation. Hemodynamic parameters and respiratory mechanics parameters were recorded. As PEEP increased at high tidal volume, blood flow was impaired at a higher PEEP (20 cmH A pulmonary shunt is a pathological condition that results when the alveoli of the lungs are perfused with blood as normal, but ventilation fails to supply the perfused region. Excessive cardiac output (CO) can cause intrapulmonary shunts  (Lynch eThe ventilation/perfusion (V/Q) ratio was defined as the ratio of the amount of air reaching the alveoli per minute to the amount of blood reaching the alveoli per minute. With the progress of technology, sidestream dark field (SDF) and electric impedance tomography (EIT) visualization technology can provide us with a method to observe the changes in pulmonary ventilation and pulmonary microvascular blood flow directly. In particular, these technologies provide a useful tool for the real-time and visualized evaluation of the V/Q ratio at the bedside.In this study, SDF and EIT visualization technology were used to study the effects of CO and PEEP on pulmonary ventilation and blood flow under physiological conditions by mechanical ventilation. The aim was to clarify the pulmonary microcirculation perfusion performance when V/Q changed due to CO and PEEP, which may contribute to a better understanding and guide mechanical ventilation strategies at different critical pathophysiological stages. The explanation of these mechanisms provides a theoretical basis for the protection of pulmonary circulation under mechanical ventilation.This study is an animal experiment that did not involve any patients.2O, oxygen inhalation (FiO2) = 30%, and SpO2 ≥ 95%, maintained by monitoring tongue oxygen saturation.The experiments described below were approved by the Animal Care and Use Committee of Chinese PLA General Hospital (No. SQ2020052). Ten healthy male pigs that weighed 22.45 ± 2.28 kg were provided by the Animal Facility of Lvyuanweiye . They were anesthetized and paralyzed for endotracheal intubation and then mechanically ventilated in a supine position. The induction of anesthesia was performed using an anesthesia machine . Anesthesia was induced with intramuscular atropine (0.04 mg/kg), tiletamine–zolazepam (6 mg/kg), and sevoflurane (2 L/min) and maintained by continuous infusion of propofol (2 mg/kg/h), midazolam (0.4 mg/kg/h), and fentanyl (10 µg/kg/h). Oral endotracheal intubation  and placement of a nasogastric pressure monitoring tube set  were performed. Mechanical ventilation was initiated with AVEA . Ventilation was set as the volume control model using the following parameters: tidal volume (VT) = 6 mL/kg, frequency (f) = 15 per min, PEEP = 5 cmH® system; Pulsion Medical Systems, Munich, Germany). Related hemodynamic parameters were obtained directly as detected by the instruments, such as systolic artery pressure (SAP), diastolic artery pressure (DAP), mean artery pressure (MAP), heart rate (HR), central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), global end-diastolic volume (GEDV), extravascular lung water (EVLW), stroke volume variation (SVV), pulse pressure variation (PPV), cardiac output (CO), stroke volume (SV), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR). The arterial blood gases were also measured, including arterial oxygen saturation (SaO2), oxygen partial pressure (PO2), and partial pressure of carbon dioxide (PCO2), using a GEM Premier 3000 gas analyzer .A right internal jugular vein catheter  was placed percutaneously using the sterile technique. A Swan–Ganz  catheter was placed into the left external jugular vein. Femoral artery PiCCO  was catheterized through the right femoral artery and connected to a PiCCO Module . An EIT belt with 16 surface electrodes was placed around the pig’s thorax. A bolus of 10 mL 10% NaCl was injected through the central venous catheter during a respiratory pause (≥8 s). Ventilation was paused via an end-expiratory hold maneuver with the ventilator in the intubated pigs. The maneuver was repeated a maximum of once in 30 min if significant tortuosity/interruption in the regional impedance–time curve was observed. EIT data analysis was performed using customized software developed with MATLAB . The related detailed methods are described in previous studies  . Through slight movements of the probe, microcirculatory images at three different locations were collected during each examination. At each time point, the subpleural pulmonary microcirculation was evaluated at three different locations through SDF imaging . Three 5 s video clips were recorded at each time point. The video clips used for analysis underwent a quality-control test based on image resolution quality, clarity of the image, and elimination of pressure-induced artifacts. The Automated Vascular Analysis software package  was used for the analysis following expert