Only 13 to 50% of patients with metastatic melanoma respond to new commercialized therapies. The reason why the same chemotherapeutic treatments yield different responses in patients can be attributed to the degree of multidrug resistance (MDR) developed by the host tumor cells. For instance, the glycolytic metabolism of cancer cells enhances the intratumoral accumulation of lactic acid, decreases intratumoral pH and potentiates MDR. Lipid nanoparticles (LNC) have been widely exploited as carriers of MDR reversing molecules. In this study, we proposed to modify LNC with novel copolymers to impart stealth properties and to improve tumor cell entry. Modified-LNC showed in vitro pH-responsive properties characterized by an enhanced cellular uptake under acidic conditions. Moreover, surface modification led to an increased biological effect by protecting the nanocarrier from opsonization by complement activation. These data suggest that pH-sensitive LNC are promising nanocarriers to target metastatic melanoma.Despite significant advances in melanoma therapy, low response rates and multidrug resistance (MDR) have been described, reducing the anticancer efficacy of the administered molecules. Among the causes to explain these resistances, the decreased intratumoral pH is known to potentiate MDR and to reduce the sensitivity to anticancer molecules. Nanomedicines have been widely exploited as the carriers of MDR reversing molecules. Lipid nanocapsules (LNC) are nanoparticles that have already demonstrated their ability to improve cancer treatment. Here, LNC were modified with novel copolymers that combine N-vinylpyrrolidone (NVP) to impart stealth properties and vinyl imidazole (Vim), providing pH-responsive ability to address classical chemoresistance by improving tumor cell entry. These copolymers could be post-inserted at the LNC surface, leading to the property of going from neutral charge under physiological pH to positive charge under acidic conditions. LNC modified with polymer P5 (C18H37-P(NVP21-co-Vim15)) showed in vitro pH-responsive properties characterized by an enhanced cellular uptake under acidic conditions. Moreover, P5 surface modification led to an increased biological effect by protecting the nanocarrier from opsonization by complement activation. These data suggest that pH-sensitive LNC responds to what is expected from a promising nanocarrier to target metastatic melanoma. In recent years, cancer treatments have evolved considerably with the rise of new strategies for targeting the tumor microenvironment. Thus, targeted therapies and monoclonal antibodies have improved the prognosis and survival of many cancer patients. Despite these significant advances, low response rates and multidrug resistance (MDR) have been described for certain molecules, reducing their efficacy. For instance, in metastatic melanoma, it has been reported that only 13 to 50% of patients respond to these new therapies with, sometimes, a delay of more than three months . The reaMDR is associated with a wide range of pathological changes at different cellular and tissular levels. In a previous study, we found some differences in B16F10 and SKMel28 melanoma vasculature that could potentiate therapy resistance . MoreoveAmong the various hypotheses explaining MDR, recent data have suggested that the tumor microenvironment could play a key role in this therapeutic resistance . The oriP-gp as it has been reported with etoposide- or paclitaxel-loaded lipid nanoparticles in in vitro and in vivo studies [Nanomedicines have been widely exploited as the carriers of MDR reversing molecules . Moreove studies ,16,17,18 studies ,16 has p studies ,24,25,26stimuli-responsive nanocarriers, which could adapt to the intrinsic physicochemical and pathological factors to increase the specificity of drug delivery. Currently, numerous nanocarriers have been engineered with physicochemical changes in responding to external stimuli, such as pH, due to the nature of low pH inside the organelles  of cancer cells and in the tumor microenvironment, a way to overcome multidrug resistance. Indeed, positively charged nanocarriers generally exhibit a higher cell uptake due to increased electrostatic interactions between engineered nanomaterials and the negatively charged cell membrane [These last years, much progress has been motivated in membrane ,28,29. Tmembrane , pH-respmembrane ,32 as vimembrane ,34.18H37- alkyl chain able to be post-inserted at the surface of LNC. A library of copolymers of various compositions and molar masses was then used to decorate LNC  (PNVP) , which hrate LNC , and thew/w caprylic-capric acid triglycerides , 17.2% w/w hydroxystearate-PEG , 8% w/w NaCl , 1.5% w/w lecithin  and 35.58% of Milli-Q water. Three cycles of heating and cooling between 60 and 90 °C were carried out to obtain the phase inversion. Ice-cold Milli-Q water 23.72% w/w was added at the last inversion temperature leading to the formation of lipid nanocapsules.Lipid nanocapsules were formulated as described  by mixinexcitation = 456 nm; λemission = 590 nm, Fisher Scientific, Illkirch-Graffenstaden, France), was used. Prior to formulation, DIA was incorporated in Labrafac at a concentration of 7.92 mg/g, then the same formulation process as above was used to formulate LNC.To formulate fluorescent LNC, a fluorescent probe, DIA (4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide; λLNCs and polymers from ® apparatus  at 25 °C. Measurements were performed in 1 mM phosphate buffer Na2HPO4  + NaH2PO4  at different pH: 7.4, 6.8, 6.5, 6. LNC was 10 times diluted. Experiments were conducted 4 times with a measured average value calculated from 3 runs, with 12 measurements by a run for size and 20 measurements by run for zeta potential.The mean diameter (Z-average) and polydispersity index (PDI) of LNC were measured using the dynamic light scattering (DLS) method at a backscatter angle of 173°. Zeta potential was measured using the laser Doppler microelectrophoresis technique with a Malvern ZetasizerLNC was kept at 4 °C, stability in size, PDI, and zeta potential evaluated at days 0, 7, 14, 21. Experiments were repeated 3 times using the method described above.The buffering capacities of the different polymers alone and post-inserted into LNC were measured by acid–base titration according to the method described by Zhong et al. . 1 mL of2. To obtain pH-modified media, a complete culture medium was maintained for 24 h at 37 °C in a humidified atmosphere with 5% CO2. Media was then buffered to pH 7.4; 6.8; 6.5, and 6 using a NaH2PO4 2 M solution.The SK-Mel 28 human melanoma cell line, obtained from ATCC , and B16F10 mice melanoma cell line (gift from University of Brussels) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% heat-inactivated FBS (Lonza), 1% antibiotic and antimycotic solution (Sigma-Aldrich) and 1% non-essential amino acids (Lonza). Cell lines were cultured and maintained at 37 °C in a humidified atmosphere with 5% CO4 cells/well and incubated for 24 h. Cells were then treated with 100 µL/well of media containing either post-inserted LNCs at a concentration range of 10 µg/mL to 1000 µg/mL or polymers at the corresponding concentration contained in post-inserted LNCs (0.025 µM to 2.5 µM). After treatment, cells were washed with PBS. Cytotoxicity was determined by evaluating cell viability via indirect quantification of living cells using resazurin assay (Sigma-Aldrich). Briefly, 100 µL of resazurin (22.5 µg/mL) was added to each well, and the plates were incubated for 3 h under a humidified atmosphere with 5% CO2. The fluorescence intensity was then measured at 544 nm excitation/599 nm emission wavelengths using a Fluoroskan Ascent™ microplate fluorometer (Fisher Scientific). Experiments were repeated six times with three replicates per experiment.B16F10 and SK-Mel 28 cells were cultured in a 96-well plate at the density of 5 × 106 and 2.50 × 106 cells/well for B16F10 and SK-Mel 28, respectively. Then, cells were incubated for 24 h and were then treated for 2 h with 2 mL medium at different pH  with fluorescent LNCs at a concentration of 250 µg/mL. Cells were washed with PBS, collected after trypsinization and washed twice with PBS. To discriminate cell membrane adsorbed or internalized LNCs, cells were resuspended in 2.5 mg/mL trypan blue to quench extracellular fluorescence. Analyses were performed with a BD FACSCanto™ II flow cytometer . Experiments were repeated four times with three replicates per experiment.The uptake of unmodified LNC (BLK LNC) and modified LNC at different pH was measured by flow cytometry analysis. Cells were seeded onto 6-well plates at the density of 5 × 106 cells/well for B16F10 and 2.5 × 106 for SK-Mel 28 and incubated for 24 h. The cells were then treated with inhibitors for 30 min at 37 °C. Then, 1 mmol/L 5-amiloride hydrochloride (DAM), 10 mg/mL methyl-β-cyclodextrin (MβC) and 50 µmol/L chlorpromazine (chlorpr.) were used to inhibit macropinocytosis, lipid RAFT and mediated endocytosis pathways, respectively [To study the internalization pathways of pH-responsive LNC, different inhibitors were used. Cells were seeded onto 6-well plates at the density of 5 × 10ectively . 10 µg/mectively . Cells wectively  and excl4 cells/well and incubated for 24 h. The cells were then treated for 2 h with 300 µL of the medium at different pH  with fluorescent LNC at a concentration of 250 µg/mL. Cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min at 4 °C. Cells were then washed with PBS, and nuclei were stained with 4′,6-diamidino-2-phenylindole 3 µM DAPI (Sigma-Aldrich). Finally, cells were washed with PBS, and Fluoromount™ aqueous mounting medium (Sigma-Aldrich) was added to the slide. All images were collected with a Leica TCS SP8 AOBS confocal laser scanning microscope  equipped with an HC PL APO CS2 63x/NA 1.40 oil objective with 2x numerical zoom and 40x/NA 1.30 oil objective and gateable hybrid detectors (GaAsP). Images were acquired in the format 1024 × 1024 pixels, the bit depth of 8, and a scan speed of 400 Hz. DAPI and DiA were excited with a 405 nm diode laser (50 mW) and the 488 nm line from an argon laser (40 mW), respectively. Z-series optical sections were collected with a step size of 1 µm using a Super Z Galvo Type H stage and were displayed as maximum z-projections using the LAS X software.Confocal imaging of internalized cells was performed using a confocal laser microscope , equipped with a 50 mW diode laser. SK-Mel 28 melanoma cells were seeded onto 8-well polymer μ-slides  at the density of 25 × 102+ and 0.5 mM Mg2+ (VBS++) and incubated for 1 h at 37 °C with an increased concentration of LNC. The suspension was then diluted in VBS++ (1/25 v/v) and incubated for 45 min at 37 °C with sheep erythrocytes (Labor Dr. Merk) sensitized by rabbit anti-sheep erythrocyte antibodies . The suspension was centrifuged at 800× g for 10 min. The light absorption of the supernatant was then read at 405 nm with a microplate reader . The amount of serum able to lyse 50% of sensitized erythrocytes (CH50) was calculated for each sample using the formula:Complement activation was determined by measuring the lytic capacity of normal