Percutaneous rupture of lumbar facet cysts (LFC) is the only nonsurgical treatment which is effective in directly reducing cysts. However, this is not yet a common procedure, and its effectiveness, including the associated complications, remains unclear. Therefore, this study aimed to evaluate the clinical outcomes of percutaneous rupture for LFC and elucidate whether this minimally invasive procedure could become an alternative to surgeries for cases resistant to conservative treatments.  This study investigated 57 symptomatic patients with LFC for whom conservative treatments were ineffective and underwent percutaneous rupture of the LFC. All patients were followed up for >2 years posttreatment. Clinical evaluations  and recovery rate calculated using the Japanese Orthopedic Association (JOA) scores) and radiographic evaluations (size of LFC based on magnetic resonance imaging (MRI)) were performed from pretreatment to the final follow-up examination.  Successful LFC rupture, without hospitalization and general anesthesia, was achieved in 48 patients. No severe complications occurred during treatment through the last observation. Satisfactory clinical results with significant improvements in the VAS and JOA scores were obtained . A significant reduction in the LFC was also observed in all cases based on the posttreatment MRI findings. No successful rupture cases required subsequent surgical treatments, although four cases of LFC recurrence required additional percutaneous rupture treatment.  Percutaneous rupture for LFC is not only a safe and minimally invasive procedure without any severe complications or requirements for hospitalization and general anesthesia but also a beneficial procedure that can eliminate the need for surgery in cases resistant to conservative treatments. Intracanal cystic lesions that develop adjacent to the lumbar facet joint, first described in 1968 , 3. SincWith the advancements in radiographic techniques, particularly magnetic resonance imaging (MRI), lumbar facet cysts (LFC) have been recognized as a common disorder, which causes severe clinical symptoms, such as lower back and leg pain because of the direct nerve compression or canal stenosis that accompanies LFC.Janssen et al.  investigInitial nonsurgical treatments for LFC include pain medications, bracing, physical therapy, and steroid injections. However, some previous studies have reported unsatisfactory clinical results, including unchanged cyst sizes, after these conservative treatments , 6. For However, this is not yet a common procedure, particularly in our country, with only a few case reports , and itsThis was a retrospective study approved by the Institutional Review Board of Asao General Hospital; informed consent was obtained from all patients.From 2006 to 2020, 104 symptomatic patients with LFC were in our hospital, of whom were diagnosed with LFC based on MRI findings. However, neurological symptoms were observed only on the LFC side in all 104 cases, and those where severe canal stenosis was combined with LFC or where clinical symptoms were observed on the non-LFC side or both sides were excluded from this study.First, conservative treatments, including medications, braces, and physical therapy, were performed for all cases except six that required surgical treatment  immediately after the diagnosis because of severe pain and leg paresthesia. Moreover, percutaneous LFC rupture was performed for resisted cases after conservative treatments.After conservative treatments, the symptoms were gradually relieved in 41 of 98 cases (41.8%), and these 41 cases were defined as the control group.Finally, we investigated 57 cases that were resistant to conservative treatments for which “percutaneous rupture of LFC” treatment was performed , and allThis study investigated the patient's age, sex, history of previous lumbar surgery, level of LFC, rate of successful LFC rupture, the volume of contrast agent required for successful rupture, clinical results, and intraprocedural and posttreatment complications. Clinical results were evaluated using the visual analogue scale (VAS) scores and the recovery rate, which was calculated with the Japanese Orthopedic Association (JOA) scores , from prU test. Additionally, comparisons of pre- and posttreatment clinical results and radiographical findings were evaluated using paired t-tests. A P value <0.05 was considered statistically significant, and all analyses were performed using JMP 13 software .Measurement values are presented as means ± standard deviations. Comparisons of clinical results and radiographical findings between the control group and the group of cases with percutaneous LFC rupture were evaluated using the chi-square test and Mann–Whitney All procedures were performed with the patient awake, with careful attention to the patient's condition and complaints. The patients were placed in the prone position, and the right-left and cranial-caudal C-arm positions were adjusted to obtain clear views of the facet joint space. After sufficient skin disinfection, 5 mL of 1% mepivacaine  was