consensus [ s. ThrouWe use esophageal manometry to measure intrathoracic pressure. A nasogastric pressure monitoring tube set  was connected to the AVEA mechanical ventilator . We measured esophageal pressure (intrathoracic pressure) using end-inspiratory and end-expiratory occlusion. According to the formula of transpulmonary pressure ,10 (Gant2O). First, we needed to determine the baseline tidal volume required for the study and the effect of PEEP on tidal volume. Five were randomly selected for baseline V/Q measurement by EIT and hemodynamic parameters (VT = 6 mL/kg and PEEP = 5 cmH2O). The other five used a larger tidal volume (VT = 15 mL/kg and PEEP = 5 cmH2O) to measure hemodynamic and respiratory mechanics and V/Q. EIT parameter detection under different tidal volumes was completed within 30 min. Second, we needed to explore the physiological effects of different COs and PEEPs. After the EIT measurement, the high tidal volume pigs returned to the baseline ventilation state for 30 min (VT = 6 mL/kg and PEEP = 5 cm H2O). Then, all the pigs were randomly redivided into two groups: a high cardiac output group (n = 5) and a low cardiac output group (n = 5). The flowchart is shown in All 10 pigs were initially ventilated with lung protection with a small tidal volume  was used to compare means among multiple groups. The results for continuous variables that were not normally distributed are given as medians (25th and 75th percentiles) and were compared using nonparametric tests. The results for qualitative variables were expressed as percentages and compared between groups using a chi-square test. Statistical analyses were conducted by SPSS 16.0 , and a two-tailed p < 0.05 was considered significant.The results for continuous variables with normal distributions are given as the means ± standard deviations (SDs). Student’s 2O, the high VT group showed an obvious shunt concentrated in the gravity-dependent area, and the low VT group showed an increase in the dead space of the nongravity-dependent area. Since this study is a physiological model of normal lungs, both groups showed improvement in shunting with a further increase in PEEP. Shunting improvement was manifested in the low VT group, showing that shunting in the gravity-dependent area decreased with increasing PEEP. However, in the high VT group, the shunt continued to increase. When PEEP = 20 cmH2O, the peak airway pressure was high and could not be ventilated. The V/Q ratio in the low VT group was higher than that in the high VT group at PEEP = 10 cmH2O and especially at 20 cmH2O with a statistically significant difference.2O, the dead space was the main disadvantage (4.64 ± 7.13%). From the perspective of shunting, when PEEP = 0 cmH2O, the shunt of the high CO group (34.70 ± 1.63%) was significantly more obvious than that of the other two groups, and the difference was statistically significant. The shunt decreased with increasing PEEP, and when PEEP = 20 cmH2O, the degree of shunting of the high CO group was not significantly different from that of the baseline level. From the perspective of dead space, in the low CO group, the dead space increased with the increase in PEEP. When PEEP = 20 cmH2O, the dead space was as high as 4.64 ± 7.13%. The dead space of the baseline level was at its highest when PEEP = 0 cmH2O (16.53 ± 1.38%). Both the high CO and baseline levels showed relatively low dead space with increasing PEEP. From the V/Q ratio analysis, when PEEP = 0 cmH2O, PEEP = 5 cmH2O, and PEEP = 10 cmH2O, the V/Q ratio of the baseline level was better than that of the other two groups, but the V/Q ratio of the high CO group always improved with increasing PEEP. When PEEP =20 cmH2O, there was no difference in the V/Q ratio between the baseline level and the high CO group, but the V/Q ratio of the low CO group (27.29 ± 3.88%) was significantly reduced due to excessive dead space.Changes in the lung ventilation/perfusion (V/Q) ratio and microcirculation under the influence of high/low CO and different PEEPs are shown in 2O. PVD, PPV, and MFI all tended to increase with higher PEEP. At low CO, when PEEP was 0 to 10 cmH2O, TVD did not change significantly, but TVD decreased significantly with a further increase in PEEP to 20 cmH2O. PPV and PVD improved when PEEP increased from 0 cmH2O to 10 cmH2O but decreased with a further increase in PEEP. TVD, PPV, and PVD decreased to their lowest values when PEEP was 20 cmH2O, and the difference was statistically significant compared with the high CO group. MFI improved when PEEP increased from 0 cmH2O to 5 cmH2O, but there was no statistical significance. However, with a further increase in PEEP, the MFI gradually decreased, and there was no blood flow or intermittent blood flow in a few vessels when PEEP = 20 cmH2O. SDF videos under high and low cardiac output and different PEEPs are provided in We used total