Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen’s genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes. The introduction of an exotic pathogen into a new area leads to the establishment of new biotic interactions, giving rise to a process of coevolution between the host and the pathogen ,2. For tFusarium circinatum is an introduced pathogen currently established in Europe in Spain and Portugal infecting Pinus radiata and P. pinaster [P. radiata being one of the most susceptible species while P. pinaster is moderately resistant to the disease. Typical symptoms are sunken cankers on branches and stem with abundant resin, and wilt and necrosis of needles above the infection point [Fusarium circinatum also infects seedlings in nurseries causing pre- and post-emergence damping-off [F. circinatum in the EU.pinaster . This paon point . Fusariuping-off . PPC is ping-off ,8. In thFusarium circinatum populations worldwide to analyze their structure and infer the reproduction mode and pathways for pathogen introduction and migration [DNA-based studies have been carried out on igration . First sigration ,11,12. Aigration  revealedigration , ongoingF. circinatum Spanish population. Both mating types, MAT-1 and MAT-2, were found, although one of the two clusters (with predominant haplotype MLG32) only contained isolates of mating type MAT-2 located in the northeastern region (País Vasco). A further study with isolates representing the main haplotypes in each of the two clusters [Genetic diversity analysis revealed that Spanish population is structured in two clusters, each one with a predominant haplotype (named MLG32 and MLG59), a situation compatible with two independent pathogen introductions in Spain . Geneticclusters  showed tF. circinatum. Heterokaryons are originated through: (1) hyphal anastomosis of isolates of the same vegetative compatibility group (VCG) [Fusarium circinatum the sexual state has not been reported in nature [There are different mechanisms potentially associated with significant changes in genetic variability of up (VCG)  and (2) up (VCG) ,17. For n nature , and VCGn nature , while in nature . In nortn nature  revealinFusarium circinatum, haploid selfing is prevented [Fusarium circinatum, the sexual stage has not been reported in the field [Regarding selfing mechanism, it is differentiated haploid from diploid selfing  with difrevented  since threvented ,20,21. Ihe field , but outhe field ,22,23, phe field .Fusarium circinatum is extended in Spain to the Atlantic coast [Pinus radiata plantations are grown. It is the only exotic species of pine planted in Spain and cohabits in this area with P. pinaster, a native Mediterranean species. Moreover, some new environmental situations have been identified after its introduction [P. radiata more susceptible than P. pinaster [Population genetics studies provide insight into the evolutionary process and changes in gene frequencies that shape pathogen population structure ,25. Intric coast  where mooduction , such aspinaster ) that exFusarium circinatum populations infecting pine stands and to determine how this variation is distributed in a temporal and spatial scale. The aim is to understand the biological significance of the variation detected and to make inferences about the evolutionary processes that may influence the population structure.The objective of this work is to quantify the genetic diversity of Spanish Fusarium circinatum isolates were obtained from cankers of forest trees showing symptoms of PPC. Small pieces of samples were cut from the margin of symptomatic tissue and plated on Fusarium Selective Medium (FSM) [F. circinatum were identified based on morphological features and molecular methods. For morphological fungal identification, putative colonies of F. circinatum growing on FSM were transferred to plates with Spezieller Nahrstoffarmer Agar (SNA) medium [F. circinatum [F. circinatum was done by PCR using specific primers CIRC1A (CTTGGCTCGAGAAGGG) and CIRC4A (ACCTACCCTACACCTCTCACT), as described by Schweigkofler et al. [F. circinatum isolates produced a 360 bp amplicon. In total, 29 F. circinatum monosporic isolates were obtained, 5 isolates from Galicia, 9 from Asturias, and 31 from País Vasco (A survey was done in 2018–2021 in northern Spain. um (FSM) . Isolati) medium  incubatercinatum . Moleculr et al.  in a finís Vasco . A uniquTo analyze genetic variation of pathogen population in a temporal scale, we used a set of monosporic isolates maintained at the Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, collected from a first survey carried out in 2004–2011, soon after the first official report of the pathogen in Spain ,32. IsolFresh mycelia growing on PDA  plates at 22 °C was scraped and ground to a fine powder under liquid nitrogen in a mortar. DNA was extracted using the E.Z.N.A. Plant DNA kit  or NZY Plant/Fungi gDNA Isolation kit  following the manufacturer instructions. DNA obtained with the latter kit was used only for microsatellite analysis. DNA quantity and quality was measured using a spectrophotometer . For molecular marker analysis, only high-quality DNA was used, with absorbance ratio 260/280 and 260/230 > 1.8.Idiomorphs MAT-1 or MAT-2 were determined for each isolate. A multiplex PCR assay was performed using primers MAT1p1 (AGAAACTGACTGATACATCAAGGGG)-MAT1p3 (TCATAAGAAGTGTTGAAGGAATCACAG) and GcHMG1 (CTTTACCGTAAGGAGCGTCACCAT)- GcHMG2 (TGATCCGCCATCTGCTTGTAGAGT) for alleles MAT-1 and MAT-2, respectively, as described by Wallace and Cover  and SchwF. circinatum [F. circinatum . Briefly, samples were genotyped using a set of seven microsatellites previously designed for rcinatum  and selercinatum  for polyrcinatum . Each PCnit) mutants, following [nit mutants, isolates were cultured on Czapeck (Cz) medium  amended with 4% potassium chlorate at 25 °C with 12-h light cycle. Fast-growing mycelia sectors developing for 4–10 days were transferred to Cz medium. Colonies that grew as thin, expansive, and with no aerial mycelia were considered as nit mutants. To check for their stability, mutants were grown on PDA and then transferred to Cz and Cz with 4% potassium chlorate. For phenotyping nit mutants (nit1/3 or nitM), they were cultured on basal medium with no mineral added [nitM. Otherwise, they were considered as either nit1 or nit3. No attempt was done to distinguish between these two types because pairing between nit1 and nit3 mutants develops a heterokaryotic growth very slowly [nit1 (or nit3) and a nitM mutants from different isolates in all possible combinations were done to determine vegetative compatibility. Mycelial plugs placed 2–3 cm apart on Cz medium were incubated at 25 °C for 4–15 days and observed periodically for dense mycelia where colonies were in contact forming the heterokaryon. Mutant pairs that formed this dense mycelium complement each other, and thus they belong to the same Vegetative compatibility group (VCG). Pairings between a nit1 (or nit3) and a nitM mutants from the same isolate were done to check for heterokaryon self-compatibility (HIS) which has been reported in Gibberella fujikuroi [Vegetative compatibility groups were determined based on pairings between nitrate non-utilizing  and subpopulations of geographical origin (Spanish regions) where they were collected  . Additio5) [A) [Genetic diversity estimated by molecular markers was based on the number of alleles and allele frequencies at each SSR locus. For each isolate, a multilocus genotype (MLG) was defined by combining data for single SSR alleles. If the MLG had been identified previously by Berbegal et al. , the sam5) ,42,43 we5)  to accoum 0 to 1  was usedto 1 [A)  because distance . Bootstrp < 0.05. Second, the contribution of sexual reproduction to the genetic diversity was studied. MLGsim 2.0  was used to test if identical genotypes emerged independently through random mating (Psex) based on a simulation of 1000 permutations.Additionally, two more tests were done to evaluate occurrence of sexual reproduction. First, the null hypothesis of mating type ratios of 1:1, which are expected under random mating, was tested. The usual chi-squared test to evaluate departure from the null hypothesis is not recommended in this case because the observed frequencies for some mating types are less than five. Instead, the theoretical probability of the observed outcome was calculated based on the binomial probability distribution on the null hypothesis with p = q = 0.5. The null hypothesis is rejected when ST) is based on 999 permutations. For spatial variation study, a Mantel test was performed to test for correlations between geographic distance  and Bruvo’s genetic distance. The test was run in R using the function mantel.randtest in package ade4 [Genetic differentiation between subpopulations was estimated by Hedrick’s standardized index  (G’st), age ade4  and its age ade4 . Final aThe comparison between haplotypes identified in the two surveys performed on average 10 years apart indicates that MLG32 continues to be the most frequent haplotype . It is r5 = 0.961) in the whole pathogen population . Diversity and evenness decreased with time , and richness decreased as well . When including geographic origin within surveys, the expected number of MLG in Galicia decrease from 6 to 3, while in Asturias remains at 3; H, G, and E5 indices  , as well indices  decrease indices .Fusarium circinatum population. For Galicia and Asturias, ratios were 5:1 and 2:1, respectively, in isolates from survey 1, and 2:1 in those from survey 2 in both regions  and cannot be rejected. This is not concluded for MLG32.Mating types (MAT-1 and MAT-2) were found in regions of Asturias and Galicia, whereas in País Vasco only MAT-2 was identified. The mode of reproduction in each geographic origin was estimated based on standardized index of association value  in two major groups, one of them including all the isolates of MLG32 found in both surveys and all regions (No differentiation between surveys or regions within surveys were shown by AMOVA on the clone-corrected data. All variation (100%) is explained within the regions suggesting Spanish entiated . All phientiated , but not regions . Minimum regions  corroborPairwise population comparisons of genetic diversity values (G’st index)  indicatep-value = 10−4). A significant correlation was found between isolates from País Vasco (PV) and either Galicia and Asturias  , but not between the populations of Asturias and Galicia in any of the two surveys , corroborating that population from PV is genetically differentiated from northwestern populations . No gene flow was detected between any of the subpopulations defined by survey and origin region. According to Bayesass  were the most abundant . Spore germination was also higher for the most common haplotype, and produced more spores at 20 and 25 °C.This situation confirms that MLG32 is a well-adapted haplotype as it was shown in previous studies  where soFusarium circinatum is inbreeding or goes under asexual reproduction, at least in this region. Only two VCGs were found in País Vasco, which contributes to a limited exchange of genetic information and maintenance of individual identity [Exclusive presence of MLG32 in País Vasco during these 10 years