Objective: To study the effect of Yu Linzhu on ovarian function and mitochondria in natural aging mice.Methods: Female BALB/c mice were selected as normal group at 7–8 weeks and natural aging group at 9 months. The natural aging group was divided into Yu Linzhu intervention group and non-intervention group by intragastric administration once a day for 6 weeks. The morphology and blood flow of ovary were observed by ultrasound. Ovarian morphology and follicle were observed by HE staining. Hormone levels were analyzed by ELISA. Serum oxidative stress were detected by radioimmunoassay. The distribution of mitochondria in oocytes was observed by fluorescence staining. The ultrastructure of oocytes and the morphology of mitochondria were observed under electron microscope. The mitochondrial membrane potential was detected by JC-1.P = 0.0092 and P = 0.0334, respectively), FSH (Follicle stimulating hormone) increased (P < 0.0001). In the intervention group, FSH decreased (P = 0.0002), LH (luteinizing hormone) decreased and E2, AMH increased. In the non-intervention group, GSH-Px (Glutathione peroxidase) decreased (P = 0.0129), SOD (Superoxide dismutase) decreased, ROS (reactive oxidative species), MDA  increased. In the aging intervention group, ROS, MDA decreased and GSH-Px increased. In the non-intervention group, mitochondrial expression was scattered at the concentrated distribution point, the length of mitochondria was mostly long and the average volume increased, the density decreased, the number decreased and some mitochondria fused, and lesions such as swelling, vacuolar degeneration and inclusion body formation, membrane potential decreased (P = 0.0002). In the aging intervention group, mitochondria were evenly distributed, the mitochondria were basically round, the distribution density was moderate, the inner ridge was clear, and the membrane potential of the aging intervention group increased.Results: Two groups of aging mice had serious disturbance of estrus cycle. The ovarian area of the mice in the aging non-intervention group was smaller than that in the normal group, and the ovarian area of the mice in the aging intervention group recovered. The ovarian blood flow was weak or even disappear in the aging non-intervention group, and the blood flow in the intervention group was improved. The ovarian volume of mice in the non-intervention group was smaller than that in the normal group. Some ovarian tissues were adhered to the surrounding tissues. While in the intervention group, the ovarian volume increased, the degree of adhesion decreased, the infiltration of ovarian interstitial lymphocytes decreased, and the zona pellucida recovered. Granular cell arrangement returned neatly, egg cell shape recover regular and the number also increased. In the non-intervention group, E2 (Estrogen), AMH (Anti-Mullerian hormone) decreased (Conclusion: Yu Linzhu can improve the ovarian function of natural aging mice by improving the mitochondrial function of oocytes. Ovarian senescence is a biological process affected by many factors, gradually accumulating and continuously complicated, accompanied by the decrease of mitochondrial number, structural change and productivity of oocytes associated with the decrease of oxidative phosphorylation function. As the most important organelle in the cytoplasm, mitochondria helps oocyte maturation and embryonic development. Mitochondrial dysfunction and mitochondrial DNA mutation inhibit the normal development of oocytes. Mitochondria are the determinants of ovarian senescence , 2. ChanYu Linzhu is an ancient traditional Chinese medicine prescription that can treat infertility. It can warm spleen and kidney. In the formula, Four Gentlemen Decoction invigorate spleen and invigorate qi. Decoction of Four Ingredients nourish blood to fill essence. Semen Cuscute, Cortex Eucommiae and Cornu Cervi Dege Latinatum can warm and reinforce kidney yang. Pericardium Zanthoxyli can warm the body to promote follicle development and ovulation. The whole prescription promotes ovulation and ultimately improves ovarian function. Previous studies have found that Yu Linzhu can improve the serum hormone levels in POI patients . Basic sA total of 10 seven-to-eight-week-old SPF BABL/c female mice and 20 nine-month-old female mice were purchased from Beijing Vitonglida Experimental Animal Center . All mice were maintained at a constant temperature (20°C–22°C) and humidity (30%–70%) in a 12/12-hour light/dark cycle and had unrestricted access to water. All animal experiments were approved by the Ethics Committee of Animal Experiment, Capital Medical University, in accordance with the guidelines of the National Research for the Care and Use of laboratory animals.The following reagents were used: Serum follicle-stimulating hormone (FSH), anti-Muller tube hormone (AMH), luteinizing hormone (LH) and estradiol (E2) ELISA kits (Beijing Boorson Biotechnology Company). ROS, SOD, MDA, GSH-Px ELISA kits (Nanjing Jiancheng Biotechnology Company). 2 × HEPES buffer . 1 × PBS . Fetal bovine serum . Pregnant horse serum gonadotropin . 10% chloral hydrate . Mito Traker Green FM . 2.5% Gluta stationary liquid-electron microscope . Swiss-Giemsa compound staining solution . Hoechst33342 staining solution (1 mg/ml) . Incomplete MEM . JC-1 .Atractylodes macrocephala 6 g, fried Radix Paeoniae Alba 6, Eucommia ulmoides 6 g, Poria cocos 6 g, antler cream 6 g, Chuanxiong 3 g, grilled licorice 3 g. Buy from Beijing Tongrentang Technology Co., Ltd. On the basis of the ‘human and animal body surface area equivalent dose scale’ decoction, it was determined that each mouse in the aging Chinese medicine group was given 0.468 g/ml, 0.3 ml/ Chinese medicine equivalent dose per day according to body weight, stored at 4°C refrigerators.Traditional Chinese medicine preparation: Yu Linzhu ingredients include: angelica 12 g, ripe ground 12 g, dodder 12 g, ginseng 6 g, pepper 6 g, 10 female mice of adult reproductive age at 7–8 weeks were randomly selected as normal groups. Twenty aged female mice were randomly divided into 10 Yulinzhu intervention and 10 non-intervention groups. Animals in normal and non-interventional groups were given 0.3 ml saline solution once a day by intragastric administration once a day for 6 weeks. Yulinzhu intervention group was given 0.3 ml traditional Chinese medicine by intragastric administration once a day for 6 weeks.Small cotton swabs were soaked in saline, inserted into the vagina of mice approximately 5 mm, rotated clockwise 6 times, then rolled and smeared on a clean slide. The slide was naturally air-dried. The smears of vaginal secretions were collected for one week, stained with Swiss Giemsa staining solution for approximately 5 min, rinsed with water and observed under an ordinary optical microscope.The back fur of mice was removed with depilation ointment (Wei Ting), the mice were anesthetized by ultrasonic special table, and isoflurane (2.5%) was inhaled. A probe was placed 2–3 mm above the outer surface of the back of the mouse to see the appearance and disappearance of one side of the ovary as the moving area of the probe, and the other side of the ovary imaging was obtained by the same method. The color sampling frame was placed on the ovarian area corresponding to the back surface of the mouse to observe the arterial blood flow.2 asphyxiation, the ovary and uterus were taken by routine aseptic method. One side of the ovary of three mice were fixed in precooled glutaraldehyde solution (2.5%), the other side of the ovary was fixed in formaldehyde. The formaldehyde-fixed ovarian tissue was dehydrated by gradient ethanol, paraffin-embedded, sliced, dewaxed, stained with conventional HE, and observed under a light microscope. The blood samples of the mice were allowed to stand and centrifuged, and ELISA were used to detect the contents of serum hormone and oxidative stress indexes in strict accordance with the kit instructions.The mice were anaesthetized by intraperitoneal injection of 4% chloral hydrate 0.1 ml/10 g. Take 10 ml 10% chloral hydrate plus 15 ml double-steamed water, mix evenly dilute to 4% chloral hydrate. After 5 minutes, the mice were anesthetized, then the blood was collected from heart. Then the animals were sacrificed by CO2 asphyxiation 48 hours later. Ovaries were cut off and placed in preheated HEPES, 5 for fluorescence and 5 for JC-1  for 17 hours to the MII stage. We did not choose HCG (human chorionic gonadotrophin) because it does not work, no follicles fall into the fallopian tube. To avoid mistakes, we chose to incubate directly. The oocytes that took off the granulosa cells were transferred to a 400 nmol/L Mito Tracker Green FM four-well dish preheated at 37°C and stained in a 37°C incubator for 30 minutes and washed with PBS 3 times for 5 minutes each time. After the above staining, the oocytes were added to 10 μg/ml Hoechst 33342, stained with nucleus for 10 minutes, and washed three times with PBS. The stained oocytes were transferred onto a slide for observation and analysis by laser confocal microscopy.After 10/IU PMSG intraperitoneal injection of mice, they were killed by COfor JC-1 . Under a3 sized tissue blocks, fixed in 4°C refrigerator for 4 hours, washed 3 times with 1XPB for 10 minutes each time, fixed with 1% osmotic acid for 1 h, washed 3 times for 10 minutes each time, and incubated with 2% U3HAC stain for 30 minutes. Then, the samples dehydrated in gradient alcohol  for 10 minutes, 100% alcohol for 15 minutes, and 100% acetone for 15 minutes. Next, ultrathin slices with a thickness of 120 nm were prepared. The slices were stained with 4% UAC for 20 minutes and lead citrate for 5 minutes and observed with electron microscopy.The ovaries in Gluta fixation solution were cut into 1 × 1 × 1 mmAll MII stage oocytes were collected and transferred into 10 ug/ml JC-1 (Tetrachloro-tetraethyl benzimidazol carbocyanine iodide), stained for 25 min and washed 3 times with PBS. The stained oocytes were transferred onto slide and photographed under laser confocal. The fluorescence intensity of red and green light was determined by enzyme labeling instrument. The ratio of red to green fluorescence intensity represented the membrane potential. The red light excitation wavelength is 559 nm and emission wavelength 572 nm, the green light excitation wavelength is 488 nm and emission wavelength 520 nm.P < 0.05 indicated that the difference was statistically significant.GraphPad Prism 8.0 software was used for analysis. The experimental data are represented by The observation of mouse vaginal smear showed that the estrus