administered to achieve local anesthesia. Fluoroscopic images illustrating the LFC rupture procedure from this point onward are presented in After the treatment, patients were transferred to the recovery room and observed for 2 hours, monitoring their general condition, leg pain, and paralysis. All patients left the hospital after 2 hours of observation, based on the physician's assessment that no severe complications or complaints had occurred.Of the 57 cases where LFC rupture was performed, 21 (36.8%) had a history of previous lumbar surgery. The levels of LFC were L2/3, L3/4, L4/5, and L5/S in 2, 9, 36, and 10 cases, respectively. The facet degenerative changes were grades 1, 2, 3, and 4 in 5, 22, 25, and 5 cases, respectively, and degenerative spondylolisthesis was observed in 57.9% (33/57) of the cases. The grade of degenerative change and spondylolisthesis showed no significant progression by the time of the last observation posttreatment. Moreover, lumbar lordosis and the intervertebral angle on the LFC level did not change significantly until the last observation.P < 0.01).All patients complained of leg pain, and a slight leg weakness  was recognized in 21.1% (12/57) of the cases. Furthermore, significant differences in the VAS and JOA scores, size of the LFC based on MRI, were recognized after comparison with the corresponding scores of the control group . By the time of the last observation, no cases of postsuccessful rupture required subsequent surgical treatments; meanwhile, LFC recurrence accompanied by leg pain was observed in four cases within 1 year following the MRI's confirmation of reduction, after which the treatment was repeated. After the second injection, the cyst reduction was reconfirmed without further recurrence (up to 24 months after the second injection). In contrast, the rupture of the cyst failed in nine patients. In three patients, needle insertion was impossible because of severe degeneration of the facet joint. In the other six patients, despite successful needle insertion into the facet joint space, the cyst was not contrasted clearly, and the contrast agent leaked outside the joint. Of the nine failed rupture cases, seven underwent surgical treatment (microendoscopic resection) after 2–6 weeks. In the remaining patient, although the pain was not relieved, the patient did not agree to undergo surgery and preferred to undergo conservative treatments , the patient had no complaints. The JOA score improved from 14 points at pretreatment to 29 points at the last observation, with a 100% recovery rate.3.2.In the case of a 65-year-old female patient, successful cyst rupture was not achieved, and leakage of the contrast agent outside the joint and unimproved symptoms were observed. Two weeks later, microendoscopic resection was performed. The JOA score was improved from 5 points at pretreatment to 23 points at the last observation, with a 75% recovery rate.LFC most commonly occurs at the L4-L5 level due to excessive loading of the facet joint involved with chronic higher mobility of the spinal segment, which is particularly found in degenerative spondylolisthesis –5. KusakPrevious studies have also described the occurrence of LFC after posterior decompression surgery of the lumbar spine , 18, whiIkuta et al.  found thMany surgical procedures for LFC were reported previously, such as cyst resection with or without spinal fixation –9. Cyst Besides surgical treatments, percutaneous LFC rupture was the only treatment that could directly reduce cysts without hospital stay or general anesthesia induction. The systematic investigation by Martha et al.  includedNo severe LFC rupture complications had been reported previously. In our study, some patients complained of low back or leg pain during the procedure, particularly when the cyst burst; however, the pain decreased after the treatment without deteriorating to lower-extremity paralysis, and all patients left the hospital after 2 hours of rest. By the time of the last follow-up examination, no severe complications were observed in any of the cases. Furthermore, since percutaneous facet resolution may lead to complications, such as intravascular penetration, infection, hematoma, and vasovagal reactions , 23, carOur study had some limitations, particularly regarding the follow-up period and sample size. The average follow-up period in our study was 26.0 months; therefore, the long-term effectiveness, including the recurrence risk, was not elucidated. Huang et al.  reportedOur clinical results showed satisfactory effectiveness with the “percutaneous rupture for LFC” procedure since the rate of surgery prevention after this treatment was 84.2% (48/57 cases).Percutaneous rupture for LFC is safe and minimally invasive without any severe complications, need for hospitalization, or general anesthesia. Moreover, it is a very useful procedure that can prevent surgery in cases resistant to conservative treatments. Therefore, percutaneous rupture for LFC should be the first-choice treatment in cases resistant to conservative treatment instead of surgical treatments.


