The gluten-free products available on the markets are deficient in bioactive compounds and high in cost. The present study is designed to develop gluten-free biscuits with enhanced nutritional properties. The gluten-free biscuits are formulated with rice flour (RF) incorporated with Assyrian plum fruit flour (APF) and bio-waste date-pit flour (DPF) according to the following ratios; RF:DPF:APF (100:0:0)/T0, (90:5:5)/T1, (80:10:10)/T2, and (70:15:15)/T3. The results demonstrate that flour blends with different concentrations of APF and DPF incorporated in RF have high contents of protein, damaged starch, crude fiber, ash, phytochemicals, and antioxidants in contrast to 100% RF, which shows the lowest values for all these parameters. The pasting properties of the flour blends reveals that the values of peak, final, breakdown, and setback viscosities reduce from T1 to T3. Similarly, a differential scanning calorimeter reveals that the phase transition temperature of the flour blends decreases with the increasing amylose content. Moreover, the scanning electron microscopy of the biscuit samples shows a positive contribution of APF and DPF for the development of the desired compactness of the structure due to the leaching of amylose content from the starch. The total phenol content (TPC) and total flavonoid content (TFC) increase from 38.43 to 132.20 mg GAE/100 g DW and 18.67 to 87.27 mg CE/100 g DW, respectively. Similarly, the antioxidant activities of biscuits improved. The protein and fiber contents of the biscuits increased from 10.20 to 14.73% and 0.69 to 12.25%, respectively. The biscuits prepared from T3 resulted in a firmer texture with a reduced spread ratio. However, the formulation of T1 and T2 biscuit samples contributed to desirable physical and sensory properties. Therefore, the addition of DPF and APF to RF is a sustainable way to make gluten-free biscuits as they provide adequate amylose, damaged starch, and fiber content to overcome the essential role of gluten in the baked product with nutraceutical properties. In certain individuals, gluten consumption, particularly prolamin, which is a major component of gluten, can trigger an autoimmune disorder, i.e., celiac disease, mainly characterized by the disruption in the gut microbiome and causes malnourishment. The gluten protein is present in wheat, rye, and barley, which are causative agents of celiac disease . In receBiscuits are ready-to-eat, inexpensive, and convenient food items with a longer shelf life . Celiac Oryza sativa L. is the common species of rice that grows in Asia. High-energy rice cereal contains about 80% carbohydrates, is low in fat, and contains amino acids, mainly aspartic and glutamic acid. White rice is generally liked by consumers because of its appearance, taste, shelf life, palatability, ease of cooking, and hypoallergenic properties [Cordia myxa L (Assyrian plum) fruit has gained popularity due to its medicinal properties. Furthermore, its extract has demonstrated antioxidant, anti-cancer, anti-ulcer, and hepatoprotective properties in animal models [Phoenix dactylifera (date) are a rich source of dietary fibers, and polyphenols and have a significant impact on human health with anti-microbial, anti-mutagenic, anti-diabetic, and anti-carcinogenic properties [About one-half of the world’s population consume rice as a staple food item . Rice isoperties . Howeveroperties ,4,8. Corl models . On the operties . Date pioperties . Moreoveoperties ,19. TherThe nutritional and physicochemical roles of Assyrian plum fruit and date pit flours in the formulation of gluten-free rice-based biscuits have not been explored previously. Therefore, the goal of the present study is to develop nutritionally enriched gluten-free biscuits in terms of dietary fiber, antioxidants, and bioactive compounds providing the above-mentioned properties. Sugar, eggs, salt, and butter were purchased from a local market in Karachi. Non-waxy rice flour (IRRI-6) was provided by Graib Sons Private Limited. The Sulop Chemicals provided the milk and baking powders. All the chemicals used in this study were of analytical grade and purchased from Scharlau, Dae Jung, and Merck. The date pits were provided by Khairpur’s Date Industry, Pakistan, and the Assyrian plum fruits were freshly picked from the orchards of Karachi, Pakistan. The date pits and Assyrian plum were cleaned using distilled water. A hot-air oven  was used to dry the date pits at the temperature of 50 °C for 2 h. The pretreatment was given to cleaned date pits prior to milling in order to deactivate the polyphenol oxidase (PPO), which is responsible for the degradation of bioactive compounds during processing ,20.The dThe DPF and APF were added to rice flour (RF) in the following proportions (DPF: APF: RF) 100:0:0, 100:5:5, 100:10:10, and 100:15:15. Furthermore, these treatment (T) levels were indicated as T0, T1, T2, and T3, respectively. These flour blends were characterized for proximate composition, functional, pasting, thermal, antioxidants, and bioactive compound properties.All the analyses were performed in triplicates.The moisture (method 44–40), amylose (method 61–03), total starch (method 76–13), damaged starch (method 76–31), protein (nitrogen-converting factor of 6.25), ash, crude fiber, and lipid content of the RF, DPF, APF, and their blends were examined according to the AACC methods .The oil absorption capacity (OAC) and water absorption capacity of the DPF, APF, RF, and their blends were determined, as mentioned by Ahn et al. .® Micro-Visco-Amylograph , according to the AACC method 22–10 [The viscosity behavior of the flour blends at different temperatures and time combinations were determined by Brabenderod 22–10 . The parH), gelatinization conclusion temperature (Tc), gelatinization peak temperature, and onset temperature (To), were determined by utilizing Universal Analysis Software .The thermal properties of the flour samples were determined by using a differential scanning calorimeter (DSC)  . BrieflyThe antioxidant activity of the flour blends was determined by using the methanolic extract of samples . The extract was vortexed for 2 min and sonicated for 30 min in an Ultrasonicator . The content was centrifuged at 3500 rpm for 5 min and the supernatants were stored as final extracts for further analysis .The radical scavenging activity of extracts of the flour blends was determined by the method of Fan et al. . The DPP50 values (mg/mL) of the flour blends were determined by the interpretation of linear regression analysis.The ICThe FRAP of the flour blends was determined, as described by Gawlik et al. . The resFolin–Ciocalteu reagent was used to determine the total phenolic content (TPC) of the samples . The absThe total flavonoid content (TFC) of the flour blends was measured using the experimental technique of Saeed et al. . The absBiscuit samples were prepared by using RF, DPF, and APF in various combinations according to the method of Ali et al.  