cycle of the two groups of aging mice was seriously disordered. By naked eye observation, it can be seen that the vaginal secretion of normal group mice is sticky and drawn, the vagina of both aging group mice is dry and astringent, there is no obvious vaginal secretion, and the mice of aging intervention group increase vaginal secretion. Under microscopic observation, it can be seen that the normal group mice were more homogeneous in the stage of estrus, and the keratinized epithelial cells were deciduous, and the keratinized epithelial cells in the aging non-intervention group were not uniform. The keratinized epithelium in the aging intervention group was better than that in the non-intervention group .P = 0.0076), and the ovarian area in the aging intervention group was larger than that in the non-intervention group (P = 0.0210)  , Table 1 0.0210) . Pulse p 0.0210) .Our naked eye observed that compared with the normal group, the ovarian volume of the mice in the aging non-intervention group was smaller than that in the normal group, and some ovarian tissues were adhered to the surrounding tissues, and the ovarian volume of the mice in the aging intervention group increased and the degree of adhesion was reduced. Under optical microscope, it was found that the ovarian interstitial lymphocytes infiltrated, some pellucida decreased, granulosa cells were arranged in disorder, oocytes morphology was deformed, narrowed or disappeared, and the infiltration of ovarian interstitial lymphocytes in mice in aging intervention group was alleviated. Particellucida decreased, granulosa cell arrangement recovered neatly, oocytes morphology recovered regularly, size recovered moderately, and number increased , Table 3P = 0.0092 and P = 0.0334, respectively) and the FSH increased (P < 0.0001) in the aging non-intervention group compared with the normal group. The FSH of aging intervention group decreased (P = 0.0002), the LH decreased and the E2, AMH increased compared with the aging non-intervention group (P = 0.0129), SOD decreased and ROS, MDA increased compared with normal group. The aging intervention group ROS decreased and the GSH-Px increased compared with the aging non-intervention group . Compared with the non-intervention group, the aging intervention group was higher (It was observed by electron microscope that the mitochondria were mostly long and the average volume increased, the density decreased, the number decreased number of mitochondria was reduced, and the lesions such as swelling, vacuolar degeneration and inclusion body formation appeared. Compared with the non-intervention group, the mitochondria of the aging intervention group were basically round, the distribution density was moderate, and the inner ridge was clear . The exps higher , Table 6At present, the theory of oocyte aging mainly includes: aneuploidy theory, oxygen free radical-mitochondrial damage theory and so on. Mitochondria metabolize glucose into pyruvate and transport it to oocytes to generate ATP . During five-Zang also follow the failure. In the old age, the essence of the kidney is declining, which leads to the failure of other Zang functions, so it is necessary to take the method of tonifying the kidney and filling the essence and strengthening the life, so as to help generate qi and achieve the purpose of prolonging life and resisting aging.In the theory of traditional Chinese Medicine, kidney dominates growth, development and reproduction. The Ming Dynasty Li Chan’s ‘Introduction to Medicine’ said: ‘People to middle age, kidney qi to deficiency.’ In the Ming Dynasty, Yu Tuan’s ‘Zhengzhuan of Medicine’ also said: ‘Abundant Kidney Qi brings longevity, kidney qi deficiency brings reduction in life-span.’ It can be seen that the aging of the human body begins with the aging of the kidney, and the life activities of the human body depend on the promotion of the kidney qi and essence. Once the kidney qi fails, the In this study, the ovarian volume of aging mice was reduced and the blood flow decreased obviously by ultrasonic dynamic observation, which indicated that the decrease of blood flow was the characteristic of ovarian senescence. The ovarian blood flow recovered and improved ovarian function after Yulinzhu intervention in aging group. Morphological results also further confirmed that the infiltration of ovarian interstitial lymphocytes decreased after blood flow improvement, partial pellucida decreased, granulosa cell arrangement recovered neatly, egg cell morphology recovered regularly, size recovered moderately, and quantity increased. The further serum hormone level showed that E2, AMH increased and serum FSH, LH concentration decreased, indicating that hormone level improved. Also improved to delay ovarian aging, improve ovulation.Based on this, we found that the fluorescence intensity of oocyte mitochondria in aging group was lower than that in blank group, and the mitochondrial distribution of Yulinzhu intervention group was more uniform. Morphologically, oocyte maturation process was accompanied by changes in mitochondrial distribution, and lack of mitochondrial redistribution was sign of insufficient cytoplasmic maturation . The mitIn conclusion, the decline of fertility in POI women is due to the decrease of follicle number and egg quality after the decline of ovarian function. Mitochondria such as quantity, distribution and membrane potential activate oocyte and embryo development. As the most important organelle of oxidation reaction, by improving ovarian microenvironment, enhancing ovarian blood flow and restoring ovarian function, mitochondrial vitality, follicle development and fertility will be improved. Further understanding the relationship between mitochondria and ovarian senescence and exploring the mechanism of traditional Chinese medicine to improve the quality of oocytes will help to provide a more optimized strategy for the prevention and treatment of female senescence and improve its pregnancy rate.


Linum usitatissimum mucilage, a natural polymer, in developing a sustained release hydrogel for orally delivered drugs that require frequent dosing. For this purpose, nicorandil (a model drug)-loaded hydrogels with various feed ratios of Linum usitatissimum mucilage, acrylamide (monomer) and methylene bis-acrylamide (crosslinker) were prepared. The newly synthesized hydrogel formulations were probed fundamentally with respect to swelling behaviour, solvent penetration, and the release of the drug from the hydrogels. Later, the selected formulations were further characterized by Fourier-transform infrared spectroscopy, thermal analysis, X-ray diffraction analysis, and scanning electron microscopy. The swelling coefficient demonstrated a linear relation with the polymer ratio; however, an inverse behaviour in the case of monomer and crosslinker was observed. The drug release studies, performed at pH 1.2 and 4.5 and considering the dynamic environment of GIT, demonstrated that all formulations followed the Korsmeyer–Peppas model, displaying a slow drug release via diffusion and polymer erosion. FTIR analysis confirmed the successful grafting of acrylamide on linseed mucilage. Furthermore, scanning electron microscopy revealed a clear surface morphology with folds and pinholes in the hydrogel. Therefore, based upon the in-vitro outcomes, it can be concluded that a promising sustained release hydrogel can be prepared from natural polymer, Linum usitatissimum mucilage, offering many-fold benefits over the conventional synthetic polymers for oral delivery of drugs.The aim of this study was to investigate the potential of  The oral route of drug delivery is the most convenient way of administration but, at the same time, is associated with fluctuations in plasma drug concentrations that can be critical in certain diseases, for instance, cardiovascular problems . In ordeHydrogels stand at the middle of the podium when it comes specifically to sustaining release delivery because of their ability to provide spatial and temporal control over the release of drugs. The development of tuneable hydrogels such as in-situ forming, stimuli  responsive ,4,5, fatLinum usitatissimum (linseed) mucilage (polysaccharide) possesses the thickening, swelling and adhesive properties that render their potential for several applications in pharmaceutical preparations [Among the natural polymers, arations ,13,14. Iarations ,16 and carations . The swearations ,19. In oLinum usitatissimum (linseed) mucilage as a polymer with acrylamide monomer and methylene bis-acrylamide as a crosslinker for the gel frame. The composition based upon its intended characteristics was evaluated for influence on swelling, penetration of solvent and release of nicorandil, a model drug that has a very short half-life and that is an ideal candidate for the sustained release delivery.Therefore, the aim of the study was to develop graft copolymeric hydrogel by free radical polymerization, using The mucilage contains water-soluble non-starch polysaccharides, rhamnogalacturonan and arabinoxylans are the main polysaccharides and these components of linseed, having hydroxyl groups attached with its backbone. The mucilage has an insufficient amount of hydrolytic stability that can be improved by introducing acrylamides with groups such as alkyl and hydroxyl alkyl. The proposed chemical structure of graft copolymeric hydrogel is illustrated in The swelling of naturally occurring polysaccharides depends upon the presence of hydrophilic/hydrophobic groups, degree of crosslinking, and elasticity of network . ConsideComparative swelling ratios of different formulations of linseed-co-AAM hydrogel with varying monomer concentrations are shown in According to the literature, hydrogels formulated with lower total monomer concentration revealed a higher swelling, and the equilibrium mass swelling of the hydrogels decreased with increasing total monomer concentration. For the constant gel volume, the decrease in the total monomer concentration results in an increase in the degree of dilution of the matrix in the constant gel, which also results in an increase in the equilibrium water content of the gel ,22,23.Comparative swelling ratios of different formulations of linseed-co-AAM hydrogel with varying mucilage concentration at pH 1.2 and 4.5 are specified in The swelling ratio of the hydrogels depends on the chemical structure of mucilage. Hydrogels that have hydrophilic groups swell greater then hydrogels containing hydrophobic groups because these groups break down in the presence of water, thus, minimizing their interaction with the water molecule ,25. The Comparative swelling ratios of different formulations of hydrogels with varying crosslinker concentrations are shown in The literature indicates that power-law performances of swelling that increase the crosslinker lower the water absorbency. Higher crosslinking results in an increased degree of crosslinking in the polymer network, which results in low swelling ,29.The swelling index was carried out at pH 1.2 and 4.5 for all the above-mentioned formulations. Lower percentage of equilibrium swelling was obtained by increasing the crosslinker and monomer content in the hydrogel. With an increase in mucilage concentration, percentage of equilibrium swelling was increased .Percentage of equilibrium swelling (% ES) of all formulations of hydrogels with varying mucilage, crosslinker and monomer concentrations at pH 1.2 and 7.4 are specified in Quantity of drug integrated in different formulations of linseed-co-AAM is given in FTIR analysis was done to confirm AAM grafting on linseed mucilage. Secondly, to find out the loaded drug in the final formulation, discs revealed neither degradation nor reaction with the formulation. FTIR is based on the opinion that the basic components of a constituent that has chemical bonds usually show excitation and absorb infrared light at a specific peak frequency ,32.