Lactobacillus plantarum, and the resulting mixture of small peptides with less than 11 amino acids (RFP) was extracted and purified from the fermentation broth. Subsequently, the antioxidant activity of RFP was assessed using the chemical model, cell biology, and animal model methods. RFP enhanced the expression of the antioxidant enzyme genes downstream of the KEAP1−NRF2/ARE pathway by promoting nuclear factor−erythroid 2−related factor 2 (NRF2) nuclear translocation while simultaneously removing lipid oxidation products and excess free radicals. These results suggest that RFP is a potential substance for resisting aging and disease caused by oxidative stress.Oxidative stress is an important factor on both aging and disease. Among foods endowed with beneficial healthy properties, rice is a very useful material, not only because it has a good amino acid ratio and produces antioxidant peptides through microbial fermentation, but also for its inexpensive availability. In this study, rice was treated with  Oxidative stress refers to the imbalance of oxidative and antioxidant systems in the body under endogenous or exogenous stimulation, leading to the production of excessive free radicals, which results in damage to tissue cells and biological macromolecules and is associated with various diseases such as atherosclerosis, diabetes, cancer, and aging . As the Lactobacilli have evolved an efficient proteolytic system that can degrade large proteins into small peptides and amino acids, making them an important group of bacteria involved in protein degradation and active peptide generation in the fermentation process [After the oxidative stress theory was put forward, research on antioxidants followed. As a natural antioxidant, antioxidant peptides have the advantages of a comparatively simple structure, ease of absorption, high stability, and a lack of immunogenicity, for which they are drawing increasing attention in food, medicine, and other fields . Rice ha process .The chemical model method, the cell biology method, and the animal model method are the three techniques used to assess the antioxidant activity of antioxidant peptides. Only a small percentage of investigations into the antioxidant activity of peptides are conducted using cell biology or animal models; the majority use chemical model approaches . HoweverLactobacillus plantarum. First, the chemical model determined the free radical scavenging and ferric reduction ability of RFP. Then, the effects of RFP on the scavenging ability of reactive oxygen species (ROS) and malondialdehyde (MDA), and on the overall antioxidant capacity of human skin fibroblasts (HSFs) induced by UVA oxidative stress were verified. The effects of RFP on the KEAP1−NRF2/ARE pathway were also confirmed. Finally, we preliminarily detected the effects of RFP on the catalase (CAT) enzyme and total antioxidant capacity in UVA−irradiated mice and observed the changes in mouse skin through tissue sections.This study examined the antioxidant activity of the rice fermentation polypeptides (RFP) that were extracted from rice fermentation broth fermented by Lactobacillus plantarum (Orla−Jensen) Bergey, Harrison, Breed, Hammer & Hu (Lactobacillaceae) was obtained from the China Center of Industrial Culture Collection (strain number: CICC−20261); rice polypeptides (Lys−His−Asn−Arg−Gly−Asp−Glu−Phe) (RP) were purchased from Top−peptide Biotechnology Co., Ltd. ; Human Skin Fibroblasts (HSFs) were obtained from the Cell Bank of the Chinese Academy of Sciences; Cell Counting Kit−8 (CCK−8) was purchased from Biorigin (Beijing) Inc. ; Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Invitrogen ; fetal bovine serum (FBS), trypsin−EDTA and penicillin−streptomycin were purchased from Gibco ; RIPA Lysis Buffer (P0013D), nuclear and cytoplasmic protein extraction kit, trizol, BCA protein assay kits, CAT, superoxide dismutase (SOD), glutathione peroxidase (GSH−Px), total antioxidant capacity, and MDA and ROS kits were purchased from Shanghai Beyotime Biotechnology ; and all other chemicals employed were of high−purity biochemistry grade. EasyScript® One−Step gDNA Removal and cDNA Synthesis SuperMix and TransStart® Top Green qPCR SuperMix  were also utilized. A Human Nuclear Factor E2−related Factor 2 (NRF2) ELISA Kit, Human Quinone NADH Dehydrogenase 1 (NQO1) ELISA Kit, and Human Heme Oxygenase−1 (HO−1) ELISA Kit were purchased from Cusabio Biotech Co., Ltd. . All chemicals and solvents used in the experiments were of analytical grade and purchased from Sinopharm Chemical Reagents Co., Ltd. .Rice was purchased from Wuchang City, Heilongjiang Province, China; Lactobacillus plantarum suspension was added for expansion culture. The culture conditions were 37 °C and 180 rpm for 24 h. The rice was dried in an oven at 60 °C to constant weight, then crushed using a grinder and filtered through a 50−mesh screen. Deionized water was added at a material−to−liquid ratio of 1:4. After sterilization, 5% activated Lactobacillus plantarum liquid (107 CFU/mL) was added, and fermentation was performed at 180 r/min at 37 °C. After centrifugation, the polysaccharides of the supernatant were removed using the alcohol precipitation method and the ratio of ethanol to supernatant was 4:1. After freeze−drying at minus 60 °C in vacuum rotation, the freeze−dried rice fermentation liquid powder was obtained, and the yield was 0.17 g/L.The MRS medium was configured and sterilized by autoclaving for 15 min. After cooling to room temperature, the An activated ion exchanger (SP Sepharose Fast Flow)  gel was The molecular weight of the polypeptides was determined by reversed−phase high−performance liquid chromatography (RP−HPLC) . The detAn Orbitrap Fusion Lumos Mass Spectrometer was used to analyze the sequence of the peptides to obtain the original data, and then the peptide data was analyzed by database, or de novo, sequencing.•) [•+), superoxide anion (O2•−) [•OH) [The same antioxidant can have different inhibitory effects on different free radicals. Accordingly, the antioxidant capacity of the samples was comprehensively evaluated by the clearance rate of the 1,1−Diphenyl−2−Picrylhydrazyl radical (DPPH•) , 2,2′−azn (O2•−) , and hyd−) [•OH)  in vitro2 incubator  at 37 °C for resuscitation. After successful resuscitation, HSFs were washed gently in phosphate−buffered saline (PBS), and an appropriate amount of Trypsin−EDTA was added for digestion and passage, which was carried out when the cell density reached about 80%.DMEM with 10% FBS and 1% Penicillin−Streptomycin Solution was prepared in advance. HSFs were removed from liquid nitrogen and immediately melted in a water bath at 37 °C. After being blown evenly, HSFs were immediately transferred to a culture flask containing an appropriate amount of medium and placed in a 5% CO3 cells/well for 12 h, then covered with an appropriate amount of PBS and irradiated in an ultraviolet crosslinker . The UVA radiation doses were 0, 3, 6, 9, and 12 J/cm2. After irradiation, the HSFs were cultured in DMEM for 24 h, then incubated in 10 uL CCK−8 solution for 2–4 h. Absorbance was measured at 450 nm  Trading Co., Ltd., Shanghai, China) to determine the IC50 of the UVA−stimulated cell dose. RP and RFP were dissolved in DMEM at 20.00, 10.00, 5.00, 2.50, 1.25, 0.63, and 0.31 g/L. The HSFs were collected and cultured in 96−well plates for 12 h, then treated with sample solution replacement medium for 24 h, followed by incubation in DMEM containing 10% CCK−8 solution for 2–4 h. Absorbance was measured at 450 nm to evaluate the effects of the samples on cell viability. After the samples were treated for 24 h, the cells were irradiated with UVA at a dose of 12 J/cm2, cultured in DMEM for another 24 h, then incubated in 10 uL CCK−8 solution for 2–4 h. The absorbance was measured to evaluate the protective effects of the samples on UVA−damaged HSFs.The HSFs were inoculated in 96−well plates at a density of 8 × 106 cells/well for 12 h. After sample treatment and UVA irradiation, they were treated with 2,7−dichlorodihydrofluorescein diacetate (DCFH−DA), which was hydrolyzed into 2,7−dichlorodihydrofluorescein (DCFH), then oxidized by ROS to 2,7−dichlorofluorescein (DCF) with fluorescence in intracellular incubation for 1 h. They were then washed in PBS, and the fluorescence intensity was measured (excitation wavelength of 485 nm and emission wavelength of 535 nm).The HSFs were inoculated in 6−well plates at a density of 1 × 10g centrifugation  at 4 °C for 5 min, the supernatant was taken, and the total antioxidant capacity, MDA content, and the activity of SOD, CAT, and GSH−Px were determined according to the kit instructions.The HSFs were laid in 6−well plates. After sample treatment and UVA irradiation, a 100 uL cell lysate buffer containing 1 mmol/L phenylmethanesulfonylfluoride (PMSF) was added to each well. After full lysis and 12,000× p−value (P−adjust) < 0.05, |log2fold change| (|log2FC|) > 1.2 as the screening criteria, differential expression analysis was performed on the sequencing results using DESeq2 software. Goatools software was used for gene ontology (GO) enrichment analysis, and R script was used for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. When P−adjust < 0.05, the GO function or KEGG Pathway was considered to be significantly enriched.After sample treatment and UVA irradiation, the total RNA of the cells was extracted using a trizol reagent. Next, 1% gel electrophoresis, ND−2000 , and 2100 Bioanalyser  were used to analyze the degradation degree of 28S RNA and 18S RNA, determine whether there was any contamination, and detect the purity, concentration, and integrity of the total RNA to ensure its quality . The cDNA constructed according to the kit instructions was then enriched and quantified. Then, the Shanghai Majorbio Bio−Pharm Technology Co. Ltd. was commissioned to perform high−throughput sequencing on an Illumina HiSeq 4000 sequencing platform. With the adjusted −ΔΔCt. The primer sequences are listed in According to the gene sequences published in NCBI, primers were designed by PrimerExpress software, and β−actin was used as the internal reference gene for RT−PCR validation . The PCR conditions were as follows: 94 °C, 5 s; 60 °C, 15 s; 72 °C, 10 s; 94 °C, 5 s; 72 °C, 10 s; and the duration was 40 cycles. The relative expression levels were calculated using 22 rectangular neck cell culture flask with vent caps at a density of 5 × 106 cells/flask for 12 h. After sample treatment and UVA irradiation, nuclear and cytoplasmic proteins were prepared according to the nuclear and cytoplasmic protein extraction kit. They were then used to determine the NRF2 Protein content using the Human Nuclear Factor E2−related Factor 2 (NRF2) ELISA Kit.The HSFs were laid in corning 25 cm2 rectangular neck cell culture flask with vent caps at a density of 1.5 × 107 cells/ flask for 12 h. After sample treatment and UVA irradiation, the preparation of cell lysates and the detection of the protein contents of NQO1 and HO−1 were performed according to the Human Quinone NADH Dehydrogenase 1 (NQO1) ELISA Kit and the Human Heme Oxygenase−1 (HO−1) ELISA Kit.The HSFs were laid in corning 75 cmFor specific test protocols, refer to the kit instructions.2/d, and the experiment lasted for 30 days [Twenty female Kunming mice, weighing 18 ± 2 g, were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd.  and randomly divided into the control group (double steam water), model group (double steam water + UVA), sample group (1 g/L RFP + UVA), and positive control group (1 g/L GSH+ UVA). Each set of five mice was housed in a separate cage, with the temperature kept between 22 °C and 25 °C. The circadian cycle consisted of 12 h of light and 12 h of darkness. They consumed food and liquids ad libitum. After one week of acclimation to the environment, the experiment was carried out. Before applying the samples, the back hair of the mice was shaved (3 × 3 cm) and they were allowed to adapt to the environment for 1 h. The sample volume was 1 mL/d, UVA irradiated the skin for 50 J/cm 30 days .g). Other skin tissues were submerged in a 4% formaldehyde solution, gradient dehydrated with ethanol, and embedded in paraffin. The wax blocks were sliced into 4 m−thick slices, dewaxed, and traditionally rehydrated. They were then differentiated in 1% alcohol hydrochloride for 5 s after being soaked in hematoxylin for 5 min and washed in tap water for 1 h. Next, they were washed in tap water for 30 min following a 2 min period of saturation in a lithium carbonate aqueous solution. Lastly, they were sealed with neutral gum after being submerged in a 0.5% eosin solution for 2 min. Under a microscope, the structural traits of the skin tissues in each group were examined.Tribromoethanol saturated solution was used for abdominal anesthesia, and 0.25–0.35 mL of venous blood was obtained by the tail−clipping method in an anticoagulant tube for routine blood level measurement. Afterwards, skin tissues removed from the same part of the mice’s back were divided into small pieces (100–150 mg), 1 mL of cell lysis solution was added, and the skin tissue was thoroughly homogenized using a glass homogenizer. The supernatant’s CAT activity and overall antioxidant capacity were assessed after centrifugation for 5 min  test.The first elution peak appeared at 130 mL, the second at about 470 mL, and the third at 710 mL. The content of the second peak component was low. The third peak had many impurities because the high concentration of salt solution is the regeneration condition of the column a. ConseqThe results of peptide sequencing are presented in •, •OH, and O2•− increased and stabilized with the increase in concentration . The half−maximal inhibitory concentration (IC50) of RFP was 4.269 g/L for DPPH•, and 3.806 g/L for •OH.In the concentration range of 2–10 g/L, the scavenging rates of RP with known antioxidant activity  and of Rntration a–c. The as 5 g/L d,e. More2, the survival rate decreased to 47.55 ± 0.81%, and the cell morphology changed from slender to round and even detached and dead. When pretreated with RP, RFP, and positive control  for 24 h, followed by a 12 J/cm2 dose of UVA radiation, the survival rate and state of cells were significantly improved compared with the model group  lowered the levels of ROS (2 UVA decreased significantly (p < 0.002), while RP, RFP, and GSH significantly (p > 0.033) enhanced the scavenging capacity of ABTS•+ (p < 0.002).RP, RFP, and GSH pretreatment significantly  increased and NRF2 was significantly (p < 0.001) decreased after UVA irradiation, and the UVA effects were inhibited in both the high− and low−dose RFP groups (p < 0.002) decreased, and the protein content in the nucleus was increased to a certain extent by RFP pre−treatment  increased in both the high− (4 g/L) and low−dose (1 g/L) RFP groups (p < 0.033) increased (The relative expression of glutathione S−transferase mu 1 (GSTM1) was significantly  increased in the RFP high−dose group. The expressions of NQO1  increased CAT enzyme activity and antioxidant capacity  and reduce iron ions (The antioxidant activity of antioxidant peptides is closely related to their molecular weight, amino acid composition, and sequence. As they are more likely to interact with target radicals to end their chain reactions, peptides with lower molecular weight generally have stronger antioxidant activity than peptides with higher molecular weight . Most an–1200 Da . The actron ions a–e.Subsequently, we conducted a UVA−induced oxidative stress experiment on HSF cells and found that RFP significantly reduces the content of ROS and MDA, which are the hallmark products of cellular oxidative stress damage ,32, and Transcriptome analysis, which reflects genes that are actively expressed under specific conditions, was further performed. This is an important means of studying cell functions and phenotypes which has a lower cost than Sanger sequencing, good accuracy in terms of expression levels, and whose results display a high level of repeatability. Differentially expressed genes related to the NRF2 antioxidant pathway were observed in the sequencing results . The KEALactobacillus plantarum rice fermentation broth is a blend of small peptides with 10 amino acids or less, which shows positive antioxidant activity at the chemical model, cell biology, and animal model levels. However, different peptides have different antioxidant mechanisms. Therefore, the further isolation and purification of RFP should be carried out in subsequent experiments to verify the antioxidant effectiveness of single peptides one by one.RFP isolated and purified from


Titanium (Ti) alloys used for narrow dental implants usually contain aluminum (Al) and vanadium (V) for improved resistance. However, those elements are linked to possible cytotoxic effects. Thus, this study evaluated the biomechanical behavior of narrow dental implants made with Al- and V-free Ti alloys by the finite element method. A virtual model of a partially edentulous maxilla received single implants  at the upper lateral incisor area, with respective abutments and porcelain-fused-to-metal crowns. Simulations were performed for each implant diameter and the following eight alloys (and elastic moduli): (1) Ti–6Al–4V , (2) Ti–35Nb–5Sn–6Mo–3Zr (85 GPa), (3) Ti–13Nb–13Zr (77 GPa), (4) Ti–15Zr (113 GPa), (5) Ti–8Fe–5Ta (120 GPa), (6) Ti–26.88Fe–4Ta (175 GPa), (7) TNTZ–2Fe–0.4O (107 GPa), and (8) TNTZ–2Fe–0.7O (109 GPa). The implants received a labially directed total static load of 100 N at a 45° angle relative to their long axis. Parameters for analysis included the maximum and minimum principal stresses for bone, and von Mises equivalent stress for implants and abutments. Ti–26.88Fe–4Ta reaches the lowest maximum (57 MPa) and minimum (125 MPa) principal stress values, whereas Ti–35Nb–5Sn–6Mo–3Zr (183 MPa) and Ti–13Nb–13Zr (191 MPa) models result in the highest principal stresses (the 2.7 mm model surpasses the threshold for bone overload). Implant diameters affect von Mises stresses more than the constituent alloys. It can be concluded that the narrow implants made of the Ti–26.88Fe–4Ta alloy have the most favorable biomechanical behavior, mostly by mitigating stress on peri-implant bone. Contemporary dental implants systems have reached excellent levels of predictability, as evidenced by their high survival rates ,2,3. ThiNarrow implants pose a special biomechanical challenge and, thus, need to be fabricated with highly resistant alloys to compensate for the reduction in diameter. Whereas grade 4 Ti alloys combine excellent biocompatibility and acceptable mechanical properties when used in standard implants ,9, narroEven if Ti–6Al–4V presents good biomechanical properties, its use brings some controversies. Issues with this alloy include a possible link between aluminum alloys and neurodegenerative diseases ,15,16, aEven if zirconia implants have been purported as substitutes for Ti–6Al–4V fixtures , they haThis in silico study tested a model composed of a partially edentulous maxilla restored by narrow implants and respective single crowns.A DICOM file depicting a case with a partially edentulous maxillary arch  was obtained from a tomography database  and transformed into the .step format using the Invesalius v. 3.0 . The three-dimensional .step model was exported to Rhinoceros SR8 v.6.0 software , refined, and limited to the anterior region of the edentulous maxilla. The model was designed to present a cortical bone thickness of 1 mm surrounding trabecular bone, corresponding to Lekholm and Zarb type III bone .Tridimensional images of implants and abutments were obtained by scanning them directly. Conometric connection implants  and their respective abutments  were cut with an electro-erosion machine , and each geometry was obtained through a 3D scanner , being transformed into the 0.3 dm format. The