vessel density (TVD), proportion of perfused vessels (PPV), perfused vessel density (PVD), and microvascular flow index (MFI) to evaluate pulmonary local microcirculation perfusion . OverallThe changes in pulmonary and systemic hemodynamic parameters under different cardiac outputs and PEEPs are shown in First, we set high and low tidal volumes under normal cardiac output and directly observed the ventilation and blood flow of the lung under different PEEPs. The results showed the impairment of blood flow with an increase in PEEP at high tidal volume. Although the shunt and V/Q improved, the larger the PEEP was, the more difficult ventilation was. Additionally, we used the visualization technologies EIT and SDF to clarify lung ventilation and blood flow changes and the corresponding pulmonary microcirculation perfusion after different COs and different PEEPs under physiological conditions. We revealed that transpulmonary capillary wall pressure (Pcap) determines the opening and closing of the pulmonary microcirculation, controls lung perfusion, and produces extravascular lung water. These findings may guide clinicians to set mechanical ventilation parameters accurately with different COs and achieve lung and pulmonary microcirculation protection during mechanical ventilation.2O, the shunt decreased. At PEEP = 5 cmH2O, the dead space of the low VT group was significantly reduced, and the shunt of the high VT group was significantly reduced. This is in line with the ability of PEEP to improve shunting, and it also matches the current lung protection ventilation strategy with low tidal volume and high PEEP. From the perspective of blood flow in the lung, if the tidal volume is low and the PEEP is small (PEEP = 0 or 5 cmH2O), the pulmonary microcirculation may not be ideal. The pulmonary blood vessels tend to close when the lung volume is too small and decreases below the functional residual capacity (FRC). This phenomenon may be due to the active tension in the vessel wall [The mechanism and consequences of ventilation-induced lung injury (VILI) are not airway pressure but lung hyperinflation   at high CO is significantly more than that at low CO, consistent with our previous clinical study   may become a potential indicator of pulmonary microcirculation protection for the individualized treatment of mechanical ventilation in ARDS. It may be possible to use this parameter to avoid pulmonary vascular and endothelial damage in the future.Our study also has some limitations. Firstly, this is an animal experiment. We used a limited number of pigs to complete the hemodynamic effects of tidal volume, CO, and PEEP, especially the titration of the relationship between lung local ventilation and blood flow. There may be confounding effects of the different study conditions. However, what we studied is the change in normal lung under different respiratory mechanical conditions. On the basis of pathophysiological principles, we reasoned that this effect would not be greater than that in animal models of ARDS. Secondly, the main purpose of this study was to study the physiological effects of different COs and different PEEPs. Therefore, our study only illustrates the relationship between lung ventilation and V/Q under different conditions, rather than specify a clear study endpoint. Through this study, we can explore a possible execution condition for the next step to find an optimal ventilation scheme, and then proceed to the design and verification of randomized controlled animal experiments. SDF can only be measured at the end of the experiment as it requires invasive monitoring. In the future, it is hoped that there will be a method and device capable of continuous monitoring for real-time measurement of local pulmonary microcirculation.SDF and EIT are bedside noninvasive technical means that can visualize and reflect the patient’s ventilation and blood flow status in real time. This study first visualized the matching degree of ventilation and perfusion (V/Q) and realized the perfusion of lung and pulmonary microcirculation. We define it as a phenomenon of periodic switching of pulmonary circulatory microvessels. Low tidal volume lung protection ventilation helps to protect the blood flow of the pulmonary circulation from ventilation and perfusion. An increase in the appropriate level of PEEP can reduce the intrapulmonary shunt, reduce the pressure across the pulmonary vessel wall, and avoid the formation of excessive lung water under high CO. However, the collapse of pulmonary capillaries under low CO caused uncoupled ventilation and perfusion. Transpulmonary vessel wall pressure (Pcap) should be considered in the regulation of lung ventilation and blood flow coupling, which may become a potential indicator of pulmonary circulation protection for the individualized treatment of mechanical ventilation in ARDS.