between surveys indicates identity . TemporaThe presence of a well-adapted haplotype together with evidence for clonality in País Vasco supports that MLG32 may have existed for some time and that this pathogen established first in this region, a hypothesis already suggested by Berbegal et al. . SymptomThe maintenance of the population genetics in two clusters during these 10 years between surveys supports the occurrence of clonality or inbreeding and the lack of sexual reproduction. AMOVA results on the whole population show that genetic variation is explained mostly by regions, but not by time, supporting the idea of clonal organisms. However, this structure does not appear when AMOVA is performed on the clone-corrected dataset. This discrepancy in the results is probably due to the small size of the population that results from correction and point out the importance that the clones have in the AMOVA analysis.Fusarium circinatum is structured (d)   and Psex surveys . Lack ofIn NW regions with a greater number of MLGs and VCGs, the occurrence and identification of heterokaryons is more likely as has been the case. Two isolates from the first survey, one from Galicia and other from a nursery in Asturias, have two microsatellite alleles per locus. These heterokaryons may have originated by anastomoses of hyphae or diploid selfing (processes not necessarily exclusive ). DetectFusarium circinatum in País Vasco, evidence of genetic changes was found when more intensive samplings were carried out. In an intensive sampling done in 2016 in a P. radiata plantation with symptoms of PPC, one new haplotype was detected by SSR marker analysis [Despite the genetic uniformity found in the population of analysis . Only 1 Fusarium circinatum are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to new haplotypes. The existence of haplotype MLG32 maintained on time and widely distributed across northwestern regions suggest its well adaptation to the environment and the host. Results showed that País Vasco is a region that after many years of the first disease outbreak in Spain remains clearly differentiated from other Northwestern populations. This fact was supported with no evidence of migration among regions. The low genetic diversity observed and lack of new haplotypes in País Vasco indirectly evidences that regulation within the EU territory to prevent pathogen spread is efficient. In conclusion, results presented here about the temporal and spatial variation of population structure of Spanish


This study aimed to better understand the patient perspective and treatment experience of relapsed and/or refractory multiple myeloma (RRMM).This qualitative study enrolled adult RRMM patients from 6 US clinics who had ≥ 3 months of life expectancy, ≤ 6 prior lines of therapy, and ≥ 1 treatment regimen with a proteasome inhibitor and immunomodulator, or a CD38 monoclonal antibody or an alkylating agent, and a steroid. In-person semi-structured qualitative interviews were conducted to capture concepts that were relevant and important to patients. Topics included RRMM symptoms and impacts and the mode of administration, frequency, duration, convenience, side effects, and overall experience with RRMM treatment.A total of 22 patients completed interviews. At enrollment, 59.1% of participants were using regimens containing dexamethasone, 36.4% daratumumab, 27.3% carfilzomib, and 18.2% lenalidomide. More participants had experience using intravenous or injectable therapy alone (40.9%) than oral therapy alone (18.2%). Back pain and fatigue were the most frequently reported symptoms (40.9% each); 27.3% reported no symptoms. Most participants reported physical function limitations (86.4%), emotional impacts (77.3%), MM-related activity limitations (72.7%), and sleep disturbances (63.6%). Most participants perceived treatment effectiveness based on physician-explained clinical signs (68.2%) and symptom relief (40.9%). Participants experienced gastrointestinal adverse events (59.1%), fatigue (59.1%), sleep disturbances (31.8%), and allergic reactions (31.8%) with treatment. Key elements of treatment burden included the duration of a typical treatment day (68.2%), treatment interfering with daily activities (54.5%), and infusion duration (50.0%).These results provide treatment experience–related data to further understand RRMM treatment burden and better inform treatment decision-making.The online version contains supplementary material available at 10.1007/s00520-022-06979-7. Multiple myeloma (MM) is a clonal plasma cell malignancy with an incidence of 7.1 per 100,000 persons in the USA that was projected to account for an estimated 34,920 new cases  and 12,410 deaths (2.0% of cancer deaths) in the year 2021 , 6. MultExisting and emerging MM therapies, including proteasome inhibitors (PI), immunomodulators (IMiDs), and monoclonal antibodies, have dramatically improved median survival and 5-year survival rates in the past two decades –10. AlthMore knowledge about patient experiences with treatments and their impact on everyday activities, including health-related quality of life (HRQOL), is needed for healthcare providers and patients to make informed treatment decisions –18. ThisThis was a qualitative study of adult patients (≥ 18 years) in the USA with RRMM. Eligible patients had at least 1 prior MM relapse, a physician-confirmed life expectancy of at least 3 months, and were not eligible for stem cell transplant at the time of enrollment. Patients were eligible to enroll with up to 6 lines of prior therapy; the initial protocol included patients with up to 3 lines of prior therapy, but a protocol amendment of up to 6 lines was approved by the institutional review board (IRB) to facilitate broader patient recruitment. Eligible patients had received at least 1 regimen with a PI and IMiD, or a CD38 monoclonal antibody or an alkylating agent, and a steroid. Patients were also required to be able to read, write, speak, and understand English. Patients who had received stem cell transplant following relapse, had been diagnosed or treated for another malignancy within 2 years before MM diagnosis, or had any evidence of residual disease from a previously diagnosed malignancy were excluded. Patients were also excluded if they were diagnosed with a clinically relevant medical or psychiatric condition that could interfere with completing the study or had an uncontrolled/active infection or concurrent medical condition with symptoms that could confound their description of their experience with RRMM.Purposeful sampling was used to enroll patients with RRMM from 6 community-based oncology clinics located in New York, Ohio, California, and Maryland. The participating sites were selected based on their expected ability to recruit eligible patients within the needed timeframe. Sites identified potential patients by screening medical records and databases to abstract key variables such as disease background and current treatment regimen. Potentially eligible patients were then contacted by phone or in person by site personnel using a prepared script summarizing the goals of the study and inviting them to participate in a one-time face-to-face interview. If the patient was interested, the site began the informed consent process. All participants provided written informed consent and agreed to the audio-recording and transcription of the interview. The site staff then abstracted additional descriptive demographic data about participants from the patient medical records. The study protocol was approved by Advarra IRB . Participants were compensated for time spent with a prepaid cash card presented in person at the end of the interview session.n = 5) and patients (n = 3) to identify a list of concepts used to describe disease- and treatment-related experiences  interview in a private and comfortable setting at the clinic site. Each interview lasted 60 to 75 min. The interviewer used a semi-structured interview guide with follow-up probes when needed to capture the concepts that were relevant and important from the patient perspective and to conceptualize the symptoms, impacts , treatment experiences, and treatment burden of RRMM.An iterative approach was used to conduct two sets of interviews. Version 1 of the interview guide included questions involving the symptoms and impact of the disease in addition to participants’ treatment experiences. The interviews started with questions about symptoms participants experienced prior to being diagnosed with RRMM; then asked about symptoms they experienced during treatment, including whether there were any changes in their pre-diagnosis symptoms after treatment was initiated; and finally asked about impacts and treatment burden. In the initial wave of interviews, the research team observed patients’ emphasis on the burden of treatment. Hence, in the next wave of qualitative interviews, the research team focused more deeply on evaluating the treatment burden and benefit concepts that are relevant to patients with RRMM. Version 2 of the interview guide used targeted questions regarding the mode of administration, the frequency and duration of treatment, the overall convenience of receiving MM treatment, and the side effects of treatments. Questions focused on the convenience of MM medications and possible interference with daily activities and other factors leading to a favorable or unfavorable experience with treatment. Specific questions about the participant’s overall experience with treatment regimens and a typical day of treatment were also included in the second version of the interview guide.Audio-recordings were transcribed verbatim and coded for qualitative content analysis using ATLAS.ti version 8.0 . The goal of the coding process was to identify and organize relevant concepts and expressions of study participants related to the preliminary conceptual model and interview guide. Through the review of each verbatim transcript, specific codes were developed from the participants’ words, with conceptually equivalent codes grouped to represent distinct concepts within broad domains of RRMM experience  and treatment experience . The preliminary coding scheme was refined until team consensus was reached, and consistency of coding was assessed through repeated consultations among coders. A subset of three transcripts was coded independently by 2 researchers; after the initial coding, coders met and compared the concepts identified and codes assigned, resolving any differences until a consensus was obtained. The coding then proceeded following the agreed upon coding scheme, and coders met regularly to discuss emerging themes and codes. Saturation of treatment experience concepts was assessed in the CE data through a process that examined the appearance of novel concepts across interview transcripts and in accordance with the order in which interviews were conducted.n = 18, 81.8%); the mean time between the most recent clinician-defined relapse and study enrollment was 1.4 years (range: 0–3.8 years). On average, participants had 3.3 comorbidities (range: 1.0–7.0). Hypertension , respiratory illnesses , and gastroesophageal reflux disease  were among the most common comorbidities.A total of 23 patients with RRMM