Background: In previous studies, it was found that the confinement to which the population was subjected during the quarantine of the COVID-19 pandemic increased the risk of anxiety and depression. Objective: to analyze the levels of anxiety and depression symptoms in Portugal residents during the quarantine of the COVID-19 pandemic. Methods: This is a descriptive, transversal, and exploratory study of non-probabilistic sampling. Data collection was carried out between 6th and 31st of May 2020. Sociodemographic and health questionnaires PHQ-9 and GAD-7 were used. Results: The sample consisted of 920 individuals. The prevalence for depressive symptoms (PHQ-9 ≥ 5) was 68.2% and (PHQ-9 ≥ 10) was 34.8%, and for anxiety symptoms (GAD-7 ≥ 5) was 60.4% and (GAD-7 ≥ 10) was 20%. Depressive symptoms were moderately severe for 8.9% of the individuals, and 4.8% presented severe depression. Regarding the generalized anxiety disorder, we found that 11.6% of individuals present moderate symptoms, and 8.4% severe anxiety symptoms. Conclusions: The prevalence of depressive and anxiety symptoms were substantially higher than those previously found for the Portuguese population and when compared with other countries during the pandemic. Younger individuals, female, with chronic illness and medicated, were more vulnerable to depressive and anxious symptoms. In contrast, participants who maintained frequent levels of physical activity during confinement had their mental health protected. Since it appeared in China in late 2019 ,4,5 becaA study developed in China found that 54% of the respondents present psychological distress related to the disease’s outbreak. Specifically, 17% reported moderate to severe depression symptoms, 29% anxiety and 8% stress . AnotherUndoubtedly, the COVID-19 pandemic and the associated measures to protect populations, such as confinement, have a strong, negative impact on the population’s wellbeing. The pandemic crisis challenges equity in access to health care. This may arise due to the scarcity of resources, which hinder both the entry into the care system itself and the access to life-sustaining resources. This situation is particularly worrying for people with chronic diseases, due to the associated comorbidities, but also because they are the ones who recurrently need health care . These aTherefore, and despite the growing evidence that COVID-19 pandemic seriously affects people’s mental health, it is still necessary to clarify this sphere and, specifically, to elucidate how confinement reverberates on Portuguese mental health, to establish protective public policies. Thus, this study aims to analyze the levels of anxiety and depression symptoms in Portugal residents during the confinement of the COVID-19 pandemic.It is a descriptive, cross-sectional, and exploratory study of non-probabilistic sampling. Data collection was carried out using the Google Forms platform sent via social networks  between 6 and 31 May 2020.Sample size was calculated a priori using G × Power 3.1.9.7 software. In the case of depressive and anxiety symptoms, the effect sizes were based on the minimal clinically important differences (MCID) for depressive symptoms (Cohen d  =  0.24)  and anxin = 920) were those with the following criteria: (a) 18 years old and more; (b) Portugal resident; (c) be confined for at least 15 days; (d) be able to read and understand Portuguese; (e) voluntarily accept to participate in the study after observing the Free and Informed Consent Term (ICF). Given the characteristics of the data collection tool, we considered as exclusion criteria low digital competence, the impossibility of accessing the internet or not having social networks and not answering at least 80% of the total questions on the form.The initial sample consisted of 929 participants. Participants with more than 20% of missing data were excluded (9 participants). Finally, included in the sample participants ; use of medications; regular physical activity (minimum 3 times a week); health facilities resort; doctor or psychologist appointment; alcoholic habits. How satisfied are you with your health? With the following answer possibilities: Very unsatisfied, Unsatisfied, Neither dissatisfied nor satisfied and Satisfied. How satisfied are you with your family economic income? With the following answer possibilities: Very unsatisfied, Unsatisfied Neither dissatisfied nor satisfied, Satisfied and Very Satisfied. With regard to spirituality, during quarantine a person is considered: Unspiritual, With some spirituality, Not too little, not too spiritual, Spiritual and Very spiritual.-Patient Health Questionnare-9 (PHQ-9), developed by Kroenke, Spitzer and Williams 2001) , validat01 , vali-General Anxiety Disorder (GAD-7), developed by Spitzer, Kroenke, Williams and Löwe (2006) , transla® software, version 26, was used for data analysis. In the statistical data treatment, in addition to the descriptive and exploratory analysis, absolute (n) and relative (%) frequencies were used for qualitative variables, as well as measures of central tendency: mean (M), standard deviation (SD), minimum (min.), maximum (max.) and range. Means were compared between groups, using the Student’s Test and the Simple Analyze of Variance (ANOVA) with Bonferroni’s correction. To identify associations between two categorical variables, Pearson’s chi-square tests were used. The results of the present study are considered statistically significant for a level of significance below 5% [p-value were presented. We used cut-off point ≥10 to GAD-7 and PHQ-9 scales to create the dichotomous variables.Statistical analysis SPSSThe sample consisted of 920 Portuguese residents, 79.2% in the southern region of the country, 72.4% were