Haloperidol (HPL) is a typical antipsychotic drug used to treat acute psychotic conditions, delirium, and schizophrenia. Solid charge transfer (CT) products of HPL with 7,7,8,8-tetracyanoquinodimethane (TCNQ) and picric acid (PA) have not been reported till date. Therefore, we conducted this study to investigate the donor–acceptor CT interactions between HPL (donor) and TCNQ and PA (π-acceptors) in liquid and solid states. The complete spectroscopic and analytical analyses deduced that the stoichiometry of these synthesized complexes was 1:1 molar ratio. Molecular docking calculations were performed for HPL as a donor and the resulting CT complexes with TCNQ and PA as acceptors with two protein receptors, serotonin and dopamine, to study the comparative interactions among them, as they are important neurotransmitters that play a large role in mental health. A molecular dynamics simulation was ran for 100 ns with the output from AutoDock Vina to refine docking results and better examine the molecular processes of receptor–ligand interactions. When compared to the reactant donor, the CT complex [(HPL)(TCNQ)] interacted with serotonin and dopamine more efficiently than HPL only. CT complex [(HPL)(TCNQ)] with dopamine (CTtD) showed the greatest binding energy value among all. Additionally, CTtD complex established more a stable interaction with dopamine than HPL–dopamine. Atypical antipsychotics or serotonin–dopaminergic antagonists, the fourth class of antipsychotic drugs, can improve the so-called positive symptoms of schizophrenia, such as hallucinations, delusions, and agitation, as well as negative symptoms, such as catatonia and flattening of the ability to feel emotion. Each agent in this group has a unique profile of receptor interactions. Almost all antipsychotics block dopamine receptors and reduce dopamine transmission in the forebrain. Moreover, atypical antipsychotics have an affinity for serotonin receptors. Atypical antipsychotics are related to chlorpromazine and haloperidol (HPL), and HPL is used to treat acute psychotic conditions, delirium, and schizophrenia . The formation of highly colored charge-transfer (CT) complexes that absorb light in the visible region is often related with molecular interactions between electron donors and acceptors ,3. CT coDonor–acceptor complexation plays an important role especially in the field of biochemical energy transfer process . The forSolid CT products of HPL with 7,7,8,8-tetracyanoquinodimethane (TCNQ) and picric acid (PA) have not been reported till date. Therefore, we conducted this study to investigate such reactions. The molecular docking software AutoDock Vina was used to investigate the interactions between ligands (HPL and synthesized CT complexes) and receptors (serotonin and dopamine). Hydrophobic, ionizability, aromatic, and hydrogen bond surfaces were studied as well as binding energy. The best molecular docking data were submitted to molecular dynamic simulation at 300 K for 100 ns to give a more effective mechanism for illustrating receptor–ligand interactions. In terms of residue flexibility, structural stability, solvent accessible surface area, structure compactness, and hydrogen bond interactions, the dynamic properties of the complexes were compared.−1 cm−1 mol−1, respectively. The low conductance values of the synthesized CT complexes deduced the formation of D+ and A− datives anions based on the association of donor–acceptor chelation. The electronic spectra of synthesized CT complexes of HPL-PA and HPL-TCNQ refer to the association of new electronic absorption bands , which did not exist in the spectra of free reactants. The infrared spectrum of HPL-PA complex was assigned upon intermolecular hydrogen bonding between the –OH group of the PA acceptor and basic oxygen atom center of HPL donor  was 1:1. The conductivities of HPL-PA and HPL-TCNQ CT complexes were 45 and 53 ΩPL donor . In the PL donor , the –OHThe bonding of the –OH group of HPL and the –OH group of PA to the –CN of TCNQ acceptors via intermolecular hydrogen bonding was confirmed by proton NMR spectra of the free HPL donor and its HPL-PA and HPL-TCNQ complexes. The activation energy (E) was used to calculate the thermal stability of both HPL-PA and HPL-TCNQ complexes using Coats–Redfern and Horowitz–Metzger techniques ,9,10.