A major shift in the “dynamic zero-COVID” policy was announced by China’s National Health Commission on December 7, 2022, and the subsequent immediate large-scale outbreak of SARS-CoV-2 infections in the entire country has caused worldwide concern. This observational cross-sectional study aimed to describe the epidemiological characteristics of this outbreak in Sichuan, China.All data were self-reported online by volunteers. We described the epidemic by characterizing the infection, symptoms, clinical duration, severity, spatiotemporal clustering, and dynamic features of the disease. Prevalence ratio (PR), Odds ratios (ORs) and adjusted ORs were calculated to analyze the associations between risk factors and infection and the associations of risk factors with clinical severity using log-binomial and multivariable logistic regression models; 95% confidence intervals (CIs) and Wald test results were reported. The prevalence rates and clinical severity among different subgroups were compared using the Chi-square and trend Chi-square tests.Between January 6 and 12, 2023, 138,073 volunteers were enrolled in this survey, and 102,645 were infected with COVID-19, holding a prevalence rate of 74.34%; the proportion of asymptomatic infections was 1.58%. Log-binomial regression revealed that the risk of infection increased among those living in urban areas. Multivariable logistic regression analysis showed that female sex, chronic diseases, older age and the fewer doses of vaccine received were associated with an increased risk of severe clinical outcomes after infection. We estimated the mean reproduction number during this pandemic was 1.83. The highest time-dependent reproduction number was 4.15; this number decreased below 1 after 11 days from December 7, 2022. Temporal trends revealed a single peak curve with a plateau pattern of incidence during the outbreak, whereas spatiotemporal clustering analysis showed that the onset in 21 cities in the Sichuan province had four-wave peaks.The peak of the first wave of Omicron infection in Sichuan Province had passed and could be considered a snapshot of China under the new control strategy. There were significant increases in the risk of severe clinical outcomes after infection among females, with chronic diseases, and the elderly. The vaccines have been effective in reducing poor clinical outcomes. COVID-19 has been a global epidemic for three years and has infected approximately 760 million people worldwide and killed 6.8 million people as of March 19, 2023, according to the statistics released by the World Health Organization (WHO) [Previous studies conducted during the Omicron wave showed that epidemiological characteristics varied in areas owing to different control policies and pre-infection and vaccination patterns in the population. A study reported that the effective reproduction number was 2.19 in the rapid outbreak of the Omicron variant in South Africa, where fewer than one in ten people were fully vaccinated . The ReaBy the end of 2022, China implemented the “dynamic zero-COVID” policy for nearly three years since the outbreak of COVID-19 , and hadThree rounds of online surveys were conducted by the Sichuan provincial and city-level Centers for Disease Control and Prevention (CDC) between December 17, 2022, and January 12, 2023, covering more than 750,000 residents in all 21 cities in Sichuan Province. The questionnaire was carefully optimized according to the results of a previous survey round; therefore, this study draws on data from the third round of surveying. The study was conducted from January 6 to January 12, 2023, and included 19 questions referring to the demographics of participants, infection status, clinical symptoms, and so on. All questions were closed-form items and did not include any personal private information. Ethical approval for the use of the questionnaire for research purposes in Sichuan Province was obtained from the Sichuan CDC Ethics Committee . We published questionnaires on the official WeChat public account of the Sichuan CDC and called on staff at all levels in Sichuan province to advertise the survey. Furthermore, some local Internet media outlets, including the Sichuan Daily and Chengdu Publishing also transmitted the survey on their platforms.All residents of Sichuan Province were recruited as volunteers for the survey, and informed consent was obtained from every participant. The first page of the questionnaire provided an introduction to the survey objective and application of the results. The participants can also proxy-report to their families; therefore, the survey further covers infants, children, and elderly groups who may be out of the reach of our questionnaire.The following 13 symptoms were investigated: fever, cough, fatigue, headache, expectoration, sore throat, muscle pain, stuffy and runny nose, loss of taste and smell, chills, diarrhea, vomiting, and bloody stool. COVID-19 infection cases referred to participants who were positive for SARS-CoV-2 nucleic acid by RT-PCR or antigen testing by nasopharynx test paper using respiratory specimens or had not yet accepted coronavirus nucleic acid or antigen testing with one or more of the clinical symptoms mentioned above since December 7, 2022.Cases of infection combined with one or more symptoms were considered symptomatic. The clinical duration was defined as the symptom duration of symptomatic patients who recovered during the survey period. Symptomatic cases, including recovered and unrecovered patients whose symptoms lasted for ≥ 14 days, were analyzed to evaluate the severity of clinical outcomes. Cases were divided into two types: mild- or medium-type, which comprised patients with fewer than five symptoms lasting < 14 days, and severe type, which comprised patients who had ≥ 5 lasting ≥ 14 days.Baseline characteristics are presented as numbers (%) for categorical variables. We stratified the infection rates by geographical location, demographics, vaccination, and smoking characteristics . We used log-binomial regression model and calculated prevalence ratio (PR) to estimate the association between region and infection. We calculated the odds ratios (ORs) and adjusted ORs for infection of the other characteristics. The adjustment used a multivariable logistic regression model to analyze the associations between risk factors and infection, and 95% confidence intervals (CIs) and Wald test results were reported. In this regression analysis, all stratification characteristics were included as categorical variables. Multivariable ordinary logistic regression was also employed to investigate the odds of clinical severity between mild- or medium-, and severe cases, adjusting for sex, age, underlying conditions , vaccination status, and smoking status. The Chi-square and trend chi-square tests were used to compare the infection rates and clinical severity of all subgroups.0” package [P-values < 0.05 were considered statistically significant.We calculated the daily incidence of symptomatic cases in different cities in Sichuan Province and implemented a spatiotemporal cluster analysis to explore different waves in various regions. Based on the assumption that the total population during the survey period was relatively stable, we also estimated the mean reproduction number and time-dependent reproduction number in this pandemic by using “R package . Hierarc package . We used In total, 138,073 participants were enrolled in the survey between January 6 and January 12, 2023. All participants information were collected in the infection risk analysis. However, after applying the exclusion criteria, 97,675 symptomatic cases were included in the spatiotemporal and dynamic features analysis, 86,384 symptomatic cases were included in the symptom and severity analysis, and 58,917 symptomatic cases who had recovered were included in the clinical duration analysis . Table Among the 138,073 participants, 102,645 were infected by SARS-CoV-2 Omicron including 79,206 cases (77.16%) detected positively of nucleic acid or antigen test and 23,439 cases (22.84%) had one or more clinical symptoms without laboratory testing, with a prevalence rate of 74.34%. Notably, the prevalence rate among urban residents was 80.09%, 32.67% higher than that among rural residents (60.73%). The prevalence rate in women was 77.72%, 11.24% higher than that in men (69.87%), and the prevalence rate in participants with chronic diseases (77.55%) was slightly higher than the average. Across all age categories, the highest incidence rate was in the 26–50-year-old age group (77.88%), followed by the 51–65-year-old age group (75.28%), whereas the remaining groups had a lower-than-average rate. Surprisingly, smokers (64.94%) had a lower prevalence rate than non-smokers (76.23%). Overall, the prevalence rate was higher in those who had been vaccinated (75.51%) than in unvaccinated populations, but the prevalence rate among those who completed four doses of the vaccine (37.02%) was below average. The differences in the prevalence rates of all the stratified characteristics mentioned above were statistically significant  times higher for urban residents than for those in rural areas. The results of the multivariable logistic regression analysis showed that female sex , chronic diseases , and being a nonsmoker  were associated with an increased risk of infection. Furthermore, compared with the 13–25-year-old age group, the 0–6- and 7–12-year-old age groups were associated with a reduced risk of causing infections, while the adjusted risk ORs for increasing infection in the 26–50-year-old, 51–64-year-old, and 65-year-old age groups were 1.68 , 1.48 , and 1.24 , respectively. For individuals who had not been vaccinated, the completion of four doses of vaccination was associated with a reduced risk, whereas the completion of three doses was associated with an increased risk of infection . For more details, see Table Of the 102,645 patients in this study, 1617 (1.60%) did not develop any symptoms after infection, and the proportion of asymptomatic infections was 1.58%. By the end of the survey, 58,917 symptomatic cases’ clinical symptoms had completely disappeared, with a recovery rate of 60.32%. The longest clinical duration in recovered cases was 36 days, with a median of 11 days . For more details, please refer to Table p < 0.001). Individuals who had chronic underlying diseases, were not vaccinated, and were non-smokers were more likely to experience severe clinical outcomes, with a prevalence of severe disease of 63.95%, 64.14%, and 57.44%, respectively, in these cases , fever (77.14%), fatigue (67.37%), muscle pain (62.05%), and sore throat (58.41%). There were 47,116 severe cases and 39,268 light (medium) cases. The incidence of severe disease among female patients was 60.26–31.51% higher than that in males and tended to increase with age , having chronic underlying diseases , and being a nonsmoker  were associated with an increased risk of severe clinical outcomes after infection with COVID-19. Moreover, older age was associated with an increased risk. For example, the risks of severe clinical outcomes in the 26–50-year-old, 51–64-year-old, and 65-year-old age groups were 2.30 times, 2.76 times, and 3.25 times that in the 13–25-year-old age group, respectively. Furthermore, vaccinations were associated with a reduced risk of severe clinical outcomes, and the higher the dose of the vaccine received, the lower the risk of severe clinical outcomes (Table  There were 83.7 million residents in Sichuan Province in 2022. We estimated the mean reproduction number (R) according to the population and prevalence rate, which was 1.8298 . We estimated the daily time-dependent reproduction number (Rt) for the outbreak period in Sichuan province using daily incidence data, assuming a mean Weibull-distributed generation time of 3.56 days and a standard deviation of 2.28 days [ The results of regional distribution showed that the prevalence rate varied greatly from different regions, with more than 50% of the 21 cities in Sichuan province had a rate above 80%. Temporal trends revealed a single peak curve with a plateau pattern of incidence during the outbreak, and the onset of cases was concentrated between December 12 to December 23, 2022. However, the results of the spatiotemporal clustering analysis showed that onset in the 21 cities in Sichuan Province had four-wave peaks. First, onset in Dazhou, eastern Sichuan peaked on December 8. Second, onset in Chengdu, Suining, and the other 5 cities peaked between December 12 and December 15, 2022. The third wave was in Nanchong; onset in the other seven cities peaked between December 15 and 19. The last wave was in Yibin, and onset in the remaining eight cities peaked between December 18 and 20 Fig. .Fig. 4 TSince January 20, 2020, COVID-19 has been monitored as a class A communicable disease in China. It requires that all medical institutions and laboratory testing organizations report COVID-19 cases through the National Information System for Disease Control and Prevention within 2 h of detection. However, due to the home isolation of patients with mild or asymptomatic disease and the overloading of medical facilities during the outbreak, we cannot grasp the infection data precisely, which is detrimental to controlling the epidemic. Therefore, we conducted this urgent survey to describe the characteristics of a large-scale outbreak caused by the Omicron variant in Sichuan Province, China after adjusting for the COVID-19 response strategy.Our Omicron prevalence rate was higher than that of the first wave of Omicron outbreaks in other areas, including South Africa (58% in urban areas by April 2022)  and DemaThe risk of infection among urban residents was 1.33 times higher than that of rural residents; Similarly, in the United Kingdom, the risk of infection among urban residents was 1.24 times that of rural residents , which mThis study found that the rate of asymptomatic infections among infected residents was only 1.58%, which is much lower than that in Zhuhai City in Guangdong Province (76%)  and NingCough, fever, and fatigue were the most common clinical symptoms among symptomatic cases, which differs from the initial results of studies conducted in South Africa, the United Kingdom, and other countries –33. TheiSimilar to many previous studies –37, we fInterestingly, we found that smokers had a lower risk of both infection and severe clinical outcomes. The role played by smoking in COVID-19 infection has remained an open debate since the pandemic. Lippi G et al. published two articles in the European Journal of Internal Medicine to discuss this paradox. They summarized some large cohort studies or meta-analyses published on this topic in recent years –45 and cHowever, completion of the four doses of the COVID-19 vaccination significantly prevented infection and severe illnesses. It may result in that the four-dose COVID-19 vaccine strategy is a reinforced vaccine strategy specifically developed against the Omicron variant. The general population in China began to receive it on December 13 ; therefore, better immune effects were expected.However, this study had some limitations. First, this survey was conducted online, and participants were biased toward highly educated adults who were proficient in using the internet, with higher participation from females and lower coverage of infants, young children, and the elderly. The age structure of the surveyed population differed from that of Sichuan Province, leading to information bias. Second, although the total population was large, the information obtained was self-reported. Particularly, for cases defined based on clinical symptoms, the survey may have included a small number of individuals with fever and cough caused by other respiratory viruses, thereby reducing the reliability of the survey results. Third, owing to the lack of data on hospitalization and mortality cases during the outbreak, the disease burden of COVID-19 was inadequately seized. Fourth, for the evaluation of COVID-19 vaccine effectiveness, only the number of COVID-19 vaccine doses received by residents was surveyed, and detailed information on the type and timing of the COVID-19 vaccine received was not obtained. Therefore, extrapolation of our conclusions about vaccine effects should be cautious. Further research is warranted to identify appropriate correlates of protection for inactivated COVID-19 vaccines.The peak of the first wave of Omicron infection in Sichuan Province had passed and could be considered a snapshot of China under the new control strategy. Asymptomatic infections were rare in this large-scale outbreak and living in urban areas was associated with a higher risk of infection. Furthermore, there is a significant increase in the risk of severe clinical outcomes among females, those with chronic diseases, and the elderly. The vaccines have been effective in reducing poor clinical outcomes. What’s more, routine surveillance systems must be improved and optimized under the extreme conditions of highly infectious diseases.


The occurrence of optical loss on the surface of solar cells is inevitable due to the difference in the refractive index between air and glass, as well as the insufficient absorption of the active layer. To address this challenge, micron-sized geometry arrays, such as hemispheres and hemisphere pits, are prepared on quartz glass through the advanced indirect patterning technology of UV-LIGA. These geometric arrays exhibit multiple mechanisms for controlling light waves, including multiple rebounds, diffraction scattering, and total internal reflection. These synergistic effects suppress optical losses at the device’s surface and prolong the photon propagation path in the active layer. After being patterned with this structure, the average transmittance and haze of the quartz glass reach 93.91% and 75%, respectively. Compared to their flat counterpart, the decorated monocrystalline silicon solar cells demonstrated an apparent improvement in photocurrent and produced a 7.2% enhancement in power conversion efficiency. Energy scarcity and ecological contamination have always been persistent concerns for humanity. Driven by the pursuit of sustainable energy options, significant resources have been devoted to unlocking the vast benefits of solar power, characterized by its environmentally sound and renewable attributes as well as its cost-effective potential ,2,3. Amo3N4, MgF2, with an optical thickness equal to one-quarter of the wavelength, are effective at reducing reflection at specific wavelengths and angles of incidence [Although dielectric thin films like Sincidence ,12, thesncidence , biomimencidence , randomlncidence , hemisphncidence , and golncidence . These mncidence . To enabncidence . ConsequIn this report, we present an alternate approach to UV-LIGA, which enables the development of geometric arrays at air/glass interfaces. Here, UV refers to ultraviolet, while LIGA is the German acronym for “Lithographie, Galvanoformung, Abformungto”, which denotes the process of etching, electroforming, and duplicating micro-scale patterns . The staFor this experiment, the substrate of choice was quartz glass. The process of fabrication is illustrated in −1 for a duration of 5 s, which was then swiftly followed by an elevated velocity of 1000 r min−1 for a remarkable period of 30 s. As a result of this meticulous process, the photoresist film achieved a thickness of ~11 μm. Subsequently, the sample underwent a pre-baking process on a heated stage. This step aimed to eliminate any excess water and solvents, as well as solidify the photoresist. The pre-baking temperature was set at 100 °C, with a duration of precisely 3 min. The aperture of the photoresist mask was expertly etched to achieve a diameter and period of 10 μm and 12 μm, respectively. The exposure procedure employed a contact mode with an exposure energy of 60 mJ cm−2. For the development step, a solution comprising NaOH with a concentration of 5% was selected as the developer, with a development time of 30 s. Following the photolithography process, a cylindrical array with a diameter of 10 μm and a periodicity of 12 μm was achieved on the surface of the quartz glass, as depicted in (1) Lithography. The choice of photoresist plays a vital role in determining the outcome of an experiment. In this study, a commercially available positive photoresist  was employed. First, a pristine layer of photoresist was spin-coated onto the immaculately cleansed surface of the quartz glass. This process was initiated with a gentle rotation speed of 500 r min(2) Thermal reflux. The temperature and duration of the thermal reflux process were set at 150 °C and 3 min, respectively. Under the influence of high temperature, the cylindrical morphology of the polymer material transitioned into a partially molten state. Driven by surface tension, these cylindrical entities gradually diffused and transformed into hemisphere-shaped geometries, as illustrated in 2SO3)2·4H2O (450 mL), NiCl2·6H2O (5 g), H3BO3 (35 g), LN-MU (7 mL), LN-MA (1 mL), and surface additive (6 mL). The plating bath was maintained at a pH of 4 and heated to 47 degrees. A current density of 1 A dm−2 was applied to the cathode, while the anode’s current density was set to 2 A dm−2. The metal ion migrated toward the cathode due to the electrical potential difference between these two electrodes. Consequently, the ion grew into hemisphere structures that were consistent in shape with the cathode. Following the stripping process, a layer of nickel with a shape that was inverse to the hemisphere, namely a hemispherical pit, was obtained, as depicted in (3) Micro-electroforming. First, a 100 nm-thick layer of nickel was thermally evaporated onto hemisphere arrays to serve as a seed for electroforming. The electroforming process entailed the microstructure to be utilized as the core mold (cathode) and nickel as the electroplating material (anode). The composition of one liter of the plating solution is as follows: Ni Nanoimprint. To commence this process, a layer of ultraviolet nanoimprint resistance was applied onto the target quartz glass through spin coating. Subsequently, the prepared nickel template was superimposed onto the imprinting layer. Through the application of precise heat and pressure, the imprinting layer was diligently prompted to saturate every crevice of the hemispherical pits within the nickel template. Following this, the UV-exposure-induced curing process was conducted on the imprinting adhesive, with the light source energy set at 20 mJ cm−2, while the temperature and relative humidity were carefully regulated at 25 °C and 50%, respectively.The surface morphologies of the geometric arrays were characterized through the utilization of scanning electron microscopy . The wetting properties of the sample surface were characterized using a contact angle measurement instrument , with the solution employed being water. The transmission, reflection, and haze of the samples were measured using the UV-VIS spectrophotometer . The current–voltage characteristics (I-V curve) of the solar cells were measured using a solar