human serum after exposure to the LNC, according to Passirani et al. . BrieflyTo compare the different modified LNC, the complement consumption was plotted as a function of the surface area. The surface area was calculated using the formula described previously .p < 0.05.For the statistical analysis, the results were analyzed using a Kruskal–Wallis test followed by a Dunn’s post hoc test with a Hochberg correction using the R software  with the PMCR package . The lev18H37-P(NVP-co-Vim)) has been performed with different compositions to potentially modulate the surface charge of the LNC (The synthesis of a series of hydrophilic NVP and Vim-based copolymers (data not shown) bearing a hydrophobic aliphatic chain in α-position  (p < 0.05) at pH 6.5 and pH 6 with an increase of around 12 mV between pH 7.4 and 6.Post-inserted LNC with polymers P3, P4 and P5 showed significant differences in their mean hydrodynamic diameters, compared to unmodified LNC (BLK) at the same pH ( and P5) A. The di and P5) . Under n and P5) B. SurfacThe ability of LNC to escape endosomes efficiently was evaluated by studying the buffering capacities of each polymer and modified LNC through acid–base titration, as reported in 2/mL), LNC post-P5 presented a higher consumption of CH50 units: 44% compared to 20~25% for all other post-inserted LNC and 11% for BLK LNC.Complement consumption for each LNC was studied, and it increased with LNC concentration . BLK LNCDue to the heterogeneity between human and murine cell lines, the impact of polymers on cell viability was evaluated on B16F10, a murine melanoma cell line, and on SK-Mel 28 cells, a human melanoma cell line. The impact of modified LNC is reported in p < 0.01). Interestingly, this reduction was not observed with the LNC post-P5 (p > 0.05). Compared with BLK LNC, LNC post-P5 showed a significant increase of internalization at pH 6 and 6.5 for B16F10 cells A and at +/H+ exchanger involved in macropinocytosis [p < 0.001). Cellular uptake of LNC post-P5 in B16F10 was significantly decreased (p < 0.05) by approximately 50% after pretreatments with DAM and MβC. For SK-Mel 28, cellular uptake was significantly reduced (p < 0.05) by 60% and 80% after pretreatments with DAM and PMA, respectively. At 4 °C, energy-dependent pathways, such as endocytosis and pinocytosis are blocked. Inhibition of cell uptake observed at this temperature highlighted active internalization of LNC post-P5 in these melanoma cell lines, rather than diffusion across the cell membrane.Taking into consideration the efficient internalization of LNC post-P5 in the cytoplasm under acid conditions, the cellular uptake mechanism of these modified objects was investigated at pH 6. Control condition received only LNC post-P5, while other conditions were pretreated with different uptake inhibitors. DAM inhibits the Naocytosis . MβC disocytosis ,45. Thisocytosis . PMA inhocytosis , and chlocytosis . AdditioInterestingly, this study allowed developing new nanovectors sensitive to the variations in pH described at the tumor level. This project opens up interesting prospects for improving the targeting of metastatic melanoma cells resistant to various therapies.® HS 15) , was realized with the C18H37-P(NVP-co-Vim) copolymers reported in 18 mono carbon chain. The post-insertion of polymers at the LNC surface was confirmed by an increase in their hydrodynamic diameter and a change in their surface charge. Surface charge is a key parameter for stability, circulation time and cellular uptake. As expected, the zeta potential of the pH-responsive LNC was driven by Vim protonation and increased according to the Vim content in the polymers . With a pKa value of our polymers around 6, following literature [Surface modification of lipid nanocapsules is usually done by post-insertion of lipid polymers composed of double carbon chains, for example, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) ,49. In tterature ,51, and terature , Vim groFurthermore, the protonation of Vim groups in an acidic environment can bring buffering property to LNC. The buffering capacity of pH-responsive polymers plays a key role in preventing degradation of encapsulated therapeutics, such as nucleic acid, for example, by escaping the endosome through the “proton sponge effect” . Some stOnce intravenously injected, a possible neutralization of our LNC by the immune system of the receiving host or by natural killer-associated microvesicles could occur. The accelerated blood clearance (ABC) phenomenon is one of the pharmacokinetic consequences of this immune reaction, as observed with pegylated nanocarriers. This phenomenon was not observed with PNVP that did not potentiate any IgM production after a second intravenous injection ,59, maki−1)  . This mol−1 PVim . Cellulal−1 PVim . These rThese results were confirmed by confocal microscopy. Due to the highest pH-responsive behavior of LNC post-P5 compared to other post inserted LNC, we investigated the internalization mechanism of such modified LNC. Interestingly, B16F10 and SK-Mel 28 showed different mechanism pathways. The B16F10 cell line showed a reduction of cell uptake with DAM and MβC, suggesting that LNC post-P5 used macropinocytosis, whereas cell uptake of LNC post-P5 by SK-Mel 28 occurred through a balance between macropinocytosis and caveolin-dependent endocytosis. Cellular uptake pathways can directly impact the fate of nanocarriers in the cytoplasm. Compared to macropinocytosis and clathrin-dependent endocytosis, caveolae-mediated endocytosis is a preferred pathway as the caveosome cellular compartment is not subject to pH and enzyme degradation as observed in endosomes and lysosomes. Cell uptake pathways can differ with the cell line. The “proton sponge effect” of these modified LNC should, therefore, provide the capacity to escape endosomal degradation if cellular uptake occurs through macropinocytosis and clathrin-dependent endocytosis pathways.The MDR phenotype on the membrane level is characterized by three main features; reduction of the transmembrane diffusion of the hydrophobic drugs, efflux of the membrane entrapped drug molecules by the ABC transporters and impairment of the endocytic function. Our data suggest that, due to our formulation strategy, pH-sensitive nanovectors would be able to reverse MDR phenotype to improve cancer treatment. Furthermore, as previously studied by our group ,63,64,65The surface coating of nanomedicines by hydrophilic polymers is a common modification used to provide prolonged blood residence. With the increased presence of anti-PEG antibodies in the general human population , alterna


The aim of this study was to compare patients’ satisfaction and quality of life (QoL) when using implant overdentures vs. conventional dentures. The QoL of Saudi patients who were provided mandibular implant-supported overdentures was assessed using a structured questionnaire. Overall satisfaction; ability to speak, eat, and chew food; comfort; esthetics; stability; and satisfaction of general oral health were measured. A total of 48.3% vs. 6.9% were, overall, very satisfied with their implant overdentures and conventional dentures, respectively. A total of 37.9% of the patients were very satisfied regarding speaking with their implant overdentures vs. 17.2% with conventional dentures. Furthermore, 41.4% were very comfortable with their implant overdentures vs. 5.2% were very comfortable using conventional dentures. However, only 15.5% were very satisfied with the esthetics of the conventional dentures compared to 43.1% being satisfied with implant overdentures. Only 1.7% were very satisfied with the stability of conventional dentures vs. 44.8% being satisfied using implant overdentures. About 8.6% of the candidates were very satisfied regarding chewing food with conventional dentures vs. 36.2% being very satisfied using implant overdentures. Approximately 10.3% were very satisfied with their general oral health using conventional dentures compared to 29.3% being very satisfied using implant overdentures. Mandibular implant overdentures had a strong impact on patients’ quality of life over conventional complete dentures and should be considered the minimum standard of care provided to completely edentulous patients. Rehabilitation of edentulous patients with removable dentures is a long-standing procedure that was first prescribed by Purmann in 1684 . Shah etConventional removable complete dentures  still fail to provide superior function and comfort for patients. In the 1990s, and after the introduction of endosseous dental implants and their use to retain and/or support removable prostheses, many authors started to evaluate the differences between conventional dentures and implant-retained overdentures ,5. Awad A study that was conducted by Cibirka in 1997 showed a significant improvement in self-image among the implant group along with better function and comfort . AnotherSeveral reports on satisfaction with implant-supported prostheses among edentulous patients have been published, showing a significant improvement in psychological self-image among the implant group along with better function and comfort ,11.In 2002, the McGill consensus declared that “two-implant mandibular overdentures should replace mandibular conventional dentures as the minimum standard of care for completely edentulous patients” . This waImplant-supported overdentures have different types of attachments, and their use is affected by multiple factors including the practitioner’s choice and preference. There have been studies that advocate the use of bar attachments over stud attachments, as reported by Boven 2019) ; however ; howeveMany forms and scales exist by which to estimate patients’ quality of life (QoL), such as the Oral Health Impact Profile (OHIP) and Oral Health-Related Quality of Life (OHRQoL). In terms of prosthodontically-related evaluation, an assessment of prosthesis by McGill University in 2001 evaluated both patients’ satisfaction and QoL.Although several studies have established that implant-retained overdentures are better than conventional dentures in terms of stability, retention, comfort, and masticatory function, currently, there are no studies performed on a Saudi Arabian population. Thus, the aim of this survey was to compare patients’ satisfaction and QoL when using conventional complete dentures vs. implant-supported overdentures. The results of this study may help implement implant-retained overdentures as a first choice in the treatment plan of edentulous patients in Saudi Arabia.Fifty-eight edentulous individuals who were provided mandibular implant-supported overdentures in the Kingdom of Saudi Arabia were included. Quality of life (QoL) was assessed by using a structured questionnaire, self-filled by participants who were selected by convenience sampling. Each respondent was ensured of their confidentiality and anonymity, with sufficient time given to fill the questionnaire. Illiterate participants were interviewed by questionnaire distributors or any of the caregivers.The inclusion criteria included edentulous patients who use mandibular two-implant-supported overdentures for