−1 and 98 kJ mol−1, respectively, and the variant data might be influenced by the acceptor type. The activated complexes had a more ordered structure than the reactants, and the activation of entropy (ΔS ∗) had negative values, indicating that the reaction rates were slower than normal.The average activation energies for the [(HPL)(PA)] complex and the [(HPL)(TCNQ)] complex were 132 kJ molg), which refers to the minimum transition energy, was determined based on the electronic absorption spectra. Optical absorption near the edge of the absorption band can be used to estimate Eg and confirm the formation of CT complexes. The absorption coefficient (α) can be estimated from the transmittance (T) of the complex according to the following equation:g based on the following equation  and [(HPL)(TCNQ)] against serotonin (PDB ID: 6A94) and dopamine (PDB ID: 6CM4) were determined. For comparison, HPL was employed as the control. CT complexes have a larger potential binding energy than HPL in both receptors . Among tThe illustration of molecular docking for ligand–receptor interactions depicted in Molecular docking of HPL drug with serotonin and dopamine revealed the potential binding energies as −10.0 and −10.9 kcal/mol, respectively. The higher binding energy value of HPL–dopamine (HPLD) signifies stronger interaction with dopamine compared to that with serotonin. The best docking position with dopamine (HPLD) is shown in The best docked location of HPLD and CTtD with the highest docking score at 100 ns molecular dynamics (MD) generated by AutoDock Vina was used. Only the best docking output was used to build up this method in a high-throughput manner for analyzing the binding mechanism of the ligand at the active site of protein under clearly defined aqueous conditions. The root mean square deviation was computed to determine structural stability from MD data (RMSD). HPLD and CTtD formed stable conformation after ~80 ns and ~65 ns, respectively, with an appropriate RMSD value of 3.04 and 2.41 Å, respectively, as seen in the RMSD plot .The RMSD value range of <3.0 Å is the most acceptable . CTtD prRR Distance Maps creates a distance map through Chimera 1.15 software by a structural comparison tool. The map can display Cα-Cα distances within a single protein chain, as well as averages and standard deviations for many chains. The white diagonal on the map represents no distance between the two residues, but the red and blue on the map reflect residue pairings with the biggest distance differences between the two conformations .The average radius of gyration (Rg) values for HPLD and CTtD were 22.647 and 22.586 Å, respectively. Along the simulation time, Rg decreased, indicating that the structures became more compact .The number of hydrogen bond interactions between HPLD and CTtD was displayed against time using a grid-search on a 27 × 18 × 21 grid with rcut = 0.35 . There wOverall, the receptor–protein interaction enhanced the number of hydrogen bonds substantially, and it was more in CTtD.The values of solvent accessible surface area (SASA) altered as the ligand bound to the receptor . When th3OH was allowed to react with 3 mmol of each acceptor (PA and TCNQ) in 10 mL CHCl3 solvent, and both mixtures were stirred at room temperature for 45 min. Following this, the yellow and green solid complexes were isolated, washed three times with a minimum amount of CHCl3 solvent, and dried under vacuum over anhydrous CaCl2.Previously, synthesis and characterizations  of the two solid HPL CT complexes were performed . For preA Perkin–Elmer Precisely Lambda 25 UV/Vis spectrometer using a 1 cm quartz cell was used to measure the electronic absorption spectra of charge transfer complexes produced in the presence of methanol at the range of 200–800 nm. The Jenway 4010 conductivity device was also used to measure the molar conductivity in the presence of newly prepared dimethyl sulfoxide solutions. http://openbabel.org/wiki/Main Page; accessed 1 March 2022) [The structures of [(HPL)(PA)] and [(HPL)(TCNQ)] were obtained in PDBQT format using the OpenBabelIGUI tool ch 2022) . The RCS . The RCch 2022) . The Geich 2022) . The resSimulations were conducted using processor Intel(R) Xeon(R) CPU E5-2680 v4 @ 2.40GHz, 64 bit. MD simulation was performed using the optimal receptor–ligand complex position and an evaluation of the conformational space and inhibitory potential. MD simulation analysis with the GROMOS96 43a1 force field was performed using the Gronningen machine for chemical simulations .