surface of each component was opacified with penetrant ink to capture their details . Once the geometries of the implants and abutments were obtained, they were scaled, obtaining implants with a diameter of 2.7 and 2.9 mm, both 10 mm in length.In turn, the models received the implants at the position of the missing upper lateral incisor, in compliance with recommendations for narrow implants. All models had the implants placed at the same angulation and at bone level. Subsequently, each implant received a solid one-piece abutment and cement-retained porcelain-fused-to-metal single crown (Co–Cr framework covered by feldespatic ceramic). The latter was shaped according to the expected natural shape of an upper lateral incisor.The simulations were divided into groups according to the two diameters and eight alloys to be analyzed as implant constituents, as well as abutments: (1) Titanium grade V ; (2) titanium–niobium–tin–molybdenum–zirconium (Ti–35Nb–5Sn–6Mo–3Zr); (3) titanium–niobium–zirconium (Ti–13Nb–13Zr); (4) titanium–zirconium (Ti–15Zr); (5) titanium–iron–tantalum (Ti–8Fe–5Ta); (6) titanium–iron–tantalum with a higher concentration of iron (Ti–26.88Fe–4Ta); (7) titanium–niobium–tantalum–zirconium–iron–oxygen (TNTZ–2Fe–0.4O); (8) titanium–niobium–tantalum–zirconium–iron–oxygen with higher oxygen concentration (TNTZ–2Fe–0.7O). The modulus of elasticity and Poisson’s ratio of each material were obtained from the literature .Finite element model simulation was performed by exporting the models to Ansys Workbench version 19.0 software . All structures were considered to be isotropic, homogeneous, and linearly elastic. To simplify data analysis, only half of the model was analyzed by the software, divided by the mid-sagittal plane, i.e., the section plane passed symmetrically through the mesial contact of the upper central incisors and median palatine suture. Subsequently, the mesh was refined mainly at the bone–implant interface and on the prosthetic components. The gingiva and the cement were not considered in the models due to their null influence on the analysis . All abuThe total deformation of each model was initially evaluated and convergence tests were performed to check analytical accuracy. For quantitative analysis, the implants and abutments were assessed with von Mises equivalent stress (VMES), while the bone was analyzed by maximum and minimum principal stresses. The graphic display of color maps was used to compare the models qualitatively. All graphic scales were standardized according to each model. Thresholds of 100 MPa for tensile or maximum principal stresses and 170 MPa for compressive or minimum principal stresses were considered as evidence of bone overload .The stress values observed in the peri-implant bone, implant, and abutment for each alloy in the diameters tested are summarized in Models of Ti–15Zr, TNTZ–2Fe–0.4O, and TNTZ–2Fe–0.7O alloys show maximum and minimum principal stresses values comparable to Ti–6Al–4V (control), while Ti–8Fe–5Ta has lower principal stress values.The lowest maximum and minimum principal stress values are identified with the two models of the Ti–26.88Fe–4Ta alloy. The highest principal stresses at the peri-implant region are observed with the models of Ti–35Nb–5Sn–6Mo–3Zr and Ti–13Nb–13Zr alloys . The comTi–35Nb–5Sn–6Mo–3Zr and Ti–13Nb–13Zr reach the lowest VMES in the implants, regardless of the diameter. Minimum VMES in abutments is observed in the Ti–13Nb–13Zr and Ti–15Zr alloys for the implants of 2.7 mm and 2.9 mm, respectively. Regarding the implants, the highest VMES is identified in the following two combinations: Ti–26.88Fe–4Ta with 2.7 mm diameter, and Ti–15Zr with 2.9 mm diameter . For theIn the ductile materials , the highest VMES of all the models is identified in the joint of the implant–abutment . Also, nThis study was a first step to identify alternative alloys for narrow dental implants, without aluminum and vanadium. The null hypothesis was rejected, given that some of the tested Al- and V-free alloys improve stress distribution in narrow dental implants.The present finite element analysis demonstrates that some alloys can be used in lieu of the traditional Ti–6Al–4V and, thus, have acceptable biomechanical behavior in narrow implants, at least for specific indications  ,40. WithThe alloys made of Ti–15Zr, Ti–8Fe–Ta, Ti–26.88Fe–4Ta, TNTZ–2Fe–0.4O, and TNTZ–2Fe–0.7O show values within physiological ranges, in compliance with the bone overload risk values of tensile (100 MPa ) and comThe alloys of Ti–15Zr, TNTZ–2Fe–0.4O, and TNTZ–2Fe–0.7O show similar maximum and minimum principal stresses values as Ti–6Al–4V (control) in both implant diameters. It means that these alloys can be as alternatives to replace Ti–6Al–4V, due to their similar biomechanics’ behavior in narrow dental implants.Also, this research shows that in the alloys with a higher elastic modulus, higher magnitudes of von Mises stress are observed in the abutments and implants, but a better distribution to the surrounding bone, which concurs with Datte et al. . This isOur study confirms that adding Fe to titanium alloys is positive for resistance, even though the diameter is reduced. As previously described, Fe increases the yield strength and resistance in vitro . HoweverThe analysis of this study was static, however, whereas a dynamic analysis may display values 10–20% higher . In thisA limitation of this study was the focus on a single implant length. However, implant diameter is a more effective factor than implant length to improve the stress distribution in the surrounding bone ,48. It iIt is important to highlight that this study tackles a specific aspect , but it has relevant results as the first step for further analysis of the proposed alloys to be used in narrow implants. Considering the limitations of this in silico study, further studies are necessary to determine the most suitable alloy to be used in narrow implants, considering the feasibility in its manufacture, as well as their in vitro biomechanical behavior and its biological safety. The long-term success of dental implants smaller than 3 mm is not documented in the literature ,49, evenConsidering the distribution of the stresses to the bone surrounding narrow dental implants, Ti–26.88Fe–4Ta is the most favorable alloy, whereas the Ti–35Nb–5Sn–6Mo–3Zr and Ti–13Nb–13Zr models surpass the bone overload threshold. The Ti–26.88Fe–4Ta alloy has promising use for the rehabilitation of small diameter teeth and should be further tested for dental applications.


This study seeks to improve the effectiveness of the pretreatment stage when direct furfural production is integrated into the concept of a lignocellulosic biomass biorefinery. First of all, the catalytic effects of different phosphorus-containing salts ₂, FePO₄, H₃PO₄, NaH₂PO₄) were analysed in hydrolysis for their ability to convert birch wood C-5 carbohydrates into furfural. The hydrolysis process was performed with three different amounts of catalyst  at a constant temperature (175 °C) and treatment time (90 min). It was found that the highest amount of furfural ) was obtained when H₃PO₄ was used as a catalyst. The best furfural yield among the used phosphorus-containing salts was obtained with NaH₂PO₄: 40 ± 2%. The greatest impact on cellulose degradation during the hydrolysis process was observed using H₃PO₄ at 12–20% of the initial amount, while the lowest degradation was observed using NaH₂PO₄ as a catalyst. The yield of furfural was 60.5–62.7% t.p.y. when H₃PO₄ and NaH₂PO₄ were combined ; however, the amount of cellulose that was degraded did not exceed 5.2–0.3% of the starting amount. Enzymatic hydrolysis showed that such pretreated biomass could be directly used as a substrate to produce glucose. The highest conversion ratio of cellulose into glucose (83.1%) was obtained at an enzyme load of 1000 and treatment time of 48 h. Lignocellulosic biorefining technologies are expected to become more important for several decades to come. Today, their economic viability is affected by the efficient conversion of all three major constituents of lignocellulosic (LC) biomass  into value-added biobased chemicals and biofuels. Despite the fact that the lignocellulosic biorefinery concept has a clear value proposition, commercial success at the industrial scale is still inadequate.One of the reasons is that lignocellulosic biomass cannot be converted selectively into target products without a loss of valuable components, mainly hemicellulose and cellulose ,2. HemicFurfural production is one of the valuable ways to utilise hemicellulose. Furfural itself is an important feedstock for various organic platform chemicals, such as furfuryl alcohol, furoic acid, phenolic resin, etc. ,6. UnforIn a previous study , we founTherefore, the aim of this study was to determine the effect of phosphorus-containing salts ₂, FePO₄, NaH₂PO₄) as catalysts for the production of furfural from birch wood chips, and to measure the amount of cellulose remaining in the solid lignocellulosic residue for further processes.2SO4) (95–97%), phosphoric acid (H₃PO₄) (99%), aluminium phosphate (AlPO₄) (≥93%), calcium phosphate (Ca₃(PO₄)₂) (96%), iron(III) phosphate (FePO₄) (96%), and sodium phosphate (NaH₂PO₄) (96%) were purchased from Merck  and used without further purification.D-(+)-Xylose (≥99%), Cellobiose (≥99%), D-(+)-glucose (≥99%), L-(+)-arabinose (≥99%), D-(+)-mannose (≥99%), D-(+)-galactose (≥99%), formic acid (≥95%), acetic acid (≥99%), levulinic acid (≥98%), 5-hydroxymethylfurfural (≥99%), 2-furaldehyde (≥99%), sulfuric acid . The moisture content of the collected wood chips was 49 wt.%. To avoid unnecessary wood degradation during storage, the wood chips were air-dried to reduce the moisture content below 15 wt.