women. Average age was 42.6 years , 42.0% were married and 30.8% single. A total of 41% were health professionals and from that, 74.3% were nurses.Regarding the period of quarantine, a mean value of 40,1 days was found . In total, 37.9% of the participants were teleworking, 17.6% kept working as usual, and 14.9% worked with reduced/adapted schedule. Most of the participants (85.5%) reported not having any family or friends with COVID-19 in this period.Concerning the clinical history, 74.9% of the individuals reported not having chronic disease and 61.8% reported not using any type of medicine daily. A total of 2.7% (SD = 2.4) refer the use of medicines and 2.6 (SD = 2.3) the use of prescription medicines. In total, 85.4% did not resort to a health service, neither a doctor nor a psychologist (88.7%).Analyzing this period, it was found that 56.3% of the respondents did not participate in regular physical activity. In addition, 55.0% did not consume alcoholic beverages, but 16.6% had increased their alcohol consumption.Regarding the main variables, a mean of 8.13 was found for the depressive symptoms (SD of 5.89); concerning the generalized anxiety disorder, the mean found was 6.24, with SD of 4.95 .In the sample studied, 68.2% (PHQ-9 ≥ 5 score) of the participants had depressive symptoms. These symptoms were moderately severe for 8,9%, and 4,8% presented severe depression symptoms . For theAfter data analysis, relationships between different variables were clarified. p = 0.014), the 37-years-old or less group had the highest mean (8.8); for sex , women had the higher average value (8.7); also, for the health professional type , the Diagnostic and Therapeutic Technicians (DTT) had the highest mean value (11); chronic diseases patients  had a mean value of 8.9; those who daily used medicines and prescribed drugs  showed mean values of 9.4 and 9.3, respectively; in physical activity, we found higher mean values  in individuals who did not practice it regularly; the respondents who reported having resorted to health facilities during the quarantine also have higher mean values   .p < 0.0001), 37-years-old or less group showed higher mean values (6.8); sex , women are most vulnerable with a mean value of 6.7; type of health professional , observing a higher score in DTT (8.1); daily use of medicines and prescribed drugs , with a mean value for both variables of 6.9; for physical activity during the quarantine, we found that participants who reported not having practiced it regularly have high mean values ; individuals who resorted health facilities presented a mean value of 7.1   .Through multivariate analysis, predictors of depressive and anxiety symptoms were determined. As predictors of depressive symptoms, age, sex, satisfaction with health and economic income, as well as spirituality, were found. Depressive symptoms were more likely to be present in younger people, women, people who were dissatisfied with their health and economic performance and reported being unspiritual.Predictors of generalized anxiety symptoms in this sample were age, sex, satisfaction with health and economic income. That is, anxiety symptoms were more likely to occur in younger people, women and in people who are dissatisfied with their health and economic performance and unspiritual .This investigation aimed to analyze the levels of generalized anxiety disorder and depressive symptoms during COVID-19 confinement in Portuguese residents. Additional objectives meant to explain how sociodemographic and health variables were associated with these mental health indicators.The global dissemination of COVID-19 had an important impact on individuals’ lives. It threatened individuals’ physical health, installed fear of contagion and possible transmission to more vulnerable family members and gradually affected other dimensions of individual and collective health. In recent history there is no record of anything comparable to the need for prophylactic social isolation, extended to millions of people and without a definite end, a fact that places mental health at risk . AlthougPortugal has an annual prevalence of mental illnesses of 22.9% ,30, amonThese values are substantially higher than those found in other European countries, a fact that has not yet been fully explained and which may eventually be associated with greater exposure to factors that cause vulnerability or less exposure to protective factors . It willWe accessed mental health by measuring depressive symptoms and generalized anxiety disorder. Regarding this last construct, we found that 60.4% of the respondents had symptoms of anxiety (GAD-7 ≥ 5), 11.6% moderate symptoms and 8.4% severe anxious symptoms. Thus, we found a prevalence of 20% (GAD-7 ≥ 10) of relevant anxiety symptoms, a higher value than the one found in the study  that compared the prevalence of anxiety disorders in Portugal, some countries in Europe and the USA, where Portugal presented the second highest value in the group (16.5%) . The valContrasting our findings with research already carried out in this pandemic context, we find that Portuguese values (GAD-7 ≥ 10) are also higher than those evidenced in other studies, such as the investigations in Hong Kong with 14% , and in Depressive symptoms were found in 68.2% of the participants: 8.9% moderately severe; 4.8% severe. Thus, we can affirm that in the studied sample the prevalence of relevant depressive symptoms was 34.8% (PHQ-9 ≥ 10) and 13.7% (PHQ-9 ≥ 15). These values, in line with the findings related to the generalized anxiety construct, are also higher than the 7.9% prevalence of depressive disorders presented by DGS .For this variable, the values found in our sample are also higher than other studies in the same context, namely