Table 1Table 1. The images of samples were taken at 500 X by Analytical Scanning Electron Microscope, JSM-6380 A, JEOL, Dearborn, MI, USA, available at the Centralized Science Laboratory in the University of Karachi.The following analyses were performed in triplicates.The same methods as those described above were used to evaluate the DPPH, FRAP, TPC, and TFC in order to evaluate the antioxidant capacity and bioactive components of biscuit samples ,26,27,28A Vernier caliper was used for measuring the diameter and thickness of the biscuits. The spread ratio of the biscuits was calculated by dividing the values of the diameter/thickness .A texture analyzer  was used to determine the breaking force (Hardness) of the biscuits by operating the three-point bending test rig technique  .L*, a*, and b* were determined by using an NH300 Portable Colorimeter [L* value indicated the colors: 100 for perfect white and 0 for black, while a* indicated (+) redness/(−) greenness and b* (+) yellowness/(−) blueness, which were the chromaticity values.The color values orimeter . The colThe moisture (method 44–19), protein (6.25 nitrogen-converting factor) (method 08–01), lipid (method 30–25), crude fiber (method 32–10), and ash (method 46–10) contents of the biscuit samples were determined according to the AACC method . The totThe sensory evaluation of the biscuit samples was conducted in the sensory laboratory according to the method of Ali et al. . The samp ≤ 0.05. A correlation test was performed to identify the nature of correlations between antioxidant activities (DPPH and FRAP) and bioactive compounds (TPC and TFC) by using OriginPro 2022 software . To display the results, correlation graphs (Correlograms) were generated. Principle Component Analysis (PCA) was used to correlate the experimental values of proximate composition, functional, pasting, thermal and antioxidants properties, and bioactive compounds of the flour blends. Similarly, PCA was also applied to the data of the biscuit samples  by XLSTAT .Statistical Package for the Social Sciences (SPSS) software  was used to analyze the data by Analysis of Variance (ANOVA). Duncan’s multiple range tests were used to determine any significant differences between the treatments at Cordia myxa promotes increased water absorption. One of the main advantages of the addition of DPF in the flour blends was the high content of proteins and fibers [The proximate compositions of RF, DPF, APF, and their blends, presented in d fibers , which ad fibers . As a red fibers . A simild fibers  when difd fibers . The grid fibers . Moreoved fibers . In compd fibers . In thisCordia myxa contains stable polysaccharides, which play an important role in enhancing these functional properties through their β glyosidic linkage bonds [The functional properties of flour are strongly affected by the composition of the flour and may have a direct impact on the final product’s characteristics. The functional properties of rice flour blends utilized for biscuit manufacturing are presented in ge bonds . TherefoCordia myxa fruit gum was used in a food system. The breakdown viscosity has a direct relation with the stability of starch molecules under high-heat and shear forces [The viscometric properties of the flour blends have a direct impact on their textural and sensory properties, i.e., the mouthfeel of food products. Samples T1 and T2 showed higher values of peak and final viscosities, while T3 showed lower values . Moreover forces . Moreover forces .Tc), peak temperature, and onset temperature (To) of gelatinization decreased with the increase in the concentration of APF and DPF in the RF  gradually increased with the level of fortification from 6.11 to 6.82 J/g. The increase in ΔH was attributed to the presence of hydrophilic non-starch components in APF and DPF, such as soluble fibers and proteins, which competed for the available molecules of water and reduced the hydration and mobility of starch polymers, and ultimately resulted in a higher energy requirement for melting [It was observed that the conclusion temperature  and their biscuits (360.10 mg/mL).The antioxidant activities of the flour blends and their biscuit samples are presented in p ≤ 0.05) with an increase in the levels of DPF and APF in the flour and biscuit samples. The biscuits produced from T3 flour blends showed the highest FRAP  compared to T0. The antioxidant potential of flour blends and biscuit samples was due to the presence of antioxidants and bioactive compounds in DPF and APF. Furthermore, Habib et al. [A similar trend of antioxidant activity was observed for the ferric reducing antioxidant power (FRAP) as mentioned for DPPH inhibition activity . The FRAb et al.  and El-Mb et al.  suggesteb et al. . Furtherb et al. . A similb et al.  when varThe nutraceutical value of any substance can be correlated with the presence of polyphenolic compounds, which are bioactive compounds  (Saeed ep ≤ 0.05) compared to flour blends. The maximum increase in TFC was identified in biscuit samples prepared from T3, which showed a 40.41% increase in TFC during baking, and a minimum increase was observed for T1 biscuits, which showed a 36.43% increase. The observed variations in the values of TFC may be due to the formation of brown-colored pigments, i.e., melanoidins, which are the byproducts of the Maillard reaction generally developed during the baking of biscuits [Flavonoids are one of the broad groups of phytochemicals with antioxidant and radical scavenging properties . From thbiscuits   were not included in the correlogram  with the level of DPF and APF. From L*, a*, and b* values progressively decreased, increased, and increased, respectively, following the increase in DPF and APF in the biscuit samples. The lower L* and higher a* values indicated that the T1, T2, and T3 samples were darker than the T0 samples. Furthermore, the DPF and APF were darker in color (brownish) than rice flour due to the fact that they are rich in polyphenolics and mineral content, which affected their color. Moreover, biscuit samples  with higher protein contents promoted non-enzymatic browning via Maillard reactions [The physical properties of the biscuit samples are presented in eactions . Furtherp ˃ 0.05) different from one another. Moreover, the literature reveals that gluten-free products are generally deficient in protein and dietary fiber contents and higher in carbohydrate and fat contents [The chemical composition of rice-based gluten-free biscuits with different levels of DPF and APF is presented in contents . HoweverCordia myxa, and concluded that bread made with higher percentages of Cordia myxia produced a firmer texture and darker color. Another study reported the increased firmness and reduced overall acceptability of gluten-free biscuits when the concentration of resistant starch increased [The effect of the incorporation of DPF and APF on the sensorial properties of gluten-free biscuits was evaluated  and the ncreased .To, Tp, Tc, and ΔH) in PC1. Furthermore, PC2 positively correlated to setback viscosity and final viscosity. However, the breakdown viscosity negatively contributed to the variation among the flour blend samples.The differences and similarities between the samples after the incorporation of different concentrations of DPF and APF in rice flour and its biscuit samples were evaluated by PCA . The pasL*) and nutritional  characteristics for these gluten-free biscuit samples. The average values of the sensory characteristics of the gluten-free biscuit samples were also analyzed by performing PCA . Furthermore, the protein and fiber contents of the biscuit samples increased. The T1 and T2 biscuit samples showed promising results with the most acceptable physical, textural, and sensorial characteristics. The study also highlights the possibility for the utilization of date seed bio-waste in gluten-free food-processing industries for cost-effectiveness, the enhancement of nutritional profile, and waste management by providing value to date-processing industries for commercialization. Therefore, in the future, the availability of gluten-free biscuits fortified with DPF and APF will expand the market/business of nutraceutical gluten-free products.