−1 (OH stretching), 2870 cm−1  and 1722 cm−1 (carboxylic acid C=O stretching), which was probably due to acidic fractions present in linseed mucilage [−1 indicates carbonyl C=O stretching [−1 indicates –NH stretching of the AAM unit. Carbonyl moiety of the AAM unit is found at the peak at 1615 cm−1 [−1 (C-H stretching) and a quite low-intensity peak at 3518 cm−1 (C-O stretching) is at 1042. The N-H intense peak at 1605 cm−1, the presence of NH and C=O vibrations confirm the presence of grafting of AAM with linseed mucilage [In regards to linseed mucilage FTIR, in mucilage . In the retching . In the 615 cm−1 , while tmucilage .−1 represents NH bending, the absorption band at 1663 cm−1 for  bending and peak at 1391 cm−1 for CH2 and 1597 cm−1 for the Pyridinium ring [The FT-IR spectrum of nicorandil is shown in ium ring .−1 (aromatic C=C bending) and 1389 cm−1 (aromatic C-H bending) confirmed that the drug did not show any chemical interaction with the hydrogel preparation. By recording the FTIR, the spectra of the drug confirmed that the model drug was evenly distributed within hydrogels. The FTIR spectra of the crosslinker, the monomer, and the polymer were also recorded to determine the uniform distribution of all these ingredients in linseed-based hydrogel [In nicorandil, the loaded linseed-co-AAM spectra absorption peaks at 1661 cmFor evaluating morphological features of prepared hydrogels, scanning electron microscopy was performed on grafted linseed-co-AAM hydrogels. Clear surface morphology was detected with folds and pinholes. As the concentration of AAM increased, folds and holes were denser ,36. In fThe in-vitro drug release of linseed-co-AAM hydrogels with a varying monomer, linseed mucilage and crosslinker ratio is given in Formulations with varying contents of linseed mucilage  show higher release of nicorandil, i.e., 75.1, 80.4 and 85.1, respectively, as shown in As for F6 and F7, the percentage of nicorandil release after 12 h was 69.1% and 63.5%, respectively. Formulations with varying crosslinker concentrations showed a decrease in drug release with increasing crosslinker content because swelling and the interaction of the drug molecule with the physiological medium decreased, so drug release also decreased.By using DD Solver software assessment of the drug release kinetics of numerous kinetic models, zero order, first order, Higuchi, Hixson–Crowell, and Korsmeyer–Peppas models were applied. The effects of different concentrations of crosslinker, monomer, and linseed mucilage concentrations on the kinetics of drug release from all linseed-co-AAM hydrogel formulations can be observed by the correlation coefficient (R2) values and the release rates mentioned in From the values of R2 of the line graph, it can be seen that the Korsmeyer–Peppas model best fits all formulations. This model is applied to determine the release mechanism of drugs, i.e., Fickian diffusion or non-Fickian diffusion. If the value of (n) for cylindrical hydrogel discs equals 0.45, it corresponds to the Fickian diffusion, whereas, when the (n) value is between 0.45 and 1.0, it represents the non-Fickian diffusion. Values of (n) for F6, F7, F1 & F2 preparations in this study were less than 1 and greater than 0.45, showing non-Fickian diffusion, while F3, F4 & F5 shows Fickian diffusion, as the (n) value is equal to 0.45 .Accordingly, formulation F5 exhibited comparatively higher drug release due to greater swelling ratios. A decrease in the crosslinking density provides a porous network that aids the influx of water, followed by swelling. Drug release characteristics were found to be directly proportional to the swelling capacity and inversely proportional to crosslinker and monomer concentration.Linum ussitatissimum mucilage were successfully designed, with the ability to demonstrate a sustaining mechanism for drug delivery. Through a thorough analysis, it was observed that an increase in the monomer and crosslinker concentration resulted in a decrease in the swelling, as well as the rate and extent of drug release. However, increasing the mucilage concentration had the opposite impact, i.e., swelling and an increase in the rate and extent of drug release. Moreover, linseed-Co-AAM hydrogel depicts pH-independent swelling and drug release behavior. With particular emphasis on the sustained release delivery systems, formulations F2 and F5 were considered to be superior to other formulations; a deeper analysis involving in-vivo investigation is highly recommended.Within the current study, hydrogels based on Linum usitatissimum were purchased from the local food market of Sargodha, Pakistan. Acrylamide, potassium persulfate, sodium hydroxide, and potassium dihydrogen phosphate were from Sigma Aldrich, Germany. N,N-Methylene bis-acrylamide was obtained from Fluka, Switzerland. Hydrochloric acid, absolute ethanol, and n-hexane were gained by Riedel-de Haen, Germany. Nicorandil was obtained as a gift sample from GETZ Pharma Pakistan (Pvt) Ltd., Karachi, Pakistan.Seeds of Linum usitatissimum seeds were soaked in 600 mL of purified water with mild stirring at 25 °C. After a period of 24 h, the soaked seeds were incubated in a dry heat oven at 80 °C for 30 min. Mucilage was extracted via vacuum filtration and was washed with n-hexane using a cotton cloth, whereby n-hexane passed out very easily along with impurities and the filtrate was obtained in the form of mucilage. The purified mucilage was transferred to petri dishes and dried in an oven at 60 °C; the obtained scales were ground until a fine powder was obtained [Following the careful screening process, 200 g of obtained .As shown in Briefly, linseed mucilage powder was dissolved in distilled water with continuous stirring at 70 °C. Once dissolved, potassium persulfate solution (in water) was added to it and stirring was continued for another 10 min. Afterwards, crosslinker  solution and monomer were added to polymer-initiator solution at room temperature; the final weight was adjusted using distilled water. The above mixture was incubated in a water bath at 50 °C for an hour, and then temperature was gradually increased up to 80 °C until transparent hydrogels were formed. Cylindrical hydrogels were cut into 0.5 cm diameter discs, washed in an ethanol-water solution to remove the unreacted contents and dried in an oven at 50 °C for 24 h. Dried discs were stored in airtight containers for further characterization of hydrogels .d = weight of hydrogel in dry form [Swelling studies were performed using the primly weighed dry hydrogel discs. The dry hydrogel discs were immersed in medium (having pH 1.2 and 4.5) and were allowed to swell until swelling equilibrium. At predetermined time intervals, swollen hydrogels were taken out of the medium; weight was noted after removing the excess water with filter paper that, ultimately, was placed back in same media. The following equation was used to calculate swelling behaviour:dry form ,36,44.d is the weight of dried gel discs [The swelling was continued until each gel achieved a constant weight. Percentage of equilibrium swelling (%ES) or equilibrium water content (EWC) was determined by the following equation:el discs .L = weight of dried drug loaded disc, WU = weight of dried unloaded disc [Nicorandil was used as a model drug for the development of sustained-release formulations for the treatment of hypertension . Drug loded disc ,47.2) to make the disc; spectra were recorded at a wavelength of 4000–500 cm−1.The Fourier transform infrared (FTIR) spectra were recorded on an FTIR (prestige-21 Shimadzu) spectrometer for the pure model drug, unloaded hydrogel and drug-loaded hydrogel in order to identify formation of any new bond . For thiSEM is a widely applied technique for the evaluation of shape and surface morphology of hydrogels. Dried hydrogel discs were cut to specific sizes and were fixed on an aluminium stub. Hydrogels were freeze-dried and then coated with gold in a high vacuum evaporator. The coated samples were scanned and examined under an electron microscope to expose surface morphology ,50.In-vitro drug release studies were performed using USP-dissolution apparatus II at 37 ± 0.5 °C to evaluate the release behavior of all hydrogel formulations. Every disc was placed in dissolution medium, maintained at a temperature of 37 °C and stirred at a rate of 50 rpm to maintain a uniform drug concentration in the medium. An aliquot of 5 mL was withdrawn at specified time points, i.e., 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 12 h and absorbance of nicorandil was measured at a wavelength of 262 nm. In order to keep the dissolution medium volume constant, samples were replaced with an equal volume of fresh buffer maintained at 37 ± 0.5 °C. Standard calibration curves of nicorandil were obtained and absorbance was taken at 262 nm. Release kinetics of nicorandil from hydrogels was evaluated by dissolution data modeling by using DD Solver software .t = release of drug at time t, Fload = amount of drug loaded in disc.Percentage of drug release in hydrogels was determined by using the following equation:To evaluate the release pattern of nicorandil, and zero-order, first order, Higuchi, Hixson–Crowell, Korsmeyer–Peppas kinetic models were applied.


