Soil-transmitted helminths (STHs) are widely distributed throughout the world. Various factors, including the environment, socioeconomic characteristics, and access to water and sanitation, play an important role in the spread and persistence of these parasites within communities. They, in turn, affect the growth and development of members of the community, especially children. Studies in the northern provinces of Argentina have shown variable prevalence of STHs, but the factors associated with their presence have not been completely elucidated.This cross-sectional study aimed to identify the socioeconomic and environmental factors related to STH infection in indigenous villages located in Puerto Iguazú (Misiones), Argentina.Between 2018 and 2019, stool samples were collected from individuals ≥1 year residing in 3 villages: Mini-Marangatú, Yriapú, and Fortín Mbororé. Standard parasitological methods were used to determine STH prevalence. Standardized questionnaires were used to assess participants’ habits, customs, and household characteristics, and environmental data were obtained through satellite imagery. Multilinear regression with Akaike information criterion stepwise variables was used to explore relevant associations.P=.02). The analysis highlighted the significant influence of specific environmental factors on STH presence and spatial distribution, particularly in relation to hookworm infection. Vegetation patterns represented by the Vegetation Heterogeneity Index, created ad hoc for this study, emerged as a critical factor, with 2 significant predictors related to it (P=.002 and P=.004) alongside impervious surface density with a significant predictor (P<.001). The multilinear regression model yielded a high F test score , indicating a strong fit (R2=0.5465). Furthermore, socioeconomic factors, including walking barefoot in houses with dirt floors and overcrowding, were significantly correlated with hookworm infection intensity . We also used the multilinear regression model to calculate hookworm infection intensity .A total of 342 individuals from the 3 villages participated in this study. The prevalence of STHs varied across villages: 89.6% (43/48), in Mini-Marangatú, 80.8% (101/125) in Yriapú, and 68.5% (115/169) in Fortín Mbororé. Notably, there was a significant difference in hookworm infection among the villages (Our study underscores the complexity of STH transmission, as villages with similar living conditions and environmental characteristics displayed varied STH prevalence and spatial distribution. Specific environmental factors, such as vegetation pattern and impervious surface density, played major roles in STH presence, demonstrating the crucial relationship between environmental factors and hookworm infection distribution. Moreover, our findings emphasize the significant influence of socioeconomic factors on hookworm infection intensity. By gaining insights into this complex interplay, our research contributes to a better understanding of STH transmission characteristics, thereby informing targeted public health interventions for effective control. Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale [Strongyloides stercoralis is a common STH in Latin America and the Caribbean, it is not included in this group due to its complex life cycle and specific characteristics for diagnosis, quantification, and treatment [Soil-transmitted helminth (STH) infections are the most prevalent among the neglected tropical diseases (NTDs) worldwide, affecting over 1.5 billion people as of 2023 [uodenale . Althougreatment .Previous studies have shown the involvement of different factors in the transmission of STHs, including socioeconomic factors, such as the Human Development Index; nutritional and immunological factors; and environmental factors, such as the Normalized Difference Vegetation Index (NDVI) and the Enhanced Vegetation Index (EVI) -9. All tArgentina has a heterogeneous prevalence of STHs (between 0% and 88.9%) throughout the country, and the northeast and northwest provinces of Misiones, Chaco, or Salta are identified as endemic -16, withThe Misiones province is composed of 10,218 indigenous people from the Mbyá-Guaraní ethnic group distributed among 116 communities that live mostly in rural areas  under prGeographic information system (GIS), remote sensing (RS), and digital elevation model (DEM) technologies provide information to identify and analyze determinants of disease distribution, thereby facilitating the development of risk prediction models in relation to environmental variables. This, in turn, aids the design of strategies to identify and prevent these infections. In this study, we collected parasitological and socioeconomic data from different Mbyá-Guaraní villages of Puerto Iguazú, Misiones, Argentina, as well as environmental variables, to determine the main factors associated with STH infection in this region.Puerto Iguazu is a city located in the province of Misiones in northeastern Argentina. It is naturally divided by the Paraná and Iguazú Rivers, which act as the physical borders between Argentina, Brazil, and Paraguay. According to the most recent national census data, the city has over 42,800 inhabitants .This study was carried out in the rural area of Puerto Iguazú, around the city´s periphery, where Mbyá Guaraní indigenous communities have settled into 3 villages: Fortín Mbororé, Mini-Marangatú, and Yriapú. Previous studies in this area have shown a high prevalence of IPs, specifically STHs ,16,27. MThe family subsistence economy of the communities that participated in this study is from guided tours organized to visit the village, handcrafts, and social plans . These cStool samples were collected from participants from all 3 villages. The households were visited, georeferenced, and characterized using a questionnaire. Stool containers were provided, along with verbal instructions on how to collect the samples, and retrieved on the following day. The fresh samples were transported without a fixative in a refrigerated icebox and kept at 4 °C in the lab until analysis within 24 hours of collection. The inclusion criteria were based on age (participants had to be at least 1 year of age) and willingness to participate, evidenced through the informed consent process. Individuals who lived or worked for long periods of time outside the study area and those with conditions that impaired an understanding of the consent process were excluded from the study. Considering that the area is in the tropical forest biome, which involves deforestation of tropical and subtropical forests for agriculture, the extension of grasslands may affect the viability of STH eggs and larvae. Nevertheless, several publications have suggested that the forest mass is in the process of forest transition, where the recovery of natural systems such as forests is taking place .The institutional review board of the Ministry of Health of the Province of Misiones approved this study protocol and consent forms (171403/2018). Written consent was obtained from all the parents/guardians for children under 16 years of age. All individuals aged 16 years and older are considered adults in Argentina; therefore, written consent was obtained from them directly.The collected stool samples were analyzed using the Ritchie concentration technique, Baermann concentration, and Kato-Katz technique to measure the infection intensity of STH, as previously described . The parHabits, customs, and household characteristics were analyzed at the household level using GIS. The variables, which were collected using a standardized questionnaire, are summarized in Environmental and geographical data were collected using RS together with DEM. From the RS, the Soil-Adjusted Vegetation Index (SAVI), Vegetation Heterogeneity Index (VHI), Enhanced Normalized Difference Impervious Surfaces Index (ENDISI), and Bare Soil Index (BSI) were obtained. From the DEM, the Topographic Position Index (TPI) and Topographic Wetness Index (TWI) were calculated. The characteristics and sources of these indices are detailed in Data were analyzed using Stata 12 software (StataCorp) and RStudio . Measures were evaluated using proportion with 95% CIs and means with SDs. The chi-square test was used to compare significant associations between different variables. The age variable was categorized into 5 groups: group 1 (0-5 years), group 2 (6-10 years), group 3 (11-20 years), group 4 (21-40 years), and group 5 (>40 years).P<.05, with a 95% CI. The full statistical report is available in To determine the spatial distribution of STH infection in the study area, an algorithm based on the kernel density estimation (KDE) -40 technA total of 342 individuals from the 3 villages participated in this study and provided stool samples: 169 (49.4%) individuals from Fortin Mbororé, 125 (44.5%) from Yriapú, and 48 (14%) from Mini-Marangatú. The population distribution