Delamination is one of the most critical failure modes of a PV module during service lifetime. Delamination within a backsheet primarily imposes a safety risk, but may also accelerate various other PV module degradation modes. The main aim of this paper is to present a peel test set-up, which is more practical in sample preparation and execution than the width-tapered cantilever beam test and overcomes some issues of standard peel tests like the influence of sample geometry and energy dissipation through deformation on the peel test results. The best results with respect to accuracy and effort were achieved by using a 180° peel geometry where an additional adhesive tape is applied to the peel arm in order to avoid plastic deformation or breakage. The additional support of the adhesive tape leads to comparable peel strength values without any influence of the plastic deformation behavior of the peel arms with different thickness. The general architecture of wafer-based crystalline silicone photovoltaic (PV) modules has been developed since more than 40 years ago within the Flat-Plate Solar Array Project and has not significantly changed since then . A moderDelamination of one or more layers is one of the most critical failure modes of a PV module during its service lifetime ,12,13, sNevertheless, a proper evaluation of adhesion within a backsheet laminate is still a challenge, especially for the many different new materials that have been introduced into the market. Different adhesion tests have been reported in the scientific literature, from simply measuring the peel force using T-Peel tests directly on the laminates  to more T-peels are a form of tensile peel separation strength measurement of two flexible materials that have been bonded together such that the entire setup forms a “T” shape during testing see  left. WhBy comparison, using the width-tapered cantilever beam test see , right, The main aim of this paper is to present a simple peel test setup, which overcomes issues from the T-Peel test and is more practical in sample preparation and execution than the width-tapered cantilever beam test.Hence, an adapted set-up using a 180° peel geometry (derived from ISO 8510-1 ) has beeIn this work two different multi-layer films, which are used as PV backsheets, were investigated. The films consist of PA outer layers which are laminated to a PET core layer. Both films have the same layer composition except for the thickness of both outer PA layers, which is 25 µm for Laminate 1 and 45 µm for Laminate 2, respectively. To investigate the layer adhesion, 180° peel tests were performed according to ISO 8510-1: Part 2 . For thi○Sample width: 10 mmMethod A: Standard approach○Sample width: 20 mm○Additional adhesive tape is applied to the PA peel arm in order to avoid full-field plastic deformation or breakageMethod B: Engineering approachIn order to investigate the influence of orientation within the PA peel arm, peel test specimens cut in machine direction (MD) and transverse direction (TD) were investigated. For the delamination tests, rectangular specimens with two different geometries were prepared by cutting the peel arm with a scalpel:The peel tests were carried out with a screw-driven universal test machine  at a test speed of 50 mm/min at a temperature of 23 °C.y), stress at break (σb), and strain at break (εb) were deduced.In addition, tensile tests on the PA peel arms at both thicknesses were done to describe the elastic and plastic deformation behavior. In order to investigate the influence of orientation within the PA peel arm, specimens cut in MD and TD were investigated. Tensile tests were carried out with a screw-driven universal test machine  at 23 °C according to EN ISO 527-3 [More interestingly, for each case different deformation modes were observed. Regarding method A, the PA peel arms showed linear elongation in both measured directions. In the machine direction this effect was quite limited, resulting in a peel arm elongation between 2 and 5 mm and nearly no visible necking. In the transverse direction however, strong peel arm elongation between 25 and 50 mm as well as significant necking was observed. The effect of applying an additional adhesive tape is, along with other benefits, the restraint of this elongation. Also, in the case of method B two different deformation modes were observed. During measurement in the machine direction, no deformation of the peel arm and a linear front crack was observed. In the transverse direction on the contrary, the peel arm showed strong plastic deformation near the crack front. Furthermore, delamination between the peel arm and the additional adhesive tape was also observed. When compared to the machine direction, in the transverse direction the crack front was curved.(1)Higher plastic deformation of the peel arm—globally (method A) or locally (method B)—is associated with higher peel forces.(2)The reinforcement of the thin peel arm with an additional adhesive tape leads to higher peel force values.Two fundamental conclusions can be drawn from the measured results and the investigation of the deformation modes:Laminate 2 has the same composition as Laminate 1, with exception of the thicker outer PA layer, which is 45 µm compared to 25 µm. The peel tests revealed a more complex deformation behavior than for the Laminate 1, although in general, similar effects with some deviations were observable. At both specimen geometries a significant difference between peeling in the machine direction and peeling in the transverse direction was observable. The peel forces measured in the transverse direction were significantly higher than the peel forces measured in the machine direction.As before, for each case different deformation modes were observed, but this time also within the same method and load direction. Consequently, for method A four different peel force values were obtained with a high reproducibility. For each load direction low (L), small-field, or full-field (F) plastic deformation mechanisms were observed. In the machine direction half of the investigated specimens exhibited a linear peel arm elongation below 1 mm with limited necking and a linear crack front. The other half of the tested specimens showed higher linear elongation values and significant necking, but more important, full-field plastic deformation which resulted in a distortion of the peel arm. The same two deformation modes were observed in the transverse direction, but in both cases leading to higher plastic deformation than in the machine direction.Regarding method B, in the machine direction only one deformation mode was observed after peel testing, with no deformation of the peel arm and a linear front crack. In the transverse direction, however, localized (L), small-field, as well as full-field (F) plastic deformation mechanisms were observed. About half of the specimen showed no deformation of the peel arm and a linear front crack. The second half of the specimen exhibited a slight plastic deformation near the front crack. Furthermore, delamination between the peel arm and the additional adhesive tape was also observed. Additionally, compared to the machine direction, in transverse direction the front crack was curved. Supposedly this effect was smaller than for the 25 µm peel arm due to the higher peel arm thickness.To explain the different phenomena, tensile tests of the peel arm in both directions were carried out. The stress–strain curves of the 25 µm and 45 µm thick PA peel arms for machine and transverse direction are depicted in In machine direction, the stress–strain curve of the 25 µm film shows a ductile material behavior with more or less bilinear characteristic with an elastic modulus of 970 ± 40 MPa and a yield strength value of 29.3 ± 1.3 MPa. In machine direction, the 45 µm film forms a more pronounced yield point than the 25 µm film. Slightly lower elastic modulus value of 930 ± 40 MPa and yield strength value of 28.3 ± 1.0 MPa were obtained.Directly after the yield point strong strain hardening can be observed for both film thicknesses. This behavior can be attributed to strong orientation of the molecular chains along the machine direction during film extrusion, but due to the higher film thickness this effect is less pronounced for the 45 µm film than for the 25 µm film.In transverse direction however, a totally different deformation behavior was observed. Elastic modulus and yield strength values were slightly lower but comparable to the values measured in machine direction see . Due to In the following, the influence of the mechanical behavior of both peel arms is discussed.Laminate 1: The stress–strain curves in machine or in transverse direction explain the differences in the deformation behavior of the peel arm during peel testing. In the machine direction no or only limited elongation effects were observed due to the high molecular orientation and the resulting strain hardening. In the transverse direction, strong plastic deformations of the peel arm result from the re-orientation process of the chain molecules after exceeding the yield strength. In the case of method A, a full-field elongation of the peel arm was observed. In the case of method B, where a full-field plastic deformation of the peel arm is inhibited, plastic deformation was localized at the crack tip only, where a local stress concentration is expected due to the test geometry ,23,24.Laminate 2: The deformation modes of the peel arm during peel test for machine and transverse directions as well as for methods A and B are comparable to the modes of Laminate 1. The appearance of localized (L), small-field as well as full-field (F) plastic deformation mechanisms within the same test set-up may be explained by the scattering of the yield strength values. The plastic deformation of the peel arm during peel testing is strongly depending on the local