enrolled in the study; of these, 22 participants completed qualitative interviews (one patient was excluded because they were unfit to participate at the time of their scheduled interview). Interviews were conducted from November 11, 2019, to February 25, 2020. The mean interview participant age was 69 years, and the majority of participants were male (Table n = 13) were using regimens containing dexamethasone, 36.4% (n = 8) daratumumab, 27.3% (n = 6) carfilzomib, and 18.2% (n = 4) lenalidomide; site staff reported all treatments included in the regimen, so treatments reported were not mutually exclusive. About one-third of patients used monotherapy (31.8%) or doublet therapy (31.8%) and 27.3% used triplet therapy. The mode of administration for participants’ current treatments varied: fewer participants were using oral therapy alone  compared with intravenous or injectable treatment alone  or both intravenous/injectable and oral treatment .Study participants had received a mean of 3.1 (range: 2.0–3.8) lines of therapy  and fatigue  were the symptoms reported most frequently by participants; 27.3% of participants (n = 6) reported no symptoms , followed by emotional impacts  , activity limitations related to MM , and sleep disturbances .Example participant quotes regarding their MM experiences are shown in Table n = 15, 68.2%) perceived treatment effectiveness based on laboratory test results explained by their healthcare provider, and 40.9% (n = 9) based treatment effectiveness on symptom relief. Participants looked forward to receiving feedback about their laboratory results and not receiving it caused worry and concern. Without feedback, they were unable to tell whether the treatment was working or not and could not comment on the effectiveness of treatment with regards to clinical signs.Interview participants described their treatment experiences in three primary domains of interest: treatment effectiveness, treatment tolerability, and mode of administration Table . Two effn = 13, 59.1%) and fatigue . The next most commonly reported concepts were sleep disturbances  and allergic reactions . Eight participants (36.4%) reported no adverse events.A total of 18 treatment tolerability concepts were elicited during the interviews. Most participants described experiencing gastrointestinal adverse events , treatment interfering with daily activities , and duration of infusion  as key elements of treatment burden  and convenience . Participants mentioned the physical and mental ease and time savings  of taking pills as part of the convenience of oral treatment.Regarding mode of administration concepts, participants most commonly reported the overall duration of a typical treatment day n = 15, 8.2%, treThis qualitative study uncovered treatment experience-related concepts that study participants with RRMM reported as important. A central theme that resulted from this analysis was the burden of time dedicated to following the prescribed treatment regimen, with participant-perceived unfavorable factors including the duration of the typical day of treatment and the duration of infusions and injections of existing treatment. Participants indicated that they often plan their lives around treatment days due to the duration of infusion treatment and prolonged impacts of treatment-related adverse events that can occur. Many participants expressed experiencing gastrointestinal issues or fatigue and the need to plan their activities around when these adverse events have resolved and they have more energy.Participants’ perceptions of treatment effectiveness depended largely on symptom relief and clinical signs. While many participants utilized symptom relief to indicate the effectiveness of the treatment, some had difficulty relying solely on this as they continued to experience symptoms following treatment and others were asymptomatic. Therefore, it is particularly important that healthcare providers share the results of the clinical tests with their patients, as many participants reported using this information to determine whether the treatment is working.Participants in the first wave of interviews emphasized the burden of RRMM treatment, so we adjusted the interview guide for later waves to focus more on patient-reported treatment-related experiences than symptom-related concepts. However, the symptoms and impacts reported by study participants were consistent with the symptoms and impacts of RRMM reported in the literature. The adverse events endorsed by participants in this study were similar to those reported by participants with RRMM in the study conducted by Parsons et al.  .Our results were consistent with previous patient preference studies among patients with RRMM, in which drug administration  has been identified as the most important factor in patients’ treatment decision-making , 20. ModPrevious qualitative studies have assessed the symptom burden of MM , so in lN = 22). Although evidence of concept saturation was observed in the qualitative dataset, suggesting the robustness of the qualitative analysis, caution should be taken when interpreting results due to the small sample size. The study protocol was amended partway through data collection and broadened the number of lines of prior therapy from up to 3 lines to up to 6 lines. The study population may be skewed toward patients with fewer relapses (68.2% of our study sample had ≤ 2 relapses); only four participants (18.2%) had ≥ 4 relapses, so we may have under-sampled patients with more substantial relapse experience. However, by opening eligibility to patients with more prior lines of therapy, the study may have expanded to include patients with more severe conditions and a longer duration of living with myeloma and therefore a different perspective. These patients may have come to terms with the severity of their condition and therefore minimized their symptom experience or treatment burden. In addition, our study population was composed only of white and Black/African American participants and as such may miss the experiences of patients from other ethnicities or racial groups. Although the absolute number of Black or African American participants in our study sample was small (n = 4), the proportion was higher than the general population (18.2% vs 13.4%) [We acknowledge some limitations of our study. As is common among qualitative interview-based studies, the information reported here was drawn from a relatively small sample of participants (s 13.4%)  and mores 13.4%) , as MM ds 13.4%) . SelectiThe data collected from this study provide valuable insight into treatment experience concepts that are relevant to patients with RRMM. The results can be used to improve our understanding of the symptoms, HRQOL impacts, and treatment burden themes that are most important to patients. Specifically, these findings suggest that factors related to treatment administration time are important to patients and should be considered in patient-provider communications in clinical contexts. The results can also be used to inform future research into treatment preferences among patients with RRMM.Supplementary file1 (198 KB)Below is the link to the electronic supplementary material.


For the rectal cancer <5 cm from anal margin, extralevator abdominoperineal resection (eAPR) has been accepted widely by surgeons. However, the rate of perineal infection following up eAPR is approximately 70%. We did the study with the aim of evaluating the effect and safety of transperineal pelvic drainage combined with lateral position (TPDLP) on perineal wound in patients undergoing eAPR.Patients were randomly assigned to N-TPDLP group (standard arm) or TPDLP group (intervention arm). In the standard arm, surgery was completed after abdominal drainage tube was placed in pelvic. Comparatively, an additional transperineal wound drainage tube was applied in the experimental arm. Postoperatively, patients of both 2 groups were informed not to sit to reduce perineal compression until the perineal wound healed. But lateral position was demanded in the intervention arm. The primary endpoint was the rate of uncomplicated perineal wound healing defined as a Southampton wound score of <2 at 30 days postoperatively. Patients were followed for 6 months.P = .001). At 30 days postoperatively, 3 (10%) of 29 patients undergoing TPDLP were classified into grade 4 according to Southampton wound score, however, 16 (52%) of 31 patients were classified into grade 4 in control arm, and significantly difference was observed between randomization groups (P = .001). What's more, perineal wound pain was assessed at 30 days postoperatively, and it is discovered that the pain degree of patients in control arm was significantly more severe than the interventive arm (P = .015).In total, 60 patients were randomly assigned to standard arm (n = 31) and intervention arm (n = 29). The mean perineal wound healing time was 34.2 (standard deviation [SD] 10.9) days in TPDLP arm, which significantly differ from 56.4 (SD 34.1) in N-TPDLP arm (In the present study, we found that TPDLP generated a favorable prognosis for perineal wounds with acceptable side-effects. For the rectal cancer <5 cm from anal margin, abdominoperineal resection (APR) had been accepted widely by surgeons since the first description for this surgical approach. However, traditional APR is associated with a high rate of positive margin due to its non-cylindrical resection without mesorectal removal at the level of the pelvic floor. Therefore, the traditional APR has been gradually abandoned in recent years. In 2007, extralevator abdominoperineal resection (eAPR) as an alternative procedure was first described by Holm et al, and then it was accepted by more and more surgeons. Several studies reported that eAPR performed better outcomes in circumferential resection margin positivity, rate of intraoperative perforation, and local recurrence compared with APR.,6 However, after eAPR, a large cavity was created by resecting the anus, rectal, mesorectum, musculuslevatorani, and surrounding perineal skin. And then blood clot and exudate accumulate in the pelvic cavity, which increases the risk of pelvic abscess and perineal wound infection. Furthermore, the large cavity in perineal and the stiff structure of pelvic may increase the perineal wound tension followed by wound closure. Therefore, the rate of perineal infection in patients undergoing eAPR is approximately 70%.