simulator . During the testing process, the light source maintained an irradiance intensity of 100 mW mThe propagation and distribution of photons within geometric arrays were calculated using a three-dimensional finite difference time domain (FDTD) method. During modeling, the morphology, dimensions, periodicity, and arrangement of the geometric arrays were maintained consistently with experimental measurements. The spatial grid was discretized into cubic cells with a side length of 5 nm, while the time step was set to 0.4 ns. The light source employed a monochromatic wave emitted from the air, which vertically irradiated the surface of geometric arrays. The boundary conditions were established as follows: the direction of light propagation was defined by a perfectly matched layer (PML), while the direction of normal incident light adopted periodic boundary conditions (PBC).Evidently, the glass featuring micron-sized geometry arrays remarkably improved the transmission of visible wavelengths and slightly decreased it in the near-infrared region compared to the flat sample. The average transmission in the visible spectrum rose from 90.51% in the flat sample to 93.12% and 93.91% in the hemisphere and hemisphere pit, respectively. Interestingly, the haze increased from an initial near-zero value of 4.01% to about 75% across all measured bands. This elevated transmission and haze signified a greater number of photons traversing the air/glass interface, with the majority being diffused.For short- and medium-wave photons, the incident rays underwent multiple reflections within the micro-sized structures due to the feature size of the geometry being much larger than the wavelengths of the photons. Based on Snell’s law, the reflectance of a photon passing through the air/glass interface once is λ = 400 nm, and one could observe interference fringes between the incident and reflected light in the air. As the incident light wavelength increased to 1000 nm, the phenomenon of multiple rebounds dissipated and was replaced by conspicuous diffraction scattering, as illustrated in In the scenario where −2 for the flat surface to 39.83 and 39.87 mA cm−2 for the hemisphere and hemisphere pit structures, respectively. Furthermore, it is noteworthy that the introduction of geometric arrays, as opposed to a planar structure, does not alter the open-circuit voltage and fill factor of solar cells. This elucidates the fact that this optical modulation strategy does not undermine the inherent structure and electrical contacts of the devices. As a consequence of the increase in the photocurrent, the photoelectric conversion efficiency corresponding to the hemisphere and hemisphere pit structures augmented from 18.76% to 20.09% and 20.11%, respectively. This marks an impressive enhancement of approximately 7.2% compared to the planar one.Building upon this aforementioned understanding, these geometries were then implemented onto the surfaces of single-crystalline silicon solar cells. Subsequently, we conducted measurements on the reflection spectrum and current–voltage characteristics of these devices. As illustrated in Finally, the exposure of photovoltaic components to outdoor conditions rendered them susceptible to the detrimental effects of rainwater erosion and dust accumulation, which significantly impacted their stability and efficiency. Enhancing surface hydrophobicity stands as one of the approaches to address these issues. Taking these considerations into account, we further characterized the surface wettability of these geometric arrays. As illustrated in −2, resulting in an improvement in power conversion efficiency from 18.76% to 20.11%. Moreover, these geometric arrays offer other benefits, such as reusability, self-cleaning capabilities, and spectral independence, which enhance their potential for outdoor photovoltaic applications.In summary, our study demonstrates the production, replication, and transfer of micro-scale geometries at air/glass interfaces through UV-LIGA technology, effectively overcoming the inherent obstacle of direct patterning on glass surfaces. Distinct from traditional anti-reflective coatings and sub-wavelength structures, this sizable geometry affords numerous rebounds for short-wavelength light, while its periodic configuration facilitates robust diffraction scattering for long-wavelength light. As a result of these synergistic effects, there has been a significant reduction in optical loss at the air/glass interface and a considerable increase in haze. Encouragingly, the implementation of this design on silicon solar cells led to an increase in the photocurrent from 37.22 to 39.87 mA cm


Mitochondria are subcellular organelles involved in essential cellular functions, including cytosolic calcium regulation, cell apoptosis, and reactive oxygen species production. They are the site of important biochemical pathways, including the tricarboxylic acid cycle, parts of the ureagenesis cycle, or haem synthesis. Mitochondria are responsible for the majority of cellular ATP production through OXPHOS. Mitochondrial dysfunction has been associated with metabolic pathologies such as diabetes, obesity, hypertension, neurodegenerative diseases, cellular aging, and cancer. In this article, we describe the pathophysiological changes in, and mitochondrial role of, metabolic disorders  and their correlation with oxidative stress. We highlight the genetic changes identified at the mtDNA level. Additionally, we selected several representative biomarkers involved in oxidative stress and summarize the progress of therapeutic strategies. Mitochondria have a unique double-membrane structure and represent the central site of metabolism, displaying morphologies, dynamics, and functions specific to each cell and tissue . They arPathophysiological changes at the mitochondrial level have recently been associated with metabolic pathologies, neurodegenerative diseases, cellular aging, and cancer. The pathological mechanisms underlying these changes are still unknown. In this article, we describe the genetic changes identified at the mitochondrial level in metabolic pathologies and their connection with oxidative stress. Metabolic syndrome (MS) represents the accumulation of signs and conditions that, altogether, increase the risk of developing cardiovascular disease, stroke, and type II diabetes mellitus (T2DM). MS is present if at least three of the following five criteria are met: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein ,8,9. It 3+ to Fe2+), and from this level, it is then transferred to cytochrome c. The second electron is given to cytochrome b; subsequently, UQ is partially reduced to a molecule called the semi-quinone radical ion (Q-). In the second stage, a new UQH2 molecule is attached to complex III following the same pattern; therefore, a new electron is bound to the cytochrome c level, and the second electron is bound at the Q level with the formation of a UQH2 molecule. At the end of this process, four protons are generated in the intermembrane space. Four electrons are transferred from four cytochrome c molecules to complex IV (cytochrome c oxidase), where molecular oxygen is bound and reduced to water. Finally, at the level of complex IV, eight protons are transferred from the matrix  [The mitochondrion is a cytoplasmic organelle that consists of a double membrane, matrix, and mtDNA. The outer membrane and intermembrane space are relatively permeable in contrast to the inner membrane which has a restrictive permeability, containing the enzymes required for electron transport . Mitochoe space) . At the e space) ,14,16  1 and 2 are proteins involved in the fusion process of the outer mitochondrial membrane. The fusion of the outer mitochondrial membrane is most often achieved simultaneously with the fusion of the inner membrane, with the latter being mediated by the optic atrophy 1 protein (OPA1). The absence of Mfn cuts off the fusion phenomenon of both membranes. Mitochondrial fission is regulated by dynamin-related protein 1 (Drp1) and fission protein (Fis1) . Under vMitochondrial biogenesis is a complex process through which cells increase their mitochondrial mass and require coordination between nuclear and mitochondrial DNA. This process involves mtDNA transcription and translation processes, and the synthesis, import, and association of mitochondrial proteins encoded by nuclear DNA .Mitochondrial biogenesis dysfunction has been associated with metabolic disorders such as obesity and T2DM. A decline in the proliferator-activated receptor gamma coactivator-1α (PGC-1α), AMP-activated protein kinase (AMPK), and silent information regulator 1 (SIRT-1) signalling pathways seems to be the underlying mechanism for reduced mitochondrial biogenesis in the diabetic kidney and the diabetic heart as well, with hypoadiponectinemia being reported to impair AMPK-PGC-1α signalling .Autophagy is a natural mechanism which was highly conservated throughout evolution, by which the useless cytoplasmic material is transported to lysosomes for destruction . AutophaThe degradation of mitochondria through selective autophagy is referred to as mitophagy, a process that involves the selective sequestration of damaged or dysfunctional mitochondria into double-membraned autophagosomes for later lysosomal destruction. Mitophagy has been described in mammalian cells as being facilitated by two well-studied pathways, ubiquitin-mediated and receptor-mediated, and is essential for maintaining cellular fitness ,35.Mitophagy ubiquitin-mediated pathways are regulated by two key proteins PTEN-induced putative kinase protein 1 (PINK1) and Parkin. Normally, PINK1 is imported into healthy mitochondria via the TIM/TOM system and further degraded by proteolytic reactions. Damaged mitochondria lose membrane potential, which impairs the TIM/TOM system’s function, resulting in the accumulation of PINK1 on the outer mitochondrial membrane, which promotes the recruitment of Parkin and the activation of its ubiquitination ligase activity, leading to the ubiquitination of proteins from the outer mitochondrial membrane. Further, Parkin promotes the recruitment of autophagy adaptors, such as optineurin (OPTN) and nuclear dot protein 52 kDa (NDP52), leading to the degradation of damaged mitochondria ,37,38.The mitophagy receptor pathway is mediated by receptors embedded in the outer mitochondrial membrane, most notably by NIX (known as BCL2 interacting protein 3 like (BNIP3L)), BCL2 interacting protein 3 (BNIP3), and FUN14 domain containing 1 (FUNDC1), which are characterized by the presence of an LC3-interacting region (LIR) that can directly bind to the autophagy mediator LC3 to promote mitophagy when mitochondria are damaged ,39.Mitophagy is implicated in insulin resistance and some cardiac pathological conditions. The dysfunctional mitophagy mechanism has been linked to the development of insulin resistance . MoreoveMitochondrial dysfunctions generated by ROS production in the OXPHOS process are caused by mitochondrial and cellular component damage  . Metabol2•−) is generated. It interacts with other molecules or generates secondary ROS [2•− is subsequently transformed into a more stable compound, hydrogen peroxide (H2O2), under the action of the enzyme superoxide dismutase (SOD). The considerable presence of this enzyme at the mitochondrial level supports the importance of O2•− elimination [2O2 can be transported through aquaporins, present at the level of the inner mitochondrial membrane. Moreover, another possibility of eliminating this compound is diffusion at the cellular level, where it is neutralized and removed with the aid of several antioxidant enzymes such as catalase, glutathione peroxidase, and thioredoxin peroxidase [2O2 interacts with O2•− and generates the hydroxyl radical (OH•), a molecule that is extremely reactive and destructive at the cellular level. Furthermore, in the mitochondrial outer membrane, there is a monoamine oxidase (MAO) enzyme that acts as another source of H2O2 [2O2 elimination mechanisms using these organelles [•), the protonated form of O2•−, which, however, physiologically develops in small amounts at the cellular level [1O2) has been identified as playing an important role in the destruction of mtDNA [1O2 is a mitochondrial permeability modulator which can be generated through cytochrome c-catalyzed peroxidation, with carbonyl groups as the substrate [Oxidative stress is characterized by the imbalance between the production of ROS and the action of antioxidants, with the destructive effect of ROS. Most ROS are generated at the complex I and III levels of the mitochondrial respiratory chain by releasing electrons from NADH and FADH2 to the ETC . Free radary ROS . O2•− ismination ,45,47,48roxidase ,49,50. W of H2O2 , which eganelles . Anotherar level . The latar level . Anotherar level ,54. Moreof mtDNA . 