in the 3 communities was 53.8% (91/168) men and 46.2% (78/169) women in Fortin Mbororé, 56.8% (71/125) men and 43.2% (54/125) women in Yriapú, and 50% (24/48) for both sexes in Mini-Marangatú. The mean age of participants was 21 (SD 17.9) years in Fortin Mbororé, 10.4 (SD 11.43) years in Yriapú, and 15 (SD 12.21) years in Mini-Marangatú.T. trichiura was only detected in Mini-Marangatú village, with only 1 (2.1%) case, and no A. lumbricoides infections were detected in this village. The most prevalent STH was hookworm, reaching statistically different infection rates  between the 3 villages: Mini-Marangatú , Yriapú , and Fortín Mbororé . The descriptive characteristics of STH infections in the 3 villages are provided in The overall prevalence of IPs in the 3 villages was 95.8% (46/48) in Mini-Marangatú, 95.2% (119/125) in Yriapú, and 91.1% (154/169) in Fortín Mbororé. Protozoan infection ranged from 87.5% (42/48) in Mini-Marangatú to 81.6% (102/125) in Yriapú, while helminth infections were highest in Mini-Marangatú , followed by Yriapú  and lower in Fortin Mbororé  . The STH2χ4=27.9, P<.001) and Mini-Marangatú . Mixed infections with different STH species were also observed  were found in Mini-Marangatú, but no statistical differences were observed between the types of intensity.Hookworm infection was higher in the age groups ranging from 0 to 5 years and from 6 to 10 years in Yriapú and lower in Fortin Mbororé, especially in the western area located near the urban area of Puerto Iguazú. Moreover, statistical differences between age groups were observed within Fortin Mbororé  in Mini-Marangatú and 50% (61/121) in Yriapú. In the case of Fortin Mbororé, this figure was reduced to 35% (58/166) since 40% (66/166) of the households had cement floors. Practically the entire population, both children and adults from all 3 villages, walked barefoot. Although 22.9% (38/166) of households practiced open defecation, most had a latrine that consisted of a simple ground excavation. With respect to the source of drinking water, all the families in Mini-Marangatú obtained their water from boreholes, along with 75% (92/121) in Yriapú and 49% (81/166) in Fortin Mbororé. Family incomes were low and precarious, mostly coming from animal farming, crafts, or social plans. In the newer village of Mini-Marangatú, which branched off from Fortin Mbororé, 60% (99/166) of the families obtained their income from crafts.2χ3=8.8, P=.03). Lack of sanitation and hygiene, water source, the use of a latrine with simple ground excavation, and source of income were not related to a higher prevalence of hookworms. Walking barefoot and living in overcrowding conditions were significantly related to the intensity of hookworm infection , indicating the significance of the model (R2=0.46).Only the type of floor was observed to be associated with hookworm transmission, with a significantly higher prevalence of hookworm found in individuals from Yriapú village living in dirt floor houses  with the presence of hookworm infection, which explained 56% (248/342) of the variability observed in the distribution of cases in the study area . The predictors for hookworm intensity selected by the multivariate model were the distribution of dirt floor households with individuals that walked barefoot and households with overcrowding. Both variables showed a significant association (P<.001) with the intensity of hookworm infection and predicted 45% (153/342) of the variability observed in the distribution of the cases.The multivariate model used to identify the factors most related to the distribution of hookworm in the study area selected the following predictors: mean VHI, SD of VHI, and ENDISI and households with dirt floors and individuals who walk barefoot. In this model, only the environmental variables were significantly associated This study uncovers factors associated with the presence and intensity of STH infection, especially hookworm, in rural areas of Puerto Iguazú. The high prevalence observed coincides with previous studies in other rural areas of the region ,15,42,43In the multivariate analysis for hookworm infection, barefoot walking and overcrowding were implicated in an increase in the intensity of infection, while the type of household floor material was associated with an increase in transmission. These results coincide with those already observed in other studies ,44,45. AA. lumbricoides, the proximity to the Mbocay stream in the villages of Fortin Mbororé (eastern zone) and Mini-Marangatú could have generated different A. lumbricoides infection rates between the villages. In addition, the high density of construction observed in the western area of Fortin Mbororé, through the ENDISI, may have affected the transmission of this parasite, given that low humidity can hinder egg embryonation [Since hygienic conditions have been shown to greatly influence transmission through the fecal-oral route , as in tyonation .S. stercoralis [S. stercoralis observed in this village. Available studies also show the association of humidity and vegetation indices in the prevalence of S. stercoralis [With respect to environmental factors and their predictors, previous studies have highlighted the role of humidity, temperature, and soil type in the transmission of STH ,45,48,49rcoralis ,51. A jorcoralis .To analyze the effect of vegetation on the transmission of STH, the VHI, which was developed specifically for this study, helped identify areas with more or less vegetation with respect to each surrounding household. The algorithm is similar to the TPI; however, instead of elevation, the data source is a vegetational index . High values of this index around households were associated with increased hookworm infection, which indicates the importance not only of the vigorousness of vegetation but also its distribution pattern in the landscape of the study area. High values of this index around the Mini-Marangatú households coincided with the highest prevalence of hookworm in this area. These vegetation and humidity indices, which demonstrate the influence of vegetation on hookworm distribution, coincided with the risk of hookworm infection in areas of VHI values reported in other studies ,49,53.Previous studies suggested that infection is more probable in households surrounded by bare soil, among other factors ,54-56. FThe ENDISI aids in detecting impervious surfaces, particularly the amount of water stored. The survival of hookworm larvae depends on the soil’s water-retaining properties. When the soil dries out, the water is restricted to the thin film around individual soil particles, and the infective larval stage remains quiescent in the moisture film until it makes contact with its host . The lowIn general, villages sharing similar living conditions and environmental characteristics contributed to the high infection rates observed. This study demonstrated that living conditions play a role in the intensity of hookworm infection, and environmental variables are significantly associated with its presence. However, the specific differences observed in certain areas aid in elucidating how human development and social and sanitary conditions may influence lower infection rates among individuals and villages located in endemic areas. Evidence from previous studies and the results obtained herein show that environmental factors such as temperature, vegetation, and humidity play a role in the presence and maintenance of STHs in the soil. Therefore, environmental changes caused by climate change could modify the distribution of these parasites, although deforestation, bare soil, high temperature, and lack of humidity could restrict their presence.The limitations of this study include the sensitivity of the techniques used to detect STHs since low burdens of infection could be missed. Additionally, in this area, hookworm was the most prevalent STH; therefore, studies conducted in areas with a greater presence of other species of the group would be beneficial. Further studies in more heterogeneous communities with similar environmental characteristics could help aid our understanding of the socioeconomic and building characteristics that determine the presence and intensity of STH infections.This study, conducted in an endemic area for STHs, especially hookworms, reinforces the importance of the environment in the establishment of this group of parasites, which require passage through the soil for their development. Additionally, we observed that living conditions, like walking barefoot, having dirt floors, or overcrowding, are associated with the intensity of hookworm infection. Given that environmental variables cannot be changed, it is important to work on those aspects that can be modified, such as the characteristics of the house, the availability of water and sanitation, and periodic deworming as suggested by the World Health Organization deworming guidelines .


Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb—tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions. Long-term spaceflights adversely affect the musculoskeletal system, primarily the bones ,2,3. AboThe probable cause may involve the damage of stromal lineage cells which provide physiological and reparative remodeling of skeletal tissues. Cells of mesenchymal stromal lineage play an important role in providing homeostasis and physiological remodeling of various tissues, primarily the musculoskeletal system .According to current views, mesenchymal stem cells (MSCs) undergo proliferative and commitment events, and their progeny enter discrete lineages that result in the ultimate terminal differentiation of definitive phenotypes such as osteoblasts, chondroblasts, or myoblasts . Thus, tTo date, a set of ex vivo methodological approaches has been established, allowing to evaluate the potential of CFU-f and MMSCs .After spaceflights, a decrease in the number of CFU-f in the rat BM was noted after Bion biosatellite flights . At the With the prospect of interplanetary missions, the distance and duration of space flights will only increase, so the data from long-term ground-based simulations of spaceflight effects are in high demand. In the 1980s, the rodent hindlimb unloading (HU) model was developed to make it possible to study mechanisms, responses, and treatments for the adverse consequences of microgravity. Currently, HU is considered as one of the most appropriate analogs of spaceflight factors as a load deprivation and a headward shift of internal organs and fluid . The effIt is reasonable to assume that the changes after microgravity simulations like RPM and HU can be provoked by alterations in BM stromal lineage cells including primitive and committed precursors. The number and activity of stromal precursors in BM can differ in proximal and distal parts of the hindlimb skeleton, which will determine susceptibility to the negative effects of microgravity. In this study, we evaluated ex vivo the influence of space environment simulation in HU model on the stromal lineage precursors from the femur and tibia bone marrows after 14 days of HU and subsequent 14 days of reloading (RL).The number of total nucleated cells (TNC) was calculated after isolation of BM from the femur and tibia bones of the control and experimental animals. In the control group, the number of TNCs from the femur bones was, on average, 1.5 times higher than in the tibia bones. After HU, the cellularity significantly decreased in both bones: 2.2 and 1.7-fold in the femur and tibia, respectively. There was no complete recovery of TNC number during the 14 days of reloading (RL); there were still significantly fewer cells in both bones than in the VC. Only in the tibia, the number of TNCs after RL significantly increased compared to HU . It is well known that the population of stromal progenitors in BM is heterogeneous. It includes multipotent mesenchymal stromal cells (MMSCs) as well as their primitive precursors (CFU-f) and more committed progeny. The potentials of these cells can be evaluated ex vivo using well established methodological approaches.4 cells/mm2, as described previously /log2, where N0 and N were initial and final number of cells, respectively [To assess MMSC growth, BM cells were seeded at a density of 20 × 10ectively .3/cm2 in 35 mm Petri dishes and cultured in full culture medium for 7 days. Then, a culture medium in a half of culture dishes was replaced with Mesenchymal Stem Cell Osteogenesis Kit  to induce osteodifferentiation. On day 7, a part of dishes in both control and induced groups were used for histochemical detection of activity of early marker of osteodifferentiation—alkaline phosphatase with Alkaline Phosphatase Kit . Briefly, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, washed thoroughly with PBS and stained with solutions from Alkaline Phosphatase Kit  for 30 min according to the manufacturer’s instructions.To assess the level of spontaneous osteocommitment and response to osteoinduction, the first passage MMSCs were seeded at a density of 2 × 10The activity of enzyme was characterized by the presence of the colored product, and the intensity of staining was examined with bright field microscopy . Subsequent processing of digital images was done using the Sigma Scan Pro 5 software. The colored product had different tones of blue, and the intensity of the staining reflected the activity of the enzyme. The intensity of blue staining was divided into 256 channels and measured in each channel . In our On day 21, the mineralization of extracellular matrix (ECM) as a sign of late osteodifferentiation was assessed using Alizarin Red staining . The cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and incubated with alizarin red  for 30 min. Excess of stain was removed by washing four times with water. To determine the area occupied by the mineralized ECM, the entire culture surface was photographed using a Stemi 2000-C stereoscopic microscope , after which the image was analyzed using Sigma Scan Pro 5 software .The soluble mediators were detected in conditioned medium of MMSCs using multiplex analysis with Luminex xMAP technology. We used Milliplex xMAP system and cytokine assay kit for IL-1a, IL-4, IL-1b, IL-2, IL-6, IL-13, IL-10, IL-12p70, IFNg, IL-5, IL-17A, IL-18, IP-10, TNFa, as well as regulators of bone tissue metabolism: ACTH, OPG, Insulin, Leptin, PTH, DKK1, SOST, FGF23  according to manufacturer’s protocol.2—Real Time SYBR Green/ROX PCR master mix  and a set of lyophilized primers  QuantiTect Primer Assays . Normalization was performed according to the expression level of the reference gene Rplp1 . The expression level was assessed using the 2−∆∆Ct method according to the manufacturer recommendation .To determine the level of gene expression, total RNA was isolated using QIAzol lysis reagent , followed by a reverse transcription reaction using QuantiTect Reverse Transcription Kit  according to the manufacturer’s instructions. The obtained cDNA was used for quantitative PCR using a commercial RTFor the ex vivo experiments three independent stromal cell lines isolated from each single BM were used. Three technical repetitions were run in each experimental set. Ten randomly selected view fields were examined in morphometric assays.p < 0.05.Statistical analysis was performed using Microsoft Excel 2010 and Statistica 7.0, with analysis of variance method. Data are presented as mean +/− SEM. The differences were considered significant at


Recently, the application of the amniotic membrane (AM) in ophthalmology is gradually expanding from the anterior to the posterior segment of the eye. Its characteristics of anti-inflammation, anti-bacterial, anti-vascularization, immune regulation, anti-fibrosis, pro-epithelialization, and so forth have made it a hot topic in ophthalmic research. AM has been confirmed to repair photoreceptors, restore normal retinal structures, and close the abnormal structures in the optic disc. Currently, the application areas mainly include retinal hole, retinal detachment, optic disc pit, retinal degenerative diseases, and choroidal hole. This article reviews the current literature applying AM transplantation in the treatment of various posterior segment diseases while comparing the clinical outcomes with other techniques. Human amniotic membrane (hAM) is the innermost layer of the placenta. It can be applied to various clinical settings considering its unique structure, biocompatibility, and other biological properties. Previous studies have shown its ability to reconstruct the corneal surface in cornea diseases, such as persistent epithelial defects, partial limbal stem-cell defects, bullous keratopathy, and corneal ulcers. hAM, as an effective conjunctival substitute, has also been used in the resection of pterygium, conjunctival diseases, and repair of conjunctival defects. Moreover, AM