stress concentration in the peel arm at the crack tip . DeformaCG can be calculated according to Equation (1):extU, stored, reversible tensile strain energy sU, dissipated energy through irreversible tensile (dtU) and bending (dbU) deformation of peel arm, specimen width b and displacement of peel front (da) [The best way to assess adhesive strength would be calculation of the so-called adhesive fracture energy according to Moore and Williams ,25,28. Tont (da) ,28,30.2, which is needed for the calculations, proved to be challenging because of the high plastic deformation of the peel arms. The power law fit and the digitized approximation were found to not be suitable. The bilinear approach only worked for the 25 µm peel arm in machine direction, where a value for α = 0.0037 was found. A is calculated as ratio between the high strain modulus E2 and the elastic modules E [The calculation of the adhesive fracture energy was done using the ICPeel (2006) protocol . Even thodules E ,28. For CG, the plastic work in bending DG, the correction D/GtotG (input enery corrected for stored strain energy and tensile dissipations on the peel arm) and the calculated maximum stress for the damage zone maxσ (o).The tensile corrections were found to be negligible and bending of peel arm is dominating energy dissipation. As suspected, the high plastic deformations of the peel arms are not considered accurately, otherwise the adhesive fracture energy values would have been in a similar range for all peel arm thicknesses and measurement directions. Also, the size of the correction is between 27 and 49%, so the error in determination of adhesive fracture energy may become significant. Interestingly, the calculated maximum stress for the damage zone is in the region of the yield stress.In summary, the determination of adhesive fracture energy is not practical for evaluation of adhesive strength of investigated laminates, where the peel arms show high plastic deformation.The most meaningful results should be expected from a 180° peel test with method B. The additional support of the adhesive tape not only prevents thin peel arms from breaking before peeling ,5, but aWhen conducting peel tests without adhesive tapes, at least the anisotropy of the mechanical properties of the thin peel arms has to be considered. Therefore, peel tests have to be conducted in machine as well as transverse directions, and isotropic peel force values have to be derived. The mean values of the isotropic peel forces of method A is 4.4 ± 0.5 N/cm for Laminate 1 and 5.9 ± 1.1 N/cm for Laminate 2. This significant difference can be assigned to the different film thicknesses and therefore different plastic deformation behavior of the PA peel arm. The higher film thickness resulted in a higher plastic deformation of the peel arms and therefore higher peel force values.Delamination of one or more layers is one of the most critical failure modes of a PV module during service lifetime. A proper evaluation of adhesion within a backsheet laminate is still a challenge, as different adhesion tests have been reported in the scientific literature, from simply measuring the peel force using T-Peel tests to more complex test set ups based on fracture mechanics principles such as the width-tapered cantilever beam tests. The main aim of this paper was to present a peel test, which overcomes the issues from T-Peel test and is more practical in sample preparation and execution than the width-tapered cantilever beam test.The main objective was to make the adhesion values for different laminate constructions more comparable using a simple peel test geometry. Hence, an adapted set-up using a 180° peel geometry has been evaluated, where an additional adhesive tape is applied to the peel arm in order to avoid plastic deformation or breakage.The investigation showed the measured peel force values are dependent on several factors, such as load direction and peel arm thickness, since energy dissipation trough tensile deformation and bending of the peel arm is not considered. Not even the fracture mechanics approach of calculation of the adhesive fracture energy delivers satisfactory results which are independent from sample geometry. This is due to the high plastic deformation behavior of the polyamide peel arm, which makes it difficult to determine calculation parameters for the adhesion fracture energy.The best results by means of accuracy and effort have been achieved using the “engineering approach”, a 180° peel test with an additional polyester adhesive tape applied to the peel arm. The additional support provided by the tape prevented fracture and full-field plastic deformation of the peel arms, which subsequently resulted in comparable, isotropic peel strengths with no effect on the plastic deformation behavior of the peel arms of different thicknesses. The proposed test setup is more practical in sample preparation and execution than the width-tapered cantilever beam test and overcomes some issues typical for influencing factors of standard tensile peel test results such as sample geometry or energy dissipation through deformation.


Absolute poverty has historically been solved in China, and the focus on poor areas has shifted to addressing relative poverty. To realize the organic combination of the rural revitalization strategy and relative poverty governance, multi-dimensional relative poverty identification and governance path research at the village scale in an alpine-gorge region is required. For this study, the Nujiang Lisu Autonomous Prefecture’s research area in a typical alpine-gorge was chosen. This paper constructed an evaluation index system for the rural regional system based on location conditions, ecological environment, productive resources, economic base, and public service, based on the theory of multi-dimensional regional poverty and the human–land relationship. The level of poverty, types of poverty, and spatial distribution characteristics of 255 administrative villages were systematically analyzed, and poverty governance paths were proposed. The results show that: (1) There were 215 multi-dimensional relative poverty villages in Nujiang Prefecture, accounting for 84.31% of the total. The relatively poor villages with poverty grades I and II, which are classified as mild poverty, account for 77.21% of all poor villages; this demonstrated that the relatively poor villages in Nujiang Prefecture had a high potential for poverty alleviation. (2) There are 19 different types of constraints in poor villages. Grades III and IV poor villages were mostly found in high-altitude areas. The economic foundation was very weak, the infrastructure was imperfect, the land use type was relatively single, and traffic conditions were relatively backward. (3) The priority model accounted for 16.67% of relative poverty governance, the steady improvement accounted for 28.79%, and key support accounted for 54.54%. Relative poverty governance paths for various counties have been proposed, including rural revitalization priority demonstration, ecological environment governance, eco-tourism, modern agriculture + mountain agroforestry, and improved people’s livelihood and well-being. The findings provided scientific support and direction for future research on the mode and course of relative poverty governance in poor villages in the alpine-gorge area, as well as the rural revitalization strategy’s implementation. Poverty is a worldwide problem that has afflicted both developed and developing countries . PovertyScholars have various interpretations of the concept and connotation of relative poverty; opinions differ on the definition and dimensions of recognizing and evaluating relative poverty. Early research concentrated on the economic dimension . Over tiSeveral studies found it difficult to reduce impoverished people using traditional approaches and ideas when the government helped the poor out of poverty to a specific stage. The remaining impoverished people are concentrated in remote geographical areas with fragile ecological environments, outdated transportation infrastructure, and insufficient public facilities ,18. The The distribution of the rural poor in China is concentrated in 14 contiguous areas of extreme poverty, surrounded by mountains, gorges, karsts, and plateaus . After aOver the last decade, methods for identifying multi-dimensional poverty have evolved considerably. Based on field research data, scholars used the Alkire–Foster method , multi-dThe Nujiang Lisu Autonomous Prefecture (Nujiang Prefecture) in a typical alpine-gorge area was chosen as the research area in this paper. The rural regional system’s evaluation index system was built on location conditions, ecological environment, productive resources, economic base, and public service, based on the theory of multi-dimensional regional poverty and human–land relationship. After systematically analyzing the poverty level, poverty types, and spatial distribution characteristics of 255 administrative villages, poverty governance paths were proposed. The research results could provide theoretical support for the government’s local rural revitalization department in carrying out work in accordance with local conditions and promoting the rural revitalization.2 and is divided into four county-level divisions, 29 townships-level divisions, and 255 village-level divisions. The region’s total population was 552,700, with ethnic minorities accounting for 93.60% of the total population, which included 22 nationalities, such as Lisu, Nu, Dulong, Pumi, and others. Nujiang Prefecture is located in northwestern Yunnan’s Hengduan Mountains, which is a landform of “four mountains and three rivers” arranged vertically in sequence  and (2))  was usedijy is the standardized index value; ijx is the original value of the j-th index of the i-th dimension, jmaxx and jminx are the maximum