the investigations carried out in Austria , in FranThe present study allowed us to underline the impact of the pandemic on mental health, and made it possible to verify that certain individuals’ variables were significantly associated with it. We confirmed that the youngest participants, the women, the ones who consume prescription drugs daily, the ones who resorted health services during quarantine and the ones who did not practice physical activity on a regular basis, presented simultaneously higher values of anxious symptoms and depressive symptoms, therefore appearing to present greater vulnerability to these problems.Although it may seem unexpected that younger individuals have higher levels of anxious and depressive symptoms, this finding has also been found in other studies ,39,40,42The levels of anxious and depressive symptoms in this study are significantly associated with sex, showing important differences in the distribution of these two variables, presenting women higher values than men. Women are more vulnerable to stress-generating stimuli and their impact on mental health, as several previous investigations have documented ,44,45,46Notwithstanding, no significant differences were found in the levels of anxiety or depression due to the fact that the respondents were health professionals or not, a situation also verified in another study , we founThe presence of chronic disease and the daily intake of medication discriminated aspects related to individuals’ mental health. The persistent information in media about the increased risk associated with COVID-19 for people with comorbidities has certainly contributed to the intensification of psychological suffering, developing in these individuals a perception of insecurity and fear. This may have raised the level of anxious and depressive symptoms or even their worsening, when pre-existing, these data are validated by several studies developed in this context ,52.The participants who resorted to a health facility during confinement, reported higher levels of both anxiety and depressive symptoms which may be related to the perception of risk inherent to the contact with a health service. The data may also be related to the necessity for certain individuals with specific health needs (namely chronic illness) to use these resources more frequently and to combine, at the same time, several factors of vulnerability to psychological distress. Several studies have found similar data in previous pandemics  and the In our study, we found that the most physically active individuals during this period had lower levels of depressive and anxious symptoms. In fact, the beneficial effect of physical activity on the overall health of individuals and particularly on their mental health, well-being and cardiovascular health is consensual, in the pre-pandemic period ,55 and dAs in our study, other studies point to similar results, in which people at high risk of mental problems are women, young people, single people and those with a low educational level. Intervention in anxiety and depression in a pandemic context includes pharmacological treatment, psychological therapy, and physiotherapy .Some limitations may affect the interpretation of the results of this study, namely the large number of health professionals in the sample, as well as the data collection technique used. The fact that we used personal and professional social networks may have created this bias, which led to a sample with an important representation of health professionals. Other limitations of the present study are, we present the way which data collection was carried out, as we are aware that not all people residing in Portugal have access to e-mail, internet, or WhatsApp. Finally, another limitation is the fact that 36% of health professionals continued to work, which means that they were not in confinement, which can represent a bias. However, the situation can be seen from another perspective, because even the health professionals who continued to work were transversally prevented from maintaining their family relationships, which, we think, will not be far removed from what the confined people experienced.The research showed that during the first confinement of SARS-CoV-2, the mental health of the Portuguese participants in the study was at risk. This fact is reflected in a prevalence for depressive symptoms and for anxious symptomatology showing that our values are substantially higher than those found for the Portuguese population prior to the pandemic and in research carried out during the current situation.We were able to verify that depression and anxiety are more likely to be present in younger people, women, people who were dissatisfied with their health and economic performance and reported being unspiritual. In contrast, participants who maintained more frequent levels of physical activity during the confinement apparently had their mental health protected.These findings can be used to develop tailored intervention strategies and psychological support for fringes of vulnerable population, aiming the maintenance or upsurge of people’s mental health and preventing or minimizing emotional suffering in civilizational crises, such as the one we are experiencing. This intervention process must be a health priority considering that the implications to mental health will rest (surely) longer than the pandemic itself and its psychosocial impact may be incalculable.Further studies will be necessary to verify the consistency of these results since the pandemic brought the development of new habits, new risks, new vulnerabilities that tests people’s resilience and capacity to mobilize their internal and external resources to face the challenges that emerge.