%. After that, the chips were fractionated using the Muototerä classifier MT300 according to the SCAN-CM 40:01 standard. A sieve arrangement was utilised: oversize chips (45 mm holes) → over thick chips (8 mm slots) → large accept chips (13 mm holes) → small accept chips (7 mm holes) → pin chips (3 mm holes) → fines. A fraction of Ø13–7 mm was used for further experiments.Before determination of the carbohydrates in the raw feedstock, quantification of the extractives was performed according to the TAPPI 204 cm-07 standard. The used solvent was ethanol–benzene in the ratio 1:2. The extraction time was 6 h. The structural carbohydrates in wood samples before and after furfural production were determined according to the National Renewable Energy Laboratory TP-510-42618 standard see . The ove2SO4 as eluent at a flow rate of 0.6 mL/min. This determination method was also used for analysis of the obtained hydrolysate after phosphoric acid-catalysed hydrolysis.Concentrations of saccharides were determined via HPLC using a Shimadzu LC20AD liquid chromatograph equipped with a RI detector (Shimadzu RID 10 A) and a Shodex Sugar SP-0810 column at 80 °C, with deionised water as the mobile phase under a flow rate of 0.6 mL/min. The concentrations of hydrolysis process by-products, such as formic acid, acetic acid, levulinic acid, 5-hydroxymethylfurfural and furfural, were also analysed using the same HPLC but with a Shodex Sugar SH-1821 column at 50 °C and 0.008 M H3. The reactor was equipped with a steam jacket and a corresponding automatic control system to maintain constant temperature and pressure throughout the experiment [4, Ca3(PO4)2, FePO4, NaH2PO4 and H3PO4, as well as without a catalyst for an autohydrolysis process. To show the catalytic properties of the used chemicals, autocatalytic hydrolysis was performed as a reference process to better understand these salts’ catalytic effects.The experiments were conducted in a bench-scale reactor system that modelled the industrial furfural obtaining process. The reactor had an internal diameter of 110 mm and a volume of 13.7 dmperiment  . Before enzymatic hydrolysis, these chips were ground in a Retsch GmbH SM100 cutting mill. The used sieve was 0.75 mm. Hydrolysis was performed in duplicate at 50 °C in 0.05 M KH2PO4–K2HPO4 buffer at pH 6.7 using a Biosan ES-20/80 orbital shaker at 160 rpm for 12–48 h with a 15% solid load (w/v), and the results are presented as the mean. Reducing sugars were determined with the Shimadzu LC20AD HPLC using a Thermo Scientific HyperRez XP Carbohydrate Pb2⁺ 8 UM column at 70 °C and deionised water as the mobile phase at a flow rate of 0.6 mL/min.A preliminary study of enzymatic hydrolysis was also performed to understand the possibilities of using the obtained solid lignocellulosic residue for glucose production. Previously acid-hydrolysed birch wood chips were hydrolysed using the CellicEnzymatic hydrolysis was performed at different enzyme dosages: 0, 100, 250, 500, 750 and 1000 units/15 g dry substrate. Measurement of enzyme activity was performed according to the method by Denault et al. . The calFour different phosphorus-containing salts ₂, FePO₄, NaH₂PO₄) at three different amounts ) were used as catalysts during the hydrolysis process to produce furfural from the birch wood chips. 2 treatment), temperatures higher than 200 °C are needed. Furthermore, an additional step must be included in which hemicellulose is extracted from lignocellulosic biomass before furfural production using the previously mentioned method [The yield of furfural produced under autohydrolysis conditions was only 20.7% of the t.p.y. or 3.2 wt.%, calculated relative to o.d.m. Compared with industrial yield in conventional furfural production plants (which does not exceed 55% of the t.p.y.), this yield is approximately 60% lower and the addition of a catalyst is necessary to improve furfural yield. Adding phosphoric acid as a catalyst at 2, 3 and 4 wt.% to the reaction medium, the obtained amount of furfural increased to 62.9, 72.3 and 71.7% of the t.p.y. or 9.7, 11.1 and 11.0% of the o.d.m., respectively . Analysid method . From thA different situation can be observed when phosphorus-containing salts are used as catalysts to produce furfural . For exaIn contrast, when increasing amounts of FePO₄ or NaH₂PO₄ were added to the reaction zone, both chemicals showed catalytic properties on the conversion of birch wood pentosans into furfural. With an increase in the amount of FePO₄ in the reaction medium, the furfural yield showed a linear increase to 35.0% of the t.p.y., which is two times less than obtained furfural yield under the same hydrolysis conditions when H₃PO₄ was used as a catalyst. Using NaH₂PO₄ as a catalyst obtained the highest furfural yield among the studied phosphorus-containing salts—the furfural yield reached almost 40% of the t.p.y. (or approximately 6% of the o.d.m.). In addition, the obtained results indicated that the effect of the increase in NaH₂PO₄ in the reaction medium displayed similar furfural formation dynamics—at the catalyst amount of 3 wt.%, furfural yield increased, but at the catalyst amount of 4 wt.%, it began to decrease.Therefore, the general conclusion of the obtained results regarding the effect of AlPO₄, Ca₃(PO₄)₂, FePO₄ and NaH₂PO₄ on the conversion of birch wood pentosans into furfural is that the catalytic properties of these salts are much lower than those of H₃PO₄. Furthermore, an equivalent amount of furfural to today’s industrial furfural production amount (55% of the t.p.y.) could not be obtained by these salts.When acting on biomass with physicochemical processes, in addition to the target product, by-products are also inevitably formed. In our process formic acid, acetic acid, levulinic acid and 5-HMF were detected as by-products. As can be seen in Other by-products that indicate the destruction of cellulose and furfural during the hydrolysis process, such as formic acid, levulinic acid and 5-HMF, were produced in small quantities .From the point of view of the biorefinery concept, the pretreatment processes’ effects on the amount of cellulose retained in the produced lignocellulosic (LC) residue are also an important indicator. Therefore, the effect of AlPO₄, Ca₃(PO₄)₂, FePO₄, NaH₂PO₄ and H₃PO₄ catalysed hydrolysis, as well as autohydrolysis, on the content of cellulose in LC residue was analysed. The effects of autohydrolysis, H₃PO₄ and phosphorus-containing salts on cellulose degradation during the hydrolysis process are shown in 4 was used as a catalyst . The second-best result was achieved with NaH2PO4: 95% of the initial amount. By comparison, although H3PO4 as a catalyst gave the best results for furfural production, the amount of cellulose retained in the LC residue was the lowest. Therefore, to obtain high furfural yields while retaining a high amount of cellulose in the LC residue, it would be necessary to change the hydrolysis treatment parameters or use different catalysts.By comparing the cellulose yield in the LC residue depending on the catalyst amount, it can be seen that the highest amount of cellulose was retained when FePO3PO4 was used as a catalyst, with less than 0.4% of the oven-dried LC mass. Twice as many acetyl groups were retained in the LC residue if NaH₂PO₄ was used as a catalyst. These results are consistent with those observed when hydrolysate was analysed after furfural extraction.Another important parameter that can affect the effectiveness of biological treatment is the amount of acetyl groups in the obtained LC residue. During biological treatment, acetic acid is formed from these acetyl groups and is considered a critical inhibitor of the enzymatic hydrolysis of cellulose . When anTherefore, LC residue that is considerably better suited for enzymatic hydrolysis can be obtained using these two catalysts.3PO4/NaH2PO4) for furfural production. We tested three different ratios . The obtained results are summarised in 3PO4/NaH2PO4 tested, being higher compared to using NaH2PO4 alone as a catalyst and lower compared to using H3PO4 alone. With these catalyst mixtures  the obtained furfural yield was approximately 60% of the t.p.y.To test our hypothesis, we used a mixture of catalysts  and KH2PO4–K2HPO3 buffer (pH 6.7), we determined the enzyme activity with the neocuproine method. The determined enzyme activity, according to Denault et al. [To determine the suitability of the LC residue obtained in this way for the production of glucose via enzymatic hydrolysis, we chose to work with an LC residue sample treated with an Hellulose , while aellulose . Before t et al.  and Dyget et al. , was 346Enzymatic hydrolysis was performed with a fixed load of the enzyme complex  at different reaction times . As shown in During enzymatic hydrolysis, other carbohydrates were also detected in the hydrolysate. Their concentration is summarised in This study searched for the best phosphorous-containing catalyst for the co-production of furfural and glucose via catalytic and enzymatic hydrolysis, respectively. It was found that a mixture of NaH₂PO₄ and H₃PO₄ was the best catalyst for the production of furfural, compared to AlPO₄, Ca₃(PO₄)₂, FePO₄, or NaH₂PO₄ or H₃PO₄ alone. Using this catalyst, it was possible to produce approximately 61 ± 2% of the theoretically possible amount of furfural from birch wood and preserve approximately 97 ± 2% of the initially available cellulose in the solid lignocellulosic residue for the production of glucose. It is possible to deduce that this catalyst has selective properties. Preliminary study of glucose production from such pretreated lignocellulosic residue via enzymatic hydrolysis indicated that it was possible to reach a high cellulose–glucose conversion ratio  in a relatively short time. These findings indicate that it is possible to integrate furfural production into the framework of a lignocellulosic biorefinery, thus opening up the possibility of more efficient use of biomass. Future work must focus on optimisation of the operating conditions of catalytic and enzymatic hydrolysis to maximise the yield of both studied products.