and minimum of the corresponding index, respectively.In the formula, 1.Evaluation of rural single-dimension development potentialThe weighted sum formula was used to calculate the potential development score of each administrative village based on the standardized value of each indicator. The rural regional system’s single-dimensional evaluation result represents the village’s development potential in this dimension. The higher the evaluation score, the greater the potential for development potential. The formula is as follows:iS is the rural development potential score of dimensions i, i = 1, 2, 3, 4, 5 represents the location condition, economic base, productive resources, ecological environment, and public service; ijx is the value of the j-th index of the i-th dimension; and ijy is the weight of the j-th index of the i-th dimension.In the formula, 2.Single-dimensional relative poverty identification in villagesBecause of the adequacy development potential, villagers were able to pursue a certain standard of living at the village level. In contrast, villagers lose the opportunity to pursue that standard and eventually fall into relative poverty due to a lack of development potential ,33. If aiP is the relative poverty score of the i-th dimension; the iP = 1, indicating that the village is in relative poverty in the i-th dimension; and iZ is the critical value of the deprivation of the i-th dimension of the development potential. In the formula, The thresholds for the relative scarcity of the rural regional system’s development potential had not yet been unified. This paper compares relative poverty thresholds proposed by scholars ,35,36. T3.Multi-dimensional relative poverty identification in villagesiP) were added together to produce the multi-dimensional relative poverty score (X), which represents the village’s relative poverty level. When X = 0, the village was classified as non-relatively poverty; when X = 1, 2, 3, 4, 5, the poverty Grades were I, II, III, IV, V. The formula is as follows:The single-dimensional relative poverty scores  and (4). The natural break point classification method was used to divide the development potential of each dimension into three levels. The lowest score group was used as a relative poverty assessment criterion . In NujiThe development potential of rural locational conditions gradually decreased from the county seat outward a. The reThe village multi-dimensional relative poverty identification method yielded the multi-dimensional relative poverty score and poverty grade for 255 villages. In Nujiang Prefecture, 215 villages were found to be relatively poverty, accounting for 84.31% of all villages and distributed across four counties (cities) . Non-relIn the 215 relative poverty villages, the number of relative poverty grades I, II, III, and IV was 84, 82, 42, and 7, respectively. The relatively poor villages with poverty levels I and II, which fall into the mild poverty level, account for 77.21% of all poor villages; this demonstrated that Nujiang Prefecture’s relatively poor villages had a high potential for poverty alleviation.There were grade differences in the impact of various indicators among different poverty levels based on the poverty grades of the 215 relatively poverty villages in Nujiang Prefecture and the various factors of rural regional development mentioned above in The four indicators of location conditions revealed that the greater the degree of poverty, the worse the location conditions. In terms of location factors, the grade of poverty in relatively poverty villages is clearly related to major thoroughfares and townships. It has a significant inverse relationship with road network density .Regarding the economic base: Economic indicator values further reflected the relationship between various poverty levels and their economic bases. The current income status had a direct impact on their development potential, and the number of laborers was directly related to the village collective’s ability to develop. Furthermore, increasing village income was aided by the development of eco-tourism in alpine-gorge areas. In terms of economic factors, the poverty level of relatively poor villages is significantly negatively correlated with per capita income, labor availability, and the density of cultural and natural attractions .Regarding the productive resources: Land resources were found to be related to development potential, but their effects differed . There wRegarding the ecological environment: The alpine-gorge areas’ topography and fragile ecological background made agricultural production and daily life difficult . As ruraIn terms of public services, the “bottom line” of social security, education, and medical services was conducive to improving farmers’ and village collectives’ anti-risk capabilities. Obviously, the higher the level of public services, the lower the poverty grade .The villages were divided into four types and 19 combinations  based onSingle-dimensional constraint type. This type of poverty village made up 39.07% of all relative poverty villages. Poverty was divided into five categories: location condition constraints, economic base constraints, productive resource constraints, ecological environment constraints, and public service constraints . The ecoDouble-dimensional constraint type. This type of poverty village accounted for 38.14% of relative poverty villages, and there were nine combinations of “L-R, L-E, L-P, B-R, B-E, B-P, R-E, R-P, and E-P” . The mosThree-dimensional constraint type. This type of poverty village accounted for 19.53% of relative poverty villages, and there were seven combinations of “L-B-P, L-R-E, L-R-P, L-E-P, B-R-E, B-E-P, and R-E-P”. Three-dimensional constraint types were primarily distributed in Fugong and Lanping counties . There wFour-dimensional constraint type. There were seven of these poverty villages, accounting for 3.26% of relative poverty villages, and there are significant geographical, economic, and social disadvantages . LanpingBased on an analysis of relative poverty degree and poverty-causing types, this paper assessed the governance types of each relative poverty village. All dimensions of non-relatively poor villages’ development potential were on the rise, making them priority demonstration types of rural revitalization. When the poverty level was I or II, it indicated that the work of relative poverty governance could be completed by overcoming the single or double factors limiting the poverty alleviation of relatively poor villages, and they were thus classified as steadily increasing types. When the poverty level exceeds III, many factors affect the relatively poor villages, so these villages were classified as receiving key assistance. The following are the governance types of 255 villages in Nujiang Prefecture: priority demonstration (40), steady ascending (166), and key assistance (49) .(1)Rural revitalization priority demonstration modeAccording to research and field investigations, the constraints of impoverished villages in different counties in Nujiang Prefecture differ noticeably. This paper focuses on resolving outstanding constraints and adhering to the principle of “green development, adapting measures to local conditions, and making up for shortcomings” in order to consolidate and expand poverty alleviation achievements and lay a solid foundation for implementing rural revitalization, and explored the relative poverty governance path . GovernaThe southern part of Lushui city and parts of Lanping county were relatively flat, situated on the main line of external traffic, and served as the focal point of local economic and social development. It benefited from obvious geographical advantages, favorable environmental conditions, and high agricultural output. The area meets the criteria for establishing a priority demonstration area for rural revitalization. Combined with the Chinese government’s “Hundred, Thousand, and Ten Thousand” rural revitalization demonstration project, applied for the construction of rural complexes, boutique demonstration villages, and beautiful villages, could drive the development of neighboring villages.(2)Ecological environment governance modeThe key to governing relative poverty in Nujiang Prefecture is the co-governance of ecology and poverty. To improve vegetation coverage, it is necessary to strictly enforce the ecological protection red line and the boundary of spatial management and control, as well as to implement ecological restoration projects such as the Grain for Green Project and the governance of slope farmland. The vegetation ecosystem’s soil and water conservation function can be fully utilized to improve the ecosystem’s ability to regulate water and land resources. Geological hazard investigation and prevention must be carried out on a regular basis to increase people’s awareness and ability to prevent and reduce disasters. The governance of the ecological environment requires the participation of both the government and the people. Nujiang Prefecture must actively investigate mechanisms of stable interest connection with villagers, as well as develop rural-based ecological construction forces. Create ecological positions such as ecological forest rangers, natural forest rangers, public welfare forest rangers, and so on through the implementation of ecological industry infrastructure construction. Select and engage local villagers in ecological conservation, as well as promote stable employment for people lifted out of poverty. Consolidate and improve the professional cooperative model for environmental poverty alleviation, and encourage cooperatives to earn labor income by participating in ecological projects such as afforestation, grass planting, and forest tending.