A contrast-free ultrasound microvasculature imaging technique was evaluated in this study to determine whether extracting morphological features of the vascular networks in hepatic lesions can be beneficial in differentiating benign and malignant tumors (hepatocellular carcinoma (HCC) in particular).A total of 29 lesions from 22 patients were included in this work. A post-processing algorithm consisting of clutter filtering, denoising, and vessel enhancement steps was implemented on ultrasound data to visualize microvessel structures. These structures were then further characterized and quantified through additional image processing. A total of nine morphological metrics were examined to compare different groups of lesions. A two-sided Wilcoxon rank sum test was used for statistical analysis.In the malignant versus benign comparison, six of the metrics manifested statistical significance. Comparing only HCC cases with the benign, only three of the metrics were significantly different. No statistically significant distinction was observed between different malignancies (HCC versus cholangiocarcinoma and metastatic adenocarcinoma) for any of the metrics.Obtained results suggest that designing predictive models based on such morphological characteristics on a larger sample size may prove helpful in differentiating benign from malignant liver masses. In tHCC often develops in patients with underlying liver disease . Viral hAngiogenesis, or the formation of new blood vessels, is a common occurrence in the development and growth of neoplasms , 19. HCCIn this paper, we utilize a previously introduced  techniqu22.1This prospective study, which was conducted from August 2020 to September 2021, received institutional review board approval (IRB#: 16-009435) and was in compliance with the Health Insurance Portability and Accountability Act. A signed written informed consent with permission for publication was obtained from each enrolled participant prior to the study. A total of 22 patients, ages ranged 19 - 82 years, mean age 61.6 ± 16.2, with 29 identified hepatic mass/masses on their screening ultrasound imaging, were enrolled in this study. Details of participant selection are provided in 2.2For each participant, the ultrasound examinations were performed by one of two trained sonographers, with more than 35 and 15 years of experience, respectively. Subjects were scanned using an ultrasound system, Alpinion E-Cube 12R ultrasound machine , using a curved array transducer C1-6 operating at 3.6 MHz frequency. This system is capable of plane wave imaging and provides high-frame-rate images in the form of raw in-phase and quadrature (IQ) beamformed data for a total duration of 3 seconds, forming each frame of the data using 5-angle coherent plane wave compounding . For ima2.3For each case, the lesion of interest was manually segmented with the help of an experienced sonographer, to enable vessel quantification inside and outside the lesion separately. The output of the segmentation would then constitute a binary mask, delineating the region of interest (ROI). This mask underwent a 10mm dilation to include perilesional vascularity. This 10mm dilation was applied consistently to all of the masks ensuring sufficient area in the periphery of all lesions (regardless of their size) is included in the analysis. In some cases, where the lesions were large, or were situated in the proximity of the edges of the scanning region, the dilated mask would extend beyond the boundaries of the image, in which case we would use as much information as available within the image for quantification. The resulting mask was subsequently applied to the microvasculature image to limit vessel quantification within the ROI.Vessel quantification procedures were implemented on the ROI beginning by binarizing the image using an empirically chosen threshold to ensure an adequate balance between background noise exclusion and keeping the vascular structure intact. Several morphological operations were then employed to remove small objects that do not correspond to actual vessels, as well as to fill the spurious holes inside vascular structures that might appear as a result of the visualization filtering processes. Next, a thinning algorithm was applied to the resulting binary image to extract the centerline of the vascular networks, to obtain what is referred to as the skeleton image.VD), number of vessel segments (NV), number of branch points (NB), mean tortuosity as measured by the distance metric (meanτ), maximum tortuosity as measured by the distance metric (maxτ), mean diameter (meanD), fractal dimension (FD), mean of Murray’s deviation (meanMD), mean bifurcation angle (meanBA). More details on the quantification procedure and the computation of these morphological features can be found in . To evaluate the discrimination of the metrics in differentiating