1O2 is ubstrate .•) is produced enzymatically by means of nitric oxide synthases (NOS) from amino acids [•. NADPH and oxygen are also involved in this reaction [• catabolism, but also O2•− to O2 oxidation [+. In addition, mitochondria contain alpha-tocopherol (vitamin E) and UQH2, inhibitors of lipid peroxidation [Mitochondria are also a source of reactive nitrogen species (RNS). Nitric oxygen , manganese (Mn), zinc (Zn), and iron (Fe) are nutrients in various processes that take place at the intracellular level. Copper (Cu) is a cofactor for enzyme function and has an increased redox potential, allowing the transfer of electrons to oxygen and ROS production. An inadequate amount at the cellular level is associated with a compromised immune system, organ dysfunction, and oxidative damage . Mn is atrocytes ,62,63. Ireaction .Additionally, mitochondria can generate heat due to proton leak. The proton leak results from the activity of fatty acids on uncoupling proteins (UCPs). UCPs belong to the family of mitochondrial anion carrier proteins. Five UCPs have been identified in mammals  , and theType 2 diabetes mellitus (T2DM) is a chronic pathology that requires continuous medical care through pharmacological treatment, but also a reduction in risk factors involved in the etiopathogenesis of the disease. Currently, this condition is characterized by a permanent increase in incidence and prevalence . In rece2•− affects the translocation capacity of glucose transporter 4 (GLUT4) from the intracellular level to the plasma membrane, resulting in a decrease in insulin action at the tissue level and an increase in blood glucose amount [Hyperglycaemia and T2DM are directly linked to mitochondrial activity, function, and oxidative stress. Mitochondria produce the largest amount of ROS and ATP. With regards to hyperglycaemic status, the amount of ROS increases and triggers changes in cellular homeostasis with the generation of lesions at this level . The ince amount ,71. Hypee amount ,88. Chane amount . Fatty ae amount ,90. The e amount .In diabetic patients, at the level of mononuclear cells, increased amounts of ROS were identified, as well as spherical and hyperpolarized mitochondria, thus indicating dysfunctions at this level . The effm.8561C>G mutation in MT-ATP6/8 was identified and correlated with T2DM and hypergonadotropic hypogonadism [m.3242A>G mutation and the 10.4-kb deletion were associated with diabetes and deafness. It is considered that mitochondrial mutations can accumulate over time (an aspect that correlates with the aging process and neurodegeneration) [COX7A1 and NDUFB6 genes have been identified in people with insulin resistance and T2DM. Although they are nuclear genes, they encode subunits of OXPHOS complexes I and IV, respectively [Changes in mtDNA and genomic DNA have also been identified in patients with diabetes mellitus. A new gonadism . The m.3eration) . There ieration) . These cectively ,95,96.Obesity is associated with changes in carbohydrate and lipid metabolism, but also with insulin resistance, an increased risk of cardiovascular pathologies, and diabetes mellitus . It reprCells can release extracellular vesicles containing mitochondria. Recipient cells receive mitochondria through an extracellular vesicle–cell fusion event . The intAdipocytes transfer mitochondria to macrophages in adipose tissue, generating a new population of macrophages, which is greatly diminished in patients with obesity due to increased lipid intake because of reduced mitochondrial uptake by macrophages. Mitochondrial uptake is mediated by heparan sulphate and it manifests low levels in obese subjects. The exostosin (EXT) 1 gene and the EXT2 heterodimer are also associated with the maintenance of lipid metabolism homeostasis and glucose levels . The preIt has been shown that numerous components of the ETC have decreased expression in visceral adipose tissue in women with diabetes. A decrease in the expression of OXPHOS genes was also noted in these patients . A reducCardiovascular disease is the leading cause of death worldwide. There are a large number of factors involved in the development of cardiovascular pathologies. Mitochondrial changes produced during the aging process explain the functional deficit encountered in cardiovascular pathologies .Cardiomyocytes contain numerous mitochondria to generate a large amount of ATP . At the The largest amount of ATP is obtained at the cardiac level via the beta-oxidation of fatty acids but, depending on availability, glucose can be used as an energy source. In cardiac pathologies, insulin signalling is altered, affecting metabolic flexibility. Therefore, the amount of ATP decreases .Oxidative stress and mtDNA changes are identified in patients with cardiovascular pathologies. At the cardiac level, ROS are generated through complexes I and III in neutrophils, endothelial cells, and myocytes. The increase in ROS causes endothelial dysfunction, inflammation in blood vessels, and the generation of oxidized LDL at the arterial level. Ultimately, these changes trigger atherosclerosis, hypertension, and cardiac hypertrophy. In addition, mitochondrial dysfunctions stimulate enzyme activation and induce cardiomyocyte apoptosis ,80. It hSeveral studies indicate methylation changes in cardiovascular pathologies. Extensive methylations were found in the MT-CO1, MT-CO2, MT-CO3, and MT-TL1 genes in subjects with these conditions, with the methylation degree of these genes being a potential predictive marker of cardiovascular pathologies in obese patients .Mitochondrial dysfunctions are involved in the pathophysiological mechanisms that generate metabolic disorders. In this context, interest shown toward mitochondria can generate new optimal therapies for these pathologies. The aim is to slow the progression of mitochondrial dysfunctions and decrease ROS with a subsequent reduction in oxidative stress.Recent studies support the idea that physical activity improves insulin sensitivity and mitochondrial function in muscle tissue in patients with T2DM. Caloric restriction reduces excessive ROS production and inhibits inflammation, thus facilitating the prevention and treatment of metabolic pathologies ,114,115.Due to the multitude of discoveries in the field, interest in mitochondrion-directed therapy has increased. Although numerous action mechanisms are known, the challenge is represented by the transportation of active substances at this level due to the barriers that precede mitochondrial localization. In this regard, some of the drugs that may represent a new therapeutic solution in metabolic pathologies are selected further.Annonaceous acetogenins inhibit NADH ubiquinone oxidoreductase. Moreover, metformin is a complex I inhibitor, a drug used in diabetes that increases glucose consumption. Vitamin E analogues (α-tocopheryl succinate) and 3-bromopyruvate act on complex II. Complex III inhibitors are antimycin A and myxothiazole. Cyanide is a complex IV inhibitor. There are numerous pharmaceutical agents that act on complex V  [ETC components may represent a target in pharmaceutical interventions. Rotenone and overtin) ,118.A subfamily of mitochondrial proteins is represented by uncoupling proteins (UCPs). UCPs facilitate H+ transport, ultimately generating heat. Fatty acids stimulate UCP transport. In addition, it was highlighted that adrenaline and prostaglandins determine the upregulation of UCPs. They are involved in common pathologies, with UCP1 and UCP2 being associated with diabetes and obesity. Carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol are uncoupling agents for oxidative phosphorylation. UCPs represent a good pharmacological target for treating obesity and diabetes ,119,120.From a pharmacological point of view, sirtuins (silent information regulator proteins) are involved in the regulation of glucose and lipid metabolism at the cellular level ,122,123.Mitochondrial fission inhibitors such as mitochondrial division inhibitor 1 (Mdivi 1), P110, and dynasore have been identified as playing a role in ameliorating oxidative stress ,126,127.• forming nitrosothiols. NAC inhibits the oxidative degradation of NO•, thus decreasing the amount of nitrogen dioxide, peroxynitrite, and nitrotyrosine. NAC therapy has beneficial effects on oxidative stress, inflammation, epithelial dysfunction, and hypertension [Several studies have highlighted the beneficial effect of antioxidant use in metabolic pathologies ,131,132.rtension . Coenzymrtension .Although the beneficial effect of using conventional antioxidants in metabolic pathologies is known, in some clinical trials, the results of their benefits are contradictory. Salehi et al. highlighted the adverse effects of inappropriately using nontargeted antioxidants . Excessive vitamin A intake  has been associated with increased teratogenicity risk or birth defects. Vitamin C can be metabolized to oxalate, increasing the risk of calcium oxalate kidney stones, and vitamin E may increase prostate cancer risk . The excMitoquinone (MitoQ), mitovitamin E (MitoE), and MitoTEMPO are mitochondria-targeted antioxidants and are synthesized by attaching the antioxidant to the lipophilic triphenylphosphonium cation (TPP+). TPP+ is the most widely used lipophilic cation. To permeate the mitochondrial membrane, the substances must permit the optimal lipophilic level. If the molecule has low lipophilicity, it does not penetrate the mitochondrial membrane, and if lipophilicity is increased, it accumulates at the membrane level . Due to Liposomes are bilipid membrane vesicles used to transport certain bioactive substances. Liposome-encapsulated antioxidants are composed of cholesterol, phosphatidylcholine, phosphatidylglycerol, cholesterol, and antioxidants . Liposome-encapsulated antioxidants penetrate at the cellular level through the phenomenon of pinocytosis, the liposomal components fuse with the mitochondrial membrane, and the antioxidant is released at the mitochondrial level . MITO-PoIt represents a liposomal nanocarrier made up of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, sphingomyelin, and stearylated octa arginine peptide (R8). MITO-Porter binds at the mitochondrial level due to the interaction between R8 and negatively charged mitochondria .Mitochondria are versatile organelles, responsible for most of