Bladder cancer (BC) is the most common tumor of the urinary system. Non-muscle-invasive bladder cancer (NMIBC) has a high recurrence rate after surgery, and patients with muscle-invasive bladder cancer (MIBC) have poor quality of life after radical surgery. Understanding the molecular mechanism of bladder cancer is helpful for providing a more appropriate treatment approach. Annexins are calcium-binding proteins and play an important role in different tumor cells. However, the role of the annexin family in bladder cancer has not been studied in detail.ONCOMINE, UALCAN, TIMER2.0, Kaplan-Meier Plotter, cBioPortal, and WebGestalt were utilized in this study.ANXA2, ANXA3, ANXA4, ANXA8, and ANXA9 were significantly increased in bladder tumor tissues, while ANXA6, ANXA7, and ANXA11 were significantly decreased. ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA7, and ANXA9 had prognostic value in bladder cancer. In addition, specific annexins were specifically expressed in different subtypes of MIBC and were related to the histological morphology of bladder tumors. ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA7, and ANXA8 were highly expressed in basal-subtype MIBC, while ANXA4, ANXA9, ANXA10, and ANXA11 were mainly expressed in luminal-subtype MIBC. Finally, we analyzed the possible mechanisms of ANXAs in different subtypes of bladder cancer through GO and KEGG analyses and the correlation between ANXAs and immune infiltration in the tumor microenvironment.Taken together, our results indicate that annexins might play important roles in BC and have the potential to be used as markers for subtype classification. Bladder cancer (BC) is the most common genetic disease of the urinary system, and studies have shown that the progression of disease is related to the accumulation of multiple genetic and epigenetic mutations . The genAnnexins are a superfamily of secreted proteins that exist in the cytoplasm and attach to the phospholipid membranes of cells and are currently classified into five classes (groups A–E). Group A is present in vertebrates, and 13 group A members (annexin A1–A13) are present in human organs . ANXA1 w1 and Oncomine2 were used to analyze the expression of ANXA mRNA. The mRNA expression in tumor tissues and normal tissues was analyzed by UALCAN, and the mRNA expression difference between MIBC and NMIBC was verified with three datasets from Oncomine.UALCAN3. In this study, we compared the expression levels of ANXA family proteins in normal tissues and in tissues from bladder cancers of different grades. Protein expression were quantified using a visual grading system based on the extent of staining  and the intensity of staining . The combination of extent (E) and intensity (I) of staining was obtained by the product of EI  as well as basal subtype-related genes .Timer 2.06. According to different groups, online GO and KEGG analyses were performed using WebGestalt7.We analyzed 25 positively co-expressed genes of each ANXA protein using cBioPortalTimer 2.0 also provides a platform to perform a correlation analysis between the expression of a single gene and the infiltration of immune cells. Therefore, we analyzed the relationship between the expression of each ANXA protein and the infiltration of six major immune cells in bladder cancer.t-test, one-way ANOVA. Statistical significance was set at P < 0.05.Data are shown as means ± standard deviation (SD). All statistical analyses were carried out using SPSS . Differences between groups were analyzed using the two-tailed Student’s We used the TCGA database to analyze the mRNA expression of ANXA1–11 and ANXA13 in bladder cancer tissues and normal tissues. As shown in P = 0.049; P = 0.0014), and the expression level of ANXA1 and ANXA3 in high grade BC were significantly increased than those in low grade bladder cancer .Immunohistochemistry can reflect the expression of proteins in tissues. We used HPA to investigate differences in the expression of ANXA protein members between normal tissues and low-grade and high-grade bladder cancers. The staining degree could be divided into low medium and high. The results of staining degree were provided by HPA, which was based on the results of staining intensity and quantity. Representative sections were shown in P = 7.3e-06), ANXA2 , ANXA3 , ANXA5 , ANXA6 , and ANXA7  had shorter OS. Conversely, patients with higher expression of ANXA9  had a longer overall survival. In addition, there was no significant correlation between the mRNA expression level of ANXA4 , ANXA8 , ANXA10 , ANXA11 , or ANXA4  and overall survival.The Kaplan-Meier Plotter platform used TCGA data to analyze the prognosis of different genes. As shown in Next, we again used the UALCAN database to explore the relationship between ANXA protein expression and bladder cancer tumor stage. As shown in the In a TCGA study from 2017, MIBC bladder cancer was divided into five subtypes: luminal-papillary, luminal, luminal-infiltrated, basal-squamous, and neuronal . In the To further analyze the expression of ANXA family proteins in different MIBC molecular subtypes, we studied the correlation between the expression of ANXA proteins and specific genes of different subtypes. According to TCGA classification and A Kamoun et al.’s study, urothelial differentiation markers such as FOXA1, GATA3, and KRT20 are highly expressed in luminal-subtype bladder cancer, while FOXA1 and GATA3 are not expressed but KRT5, KRT6, and KRT14 are expressed in basal-subtype bladder cancer. As shown in the Based on the above results, ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA8, and ANXA13 were identified as potential genes related to basal-subtype bladder cancer (ANXA-B), and ANXA4, ANXA9, ANXA10, and ANXA11 were defined as luminal-subtype bladder cancer-related genes (ANXA-L). On this basis, we further explored the potential mechanisms of ANXA-B and ANXA-L in bladder cancer. The 25 most significantly positively correlated genes of each ANXA member were analyzed using the cBioPortal platform. WebGestalt was then used to perform GO and KEGG analyses of 200 co-expressed genes of ANXA-B and 100 co-expressed genes of ANXA-L to explore upregulated pathways of the ANXA proteins in the two subtypes of bladder cancer . In the Finally, we analyzed the relationship between ANXA family member expression and the infiltration of immune cells in bladder cancer . The TIMSince bladder cancer is the most common disease of the urinary system, understanding the biological mechanism of bladder cancer is crucial for urological research. In this article, we first studied the expression of different ANXA family members in bladder tumor and normal tissues, and the results showed that the mRNA of ANXA2, ANXA3, ANXA4, ANXA8, and ANXA9 were significantly increased in tumors, while the mRNA of ANXA6, ANXA7, and ANXA11 were significantly decreased in tumor tissues. Furthermore, we analyzed the expression of annexin proteins in NMIBC and MIBC. As shown in Non-muscle-invasive bladder cancer generally shows papillary characteristics, while the histological phenotypes of MIBC are more diverse. The University of North Carolina (UNC), MD Anderson Cancer Center, and TCGA have all conducted their own studies on MIBC subtype identification. The UNC team proposed that bladder cancer is similar to breast cancer and used unsupervised consensus clustering to divide samples into “basal” and “luminal” categories. Similarly, in the studies of MD Anderson Cancer Center and TCGA in 2014, MIBC was mainly divided into these two categories . Based oIn bladder cancer, NMIBC includes the Ta, T1, and CIS stages, while T2–T4 bladder cancer is characterized by invasion into the muscle layer, also known as MIBC. In this study, we also analyzed the correlation between ANXA expression and the clinical stage of bladder cancer. The results showed that the expression levels of ANXA2, ANXA5, and ANXA6 were significantly positively correlated with clinical stage. In the analysis of overall survival, patients with high expression of ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, and ANXA7 had poor prognosis, while those with high expression of ANXA9 had longer overall survival. In view of the above results, ANXA2, ANXA5, and ANXA6 have utility for the categorization of pathological stage and prediction of overall survival in bladder cancer and can reflect the progression and prognosis of bladder cancer. In Yuan’s study on gastric cancer, the survival rate of patients with positive expression of annexin A7 was lower than that of patients with negative expression . In addiIn further analysis of the function of ANXA genes, we found that the functions of ANXA-B  co-expressed genes were mainly related to cell adhesion, damage repair, cytoskeleton composition and extracellular matrix interactions. On the other hand, the enriched functions of ANXA-L  were mainly related to the regulation of immature T cells, cellular morphogenesis, binding transduction, plasma membrane function and tight junctions. Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression, and targeting annexin A2 could effectively inhibit tumor cell adhesion, migration and in vivo grafting . In addiIn this study, we analyzed the expression characteristics and prognostic utility of ANXA family proteins in bladder cancer for the first time. In addition, we also determined the expression characteristics of ANXA family members in different subtypes of MIBC. The results suggest that differences in the expression of ANXA family members are closely related to the histological morphology of bladder cancer. It should be noted that ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA7, ANXA8, and ANXA13 are mainly expressed in basal-subtype MIBC, while ANXA4, ANXA9, ANXA10, and ANXA11 are mainly expressed in luminal-subtype MIBC. These findings provide more markers for subtype identification and are conducive to further mechanistic research. It’s important to point out that our data only indicate the correlation between ANXA family proteins and BC. Further experiments are needed to verify the protein expression level in tissues and its functional role in BC. In summary, ANXA family proteins can serve as potential markers for the identification and prognostication of bladder cancer.