more than 3 months and those who had previous experience using conventional complete dentures. The exclusion criteria included candidates without previous experience with conventional complete denture use, those who do not use their implant-supported overdenture, and patients with maxillary implant-retained overdentures or with tooth-supported overdentures. Moreover, patients with less than 3 months of having an implant-supported overdenture were not questioned because the period of time was insufficient to measure QoL after receiving the treatment.The study protocol was approved by both the College of Dentistry Research Center (CDRC) and the Institutional Review Board (IRB) at King Saud University (KSU), Saudi Arabia. The study was performed in accordance with the ethical principles of the Helsinki Declaration and KSU policies and guidelines for research. All of the patients’ information from the included cases was confidential and kept anonymous. Participation was voluntary; the nature of the study was explained at the beginning of the questionnaire; no attempt was made to collect the participants’ information.The questionnaire used was an Arabic translation of the McGill University 2001 Assessment of Prosthesis [p-value of ≤0.05 was used to report the statistical significance of results.The data were analyzed using the SPSS 26.0 statistical software . Descriptive statistics (frequencies and percentages) were used to describe the categorical study and outcome variables. A non-parametric statistical test (Wilcoxon signed-rank) was used to compare the repeated 5-point scale responses of the study subjects to compare the satisfaction levels with the use of conventional dentures vs. implant overdentures. A Out of 58 study subjects who received implant-retained overdentures, 33 (56.9%) were male subjects; more than 80% of them were over 45 years of age. A total of 41 (70.7%) of the subjects were living in the central region of Saudi Arabia, and 29 (50%) had been using implant overdentures for more than 2 years. Approximately two thirds of all subjects (65.5%) had the perception that their oral health negatively affected their overall general health .The quality of life was assessed using 13 items that measured the subjects’ level of satisfaction with conventional dentures and implant overdentures. The 5-point scale  was used to assess patients’ satisfaction levels.‘ability to speak’, 17.2% and 19% of the patients were very satisfied and satisfied with their conventional denture, respectively; 37.9% and 36.2% of them were very satisfied and satisfied using implant overdentures, respectively. For the ‘comfort’ item, only 5.2% and 25.9% of the patients were very satisfied and satisfied, respectively, with the conventional denture, whereas a higher proportion (41.4% and 31%) of the patients were very satisfied and satisfied with the implant overdenture. The assessment of ‘satisfaction with esthetics’ showed that 15.5% and 5.2% of the subjects were very satisfied and satisfied, respectively, compared to 43.1% and 3.4% of the subjects using implant overdenture. For the ‘satisfaction with stability’ item, only 1.7% and 3.4% of the candidates were very satisfied and satisfied, respectively, compared with the proportion of 44.8% and 1.7% of very satisfied and satisfied patients using implant overdentures. The assessment of the ‘difficulty in chewing food’ showed that only 8.6% and 12.1% of the study subjects expressed that they were very satisfied and satisfied with the use of conventional dentures compared to 36.2% and 31% of the patients who were very satisfied and satisfied with the use of implant overdentures, respectively. For the item ‘difficulty in chewing and eating white bread’, only 3.4% and 17.2% stated that they were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 31% and 34.5% who mentioned that they were very satisfied and satisfied with the use of implant overdentures, respectively. For the item ‘difficulty in chewing and eating dates’, only 6.9% and 17.2% stated that they were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 27.6% and 31% of the subjects who mentioned that they were very satisfied and satisfied with their implant overdentures, respectively. For the item ‘difficulty in chewing and eating cheese’, approximately 10.3% and 20.7% were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 29.3% and 39.7% who were very satisfied and satisfied with their implant overdentures, respectively. For the item ‘difficulty in chewing and eating meat’, only 1.7% and 13.8% of the subjects were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 24.1% and 34.5% of them who were very satisfied and satisfied with the use of implant overdenture, respectively. For the item ‘difficulty in chewing and eating apple’, only 1.7% and 15.5% of the patients were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 17.2% and 32.8% of them who were very satisfied and satisfied with the use of implant overdentures, respectively. The assessment of satisfaction item ‘difficulty in chewing and eating lettuce’ showed that only 3.4% and 15.5% of the patients were very satisfied and satisfied, respectively, while using conventional dentures compared to a higher proportion of 32.8% and 22.4% who stated that they were very satisfied and satisfied with the use of implant overdentures, respectively.The assessment of the ‘overall satisfaction’ with conventional dentures showed that only 6.9% and 1.7% of the patients were very satisfied and satisfied, respectively. However, for implant overdenture, 48.3% and 1.7% of them were very satisfied and satisfied, respectively. The assessment of ‘ease of cleaning mouth and denture’ showed that 29.3% and 41.4% of the subjects using conventional denture were very satisfied and satisfied, respectively; 34.5% and 43.1% of those using implant overdentures were very satisfied and satisfied, respectively. For the item For the item of ‘satisfaction of general oral health’, approximately 10.3% and 1.7% of the patients were very satisfied and satisfied, respectively, with conventional dentures compared to 29.3% and 3.4% of the patients who were very satisfied and satisfied with the use of implant overdentures .p-values indicate a highly statistically significant difference in the responses of study subjects to 12 items regarding their use of conventional dentures and implant overdentures. Specifically, the responses ‘very satisfied and satisfied’ were more frequent for implant overdentures compared to similar responses for conventional dentures. In addition, the responses ‘not satisfied and not satisfied at all’ were significantly less frequent for implant overdentures compared to similar responses for conventional dentures. For the ‘ease of cleaning mouth and denture’ item, there was no statistically significant difference in the responses regarding the use of conventional and implant overdentures (p = 0.583) (= 0.583) .‘Do you think that your oral health has a negative impact on your general health? If yes, why?’. This open-ended question revealed responses such as: the mouth is a reservoir for bacteria and tooth decay; not able to chew food well; the mouth is the means for nourishment for the body; difficulty in eating and want to eat using teeth and be able to talk; it affects both physical health and psychological aspects; cannot eat unless chewing the food well; any sores in the mouth will affect eating and negatively affect the entire body; if food is not chewed well, then stomachache and constipation will occur; with improper chewing of food, indigestion will occur. Some subjects experienced ‘embarrassment while speaking’, and if one’s oral condition is bad, this affects the gingiva and the jawbone, which leads to problems in the whole body.The study subjects were asked This study was conducted to determine the effect of using mandibular implant-retained overdentures on Saudi patients’ satisfaction and QoL compared to using their previous conventional complete dentures. Several studies and consensus statements have established that implant-retained overdentures are a better option for the treatment of mandibular edentulism in terms of retention, stability, and overall QoL ,13,14. ARegarding the ease of cleaning the mouth and dentures, patients reported no significant differences between the two treatment options; however, this contradicts a study performed by Martín-Ares in 2016, who determined that patients with implant-retained overdentures reported better satisfaction with their oral hygiene compared to patients using conventional complete dentures .The patients in this study reported significantly improved satisfaction with speaking, comfort, esthetics, and the stability of the implant-retained overdentures; these results are consistent with those of Melas et al. (2001) and Heydecke et al. (2005), who reported that patients experienced less difficulty in performing daily activities such as speaking, smiling, and eating after the use of implant-retained overdentures ,23. FernThe present study suggests that mandibular implant overdentures considerably affect the ability to chew different types of food. A study by Feine et al. in 2002 documented that many patients wearing conventional dentures complained that they could not eat many types of food, particularly hard food. This issue motivated them to change their diets and made their nutrition worse than that of individuals with natural teeth . This stThe effect of general oral health on patients’ quality of life (OHRQoL) was assessed, and this revealed that the level of patients’ satisfaction improved with the use of mandibular implant overdentures compared to conventional dentures. This is in agreement with a study carried out by Heydecke and colleagues who found that mandibular two-implant overdentures provided better OHQoL and improved patients’ general health . This efThe last question in this study was an open-ended question, which was used to assess the negative impact of oral health on general health. Some patients think that there is a relationship between not chewing food properly and digestive issues; consequently, they believe that their general health will be negatively affected. These findings are in agreement with a study conducted by De Andrade et al., which showed that among elderly Brazilians, a high degree of negative impact of oral health on QoL was associated with patients’ general health . FurtherThe results of this retrospective study showed that mandibular implant-retained overdentures greatly improved the patients’ quality of life (QoL) compared with mandibular conventional complete dentures. The use of two dental implants to retain mandibular overdentures significantly improved denture stability and retention and thus positively enhanced the patients’ comfort, esthetics, and satisfaction while speaking compared with the use of conventional complete dentures. Moreover, the implant overdentures enabled patients to efficiently chew different types of food and hence improved their nutrition and well-being. Patients who participated in this study observed that their oral health has a direct impact on their general health.