the cellular chemical energy production. The structure and function of this subcellular organelle are peculiar, due to the abounding molecular mechanisms that take place at this level. They are responsible for cell survival, apoptosis, and homeostasis. These benefits coexist with the high degree of errors that can occur at the mitochondrial level through ROS. Mitochondrial changes have been identified in metabolic dysfunctions including insulin resistance/diabetes, hypertension, and dyslipidaemia, but also in cancer or neurodegenerative pathologies. Recent studies have identified changes in mitochondrial biogenesis and dynamics, as well as the presence of oxidative stress in metabolic dysfunctions.In diabetic patients, at the level of mononuclear cells, spherical and hyperpolarized mitochondria were identified, indicating dysfunctions at this level. In pancreatic β cells, changes including volume, shape modifications, and mitochondrial dysfunction have been reported as well. The reduction in ATP production and the increase in ROS at the muscle level can trigger an increase in insulin resistance and diabetes. In obesity patients, the excess of free fatty acids at the adipocyte level activates NADPH oxidase enzymes, with an increase in ROS production. Oxidative stress causes inflammation and boosts lipid peroxidation, disrupting insulin’s mechanisms of action. The increase in ROS causes endothelial dysfunction, inflammation in blood vessels, and the generation of oxidized LDL at the arterial level in cardiovascular diseases.To reduce the progression of metabolic pathologies, lifestyle interventions, physical exercises, and dietary changes are indicated. A balanced diet, including fruits, legumes, and vegetables of different colours, is recommended. Moreover, new pharmaceutical strategies that can improve the prognosis of these conditions are being investigated and developed. Several studies have highlighted the beneficial effect of using antioxidants in metabolic pathologies; vitamin E, vitamin C, coenzyme Q, and NAC administration are indicated for their oxidative stress reduction effect. However, in some clinical trials, the results of their benefits are contradictory. Due to the adverse effects of conventional antioxidants at the cellular level, the production and use of mitochondria-targeted antioxidants are becoming more relevant.The epigenetic role in this context is not fully understood, but changes in mtDNA have been identified which could be meaningful in the identification and design of the next therapeutic strategies. It is considered that mitochondrial mutations can accumulate over time. Several studies indicate methylation changes in cardiovascular pathologies, and the methylation degree of specific genes can be a potential predictive marker of cardiovascular pathologies in obese patients. Difficulties in this field arise due to the particularities encountered at the level of each individual. Large and complex studies are needed in order to identify and detail the changes at the mitochondrial level and the therapeutic approaches.


Aortic stenosis (AS) involves progressive valve obstruction and a remodeling response of the left ventriculum (LV) with systolic and diastolic dysfunction. The roles of interstitial fibrosis and myocardial steatosis in LV dysfunction in AS have not been completely characterized. We enrolled 31 patients (19 women and 12 men) with severe AS undergoing elective aortic valve replacement. The subjects were clinically evaluated, and transthoracic echocardiography was performed pre-surgery. LV septal biopsies were obtained to assess fibrosis and apoptosis and fat deposition in myocytes (perilipin 5 (PLIN5)), or in the form of adipocytes within the heart (perilipin 1 (PLIN1)), the presence of ceramides and myostatin were assessed via immunohistochemistry. After BMI adjustment, we found a positive association between fibrosis and apoptotic cardiomyocytes, as well as fibrosis and the area covered by PLIN5. Apoptosis and PLIN5 were also significantly interrelated. LV fibrosis increased with a higher medium gradient (MG) and peak gradient (PG). Ceramides and myostatin levels were higher in patients within the higher MG and PG tertiles. In the linear regression analysis, increased fibrosis correlated with increased apoptosis and myostatin, independent from confounding factors. After adjustment for age and BMI, we found a positive relationship between PLIN5 and E/A and a negative correlation between septal S’, global longitudinal strain (GLS), and fibrosis. Myostatin was inversely correlated with GLS and ejection fraction. Fibrosis and myocardial steatosis altogether contribute to ventricular dysfunction in severe AS. The association of myostatin and fibrosis with systolic dysfunction, as well as between myocardial steatosis and diastolic dysfunction, highlights potential therapeutic targets. Aortic stenosis (AS) is one of the most common valvular diseases in the Western world, with an estimated prevalence as high as 12.4% in the elderly , leadingMyocardial fibrosis is, in general, one of the hallmarks of a failing heart . Among sThe activation of profibrotic pathways, which involves a protein of the transforming growth factor-β (TGFβ) family called myostatin, is due to cardiomyocyte death with the consequent release of free fatty acids (FFAs), the recruitment of macrophages, apoptosis, and fibrosis . In LV dGlobal longitudinal strain (GLS), assessed with echocardiography, has been considered a surrogate marker for myocardial fibrosis . StudiesEctopic fat deposition in the myocardium, termed myocardial steatosis, is frequently associated with diastolic dysfunction in severe AS, ischemic heart disease, and various metabolic diseases ,18. GeneRegarding the role of myocardial steatosis in myocardial dysfunction, animal models have proved that myocardial triglyceride accumulation disrupts normal myocardial lipid metabolism and results in diastolic dysfunction . The rolAs shown thus far, both interstitial fibrosis and myocardial steatosis appear to significantly contribute to the development of myocardial dysfunction in different heart diseases and also in patients with advanced AS. However, in human studies, myocardial quality in terms of fibrosis and myosteatosis has been characterized mainly using MRI and histologically only in a few studies to validate MRI measurements ,6,15.Thus, the present study aimed to analyze the LV biopsies of patients with severe AS who had undergone surgery, fibrosis, and myocardial lipid infiltration, as well as to investigate the correlation between them and key echocardiographic parameters indicative of LV dysfunction. Furthermore, this study sought to evaluate the underlying mechanisms and pathways correlated with these histological phenomena.A total of 31 patients  diagnosed with severe AS undergoing elective cardiac surgery for aortic valve replacement were recruited as our study population. The clinical characteristics as well as the metabolic profiles and echocardiographic data of these subjects are shown in To establish the levels of fibrosis, cell apoptosis, and fat deposition, and the presence of ceramides and myostatin, in the LV samples obtained from patients with AS during surgery, we labeled and quantified these elements using different staining and detection methods, as exemplified in p = 0.007), as well as between fibrosis and the area covered by PLIN5 . Apoptosis and PLIN5 also showed a significant association . Regarding ceramides, we found only a positive trend with fibrosis (r = 0.235) and with PLIN5 (r = 0.241); however, these associations were not statistically significant.Then, we evaluated the interrelationship between the different histological characteristics of the dysfunctional LV samples obtained from AS patients, after adjustment for BMI . We intep < 0.001). A trend was also observed between PLIN5 and the MG and PG tertiles (p 0.089)   and peak gradient (PG) obtained from the echocardiography before surgery in patients with AS, dividing the patients into tertiles according to their echocardiographic parameters. As shown in tertiles . Regardip 0.089) . Finallyctively) .p = 0.025; β-coefficient = 0.464), independent from age, BMI, diabetes mellitus, statin therapy, and other histological characteristics of LV, explaining approximately 40% of its variance (R2 0.413) (data not shown in the table). Apoptosis in the model presented an association with fibrosis of borderline significance , independent from the other variables (data not shown in the table).Subsequently, we performed a multiple linear regression analysis to explore the relationship between fibrosis, which was considered as the dependent variable, and different independent variables, including age, gender, BMI, diabetes mellitus, statin therapy, apoptosis, PLIN5, ceramides, and myostatin. Fibrosis correlated with myostatin  as well as a slight negative correlation between fibrosis and GLS ; conversely, there was not any correlation between fibrosis and EF and E/A (p = 0.082) (p = 0.009), while there were no correlations between PLIN5 and GLS, EF, and DTI septal S’ , EF , GLS , the DTI septal S’ with the main histological features of the LVs of AS patients, after adjustments for age and BMI . We obse and E/A . A borde= 0.082) . Additioeptal S’ . Myostateptal S’ . FinallyIn this group of patients undergoing elective surgery for AS, myocardial fibrosis and myocardial steatosis, as evaluated histologically, collectively contributed to ventricular dysfunction. In particular, the degree of fibrosis was correlated with systolic dysfunction. and the degree of fat infiltration in the heart was related to diastolic dysfunction. In this group of patients, an increase in pressure overload, expressed as a mean or peak gradient, was related to increases in LV fibrosis and myostatin and ceramides levels. The levels of ventricular fibrosis were mainly explained by increased myostatin expression, independent from other confounding factors.AS is a disease that affects both the valve and the myocardium, as myocardial dysfunction is strictly associated with pressure overload . HoweverIn this group of patients with severe AS, myocardial fibrosis was strictly related to a reduction in systolic function. Moreover, in the regression model, the amount of fibrosis in LV biopsies was mainly explained by myostatin. Myostatin, a protein of the TGFβ family, was discovered in 1997 in skeletal muscle cells and characterized as a signal that regulates muscle growth and differentiation, as its gene deletion was responsible for the double-muscle phenotype in cattle . In addiThe role of myostatin has also been explored in humans across different types of heart diseases with cardiac remodeling and reduced LV ejection fractions, and a strong association has been found between the severity of the disease and myostatin overexpression ,13. MoreIn our study in AS patients, myostatin emerged as a significant predictor of ventricular fibrosis, independent of age, gender, BMI, diabetes mellitus, statin therapy, apoptosis, PLIN5, and ceramides, explaining approximately 40% of the variance. In a recent study using a mouse model of AS, treatment with monoclonal anti-myostatin antibodies failed to reduce cardiac hypertrophy or fibrosis . This suMyofibrosis and myosteatosis are considered two interrelated mechanisms of muscular damage associated with aging and/or obesity ,35. ThusCardiac steatosis has been linked with diastolic dysfunction in animal models and has been shown to be an independent predictor of diastolic function in diabetic and obese patients . AccordiMyocardial fibrosis and steatosis may thus contribute to LV insufficiency in AS, mainly affecting global systolic and diastolic functions, respectively. However, several studies using non-invasive imaging have shown that fibrosis could be distributed heterogeneously across ventricular walls in different cardiomyopathies ,38. A poIn our study, the histological degree of myocardial fibrosis and steatosis were significantly correlated with each other. Moreover, apoptosis resulted in being strictly related not only to myocardial fibrosis but also to myocardial steatosis. From a physiopathological point of view, it is possible to hypothesize that pressure overload causes the death of cardiomyocytes with the consequent release of free fatty acids (FFAs), the recruitment of macrophages, apoptosis, and the activation of profibrotic pathways .In the heart, as in other organs, the excess of free fatty acids starts to accumulate in the form of many different lipid metabolites such as ceramides and other sphingolipids when they cannot be stored in lipid droplets within the cells . CeramidFinally, it is known that myocardial steatosis may be due to both lipid droplets stored within cardiomyocytes and triglyceride droplets in cardiac adipocytes . We and In conclusion, our study provides data on the roles of myocardial fibrosis and steatosis in LV dysfunction, with the unique aspect of direct histological data analysis, which could be, for some aspects, more informative than non-invasive methods. However, further analyses are needed to explore the molecular mechanisms involved in these relationships. It may be important to associate IHC with other complementary methods of analysis, such as, for example, real-time PCR or Western blot, which can assess the expression of target genes or evaluate the proteins involved in the principal molecular pathways. Further studies conducted in wider samples are necessary for better exploring the possible molecular mechanisms and eventual targets of therapies.Even though our results are only descriptive and require further study, especially to establish the role of cardiac adipocytes, we can speculate that intramyocardial adipocytes may become dysfunctional due to different types of stress. Besides the canonical mechanisms that contribute to adipocytes’ dysfunction, such as inflammation, ROS production, and hypoxia, a role could be also hypothesized for mechanical stress. In a 3D adipocyte culture model, Pellegrinelli et al.  showed tWe recruited a final group of 31 subjects (19 women and 12 men) undergoing elective cardiac surgery for aortic valve replacement for AS. All patients were affected by severe AS, according to the European Society of Cardiology (ESC) Guidelines of 2012 . ImportaAll subjects were clinically evaluated with measurement of their blood pressure and heart rate. Information regarding their smoking habits, the presence of diseases, and medication intake was also collected. Venous blood samples for the metabolic assessments were obtained after overnight fasting. Plasma glucose levels were measured with a glucose analyzer . Cholesterol and triglyceride levels were determined using a Techincon Auto Analyzer , and dextran–magnesium precipitation was used to derive the HDL values. Weight with an approximation of 0.1 kg  and height using a stadiometer with an approximation of 0.5 cm  were determined for all subjects. Body mass index (BMI) was computed by dividing weight in kilograms by height in square meters. Waist circumference was obtained with a measuring tape at the narrowest circumference of the abdomen.All patients underwent a preoperative echocardiogram, performed with an echocardiograph equipped with speckle-tracking analysis (Philips IEE) in the echocardiography clinics at the Cardiology Department of the University Hospital of Verona. For each subject, the following were evaluated: systolic function (the EF of the LV of the biplane Simpson method %), the degree of the AS transvalvular mean gradient (MG), and the peak trans-valve gradient (PG). Furthermore, diastolic function indices were collected using tissue Doppler imaging, such as E/A . The pathological cut-off of altered relaxation was defined as E/A < 1 ms. Finally, GLS  was measured as an index of systolic functionality, using a two-dimensional speckle-tracking method. The GLS was shown as an absolute value, and we considered normal GLS values of 17.5 ± 3.2%.In the surgical room, myocardial specimens (interventricular septum tissue) were obtained by trained surgeons after a median sternotomy, approximately 1 h after the induction of anesthesia. The biopsies were then stored for subsequent immunohistochemical evaluation.2O2) for 10 min and non-immune serum for 60 min at room temperature, respectively. The slides were then incubated sequentially with primary rabbit antibodies for 16 h at 4 °C in a humidified chamber and washed with PBS buffer three times for 5 min each. Subsequently, the sections were incubated with the secondary antibody for 30 min at 20 °C and washed again with PBS for 5 min. DAB was used as the chromogen to visualize peroxidase activity. The sections were counterstained with hematoxylin and were finally assembled with Entellan and overlaid with coverslips.Freshly isolated ventricular myocardial muscle samples were fixed via immersion in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4 °C, and then dehydrated, cleared, and paraffin-embedded. Five-micrometer-thick serial sections were obtained from the embedded tissues. These sections were then stained with hematoxylin and eosin (H/E) to assess their morphologies prior to immunohistochemical processing. The following primary antibodies were used for the IHC analysis: rabbit anti-human perilipin-1 (PLIN1)  to detect adipocytes, rabbit anti-human perilipin-5 (PLIN5)  to mark lipid droplets inside cardiomyocytes, and rabbit anti-human myostatin . The secondary antibody was SignalStain Boost IHC detection reagent HRP Rabbit . A negative control was conducted by using the secondary antibody alone. A diaminobenzidine hydrochloride (DAB) substrate kit for peroxidase was used for the IHC . Briefly, the paraffin-embedded sections were dewaxed and subjected to antigen heat retrieval in sodium citrate buffer (pH 6.00) in a microwave oven for 3 cycles for 5 min each at 750 watt. Endogenous peroxidase activity and non-specific binding were blocked via incubation with 3% hydrogen peroxide  for 15 min each; and non-specific binding was blocked by incubating the sections with non-immune serum for 20 min at room temperature. The sections were then incubated with primary mouse IgM anti ceramide  for 16 h at 4 °C in a humidified chamber, washed with PBS buffer three times for 5 min each. Subsequently, the sections were incubated with the secondary antibody Biotinylated Goat anti-mouse IgM  for 30 min at 20 °C, washed with PBS for 5 min, and incubated with Vectastain ABC reagent  for 30 min at 20 °C. The sections were then washed again with PBS for 5 min. A negative control was conducted by using the secondary antibody alone.The sections were dewaxed and subjected to antigen heat retrieval in sodium citrate buffer at pH 6.00 using a microwave oven for 3 cycles for 5 min each at 750 watts. Endogenous peroxidase activity was blocked by incubating the sections with 3% HDAB was used as the chromogen to visualize peroxidase activity. The sections were counterstained with hematoxylin, assembled with Entellan, and overlaid with coverslips.Sections of the ventricles were stained with Masson’s trichrome to visualize interstitial fibrosis, according to the manufacturer’s protocol . The sections were deparaffined, rehydrated, stained with ferric hematoxylin according to Weigert for 10 min, incubated with picric acid for 4 min, quickly washed with distilled water, stained with Ponceau B solution for 4 min, quickly washed with distilled water, incubated with phosphomolybdic acid for 10 min, stained with aniline blue according to Masson for 5 min, quickly washed with distilled water, dehydrated, cleared, assembled with Entellan, and overlaid with coverslips.The detection of apoptotic cardiomyocytes was performed using the terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) method, according to the manufacturer’s protocol . To apply this method, myocardial tissues from the paraffin-embedded ventricles were cut into thin slices (5 µm thickness). In the TUNEL assay, apoptotic cardiomyocytes were identified by the presence of dark-blue-stained nuclei, while non-apoptotic cardiomyocytes displayed pink/red-stained nuclei.All ventricle sections were observed at 40×, 200× and 400× magnification using an Olympus BX51 photomicroscope microscope equipped with a KY-F58 CCD camera (JVC) and with Image-ProPlus software  or by using the EVOS FL Auto Cell Imaging System with an EVOS Onstage Incubator  photomicroscope.2 by dividing the cell count by the total area expressed in mm2.To quantify the number of PLIN1-positive adipocytes among cardiomyocytes, a manual counting method was used. The entire area of each ventricle was observed, and the number of PLIN1-positive adipocytes was manually counted. The total area of the observed section was then calculated using ImageJ software (ImageJ1.8.3) on the acquired images and the number of adipocytes was expressed as cells/mm2 area; range: 3.21–28.32 LDs/mm2 area). Myostatin was detected in all LV samples . All biopsies were also positive for ceramides inside the cardiomyocytes .The optical density (OD) analysis of PLIN5, myostatin, and ceramides was performed on the whole ventricle sections. Images acquired at 200× magnification were analyzed using the Color Threshold function of ImageJ software and manual threshold correction when necessary. All LV biopsies were positive for PLIN5, with a very variable range within the group  in the study population.2 and as the average number of cardiomyocytes undergoing apoptosis/10,000 cardiomyocytes [The degree of apoptosis was calculated by counting the number of dark-blue-stained nuclei in cardiomyocytes using ImageJ software on five images acquired at 200× magnification. The degree of apoptosis was then expressed as the number of dark-blue-stained nuclei/mmmyocytes . The scoThe results were reported as means ± standard deviation (SD). Nonparametric tests were used for non-normally distributed variables. Tau-b di Kendall correlations were used to test the level of correlation between the variables. Linear multiple regression was performed to evaluate the effects of the independent variables on the fibrosis-dependent variable. A significance level of 5% was always adopted. All analyses were performed using SPSS .In patients with severe aortic stenosis, myocardial fibrosis and myocardial steatosis, which were histologically evaluated, collectively contributed to ventricular dysfunction, correlating with systolic and diastolic dysfunction, respectively. Our study suggests synergistic roles of myofibrosis and myosteatosis in determining ventricular damage in patients with AS and pressure overload. Pressure overload is related to an increase in both myostatin and fibrosis in the myocardium, as well as an increase in myocardial steatosis and ceramide deposition in the ventriculum. Apoptosis is linked to both the degree of fibrosis and steatosis in the LVs of patients with AS.These associations may highlight potential therapeutic targets to be explored in other experimental models. Furthermore, cardiac adipocytes are also present in the ventricles of patients with AS, close to damaged areas, with a role that is yet to be completely explored.