has been confirmed to repair photoreceptors, restore normal retinal structures, and close the abnormal structures in the optic disc. Its utility is being explored in some other challenging posterior segment diseases, of which this study will introduce the progress .In this literature review, a non-systematic search was carried out in PubMed, Web of Science, Embase, and Google scholar using the following query conditions: “[(eye) OR (ocular) OR (fundus)] AND [(amnion) OR (amniotic membrane)] NOT [(cornea) OR (surface)]”. Articles in English or with English abstracts were retrieved. Most relevant results concerning the posterior segment of the eye were manually screened and categorized by Yang HW. The retrieval scope included all applicable literature present in the above databases without temporal limits.hAM is the innermost layer of the placenta, which is smooth, translucent without blood vessels, nerves, or lymphatic vessels, and has a certain degree of elasticity. Histologically, hAM is a five-layer membrane ranging from 0.02 to 0.5 mm, consisting of the epithelial layer, basement membrane, compact layer, fibroblast layer, and spongy layer . The epiThe anti-inflammatory mechanisms of AM are not yet completely understood. It can act as a physical barrier to inflammatory cell infiltration, attract and trap inflammatory cells, thereby reducing the release of inflammatory mediators. Meanwhile, it can produce secretory leukocyte protease inhibitors (SLPIs) and elastin with anti-inflammatory properties . AM alsoThe antifibrotic effect of AM was achieved by inhibiting the transforming growth factor-β (TGF-β) signaling and reducing the expression of TGF-β1, TGF-β2, TGF-β3 isoforms and TGF-β type II receptors. The formation of the scar tissue is mainly due to the excessive proliferation of fibroblasts, and TGF-β can activate fibroblasts. Therefore, the downregulation of the TGF-β signaling system is considered to be a mechanism to prevent scar-forming during the wound healing process. Compared to their counterparts grown in plastic or collagen gel, AM cultures inhibited TGF-β receptor expression in fibroblasts, and this inhibition remained after 1 week of culture, revealing the anti-fibrotic property of AM .Hemolytic streptococcus group A, Staphylococcus aureus, and Staphylococcus saprophyticus. Talmi et al.  and retinal hole (RH). One patient had rhegmatogenous retinal detachment (RRD), and five had recurrent RD. All of them had varying degrees of proliferative vitreoretinopathy (PVR), which is considered clinically refractory with a poor prognosis. Three months after the operations, their final BCVA  improved from 2.33 ± 0.51 logMAR  to 1.2 ± 0.62 logMAR (20/400). Two months after silicone oil (SO) removal, their BCVA improved to 0.8 ± 0.47 logMAR (20/125), with no postoperative adverse events. After a long-term follow-up of more than 1 year, there were no reports of transplant rejection .In November 2019, two cases of plaque chorioretinal atrophy with RD and paravascular RH were treated by Caporossi et al. Both vitrectomy and hAM transplantation into the hole were applied. OCT  imaging showed new tissue growth on hAM graft 2 weeks after the surgery, and it completely covered the hAM plugs 3 months after the surgery. The BCVA increased from 20/2,000 (2 logMAR) to 20/250 (1.1 logMAR). After 2 months of surgery, the SO was extracted, and no recurrence was observed. The patients' corrected vision was stable at 20/250 .In September 2020, Tizio et al. applied an improved surgical procedure using hAM graft and autologous platelet-rich plasma (PRP) transplantation to treat a case of pathological myopia with posterior scleral staphyloma and perivascular RH leading to recurrent RD. hAM graft promoted the healing of RH, and 6 weeks after the operation, an OCT scan showed RH having been covered by new tissues. The SO was removed 3 months later, and no recurrence of RD was observed at postoperative follow-up .In November 2020, six patients with RRD were treated by Saravia et al. using a more easily stored, highly bio-safe, lyophilized AM patch (LAMPatch). Post-operatively, all four patients' LAMPatch fitted well, and the BCVA ranged between 20/30 and 20/100 at 1 week after surgery. LAMPatch tamponade was missing in only one patient after one month, and one patient had partial LAMPatch detachment 5 months after surgery. No recurrence of RD and no complications such as increased IOP, cataract, or inflammation were observed in any patient . DetaileAn macular hole (MH) is an anatomic discontinuity of the neurosensory retina that develops in the center of the macula or fovea. Currently, vitrectomy is the most effective treatment for MH. However, in some cases, MH is often difficult to heal due to the large aperture, long courses of the disease, and other complex etiologies. The studies of Fekrat, Funata, and Madreperla –19 all iIn October 2019, Rizzo et al. conducted hAM tamponade and follow-up of eight patients with recurrent MH. After 1 week of the surgery, all patients had an OCT scan showing closure of the holes, with BCVA improved from 1.48 ± 0.49 logMAR (20/800) preoperatively to 0.71 ± 0.37 logMAR (20/800) at 3 months postoperatively and 0.48 ± 0.14 logMAR (20/50) at 6 months postoperatively . In ApriGiant MH is also a type of refractory MH. In April 2019, Caporossi et al. performed vitrectomy and AM tamponade in two patients with large MH with complex RD. Both patients had their vision improved from light perception (LP) to 20/400 (logMAR 1.3) at 3 months after surgery. After SO removal at 4 months after surgery, no recurrence of MH was observed, and the visual acuity was stable at 20/400 . We haveDuring AM tamponade surgery, because of tissue redundancy, AM larger than the basal diameter of the MH tends to produce folds. In the early phase of the postoperative recovery, inward migration of the retina exacerbates the fold and causes accumulation of the AM tissue at the edge of the hole, preventing further recovery of the hole. Relatively speaking, AM graft that is too small does not come into contact with the edge of the hole and will not induce the closure of it, thus affecting the patient's postoperative vision recovery. In view of this problem, in April 2020, Caporossi et al.  proposedAs compared to the inner limiting membrane (ILM) flap insertion for the MH treatment proposed by Chen et al.  in 2018,Retinal degenerative diseases (RDD) mainly include age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Current clinical treatment can only delay the progression of such diseases, and there is no clear treatment to reverse retinal degeneration or restore normal retinal function and vision. Retinal cell transplantation holds great potential for treating RDD in future, and retinal pigment epithelium (RPE) transplantation has been shown to rescue photoreceptor cells in dystrophic rats . It has Human retinal pigment epithelium (hRPE) cells transplanted on AM have been shown to adhere to it within 24 h. In addition, the cells maintained their epithelial characteristics  and proliferated. It was also found that hRPE cells growing in the AM were highly organized, which constitute a tight monolayer arrangement with clear cell-cell and cell-substrate interactions . These rAM can also be used as a source of stem cells in stem-cell therapy. Human amniotic epithelial stem cells (hAESC) express several stem-cell markers, such as SSEA-3, SSEA-4, SOX-2, and NANOG, which imparts hAESCs the capability to multi-directional differentiation , 31. PluIn RDD, AM can also be directly implanted under the retina to play its role. In the study of Rizzo et al.  in OctobThe optic disc pit is a rare abnormality in the papilla of the optic nerve. Approximately 25–75% of patients will develop a serous detachment of the central macula at some point in their lives, some with an RH, resulting in optic disc pit-associated macular diseases (ODP-M). At present, the pathogenic factors