(3)Eco-tourism modeAlthough the region’s fragile environment impedes development to some extent, it retains valuable animal and plant, ethnic, cultural, and tourism resources and is one of the world’s biodiversity hotspots . EcotourLocal governments must choose a group of towns and villages that have a solid foundation, distinct characteristics, and a strong ethnic cultural heritage, and then build distinctive tourist towns and villages. Increase investment in rural tourism infrastructure and public service facilities, as well as improve the construction of roads, parking lots, restrooms, tourist information services, and other facilities in popular tourist towns and villages. Create a group of rural tourism projects in these distinctive tourist towns and villages, such as agritainments, farmsteads, homestay inns, boutique inns, and leisure agricultural areas. Local villagers are the primary participants in ecotourism, but the majority still lack tourism management skills. The government’s regular rural tourism training has strengthened the training of rural tourism management cadres, village officials, the rural Wealth-Leader, managerial force, agriculture proprietors, and employees.(4)Modern Agriculture + Mountain agroforestry modeIn the alpine-gorge areas, the prevailing status quo was a scarcity of cultivated and garden land resources and an abundance of forest land resources. Traditional agricultural development has had a limited impact on increasing farmers’ economic income.Utilizing forest land resources to their full potential and developing mountain agroforestry planting models have high ecological and economic value ,42. The The government encourages farmers to develop moderate-scale characteristic industries based on local conditions through the “enterprise + cooperative + farmers” model, in accordance with the vertical climate of Nujiang Prefecture. Increase farmers’ income by providing more jobs. Xerothermic river valley agriculture should be developed in the Lushui city and western part of Lanping county, this area has abundant heat, abundant light, a large temperature difference between day and night, and good irrigation water conditions, making it suitable for the cultivation of citrus, citron, mango, loquat, pomegranate, and other subtropical fruits. Plateau modern agriculture should be developed in the eastern part of Lanping county. This area’s high altitude, low temperature, and long sunshine hours create ideal climatic conditions for the plateau’s signature cash crops, such as cherries, blueberries, organic vegetables, and honeysuckle flowers.2, and it was an important industry for the villagers to increase their income. Nujiang Prefecture has become China’s primary Fructus Tsaoko production area, as well as the largest Fructus Tsaoko planting area in Yunnan Province. Introduce powerful companies and enterprises to the green spice industry, conduct intensive processing and product research and development focusing on grass fruit, and establish a green spice industry chain system integrating “scientific research + planting + processing + display + sales “ from simple planting to deep processing and brand creation.Develop the green spice industry based on the advantages of regional resources. The Fructus Tsaoko planting area in Nujiang Prefecture was 74,333 hm(5)Improve people’s livelihood and well-being modeContinue to improve the “hardware” level of transportation as well as the “software” level of education and health in the alpine-gorge areas. Construction of transportation-based infrastructure could not only drive the development of local social and economic levels but also gradually mitigate the negative effects of location conditions. Simultaneously, continue to promote the implementation of education, health, and other projects to improve people’s livelihoods and well-being, which will contribute to ensuring the bottom line and preventing a return to poverty. Make up for the shortcomings in the village’s road hardening, water supply guarantee, living conditions, and public lighting using the “make up for what is lacking” principle. To create rural communities with thriving businesses, pleasant living environments, social etiquette and civilization, effective governance, and a prosperous plate.A systematic multi-dimensional indicator system for identifying relative poverty that is consistent with China’s national conditions has yet to be developed. Existing research primarily combines regional characteristics and selects dimensions and associated indicators. Villages with deep regional relative poverty and the primary restrictive factors for alleviating relative poverty have been identified, identifying key focus areas for local government departments to implement rural revitalization strategies and policies ,44. For In contrast to previous research ,48,49, aBased on basic village data from 2013, Wang et al.  analyzedThis study proposed relative poverty governance pathways suitable for different villages and provided a reference for the sustainable development of rural revitalization in Nujiang Prefecture. However, as for the relative poverty governance pathways of villages in Nujiang Prefecture, only the aspects of industry, eco-tourism, infrastructure, and agriculture were considered in this study. Actually, the suitable poverty governance modes for different villages were various. For example, villages suitable for ecological environment governance mode have significant differences in the workload of ecological environment protection and restoration. As for the villages suitable for eco-tourism mode, it is necessary to determine whether it is suitable for the farmhouse tourism or characteristic tourist attractions, or a mixture of multiple strategies, is more suitable. Additionally, the villages suitable for “modern agriculture + mountain agroforestry” mode have significant differences in the types of favorable agricultural products. Further, the villages suitable for infrastructure are also different. In addition, the time to achieve various indicators varies, and the difficulty of implementation is also different. Therefore, further studies should be carried out to make specific suggestions according to the local regional characteristics and impact mechanism between different measures in the future.This paper chose the year 2020 as the time node for eradicating absolute poverty in Nujiang Prefecture and began to incorporate relative poverty into the research time scale. Relative poverty research at the village scale can more accurately reflect the background characteristics of rural poverty. However, there are some flaws in this paper that must be addressed. Relative poverty is a dynamic process , and a l(1)Nujiang Prefecture had 215 multi-dimensional relative poverty villages spread across four counties, accounting for 84.31% of the total. Because of a lack of development potential in the ecological environment and productive resources, the number of poor villages was the highest among them. In the relative poverty villages, the number of relative poverty classes I, II, III, and IV was 84, 82, 42, and 7, respectively. The relatively poor villages with poverty levels I and II, which are classified as mild poverty, account for 77.21% of all poor villages; this demonstrated that the relatively poor villages in Nujiang Prefecture had a high potential for poverty alleviation.(2)In the poverty villages, there were 19 combinations of constraints in the poverty villages; the main impoverishing combinations were R (29) > B-E (28) > E (23) > B/R-E-P (17). When the basic situation of impoverished villages with different poverty levels was compared and analyzed, it was discovered that the relatively impoverished villages with poverty levels I and II had better economic foundations and supporting facilities. They were mostly found near the main road and the town government. The overall facilities and supporting conditions were relatively good, allowing poor households to engage in rural tourism and traditional agricultural industries thanks to certain resource advantages. The relatively poor villages of poverty grades III and IV, in the other hand, had poor natural conditions, with the majority located in high-altitude areas. Most cultivated lands had a slope of more than 25°, the land use type was relatively single, traffic conditions were relatively backward, and the county seat and town government were far away. The economic foundation was very weak, and the infrastructure was inadequate.(3)The 255 villages in Nujiang Prefecture were divided into three types of relative poverty governance: “the priority demonstration” , “the steady ascending” , and “the key assistance”  which requires more attention and assistance. The study adheres to the principle of “green development, adapting measures to local conditions, and making up for shortcomings” pathways to relative poverty governance were proposed for a number of villages, including rural revitalization priority demonstration, ecological environment governance, eco-tourism, modern agriculture + mountain agroforestry, and improved people’s livelihood and well-being. For the development of industries and environmental improvement in poverty-stricken areas, most of the villages were suitable for implementing multi-governing pathways, which highlighted the importance of multidimensional governance. Therefore, it is necessary to pay more attention to the comprehensive integration of multiple modes and form a higher-level comprehensive governance mode by integrating multiple poverty governance pathways in practice to accelerate regional development. This is the focus of the follow-up study.The purpose of this paper was to design a rural regional system for villages in Nujiang Prefecture. It examined the relative poverty level, spatial distribution characteristics, and poverty types in 255 villages using the multi-dimensional relative poverty identification method and proposed the paths of relative poverty governance.