In the females, the physical  and psychological  states also did not show significant change between T1 and T2. In both sexes, the continuance of outpatient rehabilitation might be recommended as one activity that can maintain physical and psychological states during a COVID-19-related state of emergency.This study was a sub-analysis of 20 consecutive elderly participants who underwent outpatient rehabilitation at a geriatric health services facility from January 2020 to the end of May 2020, based on our previous report. This study aimed to evaluate the longitudinal changes in their physical and psychological states with respect to gender in rehabilitation outpatients between the pre-nationwide (T1) and post-nationwide state of emergency (T2) caused by the Coronavirus disease 2019 (COVID-19). Gait speed (GS), timed up and go (TUG), handgrip strength (HG), and maximum phonation time (MPT) were measured as indices of physical status. The Japanese version of the Apathy Scale and five-level EuroQoL five-dimensional questionnaire (EQ-5D-5L) were used to assess the psychological state. Both states were measured in the male and female groups at T1 and T2 and then were compared. The final analysis was comprised of 13 outpatients. In males, the physical (GS,  The World Health Organization declared Coronavirus disease 2019 (COVID-19) a pandemic on March 11, 2020 , but heaPreviously, the physical activity of older adults was reported to have dropped significantly as a result of the COVID-19 epidemic ,5. FurthThe purpose of the present study was to determine whether changes occurred in the physical and psychological states with respect to the gender of outpatients undergoing rehabilitation at a geriatric health services facility as assessed before and after the nationwide state of emergency caused by the COVID-19 pandemic in Japan.This study was a sub-analysis of a longitudinal study of 20 consecutive elderly participants who underwent outpatient rehabilitation at the Elder Village geriatric health services facility in Kobe, Japan, from January 2020 to the end of May 2020, based on our previous report . OutpatiOutpatients attended rehabilitation at the geriatric health services facility once, twice, or three times per week for 20 min/session during the state of emergency . Each seGait speed , timed uThe Japanese versions of the Apathy Scale ,16 and tThe EQ-5D-5L is a self-reported questionnaire with which patients self-evaluate their current state of health ,18,19. TParticipants’ characteristics and their results are shown as numbers and as the mean ± standard deviation for the continuous variables. After collecting the data, we separated the physical and psychological assessment results into two groups by gender. As this study’s sample size was quite small, we analyzed longitudinal changes of the physical and psychological states separately in the male and female groups from T1 to T2 using the Wilcoxon signed-rank test. The overall level of statistical significance was set at 0.05. Statistical analyses were conducted with IBM SPSS Statistics 26 .Among the 20 patients, five were excluded because they could not attend outpatient rehabilitation due to their preference and/or their personal convenience or that of their family, and two patients could not walk unaided. Thus, 13 patients were included in the final analysis. Of them, the male group was comprised of six patients . Patient diagnoses included orthopedic disease in three, cardiovascular disease in two, and cerebrovascular disease in one patient. Two patients took an angiotensin II receptor blocker, three took a β-blocker, two a calcium antagonist, one a diuretic, three a statin, and two took analgesics. Exercise time per week during outpatient rehabilitation was 46.7 ± 20.6 min.In contrast, the female group comprised seven patients . Patient diagnoses included orthopedic disease in five and internal disease in two patients. One patient took an angiotensin II receptor blocker, one a β-blocker, four a calcium antagonist, two a diuretic, two a statin, and three took analgesics. Exercise time per week during outpatient rehabilitation was 37.1 ± 7.5 min.The physical and psychological status of the male participants showed no significant change between T1 and T2 .Similar to the findings in the male participants, the physical and psychological status of the female participants also showed no significant change between T1 and T2 .This is the first study, to the best of our knowledge, to investigate changes in physical and physiological states by gender of outpatients participating in rehabilitation at a geriatric health services facility from before to after the state of emergency caused by the COVID-19 epidemic in Japan. In the present study, we lost about 25% of candidates in the survey for “convenience” reasons. This may also be a direct effect of the pandemic. Previously, Yamada et al.  reportedIn the present study, we showed that the physical and psychological states of both males and females did not change significantly between T1 and T2 , did notHowever, Sasaki et al.  also repOne reason may be that the geriatric health services facility continued outpatient rehabilitation according to their policy even during the state of emergency and took adequate safety precautions against COVID-19 infection to help protect the participants. Thus, outpatient rehabilitation as a means of social participation prior to the COVID-19 pandemic might have helped outpatients of both genders maintain their physical status during the pandemic.During the COVID-19 pandemic, mental health data were collected online by Creese et al.  from a pTherefore, if we continue outpatient rehabilitation during the policies enacted during the state of emergency by the Japanese government, the physical and psychological status of the outpatients might not decrease in either gender. In other words, the continuance of outpatient rehabilitation during the study period may have contributed to the maintenance of their physical and psychological states.The present study has several limitations. This study was a sub-analysis of a previous study, only one geriatric health services facility was investigated, the sample size was quite small, the longitudinal evaluation was a short amount of time, and there were several differences in restrictive national policies compare to other countries. Furthermore, there were no control groups, so we could not compare our results with those of patients not participating in outpatient rehabilitation during the state of emergency. Moreover, although we did evaluate longitudinal changes, we could not evaluate cross-sectional gender-related differences in physical and psychological states at T1 and T2. In fact, the female group, which was older and had a higher BMI than the male group, spent less time in rehabilitation. In a future study, we need to determine whether the females underwent less rehabilitation than the males because they were older.Additionally, we investigated exercise time during outpatient rehabilitation per week but did not evaluate other activities of the outpatients, such as recreation or sedentary behavior at the geriatric health services facility by gender. Likewise, previous studies ,21 suggeThe present sub-analysis investigation of both males and females who participated in outpatient rehabilitation at a geriatric health services facility showed that neither gender appeared to have experienced a decrease in their physical and psychological states during the state of emergency brought on by the COVID-19 pandemic in Japan.


Millions of native animals around the world are rescued and rehabilitated each year by wildlife rehabilitators. Triage and rehabilitation protocols need to be robust and evidence-based, with outcomes consistently recorded, to promote animal welfare and better understand predictors of wildlife survival. We conducted a global systematic review and meta-analysis of 112 articles that reported survival rates of native mammals and birds during rehabilitation and after release to determine intrinsic and extrinsic factors associated with their survival. We assessed survival during rehabilitation and in the short- and long-term post-release, with the hypothesis that survival will vary as a function of species body size, diel activity pattern, trophic level and study location (region of the world). We aimed to determine the direction of effect of these factors on survival to assist in decision-making during triage and rehabilitation. Results showed that mammals and birds were equally likely to survive all stages of rehabilitation, and survival rates varied between locations. Birds in North America had the poorest survival rates post-release, particularly long-term, as did diurnal and carnivorous birds in the short-term post-release. Anthropogenic factors such as motor vehicle collisions and domestic or feral animal attack contributed to morbidity and post-release mortality in 45% (168 of 369) of instances. The reasons for rescue and associated severity of diagnosis were commonly reported to affect the likelihood of survival to release, but factors affecting survival were often species-specific, including bodyweight, age, and characteristics of the release location. Therefore, evidence-based, species-specific, and context-specific protocols need to be developed to ensure wildlife survival is maximised during rehabilitation and post-release. Such protocols are critical for enabling rapid, efficient rescue programs for wildlife following natural disasters and extreme weather events which are escalating globally, in part due to climate change. Wildlife rehabilitation is practiced in many countries, resulting in the rescue, care, and release of millions of animals every year . WildlifTo identify factors influencing the success of wildlife rehabilitation, it is first necessary to define “success”, and this may vary among stakeholders. From a wildlife rehabilitator’s perspective, success could constitute recovery from initial injuries and release back into the wild . SuccessCommunities and native animals rely on volunteer wildlife rehabilitators to rescue, rehabilitate and release injured or orphaned wildlife , 15, 16.We used a systematic approach to review survival rates of native mammals and birds during rehabilitation and post-release to determine factors associated with survival. We focused on mammals and birds as these classes are commonly rescued and rehabilitated, with survival data subsequently reported in the literature. The effects of a range of intrinsic and extrinsic factors on survival were evaluated to develop a framework of key considerations for wildlife rehabilitation, and to guide future research on best-practice rehabilitation methods. Specifically, we hypothesised that survival rates during rehabilitation and post-release will vary as a function of species traits that could impact susceptibility to anthropogenic and environmental threats, such as body size, trophic level and diel activity pattern, and survival will vary between regions of the world. Thus, survival likelihood will be species- and context-specific.Animal Welfare, Journal of Raptor Research, and Journal of Wildlife Rehabilitation. Additionally, 16 relevant books, symposium proceedings, reports and theses were used. We used search terms relating to the location of the study, focal taxa, rehabilitation intervention and survival, without date limits (sysrev.com) was used to reduce the returned articles based on title, abstract and keywords. As thousands of articles were returned, they were sorted by relevance then searched until there were 50 consecutive non-selected articles . Full texts were then reviewed against selection criteria as follows: research was conducted on native mammals or birds that entered care for any reason; the sample size and a survival measure and timeframe  during rehabilitation or post-release were reported; and the article (or abstract) was available online and in English. Few studies included a control group or intervention other than rehabilitation, so this was not a requirement for inclusion. Where studies included a control group, this involved monitoring a sample of wild counterparts, uninjured animals, or known baseline survival rates for the resident population.A standard systematic search strategy, as outlined by Pullin and Stewart , was usee limits . The onlOne reviewer created a summary of study characteristics for each article detailing study species, sample size, study location, reason for entry into care, percentage of