Altogether, this work thoroughly decrypted the underlying correlations between apoptosis, ferroptosis, and pyroptosis and the formation of immunosuppressive HCC TME and, meanwhile, indicated that allogeneic Vδ2+ γδ T-cell transfer would be a promising adjuvant strategy for renormalizing circulating γδ T cell and thus achieving sound clinical efficacy against HCC.Hepatocellular carcinoma (HCC) is highly malignant and prone to metastasize due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Programmed cell deaths (PCDs) including apoptosis, ferroptosis, and pyroptosis routinely occur in the HCC TME and participate in tumorigenesis. However, how apoptosis, ferroptosis, and pyroptosis are involved in constructions of the immunosuppressive TME and their underlying cross-talk remains to be further unveiled. In this work, we deciphered the immunosuppressive landscape of HCC TME, which demonstrated high expressions of inhibitory checkpoint molecules and infiltration of protumor immune cells but low infiltration of antitumor effector immune cells. Further investigations unequivocally revealed that marker genes of apoptosis, ferroptosis, and pyroptosis are closely correlated with expressions and infiltrations of inhibitory checkpoint molecules and immune cells and that higher “-optosis” links to poorer patient prognosis. Notably, such three types of “-optosis” interact with each other at both the gene and protein levels, suggesting that they conspiringly induce the establishment of the immunosuppressive HCC TME. Interestingly, examinations of circulating γδ T cells in HCC patients revealed a noticeable dysfunction phenotype. The strikingly elevated ratio of the Vδ1 The liver is a major metabolic organ and plays a crucial role in all metabolism processes in the body. In certain circumstances, such as alcohol abuse, virus infection, and drugs, liver inflammation will occur and can deteriorate into hepatic fibrosis, hepatic cirrhosis, and eventually liver cancer. Generally, tumorigenesis is a result induced by complicated events, and one determinant factor is the long-term suppressive or dysfunctional immunity of hosts. Hepatocellular carcinoma (HCC) is well-known for its high relapse rate and poor long-term prognosis. Current routine treatments of HCC rely heavily on chemotherapeutic approaches  but have unsatisfactory results. Fortunately, immunotherapy strategies  includinThe excessive proliferation nature of cancer cells and the antitumor immunity of the host unavoidably lead to numerous types of cell death, mainly including programmed  and non-regulated  cell deaths. Currently, intensively investigated programmed cell deaths (PCDs) in HCC TME include apoptosis, ferroptosis, and pyroptosis. Apoptosis belongs to the non-immunogenic cell death and shall not induce detectable immune responses under normal circumstances. On the other hand, immunogenic deaths, ferroptosis, and pyroptosis can result in immune responses due to the release of various intracellular contents, most acting as pro-inflammatory signals. Though the role of ferroptosis and pyroptosis in HCC prognosis had been previously discussed , 7, how + T cell in the liver  but also statistically evaluated the imbalance of circulating γδ T-cell subsets in HCC patients.In the HCC TME, there are various types of infiltrated immune cells, including lymphoid and myeloid linage cells. Among them, γδ T cell occupies 6.8%~34% CD3he liver  and playhe liver  and tumohe liver , 12. Inthe liver . Howeverike CD73  and CD39ike CD73 , as wellike CD73  and TGFβike CD73 . The Vδ2 of IFNγ , 18 and  of IFNγ . For yea studies , 20. The+ γδ T cells from healthy donors might benefit patients. We further hypothesize that by taking advantage of apoptosis, ferroptosis, and pyroptosis, immune cell-based adjuvant therapy  might lead to a better prognosis than chemotherapy alone or chemotherapy combined with checkpoint blockade.Together, our present work revealed that the HCC TME is globally immunosuppressive, and three types of “-optosis”  play crucial roles in creating the immunosuppressive TME and lead to the adverse prognosis of HCC patients. Moreover, we found that the proportion of the Vδ1/Vδ2 subsets of circulating γδ T cell is functionally imbalanced in HCC patients, suggesting that the adoptive transfer of allogeneic Vδ2To reveal the proportional difference in the two subsets of γδ T cells from peripheral blood mononuclear cells (PBMCs) between the healthy population and HCC patients, the raw immune-phenotyping data of these two populations provided by the Shuangzhi Purui Medical Laboratory Co., Ltd.  were obtained and analyzed. The healthy population consisted of 170 disease-free adults . The HCC population consisted of 42 patients . The antibodies used included anti-human CD3, anti-human TCR Vδ1, and anti-human TCR Vδ2 antibodies . All examinations were conducted using BD FACSCanto cytometry .https://portal.gdc.cancer.gov/) by following the related guidelines and policies. The mRNA-seq data of 50 paired peri-tumor tissue samples, together with 226 healthy liver tissue samples from the GTEx database , were used as the control group, which described donors’ clinical information including gender, race, age, clinical stages, therapy history, and biospecimen collection. Protein–protein interaction was analyzed using the online database (https://string-db.org/). The R Project for Statistical Computing version 4.0.3 was used for graph plotting and analysis unless specified otherwise. In our analysis, HCC patients were divided into three groups  according to the clinical stage (pTNM) due to the limited amount of stage IV patients. To analyze the multi-gene correlation, the mRNA-seq dataset from NIH National Cancer Institute (https://tcga-data.nci.nih.gov/tcga/) was also included. The R software packages ggstatsplot and pheatmap were respectively used to produce two-gene and multi-gene correlation maps. Spearman’s analysis was used to describe the correlation.Tumoral mRNA-sequencing data (level 3) and corresponding clinical information of 371 HCC patients were obtained from The Cancer Genome Atlas (TCGA) dataset , CD274(PDL1), PDCD1LG2(PDL2), CTLA4, LAG3, TIGIT, HAVCR2(TIM3), and SIGLEC15 in HCC tissues were intensively inspected. To analyze the correlation between “-optosis” related marker genes and 15 checkpoint molecules, both stimulatory checkpoint molecules  and inhibitory checkpoint molecules  were included. To assay ICB among HCC subgroup samples, the potential ICB response was predicted with the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm . p-ValueTo comprehensively inspect the immune microenvironment of HCC TME, we firstly compared gene expression of immune inhibitory checkpoint molecules between tumor tissues and normal (peri-tumor) tissues, including CD274, CTLA4, HAVCR2, LAG3, PDCD1, PDCD1LG2, TIGIT, and SIGLEC15. It showed that all inhibitory checkpoint molecules except for CD274 are significantly upregulated in the HCC tumor group  . Further TIDE score analysis revealed that “G2” and “G3” have better ICB responses. However, OS probability shows no statistical difference among the three subgroups. Furthermore, 7 genes with a p ≤ 0.01 correlation with OS were used to construct a prognostic signature model using the least absolute shrinkage and selection operator (LASSO) Cox regression model. The obtained Riskscore equals (0.072) * BAK1 + (0.1257) * BCAP31 + (0.0895) * BCL10 + (0.2596) * E2F2 + (0.1424) * SNW1  * RPL8 + (0.0095) * TFRC + (0.1585) * SLC7A11 + (0.1652) * SLC1A5  further supports the analysis at the mRNA level, clearly showing the existing interactions among apoptosis, ferroptosis, and pyroptosis at the protein levels (Spearman > 0.4).In the context of HCC TME, apoptosis, ferroptosis, and pyroptosis occur in an interconnecting manner, and their cross-talk may exist at both the gene and protein levels. We thus evaluated those marker genes that have a p < 0.01 correlation with the OS rate of HCC patients and found that these three types of “-optosis” genes do show cross-talk at the genomic level  correlated with immune cell infiltration   are involved in constructing the immunosuppressive HCC TME. We found that in HCC, most marker genes of the three types of “-optosis” are significantly upregulated, implicating that apoptosis, ferroptosis, and pyroptosis indeed occur routinely in HCC TME. Notably, further inspections indicated that only 7 apoptosis marker genes, 5 ferroptosis marker genes, and 3 pyroptosis marker genes have strong correlations (p < 0.01) with the OS rate. This implies that the occurrence of “-optosis” in HCC TME can directly impact the clinical prognosis. Moreover, most of these 15 marker genes are upregulated gradually from pTNM stage I to IV, implying an increasing tendency of “-optosis” from the early to the late stage of liver cancer.+ T, and CD8+ T) as well  and “-optosis” related marker genes remain largely unknown. After thorough analysis, we found that nearly all “-optosis” marker genes (p < 0.01 correlation with the OS) are positively and significantly correlated with expressions of checkpoint molecules except BCAP31, RPL8, and NLRP6  or sub-healthy conditions  and for predicting HCC prognosis (≤4 links with better prognosis) needs further validation with a larger scale of clinical samples. Nevertheless, the imbalanced Vδ1+/Vδ2+ ratio has the potential to be used as an indicator in health checkups and clinical prognosis. Additionally, reduced NKG2D expression of the Vδ1+ population suggests depressed cell activation and probably enhanced survival ability of the Vδ1+ subset in HCC. In contrast, a significantly higher PD1 expression in the Vδ2+ population indicated the depressed cytotoxicity of the Vδ2+ γδ T cell in the HCC group  can be found in the article/Work supervision and project design: YH and YW. Experiments: DC, MH, JL, and YJL. Data analysis and bioinformatics: YW and YH. Data discussion: YW, YH, JH, LL, and ZY. Manuscript drafting, proofreading, and revision: YH and YW. All authors approved manuscript submission.ZY is the founder of the Shuangzhi Purui Medical Laboratory Co., Ltd. .The remaining 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.