Immunotherapy has changed the paradigm of treating non-small cell lung cancer (NSCLC). But, selecting patients who will achieve long-term benefits from treatment remains unsatisfactory. Here, we investigated the possible use of the soluble form of CD8 antigen (sCD8) in predicting durable disease control after PD-1/PD-L1 blockade. CD8 is a marker of the cytotoxic T lymphocytes. Its soluble form (sCD8) is secreted under activation of the immune system but also has immunosuppressive properties. The data about serum sCD8 in patients dosed with anti-PD-1/PD-L1 drugs are lacking.p < 0.001). Individuals with low sCD8.2 (≤ 4.09 ng/mL) presented longer progression-free survival  and overall survival  compared to individuals with high sCD8.2 .We included 42 NSCLC patients and collected samples at baseline and for the first 3 months of atezolizumab immunotherapy. The serum sCD8 concentrations were measured with the ELISA kit and correlated with treatment outcomes. Patients with durable (≥ 12 months) disease control presented lower serum sCD8 than those without long-term benefits. The sCD8 levels measured at the end of cycle 2 (sCD8.2) were the earliest time point that successfully differentiated patients (3.76 vs. 9.68 ng/mL, respectively, Serum sCD8 could be an early biomarker of durable disease control after anti-PD-L1 treatment. Higher sCD8 in patients with worse outcomes could suggest the inhibitory effect of sCD8 on cytotoxic T-cells activation.The online version contains supplementary material available at 10.1007/s00262-023-03377-8. Immunotherapy has changed the paradigm of treating non-small cell lung cancer (NSCLC). Introducing monoclonal antibodies targeting the programmed cell death 1 receptor/programmed cell death ligand-1 (PD-1/PD-L1) axis has significantly improved the treatment outcomes in both men and women . A recenOver the last several years, multiple biomarkers have been tested to identify patients who would benefit from PD-1/PD-L1 inhibitors . The onl+ T-cells) [αβ acts as a co-receptor for the T-cell receptor (TCR): along with the TCR, it binds to the peptide-loaded major histocompatibility complex class I (MHC-I) at the surface of the antigen-presenting cells, which enhances the TCR signaling [+ T-cells rapidly proliferate and differentiate into the effector CTLs, subsequently migrating into the target sites  [αβ, a homodimer CD8αα might negatively regulate the T-cell activation via being a TCR co-repressor [The CD8 antigen is a well-recognized marker of the cytotoxic T lymphocytes  , which aT-cells) . The CD8T-cells) , 7. Nameignaling , 6 (see ronment) . Conversepressor .+ T lymphocytes [Except for the membrane-bound CD8, also the soluble form of this protein has been identified, which originates from the alternative splicing  or membrphocytes , 15; thuphocytes , 17, rhephocytes , Graves’phocytes , or acutphocytes . On the phocytes –24, suggEven though immunotherapy aims to support the activation of the immune system , there aThis was a prospective, observational, single-center study designed to evaluate the usefulness of selected serum soluble proteins as predictors of successful immunotherapy in patients with advanced NSCLC. Between February 2019 and June 2020, serial blood samples were collected at baseline and during the first 3 months of immunotherapy from 42 adult patients eligible for therapy with ATEZO within the Polish national NSCLC drug program. The detailed inclusion criteria for the program were described previously . Subjectlong-term disease control rate, i.e., the percentage of patients with at least SD after 12 months from the start of treatment . The secondary endpoints were: progression-free survival (PFS), defined as the time from the first ATEZO dose to confirmed progression or death, and overall survival (OS), defined as the time from the start of immunotherapy to death.The anti-PD-L1 treatment (1200 mg of intravenous ATEZO every three weeks) was continued until disease progression, death, unacceptable (≥ grade 3) toxicity, or significant deterioration of the quality of life. Response to ATEZO was evaluated every 3 months according to the Response Evaluation Criteria in Solid Tumors  . Clinican = 173) were collected at baseline  and at the end of cycles 1–4. After clotting, serum was separated by centrifugation at 1700 × g for 15 min, aliquoted, and kept at − 80 °C until used.Blood samples . All experiments were performed according to the manufacturer’s instructions. The optical density was read at 450 nm with the BioTek 800TS plate reader , and the log–log regression was used to calculate the sCD8 levels. The calibration range was 3.12–100 ng/mL. Samples were prepared in duplicate, and their mean was used for statistical analyses.p value < 0.05 was considered significant. The normal distribution of variables was assessed with the Shapiro–Wilk test; the normally distributed data were then compared with the t-Student test and expressed as mean ± standard deviation (sd), while the non-normally distributed data with Mann–Whitney U test and expressed as median (interquartile range). Categorical data were compared with Fisher’s exact test and expressed as numbers (%). The changes in sCD8 level over time compared to baseline were assessed with the Wilcoxon test.The statistical analysis was performed with MedCalc 20.106 software . For all analyses, a p < 0.1 were included in the multivariable model.The receiver operator characteristic curve (ROC) analysis with the Youden index was used to determine the optimal cutoff for the sCD8 protein. The durable disease control, i.e., at least SD 12 months from the start of ATEZO treatment, was chosen as an outcome for ROC analysis. The Kaplan–Meier method was then implemented to investigate the association between low/high sCD8 levels and progression or survival, and the differences between the groups were assessed with a log-rank test. Subjects who did not experience the event of interest  were censored in Kaplan–Meier analyses. The exception was patients who died within the next 3 months after the last dose of ATEZO—all these patients were considered progressed regardless of the reason for ending immunotherapy. The univariable and multivariable Cox proportional hazard models were used to confirm the prognostic value of sCD8 protein on OS and PFS. All variables from the univariable Cox analyses with a Table EGFR  or ALK (anaplastic lymphoma kinase) genes. The PD-L1 status was unknown for most participants as PD-L1 expression in cancer tissue does not play a role in the qualification for ATEZO treatment (supplementary file in ref. [ in ref. ).Table 1Figure Two patients (4.8%) stopped immunotherapy due to severe (grade ≥ 3) toxicity (both received only one dose of ATEZO), and the other two (4.8%) due to progression and toxicity. Twenty-two patients (52.4%) discontinued treatment due to confirmed progression, three (7.1%)—because of the unacceptable quality of life, and eight (19.0%)—due to death, including seven individuals who died before their first response evaluation. The median PFS in the whole study population was 4.5 (3.0–12.1) months, and the median OS was 16.8 (4.6–28.7) months. At the time of data analysis, five patients (11.9%) were still on the ATEZO treatment, and twelve patients (28.6%) were alive. One subject (2.4%) was