–9Colorectal cancer, the third most commonly diagnosed carcinoma and the third leading cause of carcinoma-related mortality, is still one of the mainly hazards for health of human.,11 Several measures such as omentoplasty (OP), biological mesh closure of the pelvic floor, myocutaneous flaps, and incisional negative pressure wound therapy have been performed to reduce the probability of perineal wound complications for recent decades.,12–14 However, OP was difficult to implement in some cases such as previous omental resection, metastatic involvement, and contracture owing to inflammation. Therefore, it is controversial whether OP is effective or not. Blok et al indicated that OP did not promote perineal wound healing; furthermore, it may result in additional morbidity and need for reintervention. Biological mesh closure of the pelvic floor was not superior in perineal wound healing and QOL in patients after eAPR and increased the duration of surgery, although it reduced the risk of perineal hernia compared with primary closure. For myocutaneous flaps, special techniques of plastic surgery and high risk of flap necrosis restricted its application. Nowadays, incisional negative pressure wound therapy was generally accepted by surgeons and it did decrease the risk of wound infection. However, for the patients with allergies, the new therapy seemed not available due to the use of film tape.Perineal wound complications not only prolong the length of hospital stay, but also increase the cost of hospitalization. What's more, it may affect the daily life of patients undergoing APR and lead to a loss of quality of life (QOL).,17 In summary, wound adipose tissue liquefaction, remains of liquid beneath incision  and sometimes high surface tension between sutured tissues could increase the risk of infection. Therefore, to apply transperineal pelvic drainage tube may be effective for perineal wound healing through keeping the wound dry. Furthermore, the application of lateral decubitus position could reduce the incision surface tension to some extent, and it could also reduce the stimulation of the perineal wound from pelvic fluid accumulation because the liquid will flow downwards.Generally speaking, surgical wound infection was associated with microbiota diversity among the wound and microenvironment . Microbes thrived in dark, moist, and nutrient-rich environments, which resulted in the wound infection eventually. indicated that pelvic drainage tube combined with subcutaneous negative pressure drainage performed better efficacy and lower infection rate for perineal incision in laparoscopic-assisted abdominoperineal resection. Furthermore, Dinaux et al reported that prone position treatment for perineal wound during eAPR was associated with significantly lower perineal wound infection and dehiscence rates. However, there have been no studies reporting the effects of transperineal pelvic drainage combined with lateral position (TPDLP) for perineal wound in patients after eAPR. Therefore, further research is needed to evaluate whether TPDLP can promote the perineal wound healing in patients following the extralevator abdominoperineal resection.Chen et alWe did the study with the aim of evaluating the effect and safety of TPDLP on perineal wound in patients undergoing eAPR.22.1The present research was a single center prospective clinical trial, performed in Second Affiliated Hospital of Jilin University. And the approval of the study protocol was obtained from the ethics committee of Second Affiliated Hospital of Jilin University. Eligible participators were randomized assigned to primary closure of perineal defect (standard arm) and transperineal pelvic drainage combined with lateral position after primary closure of perineal defect (intervention arm). An observer, unclear to the patient allocation, evaluated perineal wound healing using Southampton wound score after 30 and 60 days postoperatively. Sonography of perineal wound or pelvic computerized tomography (CT) was performed to evaluate the wound healing and perineal complications such as presacral sinus, perineal sinus, perineal abscess, and perineal herniation. In addition, during the follow-up, all severe events of perineal wound, containing medicine or surgical interventions and severe infections, were recorded.2.2 and patients who enrolment in other trials that may affect the wound healing. After that, patients were randomized to TPDLP group and standard group. And the clinical date of patients was collected pre- and postoperative in the present research, which included baseline characteristics , surgical date , and postoperative perineal wound healing date.A patient was considered eligible when the following conditions were satisfied: Age from 18 to 75 years; the distance from carcinoma to anal verge was <5 cm according to preoperative examination such as magnetic resonance imaging (MRI), CT, or colonoscopy; pathologically confirmed carcinoma; no evidences of distance metastasis were found after CT scan or MRI examination; able to complete postoperative follow-up. And the exclusion criteria were emergency surgery, synchronous primary tumors in colorectal or other organs, severe respiratory tract, liver, kidney, or cardiovascular disease, accepting neoadjuvant radiotherapy,2.3Stratified randomization was performed in the present study, and the stratified factors were age (18–59 or 60 years or older), sex , and surgical approach (open or laparoscopic surgery). After written informed consent, patients were assigned to 8 subgroups according to the stratified factors. After that, patients from each subgroup were randomly assigned to standard arm and intervention arm. The allocation of treatment was blinded to perineal wound assessor and patients.2.4All patients were given antibiotics 2 hours before surgery according to the local trial site protocol. The operation method (such as invasive surgery or laparoscopic surgery) was left up to surgeons. The techniques of Biological mesh, omentoplasty and myocutaneous flaps had not been applied in our institution, therefore none of the patients had a mesh, omentoplasty or myocutaneous flap placed. In all patients, the principle of extralevator APR approach was adhered to, in which levator muscles were laterally resected, distal rectum and anal tube were excised completely. The coccyx was not routinely resected with the exception of surgical or carcinoma reasons. Finally, perineal defect was closed layer by layer with absorbable suture material.In the standard arm, surgery was completed after placing abdominal drainage tube. Comparatively, an additional transperineal wound drainage tube was placed in the pelvic floor in the experimental arm. Postoperatively, patients of both 2 groups were informed not to sit to reduce perineal compression until the perineal wound healed. But lateral position was demanded in the intervention arm. Drainage tube was irrigated with 100 mL 0.9% saline solution everyday postoperatively to make sure the drainage tube is unobstructed in both 2 arms. Furthermore, antibiotics were routinely applied to patients from both 2 groups postoperatively.2.5 of <2 at 30 days postoperatively. The Southampton wound score is shown in Table  Other endpoints were perineal abscess rate, symptomatic and asymptomatic perineal hernia, perineal sinus rate, and other perineal complications requiring surgical intervention. The complications mentioned above were diagnosed through clinical signs and symptoms, clinical physical examination, laboratory tests, ultrasonic examination, and/or pelvic MRI scan. And the postoperative pain was defined as >3 on the scale in the present study, considered to potentially affect emotional or physical functioning.In the present study, the primary endpoint was the percentage of uncomplicated perineal wound healing which was defined as a Southampton wound score2.6U test or t test was used for continuous variables . Chi-square test or Fisher exact test was used for comparing categorical data . Multivariate analyses were evaluated with Cox proportional hazards models. The Kaplan–Meier curve was used to assess the perineal wound healing rate, and study arms were compared using a log rank test. And P < .05 was considered statistically significant.Statistical analyses were performed using SPSS for MAC, version 26.0 . Mann–Whitney 33.1Between the first of January 2018 and the first March of 2021, 71 eligible patients were approached to participate in the present study. Of the 71 eligible patients, 65 patients consented to the trail, and of which 32 patients were randomly assigned to TPDLP group  and others to N-TPDLP group (standard arm). After randomization, 1 patient did not accept eAPR, but a Hartman procedure; 1 patient died 6 days after operation and 1 patient was lost during follow-up in the intervention group. In the control group, 2 patients were lost during follow-up and 1 patient underwent neoadjuvant radiotherapy. These 6 patients were excluded owing to the fact that these patients could not be evaluated for the primary endpoint, resulting in 29 patients in the control group and 31 patients in the standard group , and 60% (36/60) of the patients were men. The mean BMI was 23.4 kg/m2 (SD 3.4) in patients of intervention arm, which did not significantly differ from 23.8 kg/m2 (SD 3.8) in the control arm (P = .664). ASA-classification was performed in all 60 patients. 5 (16%) of the 31 patients were classified into grade 3, which did not significantly differ from 5 (17%) of the 29 patients in the TPDLP group (P = .908). The mean operation time was 195 minutes (SD 21) and 190 minutes (SD 12) separately in control group and experimental group, and no significant difference was observed (P = .273). And there was also no significant difference between N-TPDLP arm and TPDLP arm in bleeding volume during operation .The baseline characteristics of the included patients were described in Table 3.3P = .866)  in TPDLP arm, which did not significantly differ from 29% (9/31) in N-TPDLP arm (3.4P = .001). Furthermore, severity of infection according to the Southampton wound score was assessed at 30 and 60 days postoperatively. At 30 days postoperatively, the infective degree of 3 (10%) patients undergoing TPDLP were classified into grade 4 according to Southampton wound score, however, 16 (52%) patients were classified into grade 4 in control arm, and significant difference was observed between randomization groups (P = .001). At 60 days postoperatively, 11 (35%) of 31 patients in the control arm was classified into grade 2 according to Southampton wound score in the control arm, which significantly differ from 0 to 29 patients in interventive arm (P < .001). What's more, perineal wound pain was assessed at 30 days postoperatively, and the pain degree of patients in control arm was significantly more severe than the interventive arm (P = .015). However, the mean length of stay was 22 (SD 7.0) in control arm and 20.1 (SD 5.0) in intervention arm (P = .377). And no significant difference was observed between randomization groups in perineal abscess rate within 6 months postoperatively (P = .36). There was also no significant difference between 2 arms in perineal hernia rate (P = .24), persistent perineal sinus rate (P = .61), and surgical reintervention rate (P = 1.00) within 6 months postoperatively  days in TPDLP arm, which significantly differ from 56.4 (SD 34.1) in N-TPDLP arm (P < .001) was significantly associated with a better perineal wound outcome compared with other factors. In the final multivariable Cox regression model, TPDLP  independent of other factors was associated with a favorable prognosis of perineal wound. However, carcinoma locating <3 cm from anal verge  and men  independent of other factors was associated with a worse prognosis of perineal wound.To determine whether TPDLP was independent factor associated with perineal wound outcomes, a univariate and multivariate analysis was performed using the Cox proportional hazard model Table . The risFurthermore, Kaplan–Meier analyses