Despite the many new possibilities, cancer pain treatment is not always effective and often poses a challenge for practitioners. At the end-of-life care, both oral and subcutaneous drug delivery very often are not attainable. The increasing number of patients in terminal stage of chronic diseases forced us to look for the alternative ways of administration of pain treatment. In this context, the potentially rapid onset of action and ease of use make aerosolized drug delivery an attractive option in palliative care settings. The objective of this review was to identify literature on pain relief with inhaled opioids. The evidence suggests that nebulized opioids might be effective in the treatment of pain in various aetiologies; however, randomized controlled studies on nebulization therapy for cancer pain are lacking. In moderate and severe pain, the treatment of choice is opioids. Although opioids are highly effective in relieving acute pain, their clinical utility can be limited by their side effects or the inability to administer them orally.,6 The latter necessitates the search for alternative forms of drug administration. Commonly employed subcutaneous delivery might be complicated by local skin reactions and is contraindicated in patients with thrombocytopenia or blood coagulation disorders. In severe cases, subcutaneous injections may even result in phlegmon. In this context, the delivery of analgesics by nebulization might be an attractive alternative, considering that it is a preferred and effective route of opioid delivery in patients with episodic breathlessness.,9 Thus, a literature review was performed to identify studies which assessed the effectiveness of nebulized opioids in the treatment of pain.Pain is one of the most important symptoms of advanced cancer.2The MEDLINE database was searched using the following terms: inhaled, inhalation, nebulized, nebulized, aerosolized, pain, analgesia and analgesic. Studies on pain relief using nebulized opioids were included in this review. A single study on nebulized fentanyl in cancer patients was added to the review despite its absence in the MEDLINE database. Studies analysis is preceded by a short discussion on the significance of inhaled opioid pharmacokinetics. This is the literature review so the ethical approval was not necessary.3The literature search provided 11 prospective studies on nebulized morphine and 11 on nebulized fentanyl. Tables 1 and 2 list the significant information on the aforementioned trials.3.1 However, the bioavailability of nebulized opioids differs greatly depending on the type of nebulizer used. Systemic absorption of opioids, delivered by simple jet nebulizers, tends to have significant interindividual variability, as shown in pharmacokinetic studies of nebulized morphine and nebulized fentanyl. Furthermore, when using jet nebulizers, which deliver aerosols irrespective of patient respiration, one can expect significant variability in opioid deposition even between consecutive nebulization's in the same patient. This can be translated into an inconsistency of analgesia with a set dose of. It has been speculated that fentanyl, due to its lipophilic properties, is more readily absorbed through the respiratory tract than morphine. However, pharmacokinetic studies employing jet nebulizers contradict this assumption. The bioavailability of both nebulized morphine and fentanyl is low (5.5 ± 3.2%) and estimated at 10%.,15The antinociceptive effect of opioids is related to their action on the μ, κ, and δ receptors located in the central nervous system and peripheral tissues. The bioavailability of morphine delivered by AERx can be as high as 66%, whereas that of fentanyl is 67%. Furthermore, the time courses of both morphine and fentanyl plasma concentrations in AERx pharmacokinetic studies were similar to their respective intravenous infusions, and so were their side effects profile. Pharmacokinetics close to intravenous and high bioavailability were also observed when fentanyl was delivered by low mass median aerodynamic diameter and high fine particle fraction metered-dose inhaler paired with SmartMist or Staccato delivery system.,18 Another approach to opioid delivery was explored by Hung et al., who studied the pharmacokinetics of a mixture of free and liposome-encapsulated fentanyl which provided both rapid onset of absorption of the former and sustained absorption of the latter component.It is worth underlining that when opioids are delivered by modern nebulizers, pharmacokinetics changes considerably. AERx, a breath-actuated nebulizer calibrated for alveolar deposition, generates a bolus of small particle aerosol only when a proper inspiratory flow and volume are achieved.3.2 The primary end-points were a decrease in visual analogue scale (VAS) greater than or equal to 50% of its baseline value and pain resolution time. The drugs investigated were intravenous and nebulized morphine, the latter at 2 different doses (10 and 20 mg). Intravenous morphine was delivered in 2 mg boluses. Drug delivery was repeated every 5 minutes for the intravenous route and every 10 minutes for the nebulization route until the intended pain VAS drop. This study showed that nebulized morphine at both doses was non-inferior to intravenous morphine in terms of both primary endpoints. In addition, morphine delivered via nebulization produced fewer side effects, all of which were minor, with dizziness being the most frequent. The mean cumulative doses were higher for nebulized morphine that is, 11.4 mg in intravenous group, 21.2 mg in nebulized 10 mg morphine group and 36.5 mg in nebulized 20 mg group. Taking into consideration significant losses of drug aerosol when it is delivered via jet nebulizer, it is to no surprise – merely 12% of the set dose is deposited in the airways. In their double-blind RCT in 44 thoracic trauma patients, Fulda et al. showed similar results. Half of the study participants received nebulized morphine every 4 hours and continuous intravenous infusion (CIV) of 0.9% saline, whereas the other half received nebulized saline every 4 hours and CIV of morphine. Additional nebulization's and intravenous boluses were delivered on demand. If on-demand doses were dispensed frequently, both the nebulization dose and CIV rate were increased according to a predefined schedule. Mean post-treatment pain VAS scores between the 2 groups were similar that is 3.38 ± 1.8 for nebulized morphine versus 3.84 ± 2.7 for intravenous morphine. Average 4-hour nominal dose of morphine was, again, higher for nebulized morphine (11.96 mg vs 6.22 mg). Nonetheless, sedation scores were higher in the intravenous morphine group . No side effects of nebulized morphine, apart from minor sedation ranked 1 in RSS, were recorded in a prospective trial in thoracic trauma patients, conducted by Nejmi et al. The authors randomized 40 patients to receive either repeated doses of 8 mg nebulized morphine or epidural bupivacaine for 48 hours. The analgesic effect, where the primary endpoint was to obtain a pain VAS score of less than 4, was similar in both treatment arms, as well as sedation. In a prospective, uncontrolled study performed by Bounes et al, a single dose of 0.2 mg/kg nebulized morphine was unable to provide satisfactory analgesia, that is, a pain numeric rating scale (NRS) of 30/100 or lower. The study group consisted of 28 patients treated in the ED with severe acute pain that was 60 mm or higher. No side effects were noted. In contrast, when the same dose of nebulized morphine is allowed to be followed with repeated half-dose analgesia becomes effective, as shown in a convenience sample study by Lefevre et al. The effectiveness of nebulized morphine, fentanyl, and alfentanil, measured with verbal NRS, in 102 patients with acute pain who were admitted to the ED.Because of its potential ease of use in both prehospital and emergency department (ED) settings, there have been a few studies showing the usefulness of aerosolized morphine in reducing acute and traumatic pain. In their double-blind, randomized, controlled trial (RCT), Grissa et al compared 3 groups of 300 patients who were admitted to the ED due to a recent trauma. However, the nebulized morphine group achieved verbally reported pain relief later (34 ± 9 vs 16 ± 3 minutes). On the other hand, a larger RCT among 52 abdominal surgery patients nebulized morphine analgesia was inferior to intravenous morphine. However, there were significant differences between the 2 groups. Surgeries in the nebulized morphine group lasted longer, upper abdominal surgeries were more frequent, and the initial pain VAS score was higher. Even though the nebulized morphine group received larger nominal dose of morphine (25.4 ± 1.1 mg vs 23.4 ± 0.8 mg), serum morphine concentrations were higher in the intravenous group. It comes as no surprise that side effects, including sedation, were more frequent in the latter. However, because of the low sedation rate, postoperative restlessness was more frequent in the nebulized morphine group. In the other 2 RCTs, in patients after cardiac surgery, Chrubasik et al showed that nebulized morphine can be effectively used as an analgesic. In the first study, a pilot study of 40 cardiac surgery patients showed that nebulized morphine was clearly superior to nebulized saline, whereas in a subsequent study, in 30 patients, nebulized morphine provided a similar analgesic effect as intravenous morphine but with less side-effects. In both studies, analgesia was assessed using additional meperidine consumption. However, 7 patients in the nebulized morphine group required additional diazepam because of restlessness. Other authors have shown the possible efficacy of nebulized morphine in pre-emptive analgesia before septoplasty or septorhinoplasty. In their double-blind RCT, Onal et al randomized 80 patients to either nebulized 65 μg/kg morphine or nebulized saline. Nebulization, delivered by a jet nebulizer, was performed 10 minutes prior to induction. It was noted that the first analgesia requirement emerged significantly later in the nebulized morphine group that is231.72 versus 48.75 minutes. No significant difference was detected between the frequency of postoperative nausea or vomiting.Nebulized morphine treatment was also explored in an anesthetic setting. Chrubasik et al in their randomized controlled trial in 20 patients after abdominal surgery showed that continuous and on-demand nebulized morphine can be as effective as continuous and on-demand intravenous morphine with fewer side effects. In this study, 89 patients were divided into 4 treatment arms: a) nebulization of 1 unit dose containing 2.2 mg of morphine, taking bioavailability into consideration, 1.4 mg would be delivered systemically, b) nebulization of 3 analogous unit doses, c) 4 mg of intravenous morphine, and d) placebo. Rescue medication, that is, 2 mg of intravenous morphine, was available in all treatment arms. As expected, 4 mg of intravenous morphine provided a similar analgesic effect, including onset time, as nebulization of 3 unit doses.As mentioned earlier, nebulized morphine pharmacokinetics depend significantly on the type of nebulizer used. The studies mentioned above exclusively employed jet nebulizers which produce low serum morphine concentrations. However, Thipphawong et al in their double-blind RCT of nebulized morphine in bunionectomy patients used an AERx nebulizer, designed to deposit most of the drug aerosol in the alveoli. Patients were hospitalized for 3 days, and pain severity was measured twice daily using the VAS. The nebulized morphine group received it at a dose of 20 mg, repeated every 10 minutes, with a maximum of 3 doses. After 3 days of treatment, nebulized morphine provided analgesia similar to oral methadone, delivered at a maximum daily dose of 45 mg, and 0.6 mg of transdermal fentanyl. The only side effect in the nebulized morphine group was dizziness, which was reported in 2 out of 30 patients. Nonetheless, according to the authors, the study was underpowered to compare the side effects profile.In the only study of nebulized morphine in palliative setting Majidinejad et al compared nebulized morphine with oral methadone and transdermal fentanyl in a RCT in 90 end-stage cancer patients.