lost to follow-up after discontinuation of ATEZO immunotherapy.In total, 173 serum samples were analyzed for sCD8 concentrations. The sCD8 levels in two samples (1.2%) exceeded the upper limit of quantitation (100 ng/mL); thus, a new (previously unfrozen) aliquot of those sera was diluted per the manufacturer’s protocol and re-analyzed. Thirteen samples (7.5%) were below the lower limit of quantitation (3.12 ng/mL), with the range of the observed concentrations 2.16–3.08 ng/mL and a median value of 2.59 ng/mL . The sCD8 concentration in all BQL (below the quantification limit) samples was at least twice the kit’s sensitivity (1 ng/mL); therefore, we decided to include all BQL samples in statistical tests. As discussed in literature , BQL datWe decided to exclude four samples from statistical analysis; the reason was a long delay in the next dose of ATEZO in one patient, which could have affected the sCD8 concentrations. The second dose of ATEZO was administered to this patient ten instead of three weeks after the first dose; thus, only the baseline serum sample from this patient (sCD8.0) was used in statistics.The median baseline sCD8 level (sCD8.0) was 7.44 (4.45–14.58) ng/mL in all patients. After the start of immunotherapy, the median sCD8 concentrations were: 9.51 (4.62–14.67) ng/mL at the end of cycle one (sCD8.1); 6.66 (4.64–14.25) ng/mL at the end of cycle two (sCD8.2); 6.00 (4.14–16.70) ng/mL at the end of cycle three (sCD8.3), and 7.41 (5.21–14.16) ng/mL at the end of cycle four (sCD8.4), i.e., about 3 months from the start of treatment.The sCD8 concentrations at baseline and during immunotherapy were independent of age (< 65 vs. ≥ 65), BMI (< 25 vs. ≥ 25), gender, NSCLC subtype (adenocarcinoma vs. others), and smoking status (current smokers vs. others) .p > 0.05 from the Wilcoxon test). However, patients who achieved long-term disease control presented significantly lower sCD8 concentrations at the end of cycles 2 (sCD8.2), 3 (sCD8.3), and 4 (sCD8.4) than patients who lacked the long-term benefits from treatment (p < 0.05 from Mann–Whitney U test). The median sCD8 values were: 5.81 versus 8.68 ng/mL at baseline (p > 0.05); 5.97 versus 9.89 ng/mL (p = 0.06) for sCD8.1; 3.76 versus 9.68 ng/mL (p < 0.001) for sCD8.2; 3.36 versus 8.70 ng/mL (p < 0.05) for sCD8.3; 4.48 versus 8.85 ng/mL (p < 0.05) for sCD8.4. There were no differences in sCD8 levels between patients who achieved and lacked the objective response during immunotherapy (data not shown).Figure p < 0.001) as a threshold with the highest sensitivity  and specificity , and further classified patients into low- and high-sCD8.2 level subgroups . The Kaplan–Meier and Cox proportional hazard regression analyses were subsequently performed to test the usefulness of the ROC-derived cutoff for PFS and OS.As sCD8 concentrations at the end of the second cycle were the earliest time point that successfully identified patients with durable disease control, the sCD8.2 levels were selected for survival analysis. The ROC analysis was performed to find the best sCD8.2 value to predict the long-term benefits from treatment . We found sCD8.2 level ≤ 4.09 ng/mL , and 89.6% lower hazard of death  compared to patients with high sCD8.2.The univariable Cox proportional hazard analyses Table  confirmep < 0.001) and OS  compared to individuals with high sCD8.2. Thus, sCD8 seems to be a promising early biomarker of successful NSCLC therapy with ATEZO. Further studies are warranted to confirm our observation in a larger cohort of patients, in other types of cancer, and other PD-1/PD-L1 inhibitors.This is the first study to demonstrate the circulating soluble sCD8 protein as a predictor of successful anti-PD-1/PD-L1 therapy in cancer. NSCLC patients with durable (≥ 12 months) disease control presented lower on-treatment levels of serum sCD8 compared to those who lacked the long-term benefits from treatment. We identified the sCD8 concentrations measured at the end of cycle 2 to be predictive in terms of treatment efficacy: individuals with low sCD8.2 (≤ 4.09 ng/mL) presented longer PFS   and high+ T-cells in vivo [Poor anti-PD-L1 treatment outcomes in patients with high serum sCD8 might be due to the inhibitory effect of sCD8 on immunological activation. Indeed, despite considering the soluble form of CD8 as a marker of immunological activation e.g., , 17, 20), 2017, 2 in vivo . A poten in vivo  or Hashi in vivo , 43. In  in vivo . Similar in vivo , and tho+ T-cells. On the other hand, the sCD8 protein binds to MHC-I [The CD8 antigen serves as a co-receptor for TCR Fig. a: it enhto MHC-I  and HLA-to MHC-I , which mto MHC-I , which mThe interesting question remains which sCD8 form is mainly secreted in NSCLC patients undergoing immunotherapy with PD-1/PD-L1 inhibitors. The in vitro studies showed that the molecular weight of sCD8 secreted by the human T-cell lines was 27 kDa , 30 kDa We acknowledge that our study had some limitations. Firstly, due to its pilot character, the sample size was small, and we could not include a validation cohort. Moreover, we did not define an adequately powered sample size a priori as the study was a pioneer in demonstrating the usefulness of sCD8 as a biomarker of successful anti-PD-L1 therapy. Also, the number of samples collected from specific patients was sometimes incomplete, as a few patients died or stopped receiving ATEZO before their first response evaluation. Lastly, the biological mechanism underlying better outcomes in patients with low sCD8 levels has not been investigated, and further experiments are necessary to reveal the role of sCD8 in successful cancer immunotherapy. If poor immunotherapy outcomes resulted from the inhibitory effect of sCD8 during T-cells activation, the agents selectively directed against sCD8 could potentially reverse that effect. This question, at the moment, remains open.Despite all these limitations, our pilot study demonstrated, for the first time, that circulating sCD8 levels could successfully indicate patients with durable disease control after PD-L1 blockade and that the topic is worth exploring. The strength of the study was its longitudinal character, which allowed us to identify the earliest time point that differentiated patients into those with and without long-term benefits based on the sCD8 levels. Also, the vast majority of samples were collected before the COVID-19 pandemic outbreak, while the sample collection process was definitely ended before the number of COVID-19 cases in Poland became significant; thus, our results were unaffected by SARS-CoV-2 infection. As sCD8 increases in viral diseases  disease control in patients treated with anti-PD-L1 drugs. Low sCD8 after two cycles of ATEZO predicted longer PFS and OS in NSCLC, suggesting a possible inhibitory effect of high sCD8 concentrations on the activation of the immune system. Further studies are warranted to confirm our observations in a larger study population, as well as in patients receiving other anti-PD-1/PD-L1 agents and suffering from other types of cancer. However, the results of our study are promising and provide a step toward a better understanding of phenomena associated with successful anti-PD-1/PD-L1 immunotherapy.Supplementary file1 (PDF 591 KB)Below is the link to the electronic supplementary material.