were performed to further analyze the association of TPDLP and prognosis of perineal wound in patients after eAPR.P < .001, Kaplan–Meier log-rank)  were considered to be associated with perineal complications. Therefore, if TPDLP was put into effect, the risk factors of perineal complications would be eliminated partly, and the prognosis of perineal wound would be better.Abdominoperineal resection (APR), as a radical surgery of rectal carcinoma <5 cm from anal verge, is the most widely accepted operation by surgeons.P = .866), the mean perineal wound healing time (34.2 SD 10.9) in TPDLP arm was significantly shorter compared with the patients in control arm (56.4 SD 34.1) (P = .001). Second, the perineal wound infection degree of the patients in intervention arm was lower compared with patients in control group at both 30 and 60 days postoperatively. Finally, the pain degree of the patients in control group was more severe than patients undergoing DPDLP at 30 days postoperatively.The present study elicited 3 main findings. First, although no significant difference was observed in uncomplicated perineal wound healing between the 2 randomization groups  in the present study, which was not consistent with the viewpoint of Pramateftakis et al. The distinction may be related to the following 2 elements. First, drainage tube as a foreign matter for body may induce aseptic inflammation around the tube, and the wound healing near the drainage tube would be delayed by 1 to 2 weeks after the tube pulled out. Second, the number of eligible patients was small, and the difference may be statistically significant if more patients enrolled in the present research.Several randomized prospective studies comparing different interventions such as omentoplasty (OP), biological mesh closure of the pelvic floor, myocutaneous flaps, and incisional negative pressure as a strategy to promote perineal wound healing have been published over recent decades. However, studies related to transperineal pelvic drainage combined with lateral position for perineal wound treatment were quite rare. The retrospective literature of Abdominoperineal Resection for Rectal Cancer: Is the Pelvic Drain Externalization Site an Independent Risk Factor for Perineal Wound HealingP < .001), however, men  and carcinoma locating <3 cm  from anal verge were associated with a poor outcome. The literature of predictors of wound dehiscence and its impact on mortality after abdominoperineal resection: data from the National Surgical Quality Improvement Program put forward that men independent of other factors was associated with a worse prognosis of perineal wound , which was concurrent with the present study. On the contrary, the research of Predictors of Perineal Wound Complications and Prolonged Time to Perineal Wound Healing After Abdominoperineal Resection indicated that hypoalbuminemia was associated with poor outcomes of perineal wound , which was not consistent with the present study. The different result may be attributed to the little enrolled patients in the present study. Hypoalbuminemia was also associated with a worse prognosis of perineal wound in this research, and the risk factor may be statistically significant if more eligible patients were enrolled. Furthermore, the present prospective cohort trial indicated that patients treated with TPDLP performed a lower severity of perineal wound infection according to Southampton wound score, which was consistent with the viewpoint of Zeng et al.In the present study, multivariate analysis was performed to further evaluate the independent factors associated with perineal wound outcomes. TPDLP was associated with a favor perineal wound prognosis for patients after eAPR , and the moist environment (such as hematocele and hydrops) where bacteria thrives may be eliminated through transperineal wound drainage, which provided theoretical basis for the present trial. In addition, transperineal wound drainage was more adequate compared with transabdominal wound drainage, after all, the perineum is the lowest point of the pelvic cavity for human with erect position. Wiatrek et al indicated that the perineal wound tension was related to the incidence of complications of perineal wounds, and lateral decubitus position should be used to reduce wound tension, which could also reduce the stimulation of the perineal wound from pelvic fluid accumulation because the liquid will flow downwards.Transperineal wound drainage tube combined with lateral position was performed in patients after eAPR in the present research. Previous clinical trial had reported that non-drainage was associated with higher risk of deep infection in patients after total hip arthroplasty.The present study had several limitations. First, this was a single central prospective clinical trial, and a multi-central study would be idealized. Second, a sample size of the present clinical trial was still small because the patients suffering lower rectal carcinoma were few during the past 3 years in Second Affiliated Hospital of Jilin University. But a sample size of 61 patients was also acceptable for prospective studies. Finally, it was challenging for evaluators to objectively assess the perineal wounds, especially when wound scoring systems were limited and not validated for every type of wounds. Therefore, the Southampton wound score was the best available method for this purpose. At last, a prospective and multi-center study with a large sample size is required to further evaluate the efficiency and safety of TPDLP in the future.5In the present study, we found that TPDLP generated a favorable prognosis for perineal wounds with acceptable side-effects. Thus, TPDLP may be a promising and exciting therapeutic strategy for patients suffering abdominal perineal resection.Jian Shi, Colorectal Section, Department of Surgery, The Second Hospital of Jilin University, No. 218, Ziqiang Dist, Changchun, 130041, Jilin.AS and SW conceived the study design. MW and YY acquired the data for the study. ZZ, DL, and YG analyzed and interpreted the data. AS drafted the manuscript. MW and YY revised the manuscript critically. The authors read and approved the final manuscript.Data curation: Zeyun Zhao, Yu Guo, Rui Qi, Yang Yang.Formal analysis: Donglin Li.Methodology: Yongping Yang.Writing – original draft: An Shang.Writing – review & editing: Shuang Wang, Min Wang.


Solanum tuberosum L) is one of the four most important crops worldwide in production and consumption. It originated from South America along the Andes, where six hotspots of diversity known as subcenters of origin are described from Venezuela to Chiloe Island in Chile, and where the greatest diversity of potatoes in the world is found. Today, the use of ancestral genetic resources has gained significant relevance, recovering and producing foods with a greater nutrient content and beneficial to human health. Therefore, native potatoes possess a set of characteristics with great potential for use in potato breeding guided primarily to produce better feed, especially potatoes of the Chilotanum Group that are easily crossed with conventional varieties. The primary objective of this study was to evaluate 290 accessions of S. tuberosum subsp tuberosum belonging to the Chilotanum Group using a set of molecular markers and correlate them to its phenotypic traits for future use in breeding programs. For this purpose, 290 accessions were analysed through 22 specific microsatellites described previously, correlating them with flesh and skin colour, total phenolic content, and anthocyanin content. A division into groups considering all the 290 accessions resulted in two clusters using STRUCTURE analysis and seven different genetic clusters using UPGMA. The latter exhibited common phenotypic characteristics as well as anthocyanin content, strongly supporting a correlation between phenotypic traits and the genetic fingerprint. These results will enable breeders to focus on the development of potatoes with high polyphenol and anthocyanin content.The potato ( Solanum tuberosum) is currently the fourth most important crop after rice, wheat, and corn. It plays a relevant role in global nutrition and for the sustainable development goals of the United Nations related to zero hunger and feed security . Chile is considered a subcenter of origin of this crop. The Chilean germplasm provides new material for researchers interested in the origin of cultivated potatoes and S. tuberosum in Chile  declared six potato hotspots of diversity along the Andes Mountains from Venezuela in the North to Chiloe Island in the South . Native in Chile . The Potin Chile . López eS. tuberosum subsp. tuberosum) using SSR markers, chemically in terms of TPC and anthocyanin content and phenotypically in terms of tuber coloration in the flesh and skin, and to determine the correlations between them.There has been extensive research to date on potatoes due to the narrow genetic diversity among the known cultivars and the urgency to broaden the genetic pool to enhance breeding programs. Potatoes might be found in different environments, and because of this plasticity, they exhibit a great potential to adapt to the future climatic change caused by global warming and water stress conditions. In addition, more sustainable cultivation methods must introduce more abiotic and biotic resistance genes from wild potatoes. The 22 simple sequence repeat (SSR) markers developed by The tubers of 290 accessions from the Potato Genebank at the UACh were screened in two seasons, according to the scale for potato pigmentation from the CIP characterization parameters . The def2, which were fertilised with N (100 kg/ha), P2O5 (180 kg/ha), and K2O (60 kg/ha) at the planting time and N (50 kg/ha) plus K2O (60 kg/ha) at ridging, according to previous soil analyses (data not shown). Insecticides and fungicides were used to control biotic damage, according to manufacturer’s recommendations. Sprinkler irrigation was implemented to satisfy water requirements of plots. The harvest of tubers was done by hand when leaves and stem were dead , according to each potato genotype. A total of 10 potato tubers were randomly selected from each accession for morphological characterisation, as well as for TPC and anthocyanin content determination. In total, 100 g of fresh potatoes (skin and flesh) was obtained from 10 different tubers, sliced, and lyophilized for chemical analysis two weeks after the harvest.All accessions were grown in 2015/2016 and 2016/2017 at the Austral Experimental Station of the UACh located in Valdivia . Two rows (each 4 m long and separated by 0.75 m) per accession were planted. The plant density was 5.3 plants/mTotal polyphenols were determined by a colorimetric assay using the Folin–Ciocalteu reagent according to the modified method of The anthocyanin content was determined as described previously  Samples −1. The anthocyanin compounds were measured through the content of anthocyanidins (aglycone anthocyanins) using the method proposed by Anthocyanin-specific metabolites were also quantified by HPLC-DAD in the flesh of different native potato accessions. The extracts were prepared as described by Total DNA from the 290 accessions was obtained from leaf tissue