for the disease remain unknown .From July 2018 to January 2019, Rizzo et al. attempted to treat three patients with ODP-M using vitrectomy combined with AM implantation in optic nerve recess and vitreous gas injection. Postoperatively, the patients were found to have decreasing subretinal fluid (SRF), and the BCVA also improved to 20/25 at the 6-month follow-up . SubsequTherefore, alternative biomaterials to seal the optic nerve excavation are the latest popular treatment option, and the ILM has also been applied by researchers to fill the excavation and achieved good results . HoweverMorning glory syndrome (MGS) is also a rare congenital malformation of the optic papilla. It may be a type of optic nerve entrance defect ; or it mIn July 2017, Zhu et al.  presenteIn recent years, more and more researchers are expanding the application of hAM to the posterior segment of the eye, making it a popular field of research. Due to the excellent biological properties of hAM, its application in the posterior segment has shown satisfactory results. However, owing to the limited sample size, most of the current studies are confined to clinical outcomes, and the mechanism is not yet clarified. There are no comparative reports with other emerging surgical studies. Therefore, more prospective, multicenter randomized controlled studies should be carried out to confirm these aspects.Although the application effect of hAM in the direction of RH and MH is promising, the current study alone cannot explain whether the holes are sealed by the regeneration of the retinal neuroepithelial layer. Moreover, in some reports, the operation has been associated with the disappearance and displacement of hAM, so how to avoid these occurrences is a major concern in future. It is also necessary to further explore the mechanism of cell growth and the growth orientation after AM tamponade as well as which side of the AM adhesion is more beneficial to the recovery of the macular and retinal structure during the operation.in vitro as a substrate or as a source of stem cells, when implanted directly under the retina, the mechanism is not clear. Whether hAM functions as a substrate promoting the development of photoreceptor cells or hAM's stem cells develop toward photoreceptor cells deserves further investigation. The advantages and disadvantages of the above three treatment options are also worthy of comparison in clinical studies.The current study has explored three aspects of hAM as a substrate, implant, and stem cell source. Although hAM can develop toward photoreceptor cells The hAM implantation in the treatment of optic disc pits is thought to have structurally closed the pits, thereby reducing SRF/IRF fluid, but the specific mechanism is still unclear. There is also a lack of research on whether hAM can remain in this closed structure intraocularly for long periods.At present, there is only one case of hAM transplantation for the choroidal hole, which is still in the exploratory stage. More research is needed to probe the repairing ability and to study the repairing mechanism of hAM on the choroidal hole in comparison with other therapeutic methods, such as scleral buckling.In conclusion, the application of hAM in the posterior segment of the eye has gradually become a heated field of research. Its superb biological properties and structures present great potential in fields such as retinal hole, macular hole, optic diseases, retinal regenerative disease, and choroidal hole. It deserves further exploration in more prospective and larger randomized controlled studies to evaluate its underlying applications in the posterior segment of the eye.HY, ZL, and WJ: study conceptualization, methodology, and original draft preparation. ZL: review and editing and figures preparation. WJ and AY: supervision. All authors have read and agreed to publish the manuscript.


Background Multiple sclerosis (MS) is an inflammatory demyelinating disease. Auditory evoked potential studies have demonstrated conduction and neural processing deficits in adults with MS, but little is known about the electrophysiological responses in children and adolescents.Objective to evaluate the central auditory pathway with brainstem auditory evoked potentials (BAEP) and long-latency auditory evoked potentials (LLAEP) in children and adolescents with MS.Methods The study comprised 17 individuals with MS, of both sexes, aged 9 to 18 years, and 17 healthy volunteers, matched for age and sex. All individuals had normal hearing and no middle ear impairments. They were assessed with click-BAEP and LLAEP through oddball paradigm and tone-burst stimuli.Results Abnormal responses were observed in 60% of electrophysiologic assessments of individuals with MS. In BAEP, 58.82% of MS patients had abnormal responses, with longer wave V latency and therefore longer III-V and I-V interpeak latencies than healthy volunteers. In LLAEP, 52.94% of MS patients had abnormal responses. Although statistical differences were found only in P2-N2 amplitude, MS patients had longer latencies and smaller amplitudes than healthy volunteers in all components.Conclusion Children and adolescents with MS had abnormal BAEP responses, with delayed neural conduction between the cochlear nucleus and the lateral lemniscus. Also, abnormal LLAEP results suggest a decrease in neural processing speed and auditory sensory discrimination response. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system – where inflammation, demyelination, and axonal loss occur as early as the initial stages of the disease. It is one of the most frequent causes of neurological disability in young individuals.MS affects mainly young adults, mostly women 20 to 40 years old. However, an estimated 30,000 children and adolescents worldwide are believed to be affected by it, totaling 2% to 5% of all cases.MS manifests as an inflammatory disease among children and youth, causing more seizures and evidence of brain atrophy, axonal damage, and accumulated lesions identified in magnetic resonance imaging (MRI) than in disease onset at adulthood.Considering these individuals' neuronal impairment, it has been recommended to use evoked potentials in batteries to diagnose MS, assess the progress of the disease, and monitor the benefits and limitations of various treatments.Auditory evoked potentials assess the neuroelectric activity in the auditory pathway from the auditory nerve to the cerebral cortex, evoked with acoustic stimuli. The brainstem auditory evoked potentials (BAEP) are one of the most used resources in clinical practice; their main objectives are to identify changes from the auditory nerve to the brainstem and estimate the electrophysiological hearing threshold.Various studies have assessed electrophysiological measures in adult MS patients.The study group (SG) comprised 17 individuals who attended the Children's Institute of the Medical School Clinics Hospital at the University of Sao Paulo (HCFMUSP), diagnosed with MS , of both sexes , aged 9 to 18 years (13.71 ± 3.01). The age of symptom onset ranged from 4 to 16 years (11.71 ± 3.51), and the diagnosis was confirmed by 6 to 17 years old (12.29 ± 3.45).The control group (CG) comprised a convenience sample of 17 healthy volunteers, matched with SG for age and sex, without developmental impairments or neurological or psychiatric complaints, recruited from local schools.None of the participants had obstructions in the external auditory meatus or conductive impairments – they had type-A tympanograms –, and all of them had normal hearing (hearing thresholds below 15 dB HL at 500 to 4000 Hz).The research was approved by the institution's Research Ethics Committee under number 1.784.31. All parents/guardians and participants respectively signed informed consent and assent forms before the study.After the complete audiological assessment, the auditory evoked potentials were obtained using the Smart EP equipment manufactured