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/WW and GJ developed the idea, data analysis, and writing original draft. LC contributed to data curation and formal analysis. HL and SX reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inositol hexaphosphate , are inositol metabolites containing high-energy phosphoanhydride bonds, involved in many key biological processes, including growth, vesicular remodeling, and energy homeostasis. The current understanding of how PP-IPs control mammalian cellular signaling networks in physiology and disease is summarized by Lee et al. [6, the most abundant IP. The plant PP-IP synthesis pathway is similar, but distinct from that of the human, reflecting differences in how molecules such as IP3 and IP6 function in plants vs. animals. In this review, they compare IP6 synthesis pathways, synthesis and regulation of the PP-IPs, and function of a specific protein domain called the Syg1, Pho1, Xpr1 (SPX) domain in binding PP-IPs and regulating inorganic phosphate sensing, providing novel insights into the biosynthetic pathway and bioactivity of these key signaling molecules in plant and human systems [6, are important stores of P in plants.Reviewing specific signaling functions for IPs in different cell types, with new information regarding their cellular roles and cellular production, Maffucci and Falasca highlight the interaction of IPs with several proteins, protein complexes, and modulation of enzymatic activity, essential in regulation of several cellular processes , and dise et al. . IPs maye et al. . Comparie et al. . While e systems . Of cour6 and/or other IPs has never been a straightforward analytical task [6, inositol(s), and IPs. However, 150 years have elapsed since the discovery of IP6 to the first descriptions of its beneficial effects. This long delay may be due to the difficulty in determining phytate and other IPs in biological media, as discussed by Grases and Costa-Bauza [Determination of IPs in biological fluids is difficult. From the early precipitation-based techniques, introduced more than a century ago, to the latest development of enzymatic bio- and nano-sensor applications, the analysis of IPcal task . Due to cal task . During 6 is recognized as a strong antioxidant due to its specific configuration and properties of inhibiting hydroxyl radical formation, very few in vivo studies exist. Aguree et al. [6 in reducing the oxidative stress in a very specific animal model of human hereditary hemochromatosis, showing that IP6 protects against oxidative stress induced by genetic iron overload and high fat diets in β2-microglobulin knockout mice [6. It has been shown that IP6 and IPs have high capacity to inhibit calcium salt crystallization and that oral or topical administration of IP6 significantly decreases the development of pathological calcifications [6 and Ins have recently received much attention for their roles in cancer prevention and treatment. A striking, consistent, and reproducible anticancer activity has been demonstrated in different experimental models and systems, targeting several key molecular targets, such as PI3K/Akt, mTOR, MAPK, PKC, and NF-κB. A few clinical studies have shown that IP6 alone or in combination with Ins is able to enhance the anticancer effect of conventional therapy, control cancer metastases, and improve quality of life. The historical perspectives, from the first experiments, concepts, and hypotheses to the first clinical observation in colon cancer patients is presented here [6 exerted its biological functions by downregulating the miR-155 expression in human colon cancer cells [6 and Ins was observed not only in cancer [6K pathway is a potential target in obesity and other metabolic diseases [6K isoforms have been proposed as possible targets for cancer therapy [Multiple beneficial effects on human health are related to IPs, and only few are highlighted here. Although IPe et al.  examine out mice . Blockinted here , and thated here . It is ited here . To better cells . Howevern cancer  but alson cancer . So, inon cancer . Targetin cancer . The IP6diseases . Further therapy . That is6, and IPs.In conclusion, there has been fascination for plants stretching back to millennia, and humans have remained dependent on plants for medicines. The plant kingdom is rich with organic compounds for traditional medicine, as well as for the development of novel agents to improve human health, as was shown for Ins, IP


Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo ( Lignin biosynthesis consists of three steps: the biosynthesis of lignin precursors (lignin monomers) in the cell, transport of the precursors from the cytosol to the cell wall, and polymerization in the cell wall. The biosynthesis and polymerization of lignin precursors have been intensively studied, whereas knowledge on the transport of lignin precursors is limited [Lignin is a major cell wall component of vascular plants and the second most abundant natural polymer on Earth. Lignin contributes to efficient water transport in xylem tissues, plant posture maintenance, and microorganism resistance in vascular plants. Lignin predominantly comprises  limited ,2,3.Arabidopsis thaliana [A. thaliana, AtABCG29, was reported to be a transporter of p-coumaryl alcohol, the monolignol of the H-unit lignin [A. thaliana have attempted to identify the ABC transporters of lignin precursors [To study the transport of lignin precursors, Miao and Liu conducted transport experiments using microsomal membrane fractions derived from rosette leaves of thaliana . Microsot lignin , althougecursors ,7,8; howWhile the transporters may be involved in the translocation of lignin precursors from the cytosol to the cell wall, it is also possible that the lignin precursors, such as monolignols, can move across membranes via passive diffusion because they are small, relatively hydrophobic molecules. A model experiment with a lipid bilayer disk showed that the phenolic compounds were partitioned into the lipid bilayer disks without transporter proteins . Additiop-glucocoumaryl alcohol, coniferin, and syringin, have been considered the storage forms of lignin precursors and the forms to be transported toward outside cells [Pinus thunbergii [Chamaecyparis obtusa) [Ginkgo biloba [1  and S2  layers; however, coniferin disappears in the cells during the formation of the S3 layer  and during the lignification stage [Monolignol glucosides, i.e., de cells . In partunbergii , Japanes obtusa) , and Gino biloba . Raman son stage ,16. Furton stage ,18,19. T+-ATPase (V-ATPase) and the H+ gradient are involved in coniferin transport, which are common in both poplar (Populus sieboldii × Populus grandidentata) and Japanese cypress [p-glucocoumaryl alcohol [A. thaliana, but little is known about the transport of lignin precursors in monocots.Biochemical experiments using microsomal membrane vesicles prepared from the actively lignifying tissues of woody plants suggest that vacuolar type H cypress . A simil alcohol . Recentl alcohol . These sIn the context of biomass production for a sustainable society, bamboo has been intensively studied, owing to its fast growth in recent years. The elucidation of lignin biosynthesis mechanisms in bamboo allows for the regulation of lignin quality and quantity, leading to efficient biomass utilization. While lignin biosynthesis in monocots has been actively studied ,24,25,26p-glucocoumaryl alcohol were identified, and it was found that their transport modes were similar to those of poplar and Japanese cypress, suggesting that the active transport of monolignol glucosides into intracellular compartments is conserved among lignifying tissues and commonly contributes to lignification in vascular plants.In the present study, we examined the transport activities of lignin monomers necessary for the lignifying tissues of bamboo. The transport activities of coniferin and The growth rate of bamboo shoots is rapid, so predicting the growth and lignification stages of each culm or each internode is difficult, especially at the early growth stages. Therefore, the elongation growth and lignification processes of bamboo shoots were elucidated to identify the lignification stage of each internode and to obtain the lignifying tissues quickly at the harvesting site.Phyllostachys pubescens, which began growing in March in Nobeoka, Japan. The elongation of bamboo shoots was observed until mid-May, when they reached lengths of approximately 16 m, which was consistent with previous reports [P. pubescens [The bamboo shoots we used in this study were  reports . At the  reports ,28. The ubescens . The lenP. pubescens [Sinobambusa tootsik [The lignin distribution in the growing bamboo culms was examined by Wiesner reaction and the lignin content in each internode was determined using the rapid thioglycolic acid method . Lignifiubescens  and Sino tootsik . Thus, tp-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, along with some of their glucosides, p-glucocoumaryl alcohol, coniferin, and syringin  of late April samples at different lignification stages  and by H+ gradient erasers (NH4Cl and gramicidin D), but it was not inhibited by an ABC transporter inhibitor (vanadate)  D. Thus, reported ,21. p-Glp-glucocoumaryl alcohol was shown in differentiating xylems of not only a gymnosperm [p-glucocoumaryl alcohol has been detected in developing bamboo, and its content peaks during the early stage of lignification [p-glucocoumaryl alcohol is used for lignification in bamboo; consequently, the p-glucocoumaryl alcohol transport identified in the present study may be involved in the lignification of bamboo.The incorporation of radiolabeled mnosperm , but alsmnosperm . Furtherfication . These rβ-glucosidase responsible for the delivery of its aglycone, coniferyl alcohol, localize in the cell walls of Pinus contorta var. latifolia [β-glucosidase of rice is also indicated to localize in the cell wall [Numerous glucosides of phenolic compounds have been investigated and recognized as vacuolar storage substances in many plant species ,35. Howeatifolia  and poplatifolia . Coniferell wall . The traell wall ,33.β-glucosidase in an A. thaliana mutant resulted in coniferin accumulation, but the lignification did not change [A. thaliana resulted in ectopic lignification [p-glucocoumaryl alcohol are highly conserved in lignifying tissues, implying the importance of the transport of monolignol glucosides in lignification.At present, the roles of monolignol glucosides in lignification are still unclear in dicots, despite several reverse genetic studies. The downregulation of coniferin t change . The ovefication . Howeverfication . Althougfication ,20,21, c+-ATPase activities from August samples  and Wako Pure Chemicals . Coniferin and syringin were provided by Dr. Noritsugu Terashima of Nagoya University. eviously ,41.P. pubescens were collected from Nobeoka (32°58′58′′ N 131°58′71′′ E), Miyazaki Prefecture, Japan. Three independent culms (a–c) of similar length were collected on each sampling day  phloroglucinol in 95% ethanol for 1 min, followed by the addition of 6 M HCl. The incubated sections were observed under a light microscope .Each bamboo sample was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After fixation, the blocks were washed with water and transverse sections (40-μm thickness) were obtained. The sections were incubated in 2% , 800 µL of the supernatant was discarded and 1 mL of distilled water was added as a wash. After centrifugation, the supernatant was discarded, 1 mL of 1 M NaOH was added, and the mixture was vertically shaken (120 rpm) for 1 h. After centrifugation , 1 mL of the supernatant was transferred to a new 1.5-mL tube. Approximately 220 µL of concentrated HCl was added and mixed by inversion. After centrifugation , the supernatant was discarded, and 1 mL of 1 M NaOH was added to dissolve the pellet. The lignin concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer  with a calibration curve prepared with the milled wood lignin of bamboo [The lignin content was determined using the rapid thioglycolic acid method . Brieflyf bamboo . The samv/v) glycerin, and 0.5% (w/v) polyvinylpolypyrrolidone, and were supplemented with protease inhibitors . The samples were then homogenized with a homogenizer  at 10,000 rpm for 70 s. The mixture was passed through doubled Miracloth (Merck) to obtain a filtrate. After centrifugation  to remove tissue debris, the resulting supernatant was ultracentrifuged  to collect microsomal fractions. The supernatant was discarded, and the pellet was suspended in resuspension buffer, which included 10 mM HEPES–KOH (pH 6.8), 1 mM EDTA, and 10% glycerin, and subjected to ultracentrifugation again. After suspending and dispensing using a potter-type Teflon homogenizer, the microsomal membrane fraction was rapidly frozen using liquid nitrogen and stored at −80 °C.The following operations were all performed at 4 °C or on ice. Small bamboo blocks without the epidermis were added to a homogenizing KOH buffer (pH 8.0), which contained 100 mM HEPES, 5 mM EDTA, 10% (4 plus 50 mM HEPES-KOH buffer (pH 7.5) supplemented with 100 µM of various lignin precursors , the supernatant was passed through a filter  to remove the precipitate and then subjected to an HPLC analysis as previously described [The incubation mixture was prepared by adding a membrane fraction to a solution of 5 mM ATP/MgSOecursors  as the sescribed .