unassisted deaths in care (i.e. excluding euthanasia), percentage survival to release from care, short- and long-term survival post-release, factors reported as affecting survival, and causes of mortality. Post-release survival was categorised as short-term (< six months) or long-term (> six months) to minimise bias between study outcomes. These time frames were selected because some studies reported survival at multiple time points, and monitoring varied from 14 days to six years. We considered six months to be a reasonable time for animals to settle into their environment and forage independently in more than one season. Location was also included in the analyses to disentangle the potential effects of the suite of species found at a location, varying rehabilitation policies and practices around the world, or other abiotic processes from the biological characteristics of species.https://eol.org/) to categorise species according to class , diel activity pattern , average adult weight  and trophic level . The adult weight classes were designed to separate species according to the Australian critical weight range, i.e. 35 g– 5.5 kg [We used the Encyclopedia of Life —and reported as frequencies. Factors affecting survival during rehabilitation or after release were categorised as being related to the event that precipitated entry into care (e.g. severity and type of injury), intrinsic or individual traits , intervention , release environment , and human-wildlife interface (e.g. hunting activity and urban expansion), and summarised. These factors were shown statistically or observationally to affect survival in the reviewed articles.I2 [Statistical analyses were performed in R (version 4.0.5) . A mixedI2 . ProbabiPublication bias can exist where small studies with small effect sizes are not published or there is selective reporting within studies. We tested for bias in METAFOR by creating a funnel plot of effect size versus sampling variance of the effect size for each survival measure . Egger’sThe literature search yielded 5617 publications, of which 187 were initially selected; after reviewing the full texts, 112 articles satisfied all inclusion criteria . SeveralMost reasons for entry into care were anthropogenic in origin, followed by non-specific causes, with a small proportion attributed to natural environmental causes . Most moThere were no differences between classes for unassisted death (p = 0.20) or survival during (p = 0.08) or after rehabilitation ; however, not all levels of predictors were present in both classes at all survival stages, so we assessed classes separately for effects of study location, diel activity pattern, trophic level, and adult weight class (referred to as the full model). No factors were significant for either birds or mammals for survival to the end of rehabilitation . The logI2 < 50%; Study location affected the unassisted death rate of birds; North America had the highest log-odds of death and Oceania had the lowest log-odds of death . Mean prIn the reviewed articles, 18 included an experimental design to assess the outcomes of different methods of rehabilitation . Fifty-eight studies statistically assessed the effect of various factors on survival, and 38 included observations of factors that authors believed affected survival rates in their study . Most faMelanitta perspicillata) showed survival of only 14.3% compared with 49.8% for unoiled non-rehabilitated scoters at five months, while unoiled scoters rehabilitated for other reasons had 77.2% survival.Only 18 studies incorporated a control group, which was usually a wild cohort that did not require rehabilitation. Most studies showed either reduced survival for rehabilitated animals compared with control groups, or similar outcomes . Oiling This systematic review supports our hypothesis that wildlife survival during rehabilitation and post-release is species- and context-specific. Most studies in this review were from Australia, Europe, North America and southern Africa. Meta-analysis demonstrated effects of species’ diel activity type, trophic level, and location of the study on survival, but not adult weight, supporting some but not all our hypotheses. Study location was a strong predictor of death in care and survival short- and long-term post-release for birds. There are various potential explanations for the effect of location, including differing triage protocols and therefore frequency of euthanasia, impacts of different threats in the environment, and varying perceptions towards the value of wildlife around the world , 119. ThNyctereutes procyonoides) and European hedgehogs (Erinaceus europaeus), both with relatively high death rates. Hedgehogs that were admitted due to trauma, parasite infections and vehicle collisions had very low recovery rates, and road casualties died very quickly in care [Sarcoptes scabiei infections experienced 57.4% and 38.9% unassisted death rates for two groups given different treatment regimens [Eudyptula minor) rescued after an oil spill event in Australia [Unassisted deaths in care can act as an indicator of ineffective triage criteria and appropriate treatment and husbandry protocols, as these are animals that die without euthanasia. The rates of unassisted death varied depending on the study location for birds, and by trophic level for mammals. This variation could indicate that the decision to euthanise is made sooner in Oceania compared with North America, or that threats in North America are more likely to result in unpredictable death in care. The only omnivorous mammals with unassisted death rates recorded in the meta-analysis were raccoon dogs , American black bears (U. americanus) and brown bears (U. arctos) in North America and white rhinoceros (Ceratotherium simum) in South Africa. Their high rate of survival could be associated with the success of the captive rearing process, as most of these animals entered rehabilitation as orphans [Falco peregrinus) and brown pelican . Anderson et al. [Pseudocheirus peregrinus) in Australia faced the same predation pressures, however, translocated rehabilitated possums were at a disadvantage in unfamiliar territory and initially had lower survival rates [In the reviewed articles, there were several large mammalian species with high short-term post-release survival rates. In our meta-analysis, large species included Asiatic black bears  survival during rehabilitation, where age and response to treatment were predictive of survival instead [Few studies have experimentally assessed factors influencing wildlife survival during rehabilitation and release , yet man instead . In many instead . The larMorus capensis) may have contributed to higher release rates after oiling than for smaller oiled bird species [Our review found that intrinsic traits of species or individuals can affect survival outcomes, yet traits of importance vary with the species and type of injury they sustain. For example, the large body size and non-migratory nature of Cape gannets Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, which currently lacks disease-modifying therapy to slow down its progression. Idebenone, a coenzyme Q10 (CQ10) analogue, is a well-known antioxidant and has been used to treat neurological disorders. However, the mechanism of Idebenone on PD has not been fully elucidated. This study aims to predict the potential targets of Idebenone and explore its therapeutic mechanism against PD. α-synuclein was used to validate the molecular mechanism.  We obtained potential therapeutic targets through database prediction, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Next, we constructed and analyzed a protein-protein interaction network (PPI) and a drug-target-pathway-disease network. A molecular docking test was conducted to identify the interactions between Idebenone and potential targets. Lastly, a PD cell line of SH-SY5Y overexpressing mutant α-synuclein degradation by suppressing the AKT/mTOR pathway. Pretreating cells with chloroquine (CQ) to block autophagic flux could diminish the pharmacological effect of Idebenone to clear α-synuclein.  A total of 87 targets were identified based on network pharmacology. The enrichment analysis highlighted manipulation of MAP kinase activity and the PI3K-AKT signaling pathway as potential pharmacological targets for Idebenone against PD. Additionally, molecular docking showed that AKT and MAPK could bind tightly with Idebenone. In the cell model of PD, Idebenone activated autophagy and promoted α-synuclein, thus providing a theoretical and experimental basis for Idebenone therapy against PD. This study demonstrated that Idebenone exerts its anti-PD effects by enhancing autophagy and clearance of  Parkinson's disease (PD) is the second most common neurodegenerative disease, whose prevalence is growing faster than any other neurological disorder . At presIdebenone, a coenzyme Q10 (CQ10) analogue with better pharmacological properties, contains a redox-active moiety, and thus it is able to transport electrons to protect cells against oxidative damage and help to preserve mitochondrial respiratory capacity . Neverthα-synuclein is a major constituent of abnormal neuronal aggregates, known as Lewy bodies (LBs), which are a pathological hallmark of PD. In addition, the neurotoxic effect of α-synuclein has been suggested to play a central role in the pathogenesis of PD [α-synucelin protein continue to gain attention [α-synuclein [α-synuclein protein and exert cytoprotective effects [Misfolded is of PD . Therefottention , 14. Macttention , have beynuclein , 17. Num effects –22. Ideb effects , an upstα-synuclein A53T mutant (SH-SY5Y-A53T). We hypothesized that Idebenone could target AKT and decrease α-synuclein levels by enhancing autophagy through inhibition of the AKT/mTOR pathway. Our study provides new insight into the use of Idebenone as promising treatment option for patients with PD.In this study, we investigated the underlying therapeutic mechanism of Idebenone on PD using network pharmacology, a novel approach integrating network biology and polypharmacology, to further our understanding of the mechanisms of Idebenone treatment , 25. Thehttp://www.chemspider.com/) and then searched for its reported pharmacological targets through Swiss Target Prediction (http://www.swisstargetprediction.ch/) [https://omim.org/), DisGeNET (https://www.disgenet.org/) [https://www.genecards.org/), using the search terms “Parkinson's disease” or “Parkinson disease”. The potential target genes were predicted by an intersection between the primary targets of Idebenone and PD, which were presented by Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/).We obtained the chemical structure of Idebenone from ChemSpider (ion.ch/) . For diset.org/) , and Genp value less than 0.05. Meanwhile, the KEGG analysis was carried out using the online tool, Metascape (https://metascape.org/gp/index.html/) [To further illustrate the biological processes of the targets systematically, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. We applied the R package, clusterProfiler , to condx.html/) . The topx.html/) . The drux.html/) .https://string-db.org/) [We submitted the identified targets to the STRING database v11.0 (db.org/)  to obtaihttp://www.rcsb.org/) and saved it in the PDB file format. Then we removed water and small molecules in PyMOL . We used Autodock v4.2.6 (http://autodock.scripps.edu/) to process the protein receptor files and ligand files, including adding hydrogens atoms, calculating Gasteiger charges, assigning AD4 type atoms, spreading charges deficit over all residue atoms, and setting rotatable bonds, and then converted them to a PDBQT format. We set a grid box to cover the active site and the molecular docking between Idebenone and hub genes using AutoDock's genetic algorithm. We chose the binding mode with lowest binding energy and visualized them with a 2D diagram using the Molecular Operating Environment  software. In general, the binding energy ≤−5 kcal/mol indicates good binding activity [We searched for X-ray crystal structures in the protein data bank  to threonine (T) as a result of a missense mutation at position 209 from G to A [We purchased the SH-SY5Y cell line from the American type culture collection . The cell line was transfected by lentivirus particles stably overexpressing SNCA (A53T) after puromycin-based screening. In the m G to A . Cells wIdebenone was purchased from a commercial reagent company , in the form of powder, stored at 4°C, and dissolved in DMSO at a storage concentration of 20 mM and stored at −20°C. The drug was diluted to the working concentration with fresh medium, and the drug was freshly prepared before each experiment.