by Intelligent Hearing System and ER 3-A insert earphones. During the assessment, subjects remained seated in a reclining chair, in an acoustically and electrically treated room. The skin surface of the forehead, mastoids, and scalp was cleaned with abrasive paste, and Ag-AgCl electrodes were then positioned with electrolytic paste and micropore tape, following the international 10-20 system . Electrode impedance was maintained below 3 kOhms in all trials.Two BAEP channels were applied using an electrode montage of Fz (active electrode), Fpz (ground), and M1 and M2 (reference electrodes). This potential was evoked monaurally through rarefaction click, at a presentation rate of 19.1 clicks per second, at 80 dBnHL, using a 100 Hz high-pass filter, 1500 Hz low-pass filter, and 12 ms recording window. Dual trials were performed with 2,048 sweeps each to check reproducibility.Waves I, III, and V were identified and analyzed regarding absolute latencies, and I-III, III-V, and I-V interpeak latencies. Based on the equipment's user manual, each person's BAEP results were classified as either normal or abnormal . Changes were classified as follows: changes in the low brainstem if there was an increase in the I-III interpeak latency; changes in the high brainstem if there was an increase in the III-V interpeak latency; or mixed if both I-III and I-V interpeak intervals had increased latencies.For LLAEP, the electrode montage was Cz (active electrode), Fpz (ground), and M1 and M2 (reference electrode). Tone-burst stimuli were presented monaurally in an oddball paradigm, at 75 dBnHL, with the standard stimulus (85%) at 1000 Hz and the target stimulus (15%) at 2000 Hz. A total of 300 sweeps were presented at 1.1 sweeps per second, with high- and low-pass filters between 1 and 30 Hz, and a 500 ms recording window.Participants were instructed to pay attention to the target stimuli and count aloud the number of times they occurred. The trial that corresponded to the target stimuli was subtracted from the standard stimuli. P1, N1, P2, and N2 components were identified and analyzed regarding latency and amplitude in the standard trial, whereas P3 was so in the target trial. P1, N1, P2, N2, and P3 latencies and P1-N1, P2-N2, and N2-P3 amplitudes were analyzed. The normality of absolute latencies followed that proposed by McPhersonQuantitative values were analyzed regarding descriptive analysis, and a no-paired t-test was used to compare SG and CG. Concerning qualitative data, the proportion of normal and abnormal results and the types of changes were analyzed with Fisher's exact test. Statistical significance was set at p-value ≤ 0.05 for all inferential analyses.Also, an analysis was carried out in order to verify the association between the main focus of alteration on MRI  and the results of BAEP and LLAEP, by means of Fisher's exact test.Absolute and interpeak BAEP latencies and absolute LLAEP latencies and amplitudes were initially compared between the right and left ears of each group. As none of the analyzed variables presented significant differences between the ears, the right and left ears were grouped for the other analyses (comparison between groups).The comparison between SG and CG revealed statistically significant differences in BAEP III-V and I-V interpeak latencies, with longer latencies in SG . It is For LLAEP, the comparison between groups revealed statistically significant differences in P2-N2 amplitude, which was higher in CG . FurtheThe comparison of normal and abnormal BAEP and LLAEP results between the groups showed a higher incidence of changes in SG than in CG, with a statistically significant difference between them .Moreover, the combination of BAEP and LLAEP in group comparison indicated that six individuals had changes in both potentials, four individuals had changed only in BAEP, and three individuals had changed only in LLAEP . Only fNo association was observed between MRI results and BAEP and LLAEP results .This study aimed to assess the central auditory pathways in children and adolescents with MS. This age range is seldom addressed in the literature, probably because the disease is more prevalent in adults.BAEP analysis revealed changes in 58.82% of patients. Increased wave V and consequently in III-V and I-V interpeak intervals indicate decreased neural conduction speed of the acoustic stimuli in the auditory pathways in the high brainstem, between the cochlear nucleus and lateral lemniscus.The scientific literature reports a great variability in the incidence of BAEP changes in adults with MS, encompassing 20%,Studies in adults reported similar results to those found in the present one, with increased wave V latency,As for children and youth, a study assessed a small group of 11 children and adolescents aged 9 to 17 years and found increased III-V and I-V interpeak latencies, suggesting changes in the high brainstem.Such results may suggest a gradual impairment of the auditory pathways, progressing from the most central region of the auditory system to future impairments in more distal regions of the central nervous system. Nevertheless, the results found in the literature remain quite variable. Moreover, there is a gap in the characterization of samples regarding MS locus and the time elapsed from the disease onset to the study. Hence, future studies that control these variables may find more systematic and consistent results concerning impairments in this population's auditory pathways.As for the cortical auditory pathways, more than half (52.94%) of MS patients in this study had LLAEP changes. Even though there were no statistically significant differences in latency values, SG had longer latencies than the healthy volunteers. Similarly, there was a decrease in response amplitudes, although a statistically significant difference was found only in P2-N2.Barbosa et al.These results suggest that MS patients may have slowed neural processing and decreased neural activity in sensory, inattentional,According to Comi et al.,In the present study, no association was observed between the main focus of alteration detected on MRI and the electrophysiological results. This result may be justified by the limited sample size, considering that the population is heterogeneous in terms of the different demyelinating lesion sites found in each patient.In a larger sample, of 32 patients, abnormal latencies in the potentials have been related to the locus of demyelinating lesions, agreeing with what was observed in the MRI.Changes in temporal resolution and auditory task memory and difficulties discriminating speech in noisy environments have been described in MSMS impact on cognitive functions is still little known – although changes in cognitive functions are known to be common in children with MS.Various otorhinolaryngological symptoms are also described in MS, including speech disorders, sleep disorders, vertigo, imbalance, dysphagia, changes in smell, and hearing loss.This study had a larger sample than the previous one that assessed auditory evoked potentials in same-age MS patients. Nonetheless, the present research had a limited sample size, which hindered other correlations concerning, for instance, the influence of age on symptom onset, disease duration, and medications used. Therefore, future research is expected to have larger samples and characterize them in further detail to control other variables that might influence electrophysiological responses.Another limitation of the study, regarding LLAEP analysis, was that it did not obtain data on the participants' school achievements. Neither was it possible to perform a behavioral assessment of the central auditory processing or a neuropsychological assessment battery to correlate with the findings of the electrophysiological assessment. Thus, future studies with larger samples that complement such data may clarify other nuances that could not be measured in the present one.