Extracellular vesicles (EVs) play an important role in the communication of cancer cells with their local microenvironment and distant organ systems, in order to promote a supportive tumor microenvironment, as well as to prepare premetastatic niches. In this study, we aimed to analyze if the EVs secreted by urological cancer cells are taken up by specific organ systems, depending on their origin. After the intravenous injection of fluorescence-labeled EVs from benign and malignant prostate, kidney, and bladder cells in immunodeficient mice, their organs were harvested and analyzed for the presence of fluorescent EVs. We could show that (i) EVs are taken up not entirely organ-specifically but in different amounts, depending on their origin; (ii) EVs from malignant cells are taken up more efficiently than EVs from benign cells; and (iii) EVs are taken up very fast. These observations hint to an organotropism in EV uptake, which needs to be further investigated.Extracellular vesicles (EVs) secreted by cancer cells have been shown to take a pivotal part in the process of local and systemic tumor progression by promoting the formation of a supportive local tumor microenvironment and preparing premetastatic niches in distant organ systems. In this study, we analyzed the organ-specific uptake of EVs secreted by urological cancer cells using an innovative in-vivo approach. EVs from benign and malignant prostate, kidney, and bladder cells were isolated using ultracentrifugation, fluorescence-labeled and injected intravenously in immunodeficient mice. After 12 or 24 h, the animals were sacrificed, their organs were harvested and analyzed for the presence of EVs by high-resolution fluorescence microscopy. Across all entities, EVs were taken up fast (12 h > 24 h), and EVs from malignant cells were taken up more efficiently than EVs from benign cells. Though not entirely organ-specific, EVs were incorporated in different amounts, depending on the entity . EV uptake in other organs than lung, liver, brain, and spleen was not observed. Our results suggest a role of EVs in the formation of premetastatic niches and an organotropism in EV uptake, which have to be examined in more detail in further studies. The observation that malignant tumors preferentially metastasize into specific organ systems is called organotropism—a phenomenon known for a very long time in cancer biology but still incompletely understood at the molecular level. For example, prostate cancer affects the bone and lymph nodes in more or less all cases of metastatic disease, renal cell carcinoma most often metastasizes into the bone and brain, and urothelial carcinoma of the urinary bladder tends to spread to lymph nodes, bone, and lungs ,2,3. OneIn the last years, several elegant studies have been performed, which identified extracellular vesicles (EVs) secreted by primary tumor cells as further important players in the process of organotropic metastasis ,10,11. TEVs are nanometer-sized particles, delimited by a lipid bilayer that are released into the extracellular space by virtually all human cells and regulate specific biological functions after binding to and being incorporated by recipient cells . They plIn this study, we aimed to gather first evidence for the contribution of tumor-secreted extracellular vesicles to organotropic metastasis in urological malignancies by investigating the organ-specific uptake of systemically injected EVs secreted by benign and malignant human prostate, bladder, and kidney cells in immunodeficient mice. Furthermore, we aimed to identify the EV-uptaking cell types in distinct organs.Cell culture: The following cell lines were used in the experiments: HCV29 , T24 , BPH1 (benign prostate cell line), VCaP (prostate cancer cell line), Hek-293 , and 786-O . All cell lines were regularly tested for mycoplasma infection and their identity was verified by STR profiling. All cells were cultured at 37 °C in a humidified environment with 5% CO2. HCV29, T24, and Hek-293 cells were cultivated in DMEM medium , supplemented with 10% fetal bovine serum . VCaP cells were cultivated in phenol red free DMEM medium , supplemented with 10% FBS. BPH1 cells were cultivated in RPMI medium , supplemented with 20% FBS, 20 ng/mL testosterone , 5 μg/mL transferrin , 5 ng/ml sodium selenite , and 5 μg/mL insulin . Additionally, 786-O cells were cultivated in a 1:1 mixture of DMEM and RPMI medium, supplemented with 10% FBS.EV isolation: EVs were isolated from the conditioned media of the above-mentioned cell clines via ultracentrifugation. To generate conditioned medium, the cells were cultivated in 300 cm2 flasks until a confluence of 40 to 50%. Then, the medium was removed, the cells were washed three times, and medium was supplemented with EV-depleted FBS, instead of normal FBS. EV-depleted FBS was made by ultracentrifugation of normal FBS ; 200,000× g, 4 °C, 18 h), with subsequent sterile filtration through a 0.22 μm filter . After 72 h of cultivation in this EV-depleted medium, the conditioned medium was removed and underwent differential centrifugation to remove cellular fragments and larger EV subpopulations, such as apoptotic bodies or microvesicles. To do so, the conditioned medium was centrifuged at 2000× g for 20 min at 4 °C. After that, the supernatant was centrifuged at 15,000× g for 30 min at 4 °C. The supernatant was then removed and used for ultracentrifugation. Ultracentrifugation was performed using a Beckman Coulter L-80 ultracentrifuge with a type 70 Ti rotor . After ultracentrifugation, the EV-containing pellet was resuspended in phosphate-buffered saline or lysis buffer, depending on the experiments EVs were used for.EV characterization: EV characterization was performed by Western Blot analysis, nanoparticle tracking analysis (NTA) and transmission electron microscopy, as previously described [escribed . WesternFluorescence labeling of EVs: EVs isolated by ultracentrifugation were labeled with the lipid-intercalating fluorescent dye PKH26 (red) using the PKH26 red cell linker kit , according to the manufacturer’s instructions. Briefly, EVs were resuspended in 100 μL PBS after ultracentrifugation, and 500 μL Diluent C was added. In another reaction tube, 500 μL Diluent C was mixed with 2 μL of PKH26 dye (1 mM). Then, both solutions were pipetted together, mixed, and incubated on ice for 5 min. During this incubation time, the solution was repeatedly, gently mixed. After the incubation time, the staining reaction was stopped by the addition of 1ml EV-depleted FBS. To remove excessive unbound dye, the fluorescence labeled EVs were again ultracentrifuged at 100,000× g for 90 min at 4 °C and resuspended in new PBS. These labeled EVs were stored at 4 °C and used for intravenous injection in mice within 24 h.Animals: For the animal experiments, male (for prostate cell lines) or female (for bladder and kidney cell lines), 6–8 weeks old Balbc/nude mice  were used. The mice were kept in isolated ventilated cages under specific pathogen-free conditions in a temperature- and humidity-controlled, 12 h dark/light environment at the animal care facility of the institute for clinical and experimental surgery at Saarland University. The animals had free access to tap water and standard pellet food. Their health status was monitored daily. All experiments were approved by the local governmental animal care committee (No. 28/2014 and 30/2015) and conducted in accordance with the German legislation on the protection of animals and the National Institutes of Health guide for the care and use of laboratory animals.EV injection: A total of 20 μg of fluorescence labeled EVs diluted in 200 μL PBS was injected per mouse and cell line via the retrobulbar route; 12 or 24 h after injection, the mice were sacrificed and various organs  were harvested for microscopic examination (n = 2 for each cell line and time point). These organs were immediately stored in liquid nitrogen until further processing. Two animals per time point (12 and 24 h) were injected with PBS only as negative control.Histology and fluorescence microscopy: Cryonconserved organs were cut into 4 μm thin slices using a Leica 3050 cryostat  and transferred onto SuperFrost Plus slides . Afterwards, the tissue slices were washed with PBS (3 × 5 min) and incubated for one hour at room temperature with Alexa488-labeled Phalloidin  to label the cytoskeleton/microtubules of the cells and get a better impression of the tissue architecture. Then, the slides were washed with PBS again (3 × 5 min) and mounted with DAPI-containing Vectashield® antifade mounting medium . Within 24 h, the stained tissue slides were analyzed for the presence and quantity of red-fluorescent EVs using a Carl Zeiss LSM780 laser scanning microscope . To quantify the number of EV-positive cells, cells with positive EV signals per high power field (HPF) were counted using the Cell-Profiler software, in five representative HPFs per slide. For each time point, cell line and organ, two slides were stained and examined by fluorescence microscopy.Immunostaining: To identify uptaking cell types, several immunofluorescence stainings were performed using the above-mentioned tissue slides. The slides were washed three times in PBS and unspecific protein binding sites were blocked by incubation with a blocking solution  and 0.3% Triton X-100 ) for one hour at room temperature. Thereafter, the slides were incubated with the following primary antibodies at 4 °C overnight: CD31 , EpCAM , F4/80-Alexa488 , Vimentin-Alexa488 , and Sodium-Potassium-ATPase-Alexa488 . In cases when the primary antibody was not directly fluorescence labeled (CD31 and EpCAM), this was followed by PBS washing and secondary antibody incubation for two hours at room temperature . After washing in PBS, the slides were mounted with DAPI-containing Vectashield® antifade mounting medium  and analyzed within 24 h using a Car Zeiss LSM780 laser scanning microscope .Software/Statistics: For the analysis of microscopic images, the Cell Profiler (Broad Institute), ImageJ (NIH) and Zen 2012 Lite  softwares were used. Quantitative data are presented as medians +/− standard deviations.The morphology and size of the isolated EVs was analyzed by transmission electron microscopy. In all cases, a considerably pure fraction of small EVs with a size range between 50 and 150 nm was isolated. Transmission electron microscopy showed the typical ring-like structures delimited by a lipid bilayer. Furthermore, Western Blot analyses of all cell lines and their EVs and Nanoparticle Tracking Analyses of all EVs were performed A,B. In aEV-uptake was detected in the brain, liver, lungs, and spleen to different amounts in all cases, depending on the time point after injection and the cell line of origin. Representative fluorescence microscopy images of uptaking organs with 786-O EVs are shown in While in the brain, liver, and spleen EVs showed as typical dot-like, distinct signals; they often appeared as lines along the alveolar surface in the lungs, especially 24 h after injection  row 3. Op < 0.01 for liver and lung after 12 and 24 h, p < 0.05 in the brain after 12 h) and the brain (after 24 h). For all organs and cell lines, there were more EV signals 12 h after injection, compared to 24 h after injection.Prostate EVs were detected in the lung, liver, brain, and spleen after intravenous injection . EVs from the tumor cell line were taken up more efficiently than EVs from the benign cell line.EVs from the renal cell carcinoma cell line 786-0 and from the benign kindey epithelial cell line Hek293 could be detected in the brain, lung, liver, and spleen after intravenous injection. The majority of EVs were seen in the brain and lung, followed by the spleen and the liver . As for p < 0.01 after 12 h and 24 h), and more EVs were detectable after 12 h than after 24 h. One exception is the uptake of EVs in the brain after 24 h, were there were significantly more signals for HCV29 EVs than for T24 EVs. In contrast to all other cell lines, HCV-29 EVs were found in the kidneys and adrenals at very low numbers  and HCV-29 , the strongest EVs signals appeared in the lung, followed by the liver, brain and spleen  or subcellular structures (Na-K-ATPase: cell membrane) and investigated the colocalization of their signals with those of fluorescence labeled EVs. In the lung, colocalization of EVs with epithelial cells and macrophages could be observed a,b. Of nIn this study, we investigated the organ-specific uptake of extracellular vesicles (EVs) from benign and malignant prostate, bladder, and kidney cells after fluorescence labeling and intravenous injection in immunodeficient mice. We observed that the EVs from all cell lines were taken up in the brain, liver, lungs, and spleen in different amounts. This reflects clinical metastatic patterns of prostate, bladder, and kidney cancer to some extent but not in all points. For example, liver and lung metastases are a common finding in bladder and kidney cancer patients; however, they are rarely observed in men affected with metastatic prostate cancer. The metastatic potential of the three used malignant cell lines is known to some degree from xenograft studies: orthotopic