μl of the CCK8 reagents was added to each well and the plate was incubated for 2 h at 37°C. The spectrophotometric absorbance of each sample was measured at 450 nm using a microplate reader . The cell viability was expressed as the percentage of the control group.Cell growth was detected by the Cell Counting kit 8 . Cells were seeded in a 96-well plate with density reaching at least 2 × 104 cells per well. The cells were treated with Idebenone at different concentrations for 18 h. Then, 10 In order to verify the autophagy-enhanced effects of Idebenone, chloroquine  was used to inhibit autophagy as previously described .g for 10 min. The supernatant was collected, and the protein concentration was determined using a BCA assay . Protein lysates were loaded onto a 6–15% gel for sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% nonfat milk for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: rabbit anti-α-synuclein , anti-β-actin , SQSTM1/p62 , AKT , Phospho-AKT , mTOR , Phospho-mTOR , and mouse-anti-LC-3B . The following day, the membranes were washed with 0.1% TBST for 15 minutes and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, including goat anti-rabbit IgG  and goat anti-mouse IgG . After that, the incubated membranes were washed three times with TBST buffer. Finally, the membranes were imaged with an enhanced chemiluminescence (ECL) detection reagent and captured by Bio-Rad ChemiDoc MP system. The bands were quantified by ImageJ software. All experiments were repeated in triplicate.Cells were lysed in ice-cold RIPA buffer  for 1 h, and then the lysates were centrifuged at 14000 × t-test was used to compare two independent groups, while one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied to compare multiple groups. A p value < 0.05 was considered to be statistically significant.GraphPad Prism 8.0 was used to perform statistical analysis. All data were expressed as mean ± standard deviation (SD). Student's The structure of Idebenone is shown in To identify the function of the potential targets, we performed GO and KEGG pathway enrichment analysis of the 87 target genes. The GO analysis included biological process (BP), cellular component (CC), and molecular function (MF). The top 20 most significant terms are shown in Figures p values are shown in the bubble gram  and found that the inhibitory effect on the expression of α-synuclein was stable and highly significant at an Idebenone concentration of 2.0 μM. To determine whether the clearance of α-synuclein was related to autophagy, the related indicators of LC3, p62 were detected. The results indicated that LC3-II increased at Idebenone concentrations ranging from 0.5 μM to 2.0 μM, while p62 did not change  mutant strain using Western blot analysis . Then, tt change .α-synuclein was cleared by Idebenone-induced autophagy, 10 μM CQ was first used to pretreat SH-SY5Y-A53T cells for 24 h, after which Idebenone was added to a final concentration of 2 μM. CQ could inhibit the fusion of autophagosomes and lysosomes, thus leading to the accumulation of autophagosomes and interrupting autophagy flux. As shown in α-synuclein increased significantly compared to pretreatment with Idebenone only, suggesting that CQ could eliminate the clearance effect of α-synuclein by Idebenone.To further confirm that Based on the previous pathway enrichment analysis and docking results, PI3K-AKT pathway was considered to be a potential mechanism for explaining the autophagy-enhancing effect of Idebenone. The PI3K-AKT pathway and its related genes are presented on α-synuclein degradation may contribute to the therapeutic effect of Idebenone against PD.Recently, it has been proposed that Idebenone exerts cytoprotective effects by activating fundamental pathways, rather than by functioning as a direct antioxidant agent, thus providing the possibility of discovering a new mode of action . Previouβ [We obtained 87 PD-relevant targets through database prediction, among which APP, AKT1, PIK3CA, MAPK1, EGFR and AKT2 were regarded as hub genes in the PPI network analysis. After that, we conducted molecular docking and the results suggested that MAPK1 and AKT1 exhibit stable binding modes with Idebenone. MAPK, including the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38, plays an important role in the neuroinflammation process, and an excessive inflammation response confers detrimental effects and contributes to neurodegeneration , 36. Yanβ .α-synuclein decreased significantly at an Idebenone concentration of 2 μM. Based on network pharmacology results, Idebenone can bind tightly with MAPK and AKT, which are both related to autophagy. Activating autophagy has been a therapeutic strategy to attenuate α-synuclein toxicity in PD pathogenesis. Thus, we postulated that autophagy may contribute to the clearance of α-synuclein. In this regard, we detected autophagy-relevant proteins and the results revealed that the levels of the autophagosome marker LC3-II increased after treatment, suggesting the activation of autophagy. To further verify whether autophagy was necessary for the therapeutic effect of Idebenone, we blocked autophagic flux using CQ and found that the clearance of α-synuclein was diminished.To determine whether Idebenone exerts its protective effects in PD in vitro, we treated SH-SY5Y-A53T cells with Idebenone. Given that Tai et al.  reportedNotably, one of the most characterized substrates of selective autophagy, p62 , did not change after drug treatment. On the one hand, autophagy is not the only way to degrade p62 , 44, whiμM, while insulin independently enhanced p-AKT levels at Idebenone concentrations higher than 5 μM. For reference, the concentration of Idebenone applied in our study ranged from 0.5 μM to 2.0 μM. To our knowledge, Idebenone may play a bilateral role on AKT activation depending on the drug concentration and pathological conditions. To some extent, Idebenone likely acts as a “regulator” to rescue cells from a pathological state.In order to further understand the biological mechanisms of Idebenone therapy in PD, we referred to the results of the KEGG pathway enrichment analysis and postulated that the AKT/mTOR pathway may contribute to the activation of autophagy. Our experimental results suggested that Idebenone could suppress AKT phosphorylation (Ser473) and mTOR phosphorylation (Ser2448) in a dose-dependent manner. This appears to contradict the results of the study by Tomilov et al. , which sα-synuclein levels in the SH-SY5Y-A53T cell line, whether administration of Idebenone can reach the optical concentration in the blood in the central nervous system in vivo and exert its autophagy-enhancing effects remains unclear. Lastly, more experiments are needed to verify other predicted outcomes, including the potential targets and pathways.There are several limitations in this study. Firstly, in the network pharmacology analysis, the potential targets from the database prediction are based on established knowledge, which restricts the power to identify novel target. Thus, we call for further exploration with the application of sequencing technology. Secondly, although we demonstrated that Idebenone can decrease α-synuclein through the activation of autophagy. The underlying mechanism may relate to the suppression of the AKT/mTOR pathway. Our work provides new insights on the therapeutic mechanism of Idebenone treatment against PD.In summary, we applied network pharmacology to identify 87 related targets of Idebenone against PD. Further analysis revealed that MAPK1 and AKT1 could bind tightly with Idebenone. In addition, in vitro experiments showed that Idebenone may reduce the level of


Granum is a basic structural unit of the thylakoid membrane network of plant chloroplasts. It is composed of multiple flattened membranes forming a stacked arrangement of a cylindrical shape. Grana membranes are composed of lipids and tightly packed pigment-protein complexes whose primary role is the catalysis of photosynthetic light reactions. These membranes are highly dynamic structures capable of adapting to changing environmental conditions by fine-tuning photochemical efficiency, manifested by the structural reorganization of grana stacks. Due to a nanometer length scale of the structural granum features, the application of high-resolution electron microscopic techniques is essential for a detailed analysis of the granum architecture. This mini-review overviews recent approaches to quantitative grana structure analyses from electron microscopy data, highlighting the basic manual measurements and semi-automated workflows. We outline and define structural parameters used by different authors, for instance, granum height and diameter, thylakoid thickness, end-membrane length, Stacking Repeat Distance, and Granum Lateral Irregularity. This article also presents insights into efficient and effective measurements of grana stacks visualized on 2D micrographs. The information on how to correctly interpret obtained data, taking into account the 3D nature of grana stacks projected onto 2D space of electron micrograph, is also given. Grana ultrastructural observations reveal key features of this intriguing membrane arrangement, broadening our knowledge of the thylakoid network’s remarkable plasticity. Coleochaetales and Charales as grana; however, based on the widely accepted evolutionary hypothesis, grana evolved after land colonization and therefore, are unique for plants  domains. Cyanobacteria possess unstacked photosynthetic membranes forming fascicular, radial, or parallel arrangements . Similarr plants . It is wr plants .From the structural point of view, grana might be described as stacks of discoidal-shaped thylakoids; however, such a general definition is insufficient for detailed qualitative and quantitative analysis of grana structure. A single granum stack is usually composed of 5–25 thylakoid layers with diameters between 300 and 550nm. The model granum structure is built by thylakoid membranes with the same diameter forming a perfect cylindrical shape. However, in most plant species and specific environmental conditions, the grana structures are highly irregular – with a variable diameter of thylakoid layers and their shift in the lateral plane . It is dGeneral granum definition also does not determine the boundary of the granum in the vertical plane. No limit of the maximal granum height exists; many plant species, especially shade-grown ones, can form grana composed of over 50 layers . NonetheAn accurate understanding of the granum structure enables the determination of reliable measuring protocols necessary to provide comparable results obtained by different researchers. Detailed analysis of grana structure provides important information about the thylakoid membrane remodeling forced by plant ontogenesis , light iStructural grana analysis using mutants with an aberrant composition of thylakoids is particularly helpful in understanding the role of lipid–pigment–protein components in the self-organization of different membrane shapes, such as curved, flat, and stacked regions . A neceschlorina mutants, characterized by reduced chlorophyll b content  induced the formation of lower stacks compared with wild-type plants  resulted in increased grana diameter  and core proteins of photosystem II (PSII) induce grana size remodeling. Depletion of chloroplast-encoded PSII subunits caused the formation of “super-grana” containing dozens of membranes , while lnt e.g., . Tuning nt e.g., . In conte plants , and lacdiameter .Moreover, a family of structural membrane proteins – CURVATURE THYLAKOID 1 (CURT1), were recognized to mediate the diameter of grana stacks in a dosage-dependent manner and facilitate membrane curvature at the grana margins . The grain vivo analysis but with lower resolution, and (ii) high-resolution methods requiring sample fixation.Efficient measurements of grana structural parameters require high-quality visualization of the thylakoid network. The catalog of suitable microscopy methods is limited due to the dimensions of the grana stacks. In general, these methods can be divided into two groups (i) enabling In vivo methods are mainly based on the detection of chlorophyll autofluorescence , these techniques are time- and money-consuming. For instance, visualization of the 3D structure of granum using electron tomography requires a multistep procedure composed of data