P. cantorii hemocytes in the active state and during hibernation to classify the hemocyte types and obtain the relevant cell maps. We identified the marker genes of each cell population and the differentiation pathways associated with their maturation. We also discovered different immune-related genes that might play crucial roles in hemocyte differentiation before and after hibernation. Our study aims to provide a reference dataset for the unified classification of P. cantorii hemocytes and improve our understanding of the immune system of P. cantorii.Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In this study, we isolated and performed scRNA-seq analysis on Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation.Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from  Hemocytes have significant roles in homeostasis maintenance and immunity and coagulation ,2,3, andPelochelys cantorii) belongs to the family Trionychidae and is distributed in Southeast Asia, including China, India, Bangladesh, Myanmar, Laos, Thailand, Cambodia, Vietnam, Malaysia, and Indonesia. P. cantorii has been listed as a first-class national protected animal in China since 1989 due to overhunting and habitat destruction [P. cantorii is similar to that of the Chinese soft-shell turtle (Pelodiscus sinensis); thus, P. cantorii is not always identified as a separate species, is confused with P. sinensis, and is eaten illegally [P. cantorii is easy to spot because of its large size, and its long period of sexual maturation has also accelerated its extinction. A recent survey showed that only 13 wild P. cantorii were kept in captivity in 6 different locations [P. cantorii.The Asian giant softshell turtle  is a new technology for high-throughput sequencing analyses of genomes, transcriptomes, and epigenomes at the level of a single cell. ScRNA-seq can annotate unclassified cells according to the mRNA expression pattern of each cell and, thus, is a particularly powerful tool for analyzing cell diversity and heterogeneity, predicting the functions of single-cell populations, and determining functional roadmaps of cell differentiation ,19,20. SP. cantorii hemocytes in the active state and during hibernation to classify the hemocyte types and obtain the relevant cell maps. We identified the marker genes of each cell population and the differentiation pathways associated with their maturation. We also discovered different immune-related genes that might play crucial roles in hemocyte differentiation before and after hibernation. Our findings provide a reference dataset for the unified classification of P. cantorii hemocytes and improve our understanding of the immune system of P. cantorii.Here, we isolated and performed scRNA-seq analysis on P. cantorii specimens were acquired from the Asian giant softshell turtle artificial breeding base of the Ministry of Agriculture and Rural Affairs, Foshan, Guangdong, China. The mean weight of P. cantorii was 650.82 g during the active phase of the year (summer 2022) and 666.57 kg during the hibernation period (January 2022). Individuals during the active phase were cultured in outdoor cement ponds with circulating filtered water. The water depth in the pool was 50 cm, and the sand thickness at the bottom of the pool was 30 cm. The cultured density was 1 animal per square meter. When the water temperature drops to about 10 °C degrees in January, put P. cantorii in a box with water in the incubator. The water depth in the box was just above the turtle’s back. Then, we reduced the temperature by 1 degree every 2 days and kept it at 4 °C for 2 weeks before sampling. At this time, the turtles were in the maintenance period of hibernation. To reduce the chance brought by individual differences, 5 mL blood of 5 P. cantorii specimens in the active state and 5 mL blood of these identical 5 P. cantorii specimens during the hibernation period were collected from the jugular vein using an anticoagulant solution, respectively. There were two biological repeats in the active state and hibernation period of individuals. The blood and lymphocyte separation solution was placed in a 20 °C water bath for 20 min, and the whole blood was diluted with the same amount of Hank’s balanced salt solution (HBSS). Subsequently, 4 mL whole blood was slowly added to 2 mL lymphocyte separation solution so that the amount of diluted blood was superplaced on the stratified solution to keep the interface clear, and the solution was then centrifuged at 500× g for 20 min. After discarding the top layer, the mononuclear cells of the white membrane layer were absorbed along the periphery of the centrifuge tube wall and transferred to another centrifuge tube. Then, 5 mL of HBSS was added for washing and centrifugation at 300× g for 10 min, and the supernatant was discarded. The hemocyte pellet was resuspended using 1 mL of HBSS. Cell number and concentration were assessed by Countess® II Automated Cell Counter, and the cell concentration was adjusted to the ideal concentration, which was generally not less than 1000 cells/μL. All animal experiments in this research were performed according to the guidelines established by the Pearl River Fisheries Research Institute, the Chinese Academy of Fishery Sciences. The turtles used were treated humanely and ethically, and the experiments were approved by the Laboratory Animal Ethics Committee of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.Library synthesis and single-cell RNA sequencing were performed by Gene Denovo . In detail, single hemocytes were barcode-labeled and mixed with reverse transcriptase, and then wrapped by oil surfactant droplets located in the microfluidic “double cross” system to form gel beads-in-emulsions (GEMs). GEMs flowed into the reservoir and were collected. Then, silane magnetic beads were used to remove the remaining biochemical reagents and primers from the post-GEM reaction mixture. Subsequently, full-length, barcoded cDNAs were amplified by PCR with the sequencing primer R1 and P5 arms and subsequently sequenced on the Illumina 10×Genomics Chromium platform (Illumina Novaseq 6000).http://support.10xgenomics.com/single-cell/software/overview/welcome, accessed on 12 November 2021) was used to convert raw BCL files to FASTQ files and then perform alignment and count quantification. Briefly, reads with low-quality barcodes and UMIs were filtered out and then mapped to the reference genome. Reads uniquely mapped to the transcriptome and intersecting an exon at least 50% were considered for UMI counting. Before quantification, the UMI sequences were corrected for sequencing errors, and valid barcodes were identified based on the EmptyDrops method. The cell-by-gene matrices were produced via UMI counting and cell barcode calling.Next, 10× Genomics Cell Ranger software , and log-transforms the results. The formula is shown as follows: gene expression level = log  × 10,000). The normalized expression matrix is then scaled and subjected to principal component analysis (PCA) for dimensional reduction. A resampling test inspired by the JackStraw procedure is then performed . We randSeurat software implements a graph-based clustering approach. Distances between the cells were calculated based on previously identified PCs. Briefly, Seurat embeds cells in a shared nearest-neighbor (SNN) graph, with edges drawn between cells via similar gene expression patterns. To partition this graph into highly interconnected quasi-cliques or communities, we first constructed the SNN graph based on the Euclidean distance in PCA space and refined the edge weights between any two cells based on the shared overlap in their local neighborhoods (Jaccard distance). We then cluster cells using the Louvain  method tTo further analyze the characteristics of transcriptional regulation patterns of each cell subpopulation and screen the marker genes specifically expressed from each subpopulation, the expression value of each gene in the given cluster was compared against the rest of the cells using the Wilcoxon rank sum test in Seurat software . SignifiMoreover, genes usually interact with each other to play roles in certain biological functions. Pathway-based analysis helps to further understand gene biological functions, and KOG is the major public pathway-related database . Pathwayhttps://github.com/cole-trapnell-lab/monocle3, accessed on 14 February 2023). The RNA velocity analysis was performed using the package velocyto with the default parameters, and the BAM files were used as inputs. For visualization, velocity vectors were plotted as locally average vector fields on the tSNE embeddings of our high-quality cells from the previous step [To calculate the cell cycle, the Seurat AddModuleScore function was used to score the possible cell cycle period based on the expression of characteristic cycle genes . Cells wous step .P. cantorii in the active state and during the hibernation period. The samples were then prepared for 3′ scRNA-seq using the 10× Genomics platform   was usedemocytes B,C. Moreemocytes . A totalemocytes . For eacemocytes . We thenemocytes A. Dot plemocytes B and vioemocytes C of the emocytes A, suggescd9, cd151, itga2b, and actb was considered the NK-cell population , was clustered into a T-cell population, and Cluster 5, which expressed cd74, cd79a, swap70, crip1, rpl21, rps27, and rps29 belonged to B-cell population  cell-related genes pulation . The neund gata1 . Clusterpulation . In Clusonocytes .stat1, traf3, and socs6. As shown in stat1 was upregulated in NK cells and erythrocytes, traf3 was upregulated in neutrophil cells, erythrocytes, T cells, and monocyte populations, and socs6 was upregulated in neutrophil populations. Moreover, we also identified 62 differentially expressed heat-shock genes  such as dnajb1, dnajb6, dnajb14, dnajc21 and dnajc25, and HSP temperature-responsive genes (HSPs) such as hspa2, hspa4, hspa4l, hspa5, hspa8 and hspa13  and HSP temperature-responsive genes (HSPs) were differentially expressed between hemocytes in the active state and during the hibernation period (cirbp, traf3, dnajb1, dnajb6, dnajb14, dnajc21, dnajc25, hspa2, hspa4, hspa4l, hspa5, hspa8 and hspa13, may play a critical role during cell stress to prevent the appearance of folding intermediates that lead to misfolded or otherwise damaged molecules [In addition, we also found that many genes encoding heat shock genes related to the stress response including Hsp40 family chaperone genes (n period C, which olecules . P. cantorii. This may be a response to protein damage caused by stress during hibernation. We also found that several metabolism-associated pathways such as “endocytosis”, “platelet activation”, and “regulation of actin cytoskeleton” were abundant among the downregulated DEGs (P. cantorii stays in the hibernating refuge, the expression of genes involved in metabolism-associated pathways were suppressed to save energy.Moreover, most of the pathways significantly annotated by upregulated genes were enriched in “proteasome” and “nucleocytoplasmic transport” pathways , indicatted DEGs . When thP. cantorii hemocytes in the active state and during hibernation to classify the hemocyte types and obtain the relevant cell maps. Our study aims to provide a reference dataset for the unified classification of P. cantorii hemocytes and improve our understanding of the immune system of P. cantorii.In this study, we isolated and performed scRNA-seq analysis on