VCaP xenografts were shown to produce lymph node, lung, and bone metastases ,19. AddiAcross all cell lines and organs, EVs from malignant cell lines were taken up more efficiently than EVs from benign cell lines, and the number of EV positive cells in target organs was higher 12 h after intravenous injection than 24 h after intravenous injection. This corresponds well with data from the literature, which showed that cancer-associated EVs are taken up very efficiently by recipient cells, and their intracellular turnover is very fast ,28,29. HOf interest, EVs were not taken up by their organs of origin, i.e., VCaP EVs were not taken up in the prostate, 786-O EVs were not taken up in the kidneys, and T24 EVs were not taken up in the bladder. One might assume that EVs circulating in the bloodstream should have a higher affinity towards the tissue type from which they were secreted. Analogously to that—coming back to Paget´s seed and soil hypothesis—it could be hypothesized that circulating tumor cells are preferentially taken up by their tissue of origin, as this might provide the best soil for the seed to grow out. However, in fact, metastases in the same organs of the primary tumor are only very rarely observed. While urothelial carcinoma of the urinary bladder and prostate cancer are often multifocal, this can be regarded as a result of field cancerization with the development of independent tumors, rather than intra-organ metastases ,33. HencWhy were EVs taken up to different amounts in the examined organs, depending on the cell line of origin? Several factors might influence the specific uptake of EVs by recipient cells, which might play a more or less important role in the individual physiological or pathological condition looked at. One explanation is the different distribution of surface molecules on the membrane of extracellular vesicles, which are involved in receptor-mediated uptake. Here, for example, integrins could be shown to be involved ,12, as wRegarding the investigation of uptaking cell types, we could identify macrophages in the liver and macrophages, as well as epithelial cells in the lung, to be colocalized with fluorescence labeled EVs. However, only a small fraction of EVs were taken up by the examined cell types, as indicated by many distinct, red EV signals representing non-colocalized EVs, alongside the yellow signals representing colocalization. In studies with other tumor entities, the uptake of EVs by resident macrophages in the liver, termed Kupffer cells, was also observed ,11. FurtStrengths of our study include the parallel use of benign and malignant cell lines from three tumor entities, which has never been performed before (to our knowledge). Moreover, we analyzed different time points after EV injection (12 and 24 h) to get an impression of the time frame of EV uptake and turnover in distant organs/premetastatic niches. Additionally, we analyzed a high number of organs for potential EV uptake and used high-resolution laser scanning microscopy for the examination of harvested organs after intravenous EV injection.However, our study is not without limitations. We injected fluorescence labeled EVs intravenously via the retroorbital venous plexus. It cannot be excluded that the site of EV injection has a significant impact on the pattern of their uptake in different organs. For example, the observation of EV uptake in the brain in all cell lines in our study might also be due to the fact that this was the first organ EVs traversed after their injection. To analyze such a dependence of EV uptake on the site of injection, one would have to systemically compare different injection sites—for example, retroorbital, tail vein, and intracardiac—in the same experimental setting. As the bone is a frequent site of metastasis in bladder, kidney, and most of all prostate cancer, it would have been desirable to also analyze EV uptake in the bone. Unfortunately, this was technically not possible in our study, as we performed immediate, fresh-frozen sections of the harvested organs 12 and 24 h after intravenous EV injection. To include the bone in these experiments, one would have to first demineralize the bone for a longer time, which would probably disturb the results of fluorescence microscopy. Of note, it was already shown by several groups that primary tumor-secreted EVs might play an important role in the development of bone metastases ,46,47,48Regardless of the above-mentioned limitations, our study provides the first hints towards a role of cancer-associated EVs in organotropic metastasis in urological malignancies, which will be further elucidated future studies. In addition, a better understanding of the role of EVs in metastasis could pave the way for novel EV-associated therapeutic strategies ,51,52.In our mouse model, cancer-associated EVs were taken up not entirely organ-specifically, but in different amounts, depending on the cell line of origin. EVs from tumor cells were taken up more efficiently than EVs from benign cells and there was a clear time dependency, with more EV signals detected 12 than 24 h after injection. These results suggest that EVs are at least one of many factors involved in the process of organotropic metastasis. In future studies, the functional role of cancer-associated EVs and the underlying molecular mechanisms will be unraveled in more detail, in order to gain a better understanding of the processes of organotropic metastasis and discover new therapeutic approaches.


In 2019, a novel coronavirus, SARS-CoV-2, was first reported in Wuhan, China. The virus causes the disease commonly known as COVID-19, and, since its emergence, it has infected over 252 million individuals globally and taken the lives of over 5 million in the same time span. Primary research on SARS-CoV-2 and COVID-19 focused on understanding the biomolecular composition of the virus. This research has led to the development of multiple vaccines with great efficacy and antiviral treatments for the disease. The development of biomedical interventions has been crucial to combating this pandemic; additionally, environmental confounding variables that could have exacerbated the pandemic need further assessment. In this research study, we conducted a spatial analysis of particulate matter (PM) concentration and its association with COVID-19 mortality in the United States. Results of this study demonstrate a significant positive correlation between PM concentration levels and COVID-19 mortality; however, this does not necessarily imply a causal relationship. These results are consistent with similar studies in Italy and China, where significant COVID-19 cases and corresponding deaths were exhibited. Furthermore, maps of the data demonstrate clustering of COVID-19 mortality which suggest further investigation into the social determinants of health impacting the pandemic. SARS-CoV-2 is a novel virus that was first reported in Wuhan, China in December of 2019. The virus causes the disease commonly known as COVID-19, which, since its emergence, has infected over 252 million individuals globally and taken the lives of more than 5 million in the same time span. As of 12 November 2021, the United States has experienced the highest number of cases, 46,868,744, with India just behind at 34,414,186, with cases still on the rise. Primary research on SARS-CoV-2 and COVID-19 was initially focused on understanding the biomolecular composition of the virus. This research has been used to develop multiple vaccines with great efficacy and antiviral treatments for the disease. The development of biomedical interventions has been crucial to combating this pandemic; now, we need to address the environmental confounding variables that could have exacerbated the pandemic. In this research study, we conducted a spatial analysis of particulate matter (PM) concentration and its association with COVID-19 mortality in the United States. Studies in Italy and China have explored this research question, in locations where high COVID-19 cases and corresponding deaths were exhibited. Few ecological studies concerning COVID-19 and PM had been conducted in the United States at the time of analysis (summer 2021), and this research will contribute to filling that void of knowledge and information.Air pollution exists everywhere globally, although to a different extent, and concentrations of pollutants have been on the rise as the combustion of fossil fuels and burning of natural gas has increased. Natural events such as wildfires and volcanic eruptions can also contribute to air pollution levels. Consequently, health conditions, such as respiratory disease (RD), cardiovascular disease (CVD), nonfatal heart attacks, aggravated asthma, decreased lung function, increased infant mortality, and COPD, associated with ambient air pollution, cause 4.2 million deaths a year globally . One of Particulate matter is a combination of solid particles and liquid droplets that exist suspended in the air. These particles enter the atmosphere from either primary or secondary sources. Primary sources of particulate matter are particulates emitted directly from the source, and include construction sites, smokestacks, wildfires, and agricultural operations. Secondary sources of particulate matter are sources that emit precursors for particulate matter that undergo chemical/mechanical reactions in the atmosphere. Secondary sources of particulate matter include pollutants from vehicle emissions and power plants that include sulfur oxide, nitrous oxide, volatile organic compounds (VOCs), and ammonia . RegulatBetween 1 December and 5 December 1930, in the Meuse River Valley, Belgium, individuals began reporting symptoms of larynx irritation, coughing, chest pain, and even foaming at the mouth. Within 24 h of the first reported symptoms, more than 60 individuals had died and many more were afflicted. Air pollution from numerous factories, in combination with a temperature inversion, trapped the particulate matter, creating a smog that killed many [The health disasters that occurred in Donora and Webster initiated a chain of events that led to the passage of the Clean Air Act in the US; however, incidents continued to occur across global space. In December of 1952, London experienced one of the worst smog events ever recorded. It is estimated that there were approximately 12,000 excess deaths during the 5-day event and the subsequent 3-month period that can be directly tied to the particulate matter pollution. Similar to the previously discussed events, a temperature inversion occurred which allowed for minimal horizontal or vertical air movement near the ground. The colder temperature observed during this period exacerbated the pollution because domestic heat sources and power plants were functioning at maximum capacity to keep the citizens warm. In addition, London had just abandoned their electric trams for diesel-powered buses in July of 1952, which released an abundance of diesel emissions . Traffic10) and daily cause-specific mortality data for the 90 largest cities . The analysis concluded that daily variation of PM10 is positively associated with daily variation of mortality [2.5) concentration was conducted (defined as having an aerodynamic diameter ≤ 2.5 μm). Using advanced spatial epidemiological technologies, the research group used satellites and hierarchical Bayesian modeling to estimate PM2.5 concentrations in regions where ground monitors did not collect data. This methodology was used to study the geographical region of New York City, NY metropolitan and surrounding areas. This data collection method allowed the researchers to pull PM2.5 concentration data from multiple sources and overlay heat maps to obtain the most accurate and comprehensive PM2.5 data for the region. The use of heat maps provides great data visualization, and similar techniques are used in our research study to demonstrate where the association between COVID-19 and PM2.5 is high and low, “hot” and “cold”. In the study, the pollution data was tracked daily in concurrence with hospital admittance data for asthma, myocardial infarction, and heart failure, and the study concluded that high PM2.5 exposure is associated with increased risk of asthma, myocardial infarction, and heart failure [Previous research on particulate matter has demonstrated positive associations between particulate matter and mortality, especially mortality due to RD and CVD. A study conducted in 2003 explored the geographical variation in the effects of particulate matter on mortality. Using Bayesian statistics, the researchers divided the US into seven geographical regions  and analyzed the concentrations of coarse particulate matter ≤ 10 μm . The analysis concluded that individuals that were single, illiterate, and worked outdoors were more at risk for particulate matter-related CVD and RD than other demographic groups. Once the PM2.5 concentration reached 105 μg/m³ for nine consecutive days, the excess risk of CVD death for single, illiterate, or outdoor working individuals increased by as much as 45% [2.5 pollution. The highest causes of mortality attributed to PM2.5 pollution were for ischemic heart