acquisition, alignment, reconstruction, segmentation, and visualization . The length of grana and stroma thylakoids was measured per randomly chosen unit area (1μm2) of each chloroplast cross section. The number of thylakoids per granum was also determined , also named “Repeat Distance,” which represents the average thickness of the thylakoid with the neighboring partition gap . SRD migThe granum lateral  plane analysis requires a precise definition of the granum stack boundary. Due to the high variability of grana architecture in the lateral plane, identifying a single granum stack should be justified each time. For instance, it might be assumed that a single granum stack is characterized by (i) common height, which is shared by all stacked membranes building a particular stack, and (ii) shifted regions which are built by at least three stacked membranes . In suchFrom the diameter values of all membranes building a single granum, a parameter reflecting irregularity of the particular stack called “Granum Lateral Irregularity” (GLI) can be calculated. GLI is defined as the coefficient of variation (the ratio of the standard deviation to the mean) of membrane diameters within the granum . The minAlthough the whole thylakoid network of a single chloroplast forms a continuous arrangement, the grana stacks are structurally isolated units whose architecture, resulting from a plethora of interactions between membrane components, might be analyzed quantitatively and qualitatively. Recently, a growing number of studies have shown that the grana nano-morphology itself is a significant factor regulating light harvesting and electron transfer (reviewed in per se can bring the structural analysis of the thylakoid network to a higher level. Recently developed techniques called jointly “in situ liquid cell TEM” could, in the future, enable in vivo analysis of the thylakoid network nano-morphology (in vivo visualize single layers of grana stacks (In this mini-review, we focused on the use of 2D TEM for quantitative analysis of grana structure. So far, such measurements are mainly executed using manual methods. They are time-consuming and also susceptible to the “human eye” bias, which only partially might be reduced by blinded experiments. The rapid development of machine learning in analyzing different microscopy data  points trphology . On the a stacks . Finallya stacks , and (iia stacks  point toŁK and RM provided a conception of the manuscript and prepared figures. ŁK, RM, and AM wrote and edited the manuscript. ŁK provided microscopy images. All authors contributed to the article and approved the submitted version.ŁK acknowledges funding from the National Science Centre, Poland, under grant number 2019/35/D/NZ3/03904.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PIK3CA-mutated) and that has or has not spread to other organs. It is used after the cancer has progressed despite being treated with hormonal therapies. One of the most common side effects of alpelisib is an increase in blood glucose level (hyperglycemia). This can sometimes require reductions in the dose of alpelisib, or interruption or discontinuation of treatment. Early detection and initiation of treatment for hyperglycemia can help in controlling blood glucose levels and ensuring the best use and effects of alpelisib. Treatment can include lifestyle modifications (a reduced-carbohydrate diet) and administration of drugs used to treat diabetes. This report provides information on how to manage hyperglycemia caused by alpelisib, based on the experience of 14 cancer specialists and seven endocrinologists in managing this side effect.Alpelisib is a drug used to treat breast cancer that has certain characteristics (hormone receptor-positive (HR+), human epidermal growth receptor 2-negative (HER2–), PIK3CA-mutated locally advanced or metastatic breast cancer following disease progression on or after endocrine therapy. Hyperglycemia is an on-target effect of alpelisib affecting approximately 60% of treated patients, and sometimes necessitating dose reductions, treatment interruptions, or discontinuation of alpelisib. Early detection of hyperglycemia and timely intervention have a key role in achieving optimal glycemic control and maintaining alpelisib dose intensity to optimize the benefit of this drug. A glycemic support program implemented by an endocrinology–oncology collaborative team may be very useful in this regard. Lifestyle modifications, mainly comprising a reduced-carbohydrate diet, and a designated stepwise, personalized antihyperglycemic regimen, based on metformin, sodium–glucose co-transporter 2 inhibitors, and pioglitazone, are the main tools required to address the insulin-resistant hyperglycemia induced by alpelisib. In this report, based on the consensus of 14 oncologists and seven endocrinologists, we provide guidance for hyperglycemia management strategies before, during, and after alpelisib therapy for HR+, HER2–, PIK3CA-mutated breast cancer, with a focus on a proactive, multidisciplinary approach.Alpelisib is an α-selective phosphatidylinositol 3-kinase inhibitor used for treating hormone receptor-positive (HR+), human epidermal growth receptor 2-negative (HER2–),  PIK3CA) gene, which encodes the p110α-isoform of phosphatidylinositol 3-kinase (PI3K), occur in approximately 40% of patients with advanced HR+, HER2– breast cancer [Endocrine therapy, with or without the use of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor, is the current standard of care for patients with advanced hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) breast cancer ,2. Howevt cancer ,8,9, andt cancer .PIK3CA-mutated advanced or metastatic breast cancer that has progressed on or after endocrine therapy. Regulatory approval of alpelisib, and its inclusion in breast cancer treatment guidelines [PIK3CA-mutated disease, there was a clinically relevant and statistically significant benefit on progression-free survival (PFS), the primary endpoint, with alpelisib plus fulvestrant , 0.65; 95% confidence interval (CI), 0.5–0.85; p < 0.001) [p = 0.15) [Alpelisib is a small-molecule α-selective PI3K inhibitor that can help overcome resistance to endocrine-based therapy ,14,15,16idelines , was basidelines . In pati = 0.15) . In an u = 0.15) .PIK3CA-mutated breast cancer in the post-CDK4/6 inhibitor setting [Results from the more recent phase 2 BYLieve study indicate that alpelisib in combination with endocrine therapy (fulvestrant or letrozole) is also effective in patients with HR+, HER2–,  setting ,18. The  setting , and 46.PIK3CA-mutated breast cancer, although adverse events (AEs) associated with PI3K inhibition are common [In contrast to pan-PI3K inhibitors, development of which was limited by toxicity ,20,21, ae common ,22. Hypee common ,23, and e common ,22. The Hyperglycemia occurs early in the course of treatment  becauseThe hyperinsulinemia that occurs in patients with alpelisib-induced hyperglycemia may provide breast cancer cells with a survival mechanism and reduce the efficacy of alpelisib as demonstrated in preclinical studies ,25,28,29PIK3CA-mutated advanced breast cancer.In this report, we provide guidance for hyperglycemia management and monitoring strategies aimed at minimizing alpelisib dose disruption in patients with HR+, HER2−, The guidance provided in this report is the consensus opinion of a group of 14 oncologists and seven endocrinologists, who are all experts in oncology and endocrinology/diabetology from leading academic and non-academic institutions in 12 countries in Central/Eastern Europe and Israel. The content is based on a review of the published literature, recommendations on the management and monitoring of alpelisib-induced hyperglycemia from the alpelisib prescribing information, and shared inter-specialty experience of the aforementioned oncologists and endocrinologists in the management of alpelisib-induced hyperglycemia. Drafts of the guidance were reviewed and modified by this expert group the until consensus was reached.Pretreatment strategies for the prevention of hyperglycemia in breast cancer patients scheduled to receive alpelisib are listed in Patients should be screened for the following hyperglycemia risk factors before starting treatment with alpelisib: prediabetes, diabetes, body mass index (BMI), and age. Diabetes is diagnosed based on fasting plasma glucose (FPG) and glycated hemoglobin (HbA1c) levels according to American Diabetes Association (ADA) criteria . We consn = 113), increases in FPG occurring during alpelisib therapy were more pronounced in patients with diabetic or prediabetic glycemic status at baseline  . Physica2 or in those aged ≥ 75 years) are managed by a multidisciplinary team that includes, whenever possible, an endocrinologist or diabetologist to oversee appropriate antihyperglycemic treatment and ongoing management [We recommend that high-risk patients  . FurtherGiven the high risk of treatment-emergent hyperglycemia, which occurred in 64% of patients receiving alpelisib plus fulvestrant in SOLAR-1 , FPG andThe following glucose monitoring schedule is recommended for use in everyday practice, with more frequent monitoring considered in high-risk patients. After initiation of alpelisib, blood glucose should be monitored at least once every week in the first 2 weeks and at least once every 4 weeks thereafter by assessment of capillary blood glucose in fingerprick samples or by assessing FPG in a peripheral blood sample; HbA1c should be monitored after 4 weeks and every 3 months thereafter . FPG is Metformin is widely accepted as a standard treatment for alpelisib-induced hyperglycemia ,17,45. ASGLT2 inhibitors inhibit the glucose transporters responsible for reabsorption of glucose in the kidney . The resIn general, insulin sensitizers  are preferable to insulin secretagogues  due to the deleterious effect of insulin on the PI3K blockade, as demonstrated in animal models ,17. AlthIn SOLAR-1, metformin was used either alone or in combination with other antihyperglycemic agents in 87.1% of patients with hyperglycemia . After pAlthough diarrhea may be an AE of both alpelisib and metformin ,49, the As is recommended in patients with diabetes , antihypSulfonylureas should be avoided as primary treatment for alpelisib-induced hyperglycemia because they act as insulin secretagogues and increase insulin levels, which may blunt the primary antitumor effect of alpelisib . InsulinAfter interruption of alpelisib, short-term insulin (1–2 days) can be used as rescue medication for grade 3/4 hyperglycemia (FPG > 13.9–27.8 mmol/L (>250–500 mg/dL)) not controlled by oral antihyperglycemic medications alone  17,23,3,323,30. Whenever possible, use of insulin should be avoided in patients with alpelisib-induced hyperglycemia because of the risk of hypoglycemia as the effect of the drug subsides ,31,45,56Hyperglycemia is a reversible AE of alpelisib , with blThe management approach to hyperglycemia upon discontinuation of alpelisib depends on the glycemic status of patients before starting alpelisib. In patients with diabetes or prediabetes, ongoing blood glucose monitoring will indicate when to change or discontinue antihyperglycemic treatment. In other patients with alpelisib-induced hyperglycemia, antihyperglycemic treatment should be discontinued once FPG levels have normalized. There is a risk of hypoglycemia if alpelisib is stopped suddenly during oral antihyperglycemic therapy. Bearing in mind that FPG levels may normalize within 24–72 h of alpelisib discontinuation, careful tapering of oral antihyperglycemic agents and insulin is required to avoid hypoglycemia [Hyperglycemia is a common, on-target AE of alpelisib caused by α-PI3K inhibition. Even mild hyperglycemia results in hyperinsulinemia, which preclinical models suggest may reduce the efficacy of alpelisib, and more severe hyperglycemia necessitates alpelisib dose interruption and reduction, and may result in permanent discontinuation of treatment. Optimal efficacy of alpelisib can therefore only be obtained when striking a perfect balance between dosage, treatment duration, and hyperglycemia management.Fortunately, alpelisib-induced hyperglycemia is predictable and manageable. Multidisciplinary collaboration between oncologists and endocrinologists or diabetologists, with close monitoring of blood glucose levels, and prompt antihyperglycemic treatment, even when FPG is only mildly elevated, will allow breast cancer patients to continue receiving alpelisib at the optimal dose, thereby achieving the maximum disease control possible.