+ and 46 Cl− ions  were administered to neutralize the systems and reproduce physiological salt concentrations  and constant pressure (1.0 bar) over 100 ns were used for simulations using the leap-frog MD complement in the NPT/NVT equilibration run . The ste0 K and cIn the present research, we looked into how haloperidol (HPL) interacts with two key neurotransmitters (serotonin and dopamine) that are vital in mental health. These findings were compared with the synthesized charge transfer complexes of TCNQ and PA with HPL. The [(HPL)(TCNQ)] coupled with serotonin and dopamine more efficiently than HPL alone. Also, [(HPL)(TCNQ)]–dopamine has a higher binding energy value than HPL–dopamine. The molecular dynamic simulation at 100 ns demonstrated that the [(HPL)(TCNQ)]–dopamine complex had a more stable interaction with the dopamine receptor than the HPL–dopamine complex.


Compared to pure CNF film, the composite CNF/LNP films exhibited higher physical properties (tensile strength of 164 MPa and Young’s modulus of 8.0 GPa), a higher maximal weight loss temperature of 310 °C, and a perfect UVB blocking performance of 95.2%. Meanwhile, the composite film had a lower environmental impact as it could be rapidly biodegraded in soil and manmade seawater. Overall, our results open new avenues for the utilization of lignin nanoparticles in biopolymer composites to produce functional and biodegradable film as a promising alternative to petrochemical plastics.The exploration of functional films using sustainable cellulose-based materials to replace plastics has been of much interest. In this work, two kinds of lignin nanoparticles (LNPs) were mixed with cellulose nanofibrils (CNFs) for the fabrication of composite films with biodegradable, flexible and ultraviolet blocking performances. LNPs isolated from  Due to the industrial emissions induced by human activities, the ozone layer has become thinner, exposing the surface of the Earth to increasing ultraviolet radiation ,2. AccorAs the world’s most abundant resource, cellulose is an alternative to petroleum-based polymers because of its outstanding biodegradability and biocompatibility ,8. AmongLignin is one of the three major components in the plant cell wall, which contains functional groups, such as aryl, phenolic hydroxyl, and ketones, making it suitable for use as a UV shielding agent ,16. So fp-toluenesulfonic acid hydrolysis (PA). The size of the LNPs was tuned by controlling the acid treatment conditions. Then, LNPs with different contents were mixed with CNFs to prepare composite film through filtration and pressing processes. The morphology and diameter of different LNPs were first compared. Then, the morphology, physical property, thermal stability, UV shielding performance, surface chromaticity value, and biodegradation of composite films were discussed. This work is aimed at not only achieving the quantification of LNPs and CNFs in composite film, but to also elucidate the relationship between LNP diameter and film properties, which can open up broader prospects for lignin nanoparticles in food packing material, UV protection, and surface coating for vehicles.Herein, two kinds of lignin nanoparticles (LNPs) were isolated from poplar sawdust using maleic acid (MA) and p-toluenesulfonic acid and maleic acid hydrolysis were observed to investigate their morphology using FE-SEM images  (max of 243 °C), all CNF/LNP composite films possessed higher Tmax of approximately 310 °C, broadening their applications in thermal insulating and packaging materials.The thermal degradation performance of composite film is regarded as an important parameter in packaging, insulating, and gas separation materials . The weimax) b. DifferHigh transparency and strong UV absorption is important for films used in the packaging of food and medicine fields . The preThe composite film exhibited excellent biodegradability in the natural environment. For example, three composite films  and polyethylene (PE) were buried in soil at the same depth to monitor their morphology over time. After one month of burial in the soil, three composite films became fractured, likely due to the existence of