disease and cerebrovascular disease (stroke). This study in Verona was unique because of its geographical location in the center of Italy’s Po Valley. The Po Valley is a highly industrial and agricultural region, and, in conjunction with the valley feature, is one of the most polluted regions in the world. Over the past decades, PM2.5 and PM10 concentrations there have decreased; however, concentration levels are still consistently above the European standard of 25 μg/m³, making particulate matter pollution an ongoing threat to human health in that region [Comparable conclusions were made about the association between human health risk and particulate matter in studies conducted in 2017 and 2018 in Beijing, China, and Verona, Italy, respectively. However, these studies looked specifically at long-term and continuous exposure to particulate matter. Both studies analyzed mortality specifically associated with CVD and RD. The Beijing study used a generalized additive model (GAM) to estimate excess risk for CVD and RD in relation to daily mean PM2.5 effect nor short-lag temperature effect on influenza. However, analysis did show that higher daily average PM2.5 concentrations during the wildfire season were positively associated with increased influenza in the following winter influenza season. Two different analyses of differing p-values (p = 0.04 and p = 0.008) demonstrated respective 16% and 22% increases in influenza rate per 1 μg/m³ increase in average daily summer PM2.5 [In 2020, a study took place in Montana, USA to research the delayed effect of wildfire season particulate matter pollution on the subsequent influenza season. The study analyzed both short-lag (1–4 weeks) and long-lag (prior wildfire season months) effects on the following winter influenza season in Montana, USA. Using spatial regression models and a quasi-Poisson model, the study discovered no short-lag PMer PM2.5 . This anIn addition to chronic and noninfectious diseases, particulate matter has been historically associated with infectious diseases, especially coronaviruses. Coronaviruses are a family of viruses that range from the common cold to severe acute respiratory syndrome (SARS). SARS, Middle Eastern respiratory syndrome (MERS), and COVID-19 are all in this family of coronaviruses. SARS first surfaced in 2002 and 2003 in Beijing, China. In comparison to COVID-19, SARS exhibited an extremely high global case fatality rate of up to 15%. Although more fatal, SARS could only spread from asymptomatic individuals, whereas individuals with COVID-19 can infect others several days prior to exhibiting any symptoms. This is one of the reasons why SARS-CoV-2, a very similar virus to SARS-CoV, has evolved from an epidemic to a pandemic. However, the biomolecular and symptomatic similarities of COVID-19 and SARS sparked similar ecological investigations into the relationship between ambient air quality and the virus.p-value = 0.0636) between PM10 air pollution and SARS fatality. This study gathered air pollution index (API) and SARS fatality data from five geographical regions in China . Relative risk (RR) and 95% confidence intervals were calculated for the data using Statistical Analysis Software (SAS). Final data analysis demonstrated APIs in Guangdong, Shanxi, Hebei, Beijing, and Tianjin during April and May to be 75, 95, 98, 99, and 104, and the corresponding case-fatality rates were 3.84%, 5.36%, 5.58%, 7.66%, and 8%, respectively. In addition, short-term and long-term PM exposures were analyzed, and both concluded an association between PM exposure and SARS fatality [10 and NO2 with a 4- or 5-day lag. However, the study did not find any significant association between daily SO2 concentration and SARS mortality with lags up to 6 days [A 2003 ecological study following the SARS epidemic found a strong correlation  of COVID-19 in Wuhan. The temporal variation curves for particulate matter concentrations and COVID-19 CFR exhibited strong similarities. Results of the study found a positive relationship  between PM10 and PM2.5 concentrations and the COVID-19 CFR [In the first year after COVID-19 emerged, ecological studies analyzing the relationship between particulate matter and COVID-19 mortality grew exponentially, especially in China and Italy. An Italian study looked at the spatial variation and patterns between PMrovinces . A similD-19 CFR . An addiD-19 CFR . 2.5 and COVID-19 mortality within the continental US and Hawaii . The research results found in Italy’s Po Valley and China correlating increased PM pollution to increased COVID-19 mortality prompted the study here. However, both of these prior studies focused on their respective geographic locations. Analysis of the PM relationship with COVID-19 mortality in the US was still relatively absent from literature at the conception of this study. Therefore, we were motivated to explore the spatiotemporal association between PM2.5 concentration in the United States, county-level data was collected. COVID-19 data was collected from USAFacts on 7 June 2021. USAFacts collects the raw COVID-19 county data exclusively from approximately 70 different government agencies, including varying commissions, bureaus, and departments. Raw COVID-19 case numbers and total deaths were provided for each individual county [To evaluate the association between COVID-19 mortality and PMl county . The mor2.5 data was collected from the CDC National Environmental Public Health Tracking Network for the year 2016, the most recent annual data [PMual data . PopulatAll data was organized in Excel, and repopulated into SPSS  for analysis. Descriptive statistics  were calculated in SPSS for all variables. Next, a Kolmogorov–Smirnov (K–S) test was conducted to check for normality in the dataset using Equation (2).To analyze the correlation between the variables we then utilized both a Spearman’s rho and Kendall’s tau analysis. The equations for each, respectively, are represented below.2.5 annual average concentration levels, similar to a heat map. The maps were stratified by county, and each map represents the spatial distribution of an individual variable. A Spearman’s rho correlation test is for nonparametric ordinal, interval, or ratio data. This test determines the strength and direction of a correlation assuming a monotonic relationship between the two variables. Similarly, a Kendall’s tau rank correlation test was conducted due to the nonparametric nature of the dataset. Kendall’s tau assumes that the variables are either continuous or ordinal, and that the variables have a monotonic relationship. Following the primary data analysis, the Excel 3D Maps tool was used to create a data visualization. A gradient color scale was used to depict the varying levels of COVID-19 mortality and PMAll variable correlations were statistically significant at the 0.01 alpha level (2-tailed), as depicted in 2.5 annual average (r = 0.309), Spearman rho correlation analysis between COVID-19 deaths and daily PM2.5 annual average (r = 0.440), and Spearman rho correlation analysis between COVID-19 cases and daily PM2.5 annual average (r = 0.365). From the results of the statistical analysis and visual interpretation of the maps, we can conclude that there is a significant positive correlation between COVID-19 mortality rate and PM2.5 concentration. 2.5 concentration for all counties in the United States, excluding Alaska. Some correlation coefficients of particular interest include Kendall’s tau correlation analysis between COVID-19 deaths and daily PM2.5 concentration. To complete this study, the particulate matter data was stratified into low, medium, and high groups. At least 30 data points were selected from each strata and depicted via Excel 3D maps. In addition to the general study that encompassed all United States counties (minus Alaska), we conducted a substudy to identify clusters of high COVID-19 mortality and high PM2.5 as well as the mortality rate and PM2.5 at the 0.05 level. This analysis contradicts the findings of the overall study. Analyzing the maps in 2.5 is low, inversely have high case fatality rates from COVID-19. The rural nature of the region opens opportunities for further investigation into the level of access to adequate healthcare and other social determinants of health that could influence this inverse relationship between PM2.5 concentrations and COVID-19 CFR in the region.Results of a Pearson correlation found that in the high strata, there was a significant negative correlation between the CFR and PM2.5 concentrations. The first group of clusters that indicates an association between air pollution and COVID-19 mortality is located in the Upper Midwest . The small clusters in this region suggest possible associations with social determinants of health in terms of mortality correlations. The rural environment that exists in this region contributes negatively to many of the social determinants of health. Poverty, proximity to healthcare facilities, and health literacy are all associated with rural environments and are all additional variables that could contribute to increased mortality rate in their respective counties. In addition, the Upper Midwest is home to multiple Native American reservations. The Native American population is more vulnerable to COVID-19 due to the “prevalence of underlying chronic health conditions, lack of institutional resilience, the relationship between the federal government and tribal governments, and lack of social trust” [Analysis of the maps created in our study demonstrated small clusters of counties for both COVID-19 mortality and PMl trust” . Additiol trust” . Similar2.5 concentrations. Two areas with correlated clusters were in the Detroit, MI, and Philadelphia, PA cities and surrounding counties. Detroit is an urban center that has a large population. In addition to traditional urban density of activity, Detroit is a global hub for many industries. Some of the highly polluting industries in Detroit include: an oil refinery, a steel mill, a wastewater treatment plant, and coal- and gas-fired power plants [Clusters of counties were also demonstrated on the map depicting elevated PMr plants . Similar2.5 map is the extremely high concentration levels in the counties of California’s Central Valley. The valley’s geographical feature has been associated with climate inversion events that trap air in the valley, leading to increased pollution and stagnant air [One final, and greatly significant, spatial anomaly demonstrated on the PMnant air . Similarnant air . Researcnant air ,27. If t2.5 and sulfur dioxide into the air [2.5, the COVID-19 mortality rates in Hawai’i were well below the national average and what would be expected based on the results of this study. It is unclear why Hawai’i County is an outlier in this study, but confounding variables, such as population density, lifestyle habits, isolation of the island, and medical practices, could all impact the COVID-19 mortality rate. Some recent studies have found associations between vitamin D levels and COVID-19. Current research suggests that those with higher levels of vitamin D, which is to be expected in the Hawaiian population, have generally better COVID-19 outcomes. In fact, the Northern Hemisphere has borne most of the burden of cases and deaths for COVID-19, with the exception of the Nordic countries [Prior to the study, we did not anticipate that Hawai’i County would have some of the highest levels of annual particulate matter pollution. After further investigation, we discovered that, whereas Honolulu and Maui have some of the cleanest air in the country, volcanic activity from Kīlauea emits enormous amounts of PM the air . Interesountries ,30. Furtountries ,33,34,35ountries  and obesountries  have als2.5 concentrations and increased COVID-19 mortality rates.Based on the methodology of this research, we cannot attribute causation to these variables; however, the results of this study and similar studies in the US, China, and Italy warrant further investigation into a possible association between high PM2.5 concentrations and low mortality rates on the island of Hawaii warrant further investigation. Individual risk factors related to age, obesity, diet, vitamin D levels, and physical activity are all variables that could influence the inverse relationship between COVID-19 mortality and PM2.5 concentrations on the island.The results of this study add valuable information to the ever-growing data on COVID-19. However, the study does raise many questions and variables for further investigation. The clustering of high mortality rates in rural regions in the Upper Midwest and somewhat in the Southwest and South allude to the intervention of social determinants that could influence health outcomes. Specifically, social and healthcare structures on Native American reservations that are present in the Upper Midwest and Southwest regions may have been associated with the anomalously high COVID-19 mortality observed in rural counties colocated there. Investigations on how to improve health systems and communication on these reservations would be suggested in order to prepare for any future epidemics. Additionally, the high PM