the tumors, two separate analyses were performed. First, the lesions were divided into two groups, benign lesions and HCC lesions. A two-sided Wilcoxon rank sum test was applied to test for differences in distributions of the metric values for the two groups and corresponding p-values were obtained. Next, a new set was created by adding the other malignant lesions (cholangiocarcinoma (CCA) and metastatic adenocarcinoma) to the HCC cases, this time with the objective of separating benign masses from malignant ones. In all cases, p-values less than 0.05 were declared as statistically significant. All data processing and analyses were performed in MATLAB R2019a .3A total of 22 enrolled patients, consisting of 10 males and 12 females , with 29 hepatic masses  were examined by quantitative HDMI. The size of these lesions  ranged from 16.8mm to 150.3mm with a mean diameter 45.7 ± 29.3mm. Clinical diagnoses  were used to label the lesions. Among the 29 lesions, 27 ultimately underwent needle biopsy for pathologic diagnosis and 2 were radiologically confirmed based on typical contrast-enhanced CT and/or MRI imaging appearance. The 19 malignant cases included 11 hepatocellular carcinoma, 7 cholangiocarcinoma, and 1 metastatic adenocarcinoma of pancreatic origin. The 10 benign hepatic masses included 6 pathologically proven hepatocellular adenoma, 2 atypical hemangioma and 2 LI-RADS 3A labeled masses. 3.1In this section we present the visual comparison along with metric values of quantitative HDMI, for three groups of malignant and benign liver masses: 1) two larger malignant liver masses (HCC and CCA), 2) two smaller deep-seated liver masses (HCC and benign), and 3) two very small benign and malignant liver masses.meanVD, NV, NB, FD, MD, meanD, meanτ, and maxτ but higher value of meanBAin CCA compared to HCC.In group one, the conventional B-mode ultrasound and HDMI images of two larger malignant liver masses, HCC and CCA, respectively measuring 94mm and 51mm in the largest axial/lateral dimension, are shown for visual comparison in meanVD, NV, NB, FD, τ, maxτ, meanMD, and meanBAin HCC compared to the benign mass.In the second group, the conventional B-mode ultrasound and HMDI images of two deep-seated small liver masses, an HCC and a benign mass, are shown in meanVD, NV, NB, FD, τ, maxτ, and meanD in HCC compared to the benign mass.We also demonstrated the potential of our method for characterization of very small liver masses in group three. 3.2NV), number of branch points (NB), mean tortuosity (meanτ), maximum tortuosity (maxτ), fractal dimension (FD), and mean of bifurcation angle (meanBA). The corresponding p-values are 0.0035 for NV, 0.0042 for NB, 0.0260 for meanτ, 0.0035 for maxτ, 0.0231 for FD, and 0.0416 for mean.BAOut of the nine examined features in this study, six of them showed a statistically significant difference between benign and malignant lesions. These features included number of vessel segments  as well as meanBA showed significant distinctions between malignant and benign lesion groups. The current study also found that NV, NB, and maxτ have better discrimination performance than any other individual biomarker in differentiating HCC from benign. Another finding of this study is that HDMI biomarkers did not show differences between HCC and other types of liver malignancies, however, the numbers of patients are not enough to draw a statistically meaningful conclusion.This study investigated the discriminative potential of the quantitative biomarkers of contrast-free high-definition microvessel imaging (HDMI) for differentiating malignant and benign hepatic masses. Our findings show that HDMI biomarkers, HCC is a highly vascular tumor in which angiogenesis plays a major role in tumor growth and metastasis, with vascular endothelial growth factor being a major player in angiogenesis . A hallmFD as a new biomarker of tumor microvessels in contrast-free ultrasound microvessel imaging for differentiation of liver masses. In the current study, FD was found to have higher values in malignant compared to benign liver lesions. This finding is in agreement with the results of other studies, indicating that microvascular complexity calculated by FD may provide important diagnostic and prognostic information as well as insight into tumor angiogenesis  is hypovascular relative to HCC, probably due to its extensive stroma, and can be differentiated from HCC by imaging angiogenesis (Visualization and quantification of vascular structures inside cancerous lesions can be used as a tool for early detection of malignant tumors in different organs. The current study demonstrated the capability of this new quantitative method in capturing angiogeneses in very small liver lesions as small as 16mm, which is in keeping with the shown capability of this method for detecting angiogenesis in breast lesions as small as 4mm . Early oogenesis , 61.Results obtained in this work as a feasibility study indicate that there is a potential for contrast-free ultrasound microvessel imaging with subsequent morphological analysis for detection of HCCs. There is limited published experience focusing on the use of ultrasound for quantitative vascular imaging in hepatic lesions. Most of such studies either make use of contrast agents for improved resolution and contrast in the resulting images or employ available devices and technologies for blood flow imaging with limited quantitative capabilities.While patients with very early-stage HCC have excellent clinical outcomes, with 5-year survival rates over 60 to 80% after curative treatments –64, diagOne limitation associated with this study is that the sample size was small and as a result, more sophisticated statistical analysis and development of predictive models were not tenable. In the future, we plan to apply this method on a larger cohort of participants including smaller liver lesions suspected of malignancy for more accurate evaluation of its performance for early detection of HCC. Also, there is a potential for data degradation due to breathing motions. In the future we plan to utilize and expand the motion correction and denoising algorithms –72 to re5Accurate diagnosis of hepatic lesions is a challenging task and is of even greater significance when it comes to aggressive tumors such as HCC. The use of accessible and inexpensive methods for early detection of such tumors can greatly help with accelerating and streamlining the treatment process for patients. In this feasibility study, we investigated the use of a non-invasive contrast-free ultrasound microvasculature imaging technique for detection of malignant hepatic lesions and HCC in particular. By evaluating the morphological features of microvasculature visualized using this method, we were able to differentiate between the malignant and benign lesions. Results of this feasibility study indicate the potential of this technique. In the future, we will focus more on the detection of early-stage HCC as well as investigating the possibility of discriminating between different hepatic malignancies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.Conceptualization, AA and MF. Methodology, AA and MF. Software, SS and RT. Validation, AA, MF, SS, NL, MO and TA. Formal Analysis, SS. Investigation, SS, RT, AA and MF. Resources AA and MF. Data Curation, SS and RT. Writing – SS. Writing – Review and Editing, SS, AA, MF, RT, NL, MO and TA. Visualization, SS and AA. Supervision, AA and MF. Project Administration, AA and MF. Funding Acquisition, AA and MF. All authors contributed to the article and approved the submitted version.


Full-thickness skin grafting plays an important role in facial reconstruction for excessive skin defects or possible eye, nose, or lip distortion after a malignant tumor excision. For esthetic consideration, full-thickness skin grafts of the retroauricular region can be used, but the amount of skin is limited. The skin of the submental triangle has similar cutaneous features and provides a large graft. We aimed to evaluate the feasibility of using full-thickness skin grafts harvested from the submental triangle to reconstruct facial skin defects. We retrospectively analyzed 14 cases treated at a single institution to evaluate the clinical and esthetic outcomes, and patients’ satisfaction. During 6–24 months of follow-up, the wounds healed with well-matched color, texture, and contour. No obvious hypertrophies or distortions were observed. Additionally, the removal of redundant submental triangle skin resulted in the secondary gain of double chin reduction. All patients were satisfied with the appearance of both donor and recipient sites. In conclusion, the submental triangle is a good possible option for full-thickness skin grafts used to resurface facial skin defects. Using this approach, both donor and recipient sites can achieve satisfactory esthetic outcomes. Surgical-wide excision is a gold standard for treating malignant skin tumors . FollowiWhen treating facial skin defects, esthetics should be considered. In general, flaps are transferred from an area near the defect, which can provide a similar skin color and texture . CompareThe most common full-thickness skin graft donor sites include the retroauricular region, supraclavicular region, inguinal fold, buttock fold, hypothenar area, and anterior wrist fold . For estIn this study, we used a full-thickness skin graft harvested from the submental triangle to reconstruct facial skin defects and aimed to identify the clinical and esthetic outcomes.This is a single institutional, retrospective, observational case series study. We included all patients who underwent facial skin reconstruction using full-thickness skin grafts harvested from the submental triangle to resurface facial skin