Such analysis of the hypoxic environment will allow an improved understanding of the contribution of the cellular and molecular signaling pathways associated with tumor initiation, progression, and metastasis, and the subsequent knowledge will help to improve cancer treatments such as chemotherapy and radiotherapy.The Research Topic “2) is involved in many important biological functions, including metabolism, proliferation, angiogenesis, and apoptosis, while dysregulation of oxygen metabolism has been implicated in a number of cancers. Research has indicated that the pO2 varies widely between different tissues, with many being hypoxic, with a concentration range from 6 to 34 mmHg (1% to 5% O2) (2) and 150 mmHg (19.7% O2), respectively , decreasing to 40 mmHg (5.2% O2) in venous blood. Conversely, in the mouse thymus, the oxygen value was estimated at only 7.6 mmHg (1% O2) (2 is below 34 mmHg (5% O2). HIF transcription factors control the expression of thousands of genes, regulating crucial mechanisms, including angiogenesis, cancer stem cell specification, cell motility, epithelia-mesenchymal transition, extra-cellular matrix remodeling, glucose and lipid metabolism, immune evasion, invasion, and metastasis , in endocrine tumors. The relationship between oxygen tension and reactive oxygen species (ROS) has previously been established (Cui et al. identified eleven mitochondrial genes related to oxidative stress that are associated with the progression of pancreatic cancer and pancreatic neuroendocrine tumors.In this Research Topic, ablished  and Cui Pereira et al. presented an overview on the role of hypoxia in ovarian cancer, and its effects on the immune environment. Importantly, they analyzed the impact on chemoresistance and discussed the future possibilities of personalized therapies. Chen et al. further established a model based on hypoxia-related gene expression to predict prognosis in endometrial cancer, another female-specific tumor. This model is based on a genetic signature of 4 genes: ANXA2, AKAP12, NR3C1, and GPI. Finally, Quinting et al. analyzed the role of myoglobin, an oxygen-binding protein that exhibits a scavenging capacity for reactive oxygen and nitrogen species, in breast cancer. Their results strongly suggest that myoglobin supports the survival of breast cancer cells due to its scavenging properties.Women’s cancers are also frequently concerned by endocrine tumors and their hypoxic microenvironment. In a recent study, Dong et al. showed that a novel molecular classification incorporating oxidative stress and metabolism-related genes could be used for prognosis prediction and personalized medicine in stomach adenocarcinoma.The role of oxidative stress in cancer has also been extended to other types of tumors. For example, Rakotomalala et al. analyzed the different mechanisms by which hypoxia may influence the efficacy of radiotherapy in solid tumors. In particular, they detail the 6 parameters considered in the field of radiotherapy, known as the “6Rs of radiation biology”: Radiosensitivity, Repair, Repopulation, Redistribution, Reoxygenation, and Reactivation of anti-tumor immune response. This review presents innovative data concerning the effects of hypoxia during radiotherapy, and describes the results of recent clinical trials. To return to the main focus of this Research Topic, endocrine tumors, the authors analyze these effects in the case of anaplastic thyroid carcinoma.Finally, in the last part of this Research Topic, the application of hypoxia studies to cancer therapy is proposed. Indeed, it was shown that oxygen tension can considerably influence radiotherapy efficiency, with hypoxia being one of the most important causes of radioresistance. Moreover, hypoxia is associated with a poor prognosis after radiotherapy. Overall, this Research Topic presents new concepts on the role of hypoxia and oxidative stress in endocrine cancer, while also providing new tools and directions to classify the patients and to delineate the molecular mechanisms for a future optimization of the treatments and the development of a personalized medicine.BD: Conceptualization, Investigation, Validation, Writing – original draft, Writing – review & editing. RJ: Validation, Writing – original draft, Writing – review & editing.


Moreover, with increased cycles, the value of the grey-correlation degree increased. The comprehensive score of the procedure repeated nine times was significantly higher than other cycles (p < 0.05). The results of the grey-correlation degree and grade analysis showed that the best steaming process of black sesame seeds was HPS for nine cycles, followed by HPS for eight cycles and NPS after soaking in water (SNPS) for nine cycles. These findings could provide a scientific basis for replacing SNPS with HPS to simplify steaming and realize the parametric steaming of black sesame seeds, and thus, ensure the quality of black-sesame products.This study aimed to improve the steaming process of black sesame seeds. A comprehensive evaluation was conducted using the grey-correlation method based on the variation-coefficient weight to observe the treatments of normal-pressure (NPS) and high-pressure (HPS) steaming (with/without soaking in water) for nine cycles. Their effects on the contents of water, protein, fat, ash, melanin, sesamin, and sesamolin of black sesame seeds, as well as the sensory score of the black sesame pill, were determined. We found that with varied steaming methods and increased steaming cycles, the contents of the nutritional and functional components of black sesame seeds and the sensory score of the black sesame pill differed. The results of the variation-coefficient method showed that water, protein, fat, ash, melanin, sesamin, sesamolin, and sensory score had different effects on the quality of black sesame seeds with weighting factors of 34.4%, 5.3%, 12.5%, 11.3%, 13.9%, 11.3%, 7.8%, and 3.5%, respectively. The results of two-factor analysis of variance without repeated observations indicated that the grey-correlation degree of HPS was the largest among the different steaming treatments, and the following sequence was HPS after soaking in water (SNPS), NPS, and SNPS. There was no significant difference between NPS and SNPS ( Sesamum indicum L.) of the Pedaliaceae family is an important and ancient oilseed crop with more than 50% oil [Sesame  is an ancient and traditional processing method that involves nine cycles of steaming and drying. It is commonly utilized to process food and medicinal ingredients in China . NSND reCistanches Herba and Radix Aconiti) have proven that HPS technology could meet the requirement of corresponding products, save time, and be simple to operate [Chlorella vulgaris [High-pressure steaming (HPS) is a process for rapidly ripening grain by using saturated steam at high pressure . The pro operate . HPS is vulgaris . HoweverSoaking seeds is a process in which seeds absorb water and swell simultaneously, thereby softening the seed coat to facilitate processing. After soaking, grain hardness decreases and other physical properties of minor millets  change . SoakingAs a traditional Chinese health food, nine steamed–nine sun-dried black sesame seeds are regarded as a top-grade tonic . A procep < 0.05). Except for ash content, other indicators of black sesame seeds showed significant differences among the four processes.Black sesame seeds were treated for nine cycles with the process of SNPS, NPS, SHPS or HPS, respectively. Vi and Wi were calculated and are shown in Using Equations (1) and (2), 0), the protein, fat, ash, melanin, sesamin, sesamolin, and sensory score were the highest in the corresponding indicators of SNPS, NPS, SHPS, and HPS. In terms of moisture, with increased processing cycles, the contents of water decreased (0), the water was the lowest in the water contents of SNPS, NPS, SHPS, and HPS. All raw data were normalized with Equation (3). The standardized data of evaluation indices are listed in To meet quality targets, selecting the processing technologies with high contents of nutritional and functional components and excellent sensory properties for black-sesame products was desirable. Therefore, in the ideal process selected as the reference sequence  and are shown in According to Equations (5) and (6), the minimum absolute difference was 0.000, and the maximum absolute difference was 7.040.Equation (7) was used to count the correlation coefficient between the steaming technologies  and the quality indicators of black sesame seeds. Then, the calculated correlation coefficients are given in The grey-correlation degrees calculated with Equation (8) and the correlation grades (including the correlation grade within a group and correlation grade between groups) are given in According to the correlation grade within a group, with increased cycles, the value of grey-correlation degree during HPS, SHPS, SNPS, and NPS generally increased. The procedure repeated nine times was the optimum, with the statistically highest comprehensive score of black sesame seeds among the four methods . This fi36, X35, and X9. Thus, the combined score of the sample during SNPS repeated steaming for nine cycles was lower than those during HPS repeated steaming for eight and nine cycles. Comparing the four processes . Black rice, black soya bean, white glutinous rice, dry mulberry, dry wolfberry, and dry haw were purchased from a local market in Xuchang, Henan, China. The materials were cleaned by hand picking and winnowing to remove foreign matter.1–X9, respectively.