This study aimed to characterize the safety and efficacy of DC-CIK therapy in two patients with previously treated chronic lymphocytic leukemia or peritoneal cancer, respectively. Participants had received conventional chemotherapy treatment for their specific cancers, and in addition, 1–2 treatments of DC-CIK therapy were administered to subjects over the course of 1 year. Subject A received an initial dosage of 3 intravenous infusions of DC-CIK therapy on three successive days and a repeat dosage 6 months later. Subject B received an initial dosage of 3 intravenous infusions of DC-CIK therapy on three successive days and received further chemotherapy after approximately 1 year. No treatment-related adverse events were reported, and both patients experienced favorable outcomes from the treatment, including enhanced treatment response, increased chemotherapy tolerance, and prolonged survival in comparison to typical 5-year survival rates. Dendritic cells (DCs) are antigen-presenting cells of the immune system. Their functions are to capture and process tumor antigens, express lymphocyte costimulatory molecules, and secrete cytokines to initiate immune responses , 2. CytoClinical trials have suggested that DC-CIK therapy can work as an adjunct alongside chemotherapy to support the production of tumor-reducing cytokines and subsequently slow tumor progression –6. Meta-With the preclinical trial evidence, alongside the clinical trial data available, we hypothesized that treatment with DC-CIK immunotherapy as an adjunct to chemotherapy would improve patient outcomes in two forms of cancer: peritoneal cancer, and chronic lymphocytic leukemia. This is the first recorded DC-CIK treatment of either cancer available in the current literature.Two female subjects were treated at Nemocnica Malacky Hospital  for differing cancer diagnoses between April 2016-May 2020. Informed consent was obtained through the signature of an informed consent form from both patients during the enrollment stage of the study, with written consent obtained from each subject to publish the potentially identifiable data contained in the report. The study was approved by the Institutional Review Board of the Institute of Regenerative and Cellular Medicine (IRCM-2021-308). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000.in vitro following treatment with GM-CSF, IL-4, and TNFα cytokines. The expanded DC-CIK cell population was subsequently cultured with the patient-specific tumor antigen to immunologically prime the DC-CIK therapy to target the patient’s cancer.Both subjects received DC-CIK as an adjunct to traditional therapy. The subject’s mononuclear cells, including DC and T-cells, were expanded 7 cells per 5 mL, 8.65 × 107 cells per 5 mL, and 1.4 × 108 cells per 5 mL, respectively. Weekly laboratory blood tests were completed for 4 weeks to monitor for change. Leukocyte numbers dropped significantly post-treatment and remained in the 20,000–30,000-figure range until a gradual increase began in December 2017  of the B-cell series in July 2017 . The patber 2017 . There w9, 2.54 × 109, and 2.58 × 109 cells per 5 mL, respectively, delivered in a 50 cc saline infusion. There were no adverse effects reported related to the immunotherapy administration.Subject B was 70 years of age when diagnosed with secondary-malignant neoplasm of retroperitoneum and peritoneum in November 2015 . CT and 8, 2.71 × 108, and 3.35 × 108 cells per 5 mL, respectively. Over the following months, repeated CT imaging demonstrated a gradual progression of the underlying disease with increasing peritoneal metastases, malignant lymph nodes in the left pelvis, and further metastases in the spleen. It is important to note that at this stage in treatment, it was reported that the treating oncologist discouraged the patient from engaging in the trial further due to misconceptions and a lack of confidence in the immunotherapy treatment. In June 2019, there were interstitial changes in the upper lung lobes with a differential diagnosis of carcinomatous lymphangiopathy with interstitial inflammation and fibrosis. Following a diagnosis of a new hypermetabolic focus in the tail of the pancreas, the patient declined further active treatment, and a palliative care pathway was initiated. However, as of late 2022, almost 5 years after the initial diagnosis, Subject B remains alive.In May, the patient underwent blood testing, CT, and oncological review by the treating medical team. Blood counts remained unchanged, and CT findings included significant regression of findings, including size regression of the hypodense formation in the left ovary. The patient then underwent six cycles of chemotherapy and four cycles of bevacizumab. It was reported that Subject B tolerated chemotherapy better with fewer side effects and, as a result, was able to undergo further surgery. In August, a radical hysterectomy and omentectomy were completed alongside further chemotherapy. The patient received a follow-up dosing regimen of DC-CIK therapy in January 2017. Cell dosage consisted of 4.08 × 10Prior to treatment, subjects underwent screening and baseline assessments  and examination of medical records. Prior to scheduling, the subject’s medical history and records were examined by the investigator. Once enrolled, the EORTC QLQ-C30 questionnaire, disease-specific questionnaires, serological tumor markers test, the percentage of Treg cells in the peripheral blood, ultrasound, or X-ray, were administered.Subjects were tracked for 3 years to assess the safety of DC-CIK therapy and to evaluate its effect on cancer therapy. At the end of 3rd-year final questionnaires and tests  will be administered to assess the overall safety of DC-CIK therapy and effect on cancer therapy.7 to 2.58 × 109 per dose. Other studies have utilized a range of dosages and treatment regimens such as four treatments of 1.3 × 109 cells intervals of a month (10 for 18 cycles (7 every second day for 6 days (The case report describes two patients with different forms of cancer who achieved positive results when treated with a combination of chemotherapy and DC-CIK immunotherapy. As far as we are aware, this is the first report of DC-CIK therapy for CLL and peritoneal cancer, respectively. The use of DC-CIK as an adjunct to standard cancer treatments has piqued the interest of oncologists due to convincing results reported by preclinical and clinical trials in several different cancer types , 11–14.  a month , 16, 1.28 cycles  or 1.27 r 6 days . With vaSubject B notably experienced a reduction in chemotherapy side effects and increased tolerability of the chemotherapy, which further allowed her to undergo significant surgery with curative intent. This has been a trend reported in DC-CIK immunotherapy trials whereby the adverse effects of chemotherapy can be significantly reduced by this treatment. It is thought that the treatment reduced leukopenia, peripheral neuritis, gastrointestinal side effects, liver dysfunction, and myelosuppression , 7, 13. There is a significant pathophysiological difference between the cancer of the two subjects, specifically the contrast between solid and liquid cancers. Both the solid and liquid cancer subjects in this trial experienced positive outcomes in their overall cancer treatment, echoing results demonstrated by previous research in both groups , 13, 14.However, the significant disparities between the two patients in this study also present a limitation to the work. It is difficult to ascertain specific trends or model the generalizability of this treatment on either solid or liquid cancers due to the heterogeneity of the cancer types. In addition, it is challenging to isolate the contribution of the DC-CIK therapy to the therapy when dealing with diverse cancer types. Other factors related to the cancer type should be considered, such as its aggressiveness, stage, and response to standard treatments.Only around 65% of patients over 80 years are expected to survive for 5 years or more after a diagnosis of CLL , 21. ThiAs we are aware, this is the first reported case of the treatment of peritoneal cancer with DC-CIK. Although Subject B’s primary tumor was considered peritoneal, she also suffered from ovarian, pancreatic, and liver metastases, all of which have demonstrated promising results when treated with DC-CIK in previous research , 24, 25.In this report, we describe the case of two female patients with previously treated lymphocytic leukemia or peritoneal cancer, respectively, who received DC-CIK immunotherapy. The patients presented with different manifestations of cancer yet experienced similar outcomes on their treatment journey. Neither patient experienced adverse effects directly related to the DC-CIK therapy, and one patient reported significantly fewer side effects from chemotherapy post-immunotherapy administration. Subject A exceeded the 5-year survival rate for patients with her degree of disease despite being markedly older in age. Subject B is the first reported case of DC-CIK treatment for primary peritoneal cancer, and despite initiating a palliative care pathway in 2017, she remains alive 5 years later. In line with previous research, this case report supports the usage of DC-CIK immunotherapy in conjunction with standard cancer treatment and chemotherapy and highlights the need for further research and development of accessible DC-CIK therapy.The original contributions presented in this study are included in this article/supplementary material, further inquiries can be directed to the corresponding author.Written informed consent was obtained from each subject to publish the potentially identifiable data contained in the report.BM conceived, designed the study, and treatment protocol. RM set up the study, obtained the ethical approval, managed the patients, and collected data. EO’G and DoS analyzed the data, interpreted the results, and wrote the manuscript. All authors read and approved the final version of the manuscript.