–33 The increase in M6G generation might be a result of UGT2B7 glucuronidase activity, which is capable of almost equal morphine glucuronidation to M3G and M6G, and was detected in human lung specimens. Furthermore, M6G is the metabolite which is mainly responsible for morphine's analgesic effect, while M3G is mostly associated with its side effects.As shown above, nebulized morphine frequently provided analgesia similar to its intravenous route, but with limited side effects. This could be explained by the different metabolite profiles. Krajnik et al showed that when morphine is delivered via nebulization, morphine-6-glucuronide (M6G) might be synthesized in almost the same amount as morphine-3-glucoronide (M3G), whereas morphine delivered by any other route is mostly metabolized to M3G with M3G to M6G ratios ranging from 3.12 to 11.00.3.3 However, intranasal or transmucosal absorption can be limited by mucosal inflammation, increased secretion, and excessive drying; the latter is often induced by oncological treatment.,36 Dry mouth, secondary to the dysfunction of the salivary glands, may affect up to 80% of patients with advanced neoplastic disease, hindering the dissolution of buccal tablets, increasing discomfort, and reducing the effectiveness of therapy.Fentanyl, due to its lipophilicity and fast absorption rate, is already used in the treatment of acute or breakthrough pain in the home setting when delivered intranasally or transmucosally. Singh et al compared nebulized fentanyl in two doses: 3 and 4 μg/kg with 2 μg/kg intravenous fentanyl in a double-blind, randomized, controlled study in 90 patients after lower abdominal surgery, equally divided into 3 groups. The authors showed that higher doses of nebulized fentanyl produced analgesia, as measured by VAS, similar to the intravenous route, yet delayed (10 minutes vs 5 minutes) and prolonged (90 minutes vs 30 minutes). The side effects were comparable between groups. However, sedation, as measured by RSS, was always less prominent with nebulized fentanyl which can be explained by the slower rise of nebulized fentanyl peak levels. Similar findings were reported by El-Hamid et al, who replicated the study by Singh et al. In a double-blind randomized controlled trial among 87 patients after unilateral arthroscopic anterior cruciate ligament reconstruction surgery authors showed that 4 μg/kg of nebulized fentanyl provided longer (80.5 ± 11.52 minutes vs 74.7 ± 9.81 minutes), yet slightly delayed analgesia (5.13 ± 1.16 minutes vs 4.55 ± 1.18 minutes) with less sedation when compared with 2 μg/kg intravenous fentanyl. Even longer postoperative analgesia was reported by Clark et al, who tested a patient-titrated nebulized mixture of free and liposome-encapsulated fentanyl in 19 postsurgery patients. Eighteen of them achieved effective analgesia at a median time of 17 min after nebulization which lasted for a mean 3.7 hours. Postoperative fentanyl analgesia was also explored by Higgins et al, who compared the effects of 3 concentrations of nebulized fentanyl on pain VAS in a double-blind study among 30 patients after both laparoscopic and non-laparoscopic procedures. Patients who received 960 μg had better pain control (mean VAS change: −3.4 cm) than those who received 480 μg (mean VAS change: −0.8 cm) and 190 μg (mean VAS change: −1.7 cm). However, patients rated their pain only twice, before and 5 minutes after nebulization, and received intravenous morphine if pain control after 5 minutes was not satisfactory. Most patients who received lower doses of nebulized fentanyl (480 μg and 190 μg) received morphine 5 minutes after nebulization, whereas Singh showed that nebulized fentanyl analgesia sets in on average 10 minutes after nebulization.,40 Higgins et al also underlined the potential drawbacks of delivering nebulized fentanyl in the postoperative setting, that is, the requirement of constant supervision and dependence of analgesia on variable aerosol deposition if nebulization is provided via standard jet nebulizers.Another potential route of fentanyl delivery is nebulization. Nebulized fentanyl was first studied as an analgesic in postoperative patients. Worsley et al performed a small, single-blind, randomized, controlled study of 20 patients after a variety of elective surgical procedures showed that a single dose of nebulized 300 μg fentanyl significantly reduced pain VAS scores and prolonged time to alternative analgesia in 7 patients, when compared with 13 patients who received a single dose of saline or nebulized 100 μg fentanyl. In their randomized, double-blind, controlled study, Deaton et al. compared the effectiveness of nebulized fentanyl (2 μg/kg) with 0.1 mg/kg intravenous morphine in alleviating acute, non-injury, and abdominal pain in 32 patients who were admitted to the ED. It should be emphasized that fentanyl in this study was nebulized with a breath-actuated jet nebulizer, without virtually any loss of nebulized drug into the environment. Patients treated with nebulized fentanyl had both clinically and statistically significant pain relief , which was significantly larger than that in the intravenous morphine group (highest mean VAS change: −16.63 mm). Furthermore, nebulized fentanyl analgesia was more rapid and lasted longer than the 40-minute study interval. No side effects were noted in the nebulized fentanyl group compared to 7 out of 16 patients treated with intravenous morphine who required antiemetics. Treatment of abdominal pain with nebulized fentanyl was also explored by Bartfield et al, who compared its effectiveness with intravenous fentanyl in a double-blind randomized controlled study of 50 patients. Pain VAS scores were measured at baseline and at 15 and 30 minutes after the study medications. 1.5 μg/kg of nebulized fentanyl, which was delivered with a breath-actuated jet nebulizer, was shown to be non-inferior to 1.5 μg/kg of intravenous fentanyl in terms of pain VAS reduction (−16 mm vs −25 mm) and the need for rescue medication at 30 minutes post treatment delivery. As in earlier studies, nebulized fentanyl delivered analgesia with a certain delay, that is, there was a significant difference in pain VAS at 15 minutes between nebulized and intravenous fentanyl. No adverse events were recorded in both groups. In another double-blind, randomized, controlled trial, Farahmand et al compared the effectiveness of intravenous morphine (4 μg/kg) and 0.1 mg/kg of intravenous morphine in 90 patients with acute limb pain. Pain NRS was assessed at 5, 10, 15, 30, 45, and 60 minutes after drug administration. Fifteen minutes of nebulized fentanyl was shown to provide slightly stronger analgesia than intravenous morphine, although the difference was not clinically significant (highest mean NRS change: −5.2 vs −4.6). No differences in delay in analgesia and rescue dose usage were detected between the 2 groups. No adverse effects were reported in nebulized fentanyl group.Further research showed that inhaled fentanyl might be an effective alternative for acute pain in the ED setting, especially when intravenous access is not feasible. In a study by Miner et al on 41 children admitted to the ED with significant pain of various aetiology 3.0 μg/kg of nebulized fentanyl (n = 27) provided similar analgesia to 1.5 μg/kg of intravenous fentanyl (n = 14). However, 4 patients in the nebulized fentanyl group, all younger than 3 years, had significant difficulty in triggering the breath-actuated nebulizer employed in this study. Analgesia was assessed using the physician VAS score, patient VAS score (6/27 and 5/14 children), and Children's Hospital of Eastern Ontario Pain Scale score (21/27 and 9/14 children).Delivering analgesia via aerosol might be especially convenient in children in whom insertion of an intravenous cannula or intramuscular injection can be a source of significant distress. Two non-blinded, randomized controlled studies have explored this possibility. In their study in 73 children with suspected limb fractures Furyk et al showed that analgesia with 4 μg/kg of nebulized fentanyl, delivered by standard jet nebulizer, is non-inferior to 1 provided with 0.1 mg/kg of intravenous morphine that is, mean Wong and Baker faces pain scale change with nebulized fentanyl was equal −3.60 whereas with intravenous morphine: −3.00. In this open-label, single-visit, multiple-dose study, 20 patients achieved satisfactory breakthrough pain relief within a limit of up to 3 doses of 200 μg nebulized fentanyl. No serious adverse events were observed, and few patients reported dizziness, emesis, nausea and light-headedness.In a single identified trial of nebulized fentanyl for cancer pain, Boyle et al employed the AERx nebulization system.4 Despite the constant development of aerosol therapy, research, especially employing modern, efficient inhalers, is lacking. Two studies have explored the possibility of treating cancer pain with nebulized opioids, while both showed positive results.,46 The authors’ view that the potential advantages of treating pain with nebulized opioids, such as ease of use by both patients and guardians or the ability to deliver medication when other routes are not feasible, merit further research.The limited published data suggest that delivering morphine or fentanyl via nebulization might provide effective analgesia which is non-inferior to its parenteral counterparts. As shown in the studies discussed above, no serious side effects were observed, and nebulization was not complicated by respiratory symptoms. Some studies have suggested that delivering opioids by nebulization might result in a lower side effect rate, especially with regard to sedation. The claim of treatment safety is also supported by studies on treating dyspnea with nebulized opioids.


Physical intimacy is important for communicating affection in romantic relationships. Theoretical and empirical work highlights linkages between physical intimacy, affect, and physiological stress among young and middle-aged adults, but not older adults. We examine physical intimacy and its associations with positive and negative affect and cortisol levels in the daily lives of older couples.Mage = 71.6, SDage = 5.94). Physiological stress was indexed as total daily cortisol output, the area under the curve with respect to ground (AUCg).We applied actor–partner multilevel models to repeated daily-life assessments of physical intimacy (experienced and wished) and affect obtained 6 times a day over 7 consecutive days from 120 older heterosexual German couples (Physical intimacy experienced and wished were reported at the vast majority of occasions, but to different degrees at different times. Within persons, in moments when participants experienced more physical intimacy, older women reported less negative affect, whereas older men reported more positive affect. Between persons, higher overall levels of physical intimacy experienced were associated with higher positive affect and less negative affect among women and with lower daily cortisol output among men. A stronger wish for intimacy was related to more negative affect among both women and men, and to higher daily cortisol output among men.Physical intimacy is linked with mood and stress hormones in the daily life of older couples. We consider routes for future inquiry on physical intimacy among older adults. Mage = 71.6, SDage = 5.94) obtained up to seven times per day over seven consecutive days.Physical intimacy is intertwined with relational, psychological, and physical well-being, and thus constitutes an important component of close relationships . Researcaffectionate touch, defined as touch actually or typically demonstrating affection , for example, hugging, caressing, or kissing  how physical intimacy fluctuates in older couples’ daily lives and (2) how these fluctuations are associated with (a) self-reported positive affect and negative affect and (b) overall cortisol levels. To do so, we used data from 120 couples aged 56–88 years that reported momentary physical intimacy experienced and wished, and positive and negative affect. Additionally, participants provided salivary cortisol samples. Based on prior research on physical intimacy in the everyday lives of younger and middle-aged couples , we focun = 87: Participants consisted of 120 older heterosexual German couples recruited from the Socio-Economic Panel . In 2018SD = 22.84). In the closing session, participants rated the study week as typical for their everyday lives  and were compensated up to 100 Euros per person for completing all assessments. Further information on the study protocol can be found elsewhere  using an iPad, and provided saliva samples seven times per day concurrent to the questionnaires and additionally 30 min after waking  with your partner?”, answered using a 0 (“no particular wish”) to 100 (“strong wish”) sliding scale. Second, momentary physical intimacy experienced with “Since the last questionnaire, how much physical intimacy did you actually experience with your partner?”, rated on a 0  to 100 (“much intimacy”) scale.We assessed two aspects of physical intimacy. First, momentary positive affect was assessed with six items  and momentary negative affect with seven items , each answered using a 0  to 100 (“strongly”) scale. The select items cover a broad range of low and high arousal emotions that have been shown in previous studies to: (a) fluctuate from one moment to the next, (b) be associated among older adults with other important