(1) NPS after soaking in water (SNPS). First, the cleaned black sesame seeds were soaked in tap water for 24 h and then the water was drained. Second, the seeds were steamed for 2 h over boiling water in an electric steamer  for which the power was set to 1800 w. Then, the seeds were dried for 2 h in an oven  at 60 °C, and stirred once every 30 min. The steaming and drying procedure was repeated for nine cycles to collect nine samples of SNPS denoted as X10–X18, respectively.(2) NPS. Without soaking, the seeds were steamed and dried for nine cycles, following the same steps as the above section of “NPS after soaking in water (SNPS)”, to collect nine samples of NPS denoted as X19–X27, respectively.(3) HPS after soaking in water (SHPS). The SHPS processing also involved three steps with the same as the above section of “NPS after soaking in water (SNPS)”, but the second step differed. During SHPS, the black sesame seeds were steamed for 30 min at 121 °C in a high-pressure steam sterilizer  with 0.21 Mpa pressure. The steaming and drying procedure was repeated for nine cycles to collect nine samples of SHPS denoted as X28–X36, respectively.(4) HPS. Without soaking, the seeds were steamed and dried for nine cycles, following the same steps as the above section of “HPS after soaking in water (SHPS)”, to collect nine samples of HPS denoted as XThe moisture content was measured using the method of direct drying described by GB 5009.3-2016 (China). Crude protein was determined using the Kjeldahl method described by GB 5009.5-2016 (China). Fat was detected by the Soxhlet extraction method described by GB 5009.6-2016 (China). Total ash was determined by the first method described by GB 5009.4-2016 (China). Each sample was set to have three parallels.v/v), 1:50 ratio of black sesame seeds to solvent, pH 1.0, and extraction for 30 min. Each sample was set to have three parallels.Melanin was detected with an ultraviolet photometer  at 360 nm. Alcohol was used to extract black sesame pigments, and the extraction technology conditions were as follows: 50% alcohol  described by NY/T 1595-2008 (China). The chromatographic conditions of HPLC were Agilent XDB-C18 column ; column temperature 30 °C; mobile phase: CHBlack-sesame products include black-sesame paste, black-sesame pill, black-sesame cake, and so on. Among them, the black-sesame pill is a kind of honeyed pill using the steamed black sesame seeds as their main raw materials. To measure the impact of steaming and drying on the quality of the black-sesame product, the black-sesame pill was prepared with the black sesame seeds exposed to the different steaming technologies in the (1) Blend formulation and preparation of the black-sesame pillRaw material flours. First, black rice, black soya bean, white glutinous rice, and the steamed black sesame seeds were separately fried with incense. Second, the fried raw materials, mulberry, wolfberry, and haw were milled using a stainless universal crusher and sieved into fine flour of 40 mesh, respectively.Refining honey. No-crystal honey was boiled for 4 s on an induction cooker at 100 °C, and the boiling procedure was repeated three times.Blend formulation. The basic formulation of the black-sesame pill used was as follows: black sesame flour, 30 g; the refined honey, 16 g; sealwort juice, 2 g; black rice flour, 1 g; black soya bean flour, 1 g; mulberry flour, 1 g; wolfberry flour, 1 g; wolfberry flour, 1 g; white glutinous rice flour, 1 g; haw flour, 0.4 g; and sodium carboxymethyl cellulose, 0.3 g.Black-sesame pill preparation. The dry ingredients were weighed and thoroughly mixed. Sealwort juice was added and mixed until uniformity was achieved. The refined honey was added and kneaded into a ball while it was hot, and hand beaten 60 times. Then, the ball was weighed in groups. A black-sesame pill was 8 g. The pill of 8 g was initially hand rubbed in 20 circles to a smooth surface without cracks. Then, the rubbed pill was dried for 30 min in an oven at 60 °C, and flipped once at 15 min. Finally, the pill taken out from the oven was cooled to room temperature and packed using a silver paper of 8 cm × 8 cm. This was the finished product.(2) Sensory evaluation of black-sesame pillQuality indicators of black-sesame pill samples were evaluated by a panel of 10 students majoring in food according to the sensory scoring criteria . SensoryThe sensory evaluation process was as follows. First, the shape and color of the prepared black-sesame pill which was placed on a white plate were observed; second, the pill was smelt; third, tasted; then, the average of each factor was calculated; finally, the sensory score was calculated using a weighted scoring method, in which the weights of morphology, color, texture and flavor were 20%, 22%, 25% and 33%, respectively.The variation-coefficient method can avoid equal division of weight and make results more reasonable . The steiC; iC; iC; i = 1, 2, …, n; and n is the number of the indicators, and n = 1, 2, …, 8.(1) Calculating the variation-coefficient of indicator.iW is the object weight of the indicator iC; iV is the variation-coefficient of the indicator iC; and n is the number of indicators .(2) Calculating objective weight.The grey-correlation analysis is a geometric approach used to evaluate the correlation between different sequences within a design . To elimX0), and the other processing methods  were taken as the comparison sequence (jX). Except for the minimum water content, the other quality indicators of the ideal process were the highest in the corresponding indicators of SNPS, NPS, SHPS, and HPS. The equalization method was used to preprocess the raw data and convert them into dimensionless data [i is the number of the samples ; and k is the number of indicators .(1) Normalizing raw data. The ideal process was selected as the reference sequence (ess data .(3) Ci’(2) Determining the absolute difference between the comparison and the reference sequence. The absolute difference was calculated using Equation (4) .(4) ΔikThe minimum absolute difference was as follows:i is the number of samples ; and k is the number of indicators .The maximum absolute difference was as follows:i is the number of the samples ; k is the number of indicators ; and(3) Calculating the grey-correlation coefficient. The grey-correlation coefficient is defined as the correlation between the ideal and actual standardized value. It can be calculated using Equation (7) .(7)ζik=iC; iC; i is the number of the samples, and i = 0, 1, 2, …, and 36; k is the number of the indicators, and k = 1, 2, …, 8.(4) Calculating grey-correlation degree and arranging grey-relational grade. To obtain the grey-relational degree, the following Equation (8) was used:iR is the sequence arranged by the value of grey-correlation degree of the samples.The grey-relational grade p ≤ 0.05.Experimental and sensory results were subjected to analysis of variance, and means were compared using the Student–Newman–Keuls test. Significance was accepted at The four steaming processes of SNPS, NPS, SHPS, and HPS exerted different degrees of influence on the nutritional and functional ingredients of black sesame seeds and the sensory score of the black sesame pill. The influences of various steaming processes on the quality indicators of black sesame seeds were evaluated using grey-correlation analysis by analyzing the contents of water, protein, fat, ash, melanin, sesamin, and sesamolin of black sesame seeds and the sensory score of the black sesame pill. We found that nine cycles of black sesame seeds treated with steaming and drying was the optimum. Moreover, the traditional technology of NSND was reasonable. These findings indicated that HPS, as a new production process of black sesame seeds, was suitable for replacing SNPS, and the soaking before steaming process could be avoided. Overall, HPS could save time and simplify SNPS steps. The required equipment was also simple and desirable for the parametric steaming and large-scale production of black-sesame products. However, the effects of these processes on other components  in black sesame seeds were not observed. Therefore, further research is needed to assess the processing effect of black sesame seeds by examining other components, especially essential amino acids and essential fatty acids, to provide an idea for the study of traditional and modern processing technology. Overall, a method of comprehensive analysis was established to determine the effects of different processing technologies on black sesame seeds by analyzing multiple indicators. This study provided a scientific basis for the further development and utilization of new technologies.