microorganisms, which could directly digest and degrade the cellulose and lignin macromolecules in composite films . This rep-toluenesulfonic acid were purchased from Aladdin Chemical Co. Ltd. .Cellulose nanofibrils (CNFs) were purchased from ScienceK Ltd, Zhejiang, China. Woody poplar sawdust (PS) was harvested from Jiangsu, China, and stored at room temperature. Maleic acid and LNPs were isolated from PS using MA and PA hydrolysis as previously described . In brieThe CNF suspension was diluted to 0.5 wt% consistency and stirred for 1 h for pure a CNF film or mixed with different LNP suspensions for a CNF/LNP composite film. LNP content was 5 wt% on the basis of total dry weight in the composite film. The mixture was stirred for 1 h to achieve a well-uniformed suspension, then composite films with different types and contents of LNPs were prepared using vacuum filtration. The formed CNF/LNP film was placed between two commercial filter papers (with a diameter of 9 cm) and copper plates, then they were pressed for 48 h at 25 °C and 60 °C, respectively, under a weight of 5 kg.A dynamic light scattering analyzer  was used to measure the effective diameters of LNPs. Each sample with a concentration of approximately 1 g/L was measured and the results were presented as mean values.The morphology of LNPs was characterized using a cold filed-emission scanning electron microscope . The surface microstructure of the CNF/LNP composite film was measured using scanning electron microscopy . Samples were fixed onto mounts, then coated with gold before testing.The TRAPPEZIUM X type tester  was used to measure the mechanical properties (tensile stress and Young’s modulus) of the composite films. The load cell and crosshead speed was set to 500 N and 1 mm/min, respectively. Each film was tested for five measurements and the average value was reported.The thermal stability of composite films was evaluated by thermogravimetric analyzer  with a heating temperature from 50–600 °C (10 °C/min in increment) under nitrogen protection of 20 mL/min .λ is the average spectral transmittance, d(λ) is the bandwidth, and λ is the wavelength.The UV-Vis transmittance was measured using a spectrophotometer  with a wavelength from 200–900 nm. The UVA and UVB blocking rates of the composite films were calculated by Equations (1) and (2):The surface chromaticity value of the composite films was analyzed using an Xrite i1 Pro2 spectrophotometer. CIELAB consisted of three components related to color  and was specified by the International Commission on Illumination (CIE). Specifically, L represents the lightness of the color in which low numbers (0–50) and high numbers (51–100) refer to darkness and lightness, respectively. The values of a and b range from +127 to −128, where a includes green (−a) and red (+a), and b includes yellow (+b) and blue (−b).2, and 1.2 g/L CaCl2. In the biodegradation process, the composite films and polyethylene (PE) were buried at the same depth in the soil at ambient temperature or immersed in prepared sea water solution. The degradation performance of the composite films in soil and sea water were observed after one month and 18 days, respectively.The biodegradation test was carried out in soil and seawater, which simulates biodegradability under real-life conditions. A standard composition of seawater included 26.7 g/L NaCl, 0.7 g/L KCl, 2.3 g/L MgClIn summary, CNF composite films were fabricated using facile vacuum filtration and the pressing process by incorporating different types of LNPs. Although the addition of LNPs resulted in a composite film with an uneven surface and decreased mechanical property, reducing the size of the LNPs or increasing the maleic acid reaction duration could improve the tensile strength and Young’s modulus to 164 MPa and 8 GPa, respectively, compared to pure CNF film. Apart from strength enhancement, the composite film also showed excellent UV resistance and thermal stability. Compared to petrochemical-based plastics such as PE, the composite film was biodegradable in nature and friendly to the environment. Thus, this work provided a facile strategy to prepare biodegradable and multifunctional film from natural lignocellulosic biopolymers to replace conventional plastics towards sustainable fields, such as agricultural mulching films and packaging materials.