daily-life constructs, such as perceived control or health sensitivity  do you feel right now?”, momentary ty e.g., , and (c)® Cortisol, Sarstedt, Nümbrecht, Germany), labeled to indicate day of study and time of assessment. Samples were stored during the study week in participants’ home freezer, afterwards at −31°C at Humboldt University Berlin, and subsequently shipped to Dresden LabService GmbH (Prof. Clemens Kirschbaum) for cortisol assaying; extremely low and high values were double-checked. The data were screened for compliance with the collection protocol , derived from the trapezoid formula using the discrete cortisol measurements and the time between measurements (g for days on which the two first cortisol measurements (upon waking and 30 min later) and in total, at least 3 cortisol measurements per day were available. Higher AUCg scores can be interpreted as reflecting higher overall physiological stress levels  to 5 (“very good”). The utility of single-item measures of relationship satisfaction in large-scale studies has been shown elsewhere . To model between-person differences and within-person fluctuations simultaneously, we separated the repeated assessments into time-invariant between-person variables , and time-varying within-person variables .Participants provided valid data on both physical intimacy and affect on more than 9,780 occasions . As part of data cleaning, we winsorized cortisol  and imputed missing values on occasions 3 through 7  using person-and-assessment-time-specific mean cortisol values  as:We examined our research questions using repeated measures actor–partner interdependence models for distinguishable dyads, implemented in a multilevel modeling framework . For thet by woman i is a function of a person-specific intercept coefficient β i0 that indicates the expected value of the woman’s momentary positive affect; a person-specific slope coefficient β i1 that indicates the association between occasion-specific physical intimacy experienced and momentary positive affect; a person-specific slope β i2 that indicates the association between woman’s physical intimacy wished and positive affect; a person-specific slope β i3 that indicates the association between male partner’s physical intimacy wished and woman’s positive affect; and residual error, eti. Between-person differences in the person-specific intercept coefficient β i0 were modeled as:where positive affect reported at occasion i1, β i2, and β i3 were modeled as:and the person-specific coefficients β 00 indicates the expected momentary positive affect scores for the prototypical older partnered woman in the sample; γ 10 and γ 20 represent prototypical within-person associations between woman’s momentary positive affect and physical intimacy experienced or physical intimacy wished, respectively; and γ 30 indicates the prototypical association between the woman’s positive affect and her partner’s physical intimacy wished. Statistically significant two-way interactions, γ w08, γ w11, and γ w21 were identified in exploratory ways (for each outcome separately), and, in the final models, nonsignificant interactions  were trimmed. The level-2 residuals, uiw0 and uim0, the level-1 residuals, uiw1 and uim1, and uiw2 and uim2, and the level-1 residual error terms, etiw and etim, were allowed to covary,where γ Also, residuals were allowed to covary between successive occasions (autocorrelation). All equations described above were estimated simultaneously for women and men in a dyadic multilevel model.g, the within-person physical intimacy variables were configured as day-specific (instead of moment-specific) deviations from person-specific means  were on average in their early 70s, predominantly married (97%), and in a long-term relationship . Relative to men, women reported on average lower positive affect (d = 0.44), physical intimacy experienced (d = 0.26), and physical intimacy wished (d = 0.60), and exhibited lower daily cortisol levels (d = 0.31). Experiencing more physical intimacy was associated with higher positive affect among both women (r = 0.34) and men  and with lower daily cortisol levels among men (r = −0.20 p < 0.05).Descriptive statistics and bivariate correlations for the variables under study are presented in SD = 31.63, median = 30.00), mean physical intimacy wished was 36.63 ; both distributions were positively skewed (0.36–0.37). On 75% of all occasions, ratings of both experienced and wished physical intimacy were ≥ 6 (interquartile range = 59). Mean levels of both experienced and wished physical intimacy were highest between 9 p.m. and waking  followed by the time between waking up and 10 a.m. . The average within-person correlation between both intimacy variables was 0.46 (SD = 0.28). Example distribution of physical intimacy experienced and wished over the course of the study is depicted in Mean physical intimacy experienced across occasions was 36.61 , and among men, at the within-person level (γ m10 = 0.035). No significant associations between positive affect and physical intimacy wished (both actor and partner effects) were found. For the covariates, higher relationship satisfaction was associated with more positive affect among both women and men ; no significant associations were found for age, education, and BMI. Additionally, several significant interaction effects occurred. For example, older women and men who reported on average more physical intimacy wished but less physical intimacy experienced also reported lower positive affect . Women’s and men’s intercepts were correlated 0.32, and the level-1 residuals 0.18. Fixed effects explained ≈ 22% of the variability in women’s and 24% variance in men’s positive affect.Results from actor–partner multilevel models for positive affect and negative affect as outcome variables are presented in w00 = 18.883 for women and γ m00 = 17.170 for men. Among women, experiencing more physical intimacy at both between-person and within-person level was associated with less negative affect . The within-person associations of physical intimacy experienced with negative affect among women are depicted in w06 = 0.218, γ m06 = 0.190). No partner effects emerged. For the covariates, higher education among women was related to lower negative affect (γ w02 = −1.049), and higher BMI among men was related to more negative affect (γ m03 = 0.521). Higher relationship satisfaction was associated with lower negative affect . Again, significant interactions occurred. For example, participants who reported on average more physical intimacy wished and on average less physical intimacy experienced, also reported more negative affect . Women’s and men’s intercepts were correlated 0.17, and the level-1 residuals 0.24. Fixed effects explained ≈ 27% of the variability in women’s and 30% in men’s negative affect.The prototypical level of negative affect was γ w00 = 49.611 for women and γ m00 = 49.339 for men. As hypothesized, men with higher overall levels of physical intimacy had lower daily cortisol levels (γ m05 = −0.392), and men who reported more overall wish for intimacy also had higher cortisol levels (γ m06 = 0.257). Contrary to expectations, we found no significant associations of physical intimacy experienced with cortisol among women, and no within-person associations of physical intimacy experienced with cortisol among men. Considering the covariates, older age among women (γ w01 = 0.503) and higher relationship satisfaction among men (γ m04 = 5.770) were related to higher daily cortisol output. Between-person associations between physical intimacy experienced and cortisol AUCg are depicted in m08 = 0.007). Women’s and men’s intercepts were correlated 0.37, and the level-1 residuals 0.19. Fixed effects explained ≈ 1% variance in women’s and ≈ 20% in men’s physiological stress levels.Our objective was to provide further insights into the nature of physical intimacy and its associations with affect and physiological stress in the daily lives of older couples. Results revealed that in moments of more physical intimacy experienced, women reported less negative affect, and men reported more positive affect. For the between-person associations, women who experienced on average more physical intimacy reported more momentary positive affect and less negative affect. In turn, among men, more overall physical intimacy experienced was related to lower daily cortisol levels, and more overall physical intimacy wished was related to higher cortisol levels. In general, both women and men who reported on average more physical intimacy wished displayed more negative affect.n = 109) of couples reported sharing a bedroom, the moments of physical proximity in bed may serve as a context that favors engaging in intimacy. On the other hand, exchanging physical intimacy might not be possible or desired when spending time on other activities during the day; for example, in public, or when one’s partner is not physically present. This might imply that physical intimacy remains an important channel for communicating affection in older romantic relationships; however, not all physical intimacy wished were enacted by participants, and the other way around, not all moments of experiencing physical intimacy were accompanied by intimacy wished. An avenue for future research should be to investigate correlates and implications of such discrepancies.Mean levels of both experienced and wished physical intimacy dipped into the lower halves of the response scales. This suggests that, across all assessments, the levels of (experienced and wished) intimacy were relatively low, which is not surprising given the six assessments across any given day. At the same time, experiences of and wishes for intimacy were reported on the vast majority of occasions. Also, unsurprisingly, levels of experienced and wished intimacy differed by time of day, with more intimacy reported in the evenings and mornings  and within-day (occasion-specific deviation from the person-and-day-specific mean) physical intimacy variables as predictors of positive affect and negative affect . Also, considering that both partners were asked about actual behaviors, we had expected these reports to overlap more strongly. Yet, the ratings sometimes differed between partners see , as did ning see . Acknowlning see , we hyposee also . We alsoFinally, participants were in long-term, satisfying marital relationships. Thus, it is an open question whether our results generalize to less positively selected population segments. It would also be instructive to put our findings in perspective by examining physical intimacy among nonpartnered older adults. We speculate that for older singles other types of social relationships  are more important for well-being and quality of life .This study used repeated assessment data obtained across seven consecutive days from couples aged 56–88 to examine time-varying associations of physical intimacy with positive affect, negative affect, and daily cortisol levels in the everyday lives of partnered older adults. As expected, results revealed that older partners wished for and experienced physical intimacy on a day-to-day basis, and that the extent of wishes and experiences fluctuated within and across days. Additionally, in moments of experiencing more physical intimacy than usual, older women and men reported less negative affect and older men experienced more positive affect. Higher mean levels of physical intimacy experienced were associated with more positive affect and less negative affect among older women, and lower daily cortisol levels among older men. Our findings extend previous research on intimacy in old age by applying a microlongitudinal perspective and contribute to the literature by demonstrating that physical intimacy is linked with positive and negative mood and stress hormone levels in the daily life of older couples. More mechanism-oriented research is needed to better understand the intricate links between everyday physical intimacy and well-being and gender differences therein among older adults.gbac037_suppl_Supplementary_MaterialClick here for additional data file.


Neither tecovirimat nor trifluridine was immediately available; the patient was treated with naproxen. Her ocular symptoms improved, and she was discharged after 3 days with a 14-day course of oral tecovirimat and 2)a 5-day course of topical trifluridine (. In the figure on p. 1344 (2. Foos W, Wroblewski K, Ittoop S. Subconjunctival nodule in a patient with acute monkeypox. JAMA Ophthalmol 2022;140:e223742.”In the report, “Ocular Monkeypox — United States, July–September 2022,” the case report of patient E should have included a citation to a previously published case report that described the first 2 days of patient E's clinical course. On p. 1346, the last two sentences under the heading “Patient E” should have read “ p. 1344 , the tim