Long non‐coding RNA intersectin 1–2 (lnc‐ITSN1‐2) exacerbates inflammation and promotes T‐helper (Th) cell differentiation, also serves as a biomarker in critical illness diseases. However, its clinical role in sepsis remains obscure. Hence, the study aimed to explore the relationship of lnc‐ITSN1‐2 with Th cells, inflammation, disease severity, multiple organ dysfunction, and mortality risk in sepsis.Peripheral blood mononuclear cells (PBMC) were isolated from 95 sepsis patients and 50 health controls, followed by lnc‐ITSN1‐2 evaluation using RT‐qPCR. PBMC Th1, Th17 cells and their secreted cytokines in serum were detected by flow cytometry and ELISA, respectively.Z = −7.328, p < 0.001). Lnc‐ITSN1‐2 correlated with increased interferon‐gamma (p = 0.009), Th17 cells (p = 0.022), and interleukin‐17A (p = 0.006), but not Th1 cells (p = 0.169) in sepsis patients. Moreover, lnc‐ITSN1‐2 had a positive connection with C‐reactive protein (p = 0.001), acute pathologic and chronic health evaluation (APACHE) II (p = 0.024), and sequential organ failure assessment (SOFA) scores (p = 0.022). Regarding SOFA subscales, lnc‐ITSN1‐2 linked with elevated respiratory system score (p = 0.005), cardiovascular system score (p = 0.007), and renal system score (p = 0.004) but no other subscales. Besides, lnc‐ITSN1‐2 had an increasing trend, but no statistical difference, in septic deaths compared to survivors .Lnc‐ITSN1‐2 in sepsis patients was higher than it in health controls  exacerbates inflammation and promotes T‐helper (Th) cell differentiation, also serves as biomarker in critical illness diseases. However, its clinical role in sepsis remains obscure. The findings of this study indicates that lnc‐ITSN1‐2 reflects sepsis progression and unfavorable prognosis to some extent, which may serve as a potential biomarker to improve the management of sepsis patients. The recruitment criteria were set as (i) diagnosis of sepsis in terms of the sepsis‐3 criteria (2016 version)2.2Clinical characteristics were collected after admission within 24 h for further analysis, including age, gender, body mass index (BMI), smoke status, drink status, comorbidities, primary infection site, primary organism, C‐reactive protein (CRP) level, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and Sequential Organ Failure Assessment (SOFA) score. Additionally, the sepsis patients were closely followed up until death or up to 28 days, and mortality during 28‐day follow‐up was recorded.2.3PB samples of all sepsis patients were collected after admission, then peripheral blood mononuclear cells (PBMCs) and serum were separated. PBMCs of all sepsis patients were separated to detect lnc‐ITSN1‐2 by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Besides, among 60 sepsis patients, PBMCs were separated to assess the percentage of Th1 and Th17 cells  by flow cytometry (FCM) using Human Cell Differentiation Kit (Bio‐Techne China Co. Ltd.). Serum samples were separated to detect the level of interferon‐gamma (IFN‐γ) and interleukin‐17A (IL‐17A) by enzyme‐linked immunosorbent assay (ELISA) using commercial Human ELISA Kit (Bio‐Techne China Co. Ltd.). In addition, PB samples of health controls were collected after recruitment, then PBMCs were isolated to evaluate lnc‐ITSN1‐2 by RT‐qPCR. The procedures were in strict accordance with the direction of instructions.2.4™ cDNA Synthesis Kit (with random primer) (Bio‐Rad); besides, the qPCR was executed by SYBR® Green Real‐time PCR Master Mix (Toyobo). The primers were designed according to a previous study.−ΔΔCt method .The RT‐qPCR assay was performed for determining the lnc‐ITSN1‐2 in PBMCs. In brief, total RNA was extracted by PureZOL RNA isolation reagent (Bio‐Rad); then, was reversely transcribed to cDNA using iScript2.5p‐value less than 0.05.Graphics were plotted using GraphPad Prism 7.02 (GraphPad Software Inc.), and statistical analyses were completed using SPSS 24.0 (IBM). Difference of lnc‐ITSN1‐2 between sepsis patients and health controls was compared by Wilcoxon's rank‐sum test. Association of two variables was analyzed by Spearman's rank correlation test. Correlation of lnc‐ITSN1‐2 with primary infection site and primary organism was determined by the Kruskal‐Wallis H rank‐sum test or Wilcoxon's rank‐sum test. Comparison of lnc‐ITSN1‐2, Th1 cells, IFN‐γ level, Th17 cells and IL‐17A level between deaths and survivors were evaluated by Wilcoxon's rank‐sum test. Statistical significance was concluded if a 33.1Sepsis patients presented a mean age of 56.8 ± 11.8 years with 33 (34.7%) females and 62 (65.3%) males. Moreover, the median (interquartile range (IQR)) values of CRP level was 87.5 (44.3–127.0) mg/L. The APACHE II and SOFA scores were 11.9 ± 5.9 and 5.2 ± 2.4, respectively. Other clinical properties about patients were displayed in Table 3.2Z = −7.328, p < 0.001) , while lnc‐ITSN1‐2 linked to high IFN‐γ , Th17 cells  and IL‐17A  in sepsis patients  and APACHE II score  in sepsis patients  in sepsis patients , cardio vascular system score  and renal system score  ; among these, the lnc‐ITSN1‐2 was highest in patients with other infections and lowest in patients with abdominal infection   had an increasing trend in deaths compared to survivors, but did not reach statistical significance , Th17 cells  and IL‐17A level  were enhanced in deaths than those in survivors, but not IFN‐γ level   Figure .p = 0.038) was correlated with increased 28‐day mortality; meanwhile, multivariable logistic regression analysis (backward stepwise) illustrated that APACHE II score  but not high lnc‐ITSN1‐2  was independently correlated with elevated 28‐day mortality (Supplementary Table 1).Furthermore, univariable logistic regression analysis presented that high lnc‐ITSN1‐2 (odds ratio (OR) = 3.288, 4The clinical role of lnc‐ITSN1‐2 in inflammatory‐related or critical ill diseases has drawn wide attention. For instance, a study reports that high lnc‐ITSN1‐2 has been found in ankylosing spondylitis patients, meanwhile, it relates to increased IL‐1β cytokine+ T cell, induce Th17 cell differentiation, and then release the pro‐inflammatory cytokinesRecently, it is reported that lnc‐ITSN1‐2 relates to inflammation and disease severity/activity in critical ill diseases. For example, prior research presents that lnc‐ITSN1‐2 correlates with increased CRP, Ranson's score, APACHE II score in SAP patientsBesides, our study presented the relationship between lnc‐ITSN1‐2 and multiple organ dysfunction in sepsis patients, which showed that the lnc‐ITSN1‐2 related to raised SOFA score; moreover, it was positively associated with respiratory system, cardiovascular system, and renal system injury. The data might be caused by that (1) high lnc‐ITSN1‐2 regulated the inflammation (described above), which accelerated multiple organ injury in sepsis patients; (2) lnc‐ITSN1‐2 might facilitate cell injury and apoptosis,At present, lnc‐ITSN1‐2 as a prognostic biomarker for critical ill diseases has been illustrated such as AIS and SAP.The current study was cross‐sectional research, which revealed that lnc‐ITSN1‐2 might be a potential biomarker of sepsis, while the causality between sepsis and lnc‐ITSN1‐2 was hard to explore. Some limitations still existed in the current research: (1) the sample size of sepsis patients was only 95, which might affect the power in statistics, thus, subsequent study should involve more sample size to verify our conclusion; (2) the mechanism of lnc‐ITSN1‐2 facilitating Th17 cell differentiation and multiple organ dysfunction in sepsis was not clear, which should be explored in the following study; (3) the change of lnc‐ITSN1‐2 in sepsis patients was unclear after recovery, which should be explored in following study; (4) level of lnc‐ITSN1‐2 in urine and tissue apart from PBMC in sepsis patients could be investigated; (5) the quantification of lnc‐ITSN1‐2 was relative in the current study, the absolute quantification could be explored in future; (6) the level of lnc‐ITSN1‐2 in septic shock could be explored in the further study; (7) whether lnc‐ITSN1‐2 was variated by treatment regimens such as antibiotics, anti‐inflammation in sepsis could be explored in future.In conclusion, lnc‐ITSN1‐2 is highly expressed and correlates with inflammation, multiple organ dysfunction, and mortality risk in sepsis patients, indicating lnc‐ITSN1‐2 may serve as a potential biomarker in sepsis patients.The authors declare that they have no conflicts of interest.Table S1Click here for additional data file.