using the PureLink Genomic DNA kit  according to manufacturer’s instructions. The quality of each purification product was evaluated using the QuBit 3.0 fluorometer and Infinite F200/M200 NanoQuant spectrophotometer at wavelengths of 260 and 280 nm. Then, these purified DNA samples were used for allele amplification in a volume of 25 µL using the KAPA2G Robust HotStart ReadyMix PCR kit.The sequence of primers for the 22 SSRs were obtained from a previous study . PCR wasA population genetic analysis was performed with STRUCTURE software  on the 2The kinship analysis was performed using the alleles previously described by fi is the frequency of the ith amplified allele over all individuals of the population  exhibited some variation in skin pigmentation from pink to blackish, and 83 accessions showed the same color range as the secondary colour (29%), totaling up to 85% of total accessions, revealing a greater abundance of anthocyanins and phenols in the skin than in the flesh of the screened potatoes. A total of 30 accessions (10%) showed blackish colour and 56 accessions showed purple pigmentation (19%) in the skin. Regarding the secondary colour, only 2 accessions exhibited marbled distribution with a blackish pigmentation, 19 accessions showed a purple coloration, and 30 accessions showed a pink coloration. The most frequent primary colour of the skin was yellow (24%), and 59% of the screened accessions showed no secondary colour Figure 1−1 DW, with a mean of 4286 µg g−1 DW and a median of 3787 µg g−1 DW, and 39% of the genotypes had higher TPC values than the average value  and cream (3999 µg g−1 DW) flesh , and those with high anthocyanin content had purple flesh and blackish skin (920 µg g−1 DW), showing a 3066-fold difference in the content between them.The anthocyanin content in the tubers showed a broader range of variation than that of TPC, ranging from 0.3 to 920 µg gSimilar to TPC, the genotypes with purple flesh as the primary color had the highest concentration of anthocyanins, and those with pink flesh had 1.2 times more anthocyanin content than tubers with red flesh Figure 3-1 DW, followed by cyanidin (2–205 µg g−1 DW) and petunidin (2–236 µg g−1DW), and lower concentrations of peonidin (1–18 µg g−1 DW) and malvidin (7–67 µg g-1 DW). Delphinidin was the most frequent anthocyanin found in the screened potato tubers, closely followed by cyanidin and petunidin; these anthocyanins were related to purple, magenta, and dark red flesh colours, respectively. All anthocyanins, except malvidin, showed a significant relationship (p< 0.01) between their specific concentrations and the total anthocyanin content of each accession , which were arbitrarily named from A to G, and established the genetic kinship or relationship between all of them (-1 DW and TPC = 4286 µg g-1 DW). Above each histogram, the genetic clusters are shown, considering the accessions with values above the mean in each case. Thus, 58 accessions showed a higher value than the mean anthocyanin content (61.4 µg g-1 DW), which were distributed as follows among the clusters: 8 accessions in cluster A, 20 accessions in cluster B, 3 accessions in cluster C, 2 accessions in cluster D, 1 accession in cluster E, 22 accessions in cluster F, and 2 accessions in cluster G. However, 113 accessions showed values of >4286 µg g-1 DW (mean TPC of the population), which were distributed as follows: 12 accessions in cluster A, 27 accessions in cluster B, 12 accessions in cluster C, 12 accessions in cluster D, 14 accessions in cluster E, 23 accessions in cluster F, and 13 accessions in cluster G  for skin and eight categories  for flesh, according to an adapted classification proposed by the CIP, Perú. Considering all groups, the occurrence of these phenotypic traits was evaluated, and the probability of the occurrence of any of these traits in each cluster was quantified. Furthermore, the statistical significance weight for each probability was evaluated to distinguish it as a characteristic trait of the cluster. In relation to the primary skin color, there were no strong correlation between characteristics and clusters  and STM3023a, STM1104, STM1016, and STGBSS were polymorphic in a large number of genotypes in clusters A, B, and F. These clusters are important because they have high potential to include genotypes with high levels of anthocyanins and pink-like potato genotypes. These findings suggest that the presence of these SSRs is important in the expression of these phenotypes and should be considered in further investigations.Due to the narrow genetic diversity among the known potato cultivars and the need to broaden the genetic pool to enhance breeding programs, there has been extensive research on potatoes. A growing interest in functional foods has stimulated investigation on pigmented potatoes for their potential effects on human health related to their abundance of phenolic compounds and anthocyanins, as well as a plant protection effect. The biological activities of polyphenols in potatoes might be helpful for breeders in designing new varieties with numerous health benefits, for both pharmaceutical and nutraceutical industries . The incS. tuberosum subsp. tuberosum Chilotanum Group  but no petunidin. However, the highest score of petunidin also corresponds to a purple tuber, concluding that both anthocyanins could provide purple color to potato tubers. When a subset of accessions of the Potato Genebank at the UACh was examined using molecular markers of three anthocyanin synthesis genes, a greater allelic diversity was observed at the P locus that is related to the synthesis of blue/purple petunidin-based anthocyanins, were alleles correlated positively with purple pigmentation in most of the accessions , exhibited several heterogeneous genotypes and phenotypes. Even the selection of genotypes for crossing is primarily dependent on morphological traits, and genetic relatedness measured based on molecular markers help predict parental performance and improve heterotic effects. The 22 SSR markers developed by S. tuberosum Chilotanum Group in the Potato Genebank. As the allelic diversity or the mean number of alleles per locus measures the genetic variation, a comparison was made with previous investigations on native potatoes. The mean number of alleles per locus was 7.4, similar to that observed by The heterozygous autotetraploid While STRUCTURE analysis cluster into two groups with K=4, the hierarchical analysis (UPGMA dendrogram) showed 7 cluster. The reviewing of the accession’s distribution between the two approaches showed that the vast majority (> 90%) of the accessions belonging to subgroups A, B, D and F identified through UPGMA analysis were also grouped together in the first STRUCTURE cluster, while the accessions grouped in clusters E and G were mostly (>80%) grouped in the second STRUCTURE cluster. Only the accessions belonging cluster C showed a more equal distribution (55%-45%) among both STRUCTURE clusters. These results suggest that the genetic analysis approach did not resolve the degree of relatedness between the 290 accessions, and the genetic structure in the genebank, which will require further analysis. Notwithstanding the above, the simple observation of the diversity of phenotypes represented in shapes, colours, TPC and anthocyanin content in the genebank hints the high genetic variations that had not been possible to address so far. The result of the STRUCTURE analysis  can be crossed with the common potato varieties and present a source of resistance to disease and stress, as well as high contents of polyphenols and anthocyanins, which make them contributors to breeding for designing new varieties with health benefits to satisfy the nutritional requirements in human consumption and for plant health.The narrow genetic diversity observed among modern potato varieties necessitates the need for investigations like this one so that potato breeders can enhance genetic diversity in parental clones. The native potatoes of the Chilotanum Group , yellow (26%), or white (2%), and 10% of the total accessions showed purple, pink, or red flesh, whereas 46 accessions showed the same tone as the secondary color (16%). In total, 78 accessions had some anthocyanin-related colour, corresponding to 26% of total accessions. Regarding the skin colour, 164 accessions (57%) showed some variation in skin pigmentation. ranging from pink to blackish.−1 DW and the highest anthocyanin content of 920 µg g−1 DW. All anthocyanins, except malvidin, showed a significant relationship (p< 0.01) between their specific concentrations and the total anthocyanin content of each accession. Similarly, delphinidin was the most frequent specific anthocyanin found in the screened potato tubers, which was different from the published data on the anthocyanins of purple potatoes, indicating that it is a potentially interesting compound for further investigation.The TPC and anthocyanin content showed significant variation, observing genotypes with higher contents than some fruits known for their antioxidant capacity. The genotypes showing purple flesh as the primary color had the highest concentrations of anthocyanins; those with purple flesh and blackish skin had the highest TPC of 18,103 µg gGenetic diversity was evaluated using SSRs, resulting in 146 alleles with an average of 7.36 alleles per SSR and a mean PIC of 0.77; these data were consistent with the published information on other native genotypes. The most discriminatory SSRs corresponded to STM3012 and STM3023a with a PIC value of 0.91, and the largest number of alleles per SSR was observed in STM3023a, STM 0037, and STM 0030.The STRUCTURE analysis using K=4, formed two different clusters, with 195 and 95 accessions, respectively. The seven clusters obtained using the UPGMA dendrogram showed high cophenetic coefficients. The correlation of genotypes with TPC and anthocyanin content greater than the mean value indicated that clusters B (47%) and F (59%) contained the largest number of genotypes with a high content in each case. Cluster F and A had a high probability of grouping accessions with secondary deep pink colour of the skin, concluding that the clustering does present a correlation to colours, TPC, and anthocyanin. The possible SSR that differentiated the potato accessions with higher TPC and anthocyanin content was STM1016 that was present in all clusters (A–G), and also STM3023a, STM1104, STM1016, and STGBSS, the markers that were polymorphic in a large number of genotypes in clusters A, B, and F. Finally, the high variability in tuber colors, TPC, and anthocyanin content was consistent with the high genetic diversity screened using genotype-specific SSR markers, which support the use of this genetic material in potato breeding.The original contributions presented in the study are included in the article/AB and CL did the phenotypic screening and chemical analysis, MR-D did the anthocyanins analyses per HPLC. FZ and DF performed the SSR amplifications on the DNA accessions and AG developed the kinship analysis and then the association between the different groups of accessions and their anthocyanin and phenol content. All authors contributed to the article and approved the submitted version.


Purpose: In order to overcome the problem that conventional pharmacological treatments of periodontitis cannot effectively synergizing antimicrobial and immunomodulation, inspired by the critical role of toll-like receptor 4 (TLR4) in bacterial recognition and immune activation, we demonstrated a combined antibacterial-immunoregulatory strategy based on biomimetic nanoparticles.Methods: Functioned cell membranes and silk fibroin nanoparticles (SNs) loaded with minocycline hydrochloride (Mino) were used to prepare a biomimetic nanoparticle (MSNCs). SNs and MSNCs were characterized by Scanning Electron Microscope, size, zeta potential, dispersion index. At the same time, SNs were characterized by cell counting kit-8 and real-time Polymerase Chain Reaction (RT-PCR). TLR4-expressing cell membranes were characterized by RT-PCR and western blot (WB). Cell membrane coating was characterized by Transmission Electron Microscope (TEM), the Bradford staining and WB. Then, Laser confocal, flow cytometry and agar plate coating were evaluated in vitro with antibacterial effects, RT-PCR was simultaneously evaluated with immunoregulatory effects. Finally, Anti-inflammatory treatment of MSNCs was evaluated in a ligature-induced periodontitis (LIP) mouse model.Results: Successfully prepared cell membranes overexpressing TLR4 and constructed MSNCs. In vitro studies had shown that MSNCs effectively targeted bacteria via TLR4 and acted as molecular decoys to competitively neutralize lipopolysaccharide (LPS) in the microenvironment as well as inhibit inflammatory activation of macrophages. In vivo, MSNCs effectively attenuated periodontal tissue inflammation and alveolar bone loss in a LIP mouse model.Conclusion: MSNCs have good targeted antibacterial and immunoregulatory effects, and provide a new and effective strategy for the treatment of periodontitis and have good potential for application in various types of pathogenic bacterial infections. The high prevalence of periodontitis is not only a major cause of tooth loss, but also a potential risk factor for diseases such as rheumatoid arthritis, cardiovascular disease and gastric cancer . The patIt is well known that innate immunity, as the first line of defense against bacterial invasion, relies heavily on pattern recognition receptors (PRRs) . In partetc. Surface membrane biomimetic modification of nanoparticles is a novel and efficient way. The cell membrane is coated around the nanoparticle surface by physical extrusion, ultrasonic or electroporation methods, which gives the nanoparticles bionic properties and preserves the protein function of the source cell membrane  and can release antibacterial drugs to kill bacteria efficiently. Moreover, they effectively inhibited the inflammatory activation of macrophages by neutralizing LPS in the microenvironment. In vivo studies showed that MSNCs effectively suppressed periodontal inflammation and reduced alveolar bone resorption in a ligature-induced periodontitis (LIP) mouse model  cocoons were donated from Southwest University ; TLR4 lentivirus was purchased from Genechem Co. ; FITC and LPS were purchased from Sigma-Aldrich ; Cell counting kit-8 was purchased from MCE ; Sodium carbonate, lithium bromide, paraformaldehyde and hematoxylin, and eosin staining kit were purchased from Solarbio Science and Technology Co. ; acetone was purchased from CHUANDONG CHEMICAL ; Membrane and Cytosol Protein Extraction Kit, Dil, BCA Protein Assay Kit and DAPI were purchased from Beyotime Biotechnology .2 atmosphere. Gram-negative bacteria E. coli (ATCC 43888) was grown in LB medium at 37°C in an aerobic environment, and bacteria used in all experiments were in the mid-exponential growth period. The bacterial suspension was diluted to 1*106 colony forming units (CFU per mL) for experimental use after measuring the optical density at 600 nm using a microplate reader .RAW264.7 cells is a macrophage cell line  that was established from a tumor in a male mouse induced with the Abelson murine leukemia virus. And the cell line was used for macrophage membranes collection in the current study. RAW264.7 cells cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37°C in a 5% COSilk fibroin solution was prepared as previously described . BrieflyAdd the corresponding mass of Mino in SNs suspension at different mass ratios  and stir away from light overnight at room temperature. The supernatant was centrifuged at 32,000 g for 30 min, and the double-distilled water was resuspended and repeatedly washed twice before being stored at 4°C, protected from light. The supernatant was collected and used to calculate the encapsulation efficiency and drug loading efficiency. The equations of drug loading and encapsulation efficiency were described as follows:Lentiviral transfection of RAW264.7 cells was performed according to the product instructions. Briefly, RAW264.7 cells (50 000/well) were incubated for 12 h, then appropriate H-DMEM, TLR4 lentivirus (MOI = 50), and empty lentivirus were added separately to the culture medium for 10 h. After 48 h of incubation, the cells were harvested for the TLR4 mRNA levels by RT-PCR. Among them, the medium-treated group served as the control group for the experiment, the blank vector lentivirus group served as the negative control (NC) group, and the TLR4 lentivirus-treated group served as the TLR4 group. The sequences of the primers  were shown in E. coli in one group. The dialysis bags were completely submerged in PBS (PH = 7.4) and drug release was monitored at a constant temperature of 37°C for 0, 1, 4, 8, 12, 24, and 48 h.CMs extraction was performed according to Membrane and Cytosol Protein Extraction Kit. Briefly, functionalized macrophages were repeatedly rinsed with PBS after collection and cell counting. The cells were resuspended with Membrane Protein Extraction Reagent A containing PMSF. After 10 min of an ice bath, the cell suspension was homogenized using an ultrasonic disruptor and centrifugated at 700 g for 10 min, then the supernatant was collected and centrifugated at 14,000 g for 30 min, and resuspended the precipitate with PBS. Eventually, the concentration of CMs suspension was determined by the BCA method, and the suspension was stored at −80°C for use. The extracted CMs and SNs were mixed in a 1:1 mass ratio and repeatedly extruded through a polycarbonate membrane with the aid of a liposome extruder to obtain MSNCs. The morphology and structure of MSNCs were separately observed by scanning electron microscopy and transmission electron microscopy . Mean particle size and potential were detected by DLS. Finally, the Bradford method and WB were used to verify the successful coating of the CMs. After preparation of MSNCs, two equal groups of MSNCs were added into dialysis bags with appropriate amount of E. coli suspension was fixed with paraformaldehyde and washed twice with PBS. Dil-SNCs were added to the experimental group, incubated at 37°C for 1 h, stained with DAPI for 3 min, and resuspended after centrifugation to obtain the suspension. The suspension was dropped on slides for sample preparation and then images were acquired by laser confocal microscopy . Meanwhile, after paraformaldehyde fixation of E. coli, the experimental groups were incubated with FITC-SNs and FITC-SNC individually for 1 h at 37°C and washed for flow cytometry detection .The red fluorescent dye Dil and the green fluorescent dye FITC were used to label CMs and SNs, respectively. Dil -SNCs were prepared by a liposome extruder after mixing Dil with CMs for 2 h at room temperature; FITC-SNs were prepared by mixing SNs with FITC overnight away from light and purified by dialysis. E. coli were inoculated on 24-well plates, and SNCs and MSNCs were added to the experimental groups, respectively, and incubated for 24 h in a 37°C bacterial incubator. The incubated mixture was serially diluted and coated on nutrient agar plates, incubated for 24 h in a 37°C bacterial incubator, and photographed to observe the growth of colonies in the plates.RAW264.7 cells were used to detect SNCs biocompatibility and LPS neutralization. Cells were inoculated with a complete Medium in the control group and 40, 80, 120, 160, and 200 μg/mL of SNCs H-DMEM medium suspension in the experimental group, and cell viability was detected using the CCK-8 kit after 24 h of incubation. In addition, the experimental groups were incubated with LPS, LPS + SNs, and LPS + SNCs for 12 h, after being incubated in the cell incubator for 24 h. RT-PCR was performed as before.6-week-old male C57 mice were provided by the laboratory Animal Center of Chongqing Medical University. Periodontitis molds were completed by ligating the cervical portions of the bilateral maxillary first and second M of C57 mice for 1 week using 5–0 silk wire. To study the diffusion of MSNCs at the site of inflammation, we prepared FITC-labeled MSNCs and injected FITC-MSNCs into the palatal gingiva of mice with ligature-induced periodontitis, collected at 0, 4, and 24 h, and observed their diffusion in the periodontium by fluorescence distribution under a stereomicroscope. Then, all mice were divided into four groups, no treatment in the control group, no treatment in the periodontitis group after completion of periodontal ligation, and the rest of the experimental groups were administered with the effective drug concentration of 0.5 mg/ml in MSNs and MSNCs suspension respectively at the ligated tooth position by drops of 5 μl on each side for 1 week. Mice were anesthetized and executed after 1 week, and periodontal tissues were taken from bilateral ligated tooth sites for RT-PCR, WB, and Hematoxylin Eosin (HE) staining to detect the inflammatory changes. An antibody against IL-1β  was used to detect markers in cell homogenization by WB. Animal handling and surgical procedures were performed following the protocol approved by the Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University (2022 (No. 062)).post hoc test. A value of p < 0.05 was considered statistically significant .All experimental data are presented as the mean ± standard error of the mean (SEM), and the results were analyzed using a one-way analysis of variance (ANOVA) among groups followed by Tukey’s p > 0.05) to the increased concentration of SNs (p > 0.05) to the increased concentration of SNs , demonstrating that macrophage membrane camouflaging could improve the adherence to E. coli and achieve precise delivery of Mino  to the increased concentration of SNCs (p < 0.001), indicating that SNCs competitively neutralized LPS in the microenvironment and effectively inhibited the activation of pro-inflammatory macrophages , and WB assay suggested that IL-1β protein expression had significantly decreased in MSNCs  overexpressing TLR4 by genetic engineering and proposed a new antibacterial and immunoregulatory synergistic therapy for periodontitis. The TLR4 was used to achieve precise targeted drug delivery and killing of pathogens, while nanoparticles with TLR4 acted as ideal decoys for LPS to reduce the activation of immune cells. The synergistic therapy based on MSNCs made up for the defects of the current treatment and realized the effective control of periodontal inflammation.Nanoparticles are an effective platform to achieve precise drug delivery and cellular regulation . NanoparE. coli combined with MSNCs, the initial release rate of the drug slowed down significantly, but this effect diminished significantly after 8 h. And similar to Gou’s study (To achieve functions including precise drug delivery and immune escape, nanoparticles often require complex functionalization modifications . As an e’s study , the druE. coli precisely (We still verified that SNCs have good biocompatibility. We next verified that MSNCs have good antibacterial effects based on their targeted drug-release properties. On the one hand, the functionalized cell membrane on the surface of the nanoparticles could bind specifically to the LPS on bacterial due to TLR4, which made them have significant bacterial binding ability; on the other hand, MSNCs could release antibacterial drugs in a short period of time to kill recisely . LPS actrecisely . The RT-To verify the inhibitory effect of MSNCs on inflammation in periodontal tissues, a LIP mouse model was constructed in this study, and its therapeutic effect was evaluated after the topical administration of treatment. MSNCs have good local storage capacity and tissue penetration and can selectively diffuse to the periodontal inflammation site with good target diffusion of inflammation. The results showed that the level of inflammation in periodontal tissues was significantly lower and tissue destruction was significantly reduced in the MSNCs group compared with the no-treatment and MSNs groups, suggesting that MSNCs effectively reduced inflammation and inhibited bone loss in periodontitis.In summary, we constructed an engineered cell membrane-encapsulated nanosystem MSNCs for precise targeting and inhibition of bacteria, and to neutralize LPS in the microenvironment to inhibit inflammatory cell activation. MSNCs effectively inhibited periodontal inflammation and bone loss through dual regulation of antibacterial and immunity. Based on this nanoplatform, genetically engineered modifications with different ligand-receptor patterns can be constructed to respond to immune diseases infected by different pathogens. In addition, this system still has some potential for improvement, such as the drug loading rate can be further optimized and the therapeutic effect can be evaluated over a longer period of time. In conclusion, MSNCs provide a new therapeutic platform for periodontitis, and the dual functional design of antibacterial and immunoregulation provides additional consideration for the treatment of inflammatory diseases with bacterial infections.


Numerous scientific, health care, and industrial applications are showing increasing interest in developing optical pH sensors with low-cost, high precision that cover a wide pH range. Although serious efforts, the development of high accuracy and cost-effectiveness, remains challenging. In this perspective, we present the implementation of the machine learning technique on the common pH paper for precise pH-value estimation. Further, we develop a simple, flexible, and free precise mobile application based on a machine learning algorithm to predict the accurate pH value of a solution using an available commercial pH paper. The common light conditions were studied under different light intensities of 350, 200, and 20 Lux. The models were trained using 2689 experimental values without a special instrument control. The pH range of 1: 14 is covered by an interval of ~ 0.1 pH value. The results show a significant relationship between pH values and both the red color and green color, in contrast to the poor correlation by the blue color. The K Neighbors Regressor model improves linearity and shows a significant coefficient of determination of 0.995 combined with the lowest errors. The free, publicly accessible online and mobile application was developed and enables the highly precise estimation of the pH value as a function of the RGB color code of typical pH paper. Our findings could replace higher expensive pH instruments using handheld pH detection, and an intelligent smartphone system for everyone, even the chef in the kitchen, without the need for additional costly and time-consuming experimental work. Hydrogen ions concentration [H+] denoted to pH scales from 0 to 14, and the most methods counted for detection are complicated, expensive, and time-consuming as microelectrodes3, acid-based indicator4, potentiometric titration1, colorimetric and fluorescence probes application10. Currently, potentiometric measurements are the most technique used in pH detection. Where the pH of the solution can be calculated by the measurement of the different voltage between the electrodes of the potentiometric device11. Despite, the high accuracy of conventional potentiometric devices, the operation and calibration process is more complicated and costly, which is not applicable to indoor or outdoor purposes. However, the easy and accessible pH strips are used as an alternative method for visual pH detection, but the strips produce lower precise results.The pH value of different solutions is a particularly important point to determine the optimized conditions and quality control for industrial, biological, chemical, and environmental science either outdoor or indoor applications12. ML is being used in chemistry such as chemical discovery13, molecular representations14, synthetic chemistry15, materials chemistry16, aquatic chemistry research18, and water pollution19.On the other hand, machine learning (ML) techniques give algorithms the ability to predict novel values from training data derived from experiments using Artificial Intelligence (AI). Thus, there are numerous regression or classification algorithms for ML that depend on hyperparameters and mechanisms to achieve their goals and give high performance for planningHere, the ML technique was used to improve the precision of common strip pH paper. ML models were trained on the 2689 experimental data which covered the whole pH range. Further, we developed a mobile/web application based on ML algorithms to predict the pH values. Therefore, the developed app could work on mobile which could be used as portable devices for anyone (whether a chemist or not) without additional costs, fast response, and is applicable for different applications.Acetic acid, phosphoric acid, boric acid, HCl, and NaOH were used without any further purification (Sigma Aldrich). Universal indicator pH paper (1–14) Q/3211821AB001-2002 (China). The pH measurements were carried out on a 3520 pH Meter . Mastech MS6612 Digital Luxmeter Illuminometer Light  was used for measuring the light intensity in the experimental workplace.20. Briefly, the stock aqueous B-R buffer solutions (pH = 2.86) by mixing equal molar ratio (1:1:1) of 0.02 mol/L from acetic acid, phosphoric acid, and boric acid. Dropwise of 0.20 mol/L of NaOH or 0.20 mol/L of HCl was used for adjusting the pH values  to cover the whole pH range.Universal Britton–Robinson (B–R) buffer was prepared as reportedR), Random Forest Regressor (RTR), K Neighbors Regressor (KNNR), Support Vector Regression (SVR), Lasso regression (L1), Ridge Regression (L2), Elastic Net regressor (ENR), AdaBoost Regression (ABR), Gradient Boosting Regressor (GBR), and Artificial Neural Network Regressor (ANNR). All models can be found in Scikit-learn in the class model21. In addition, the data visualization of exploratory data analysis and heatmap figures were created using the seaborn package based on python code22.Regression is a technique used for prediction continues pH values learning and figuring out causal relations between the actual and prediction pH values. Eleven supervised machine learning regression models were applied to the data collected and choose the best model that fits with the selected problem, including Linear Regression (LR), Decision Tree Regressor (DT2), Mean Squared Error (MSE), Mean Absolute Error (MAE), and Root Mean Square Error (RMSE) can be calculated Scikit-learn in class metrics according to Eqs.  positions  to cover the whole image as illustrated in Fig. To extract the color code (RGB) from images, we used a Python 3.7 code based on the OpenCV package to extract the RGB for each image27. We randomly separated the data into training data  and testing data . In the inference model training phase, the testing data was completely excluded. Furthermore, in machine learning, hyperparameters are those parameters that are explicitly provided by the user to influence the learning process and improve the learning of the model. Thus, we trained our models using a series of integer number , and as a result of that the optimal hyperparameters  was found when we used K = 5.We exploited a KNN regression model-based machine learning algorithm to study 2689 collected sample data using Python 3.7 and the scikit-learn packageFigure To better understand the observed results in the different workplaces, Exploratory Data Analysis (EDA) of color code RGB against pH values with respect to different light intensities at 20, 200, and 350 Lux, was illustrated in Fig. The color code points were collected in three parts in a wide pH range. The significant changes in the color code of Red and Green or even Blue were in the range of (2.5: 9) pH values at the three different investigated workplaces of light intensities at . It is worth mentioning, that the blue color code at low-intensity light of 20 Lux (a little dark workplace) deviates from those obtained in higher or medium light intensity, which suggests avoiding future testing in low light conditions. In contrast, the results revealed no significant difference between the behavior of Red or Green colors at light intensity. The results show the increase in basicity (> 9) or increase in acidity and (< 2.5) could interpret the color and may produce less accurate prediction in that part of the pH range. Thus, this finding may encourage the scientific community to prepare higher sensitive material to work in strong acid and/or Strong base medium.x,y) between the pH parameters were investigated based on the following Eqs. ::x,y) betThe correlation between the pH parameters was presented with a heatmap in Fig. 30 to estimate coefficients of determination (R2) and the minimum errors of the corresponding regression evaluation metrics concerning root mean squared error (RMSE), mean absolute error (MAE), and mean squared error (MSE) as shown in Fig. Using experimental data, a preliminary analysis of machine learning regression techniques was performed with optimal hyperparameters on K-Nearest Neighbors (KNN), Linear, Lasso, Elastic Net, AdaBoost, Neural Network, Random Forest, and Support vector machine (SVM), and Gradient Boosting Regressor algorithms2 (0.993) combined with the lowest errors of MSE, RMSE, and MAE  compared to other models. In addition, the coefficient of the variation of the root means square error (CVRMSE) of KNN models shows a higher stability performance of 4.077 compared to other models. Further, the cross-validation with K-fold of  was tested for confirming the stability of the models. However, no significant difference was found between the results, which verified the KNN models.It's obvious that the KNN model with optimal hyperparameters of five points performs significant result of RTo deepen understanding, further investigation showed that the results of the model's prediction (based on test data) vs the experimentally obtained pH values are represented in the scatter plot in Fig. It is now clear that the KNN model can successfully show the underlying patterns of the color RGB code in the pH value estimation based on experimental data collections. Thus, the machine learning approach based on this model was further expanded and used to develop a versatile platform able to predict the pH value using common pH paper with high accuracy. The online mobile application of the prediction model was developed using python code and streamlit cloud (freely available) and permits the highly predicted determination of the pH value as a function of the RGB color code of common pH paper.As illustrated in Fig. Our study has a significant advantage over what is already used, Fig. Furthermore, Fig. 31 studied pH strips colorimetric detection using ML, as presented in Table However, Solmaz et al.However, four different types of smartphones were used to check the accuracy of pH value predictions for three buffer solutions . The default setting was used to avoid any smartphone effects. As shown in Fig. Furthermore, Table Overall, the present findings solve the problem of pH accuracy using common pH paper without the need for additional costly and time-consuming experimental work. However, our approach solves the problems of excessive cost and maintenance required for traditional pH meters.2 (0.993) results along with the lowest MSE, RMSE, and MAE errors . This paper also demonstrated the potential of the ML approach to estimate the pH value of solutions using common pH paper. We developed a freely available application that supported mobile devices to predict the pH value based on ML and using common pH paper with precise results. Future research should consider the preparation of new optical material with extremely sensitive color changes in a strong acid/base medium.The findings demonstrate a strong negative association between pH values and both the red color (−0.77) and the green color (−0.38). The blue color will have an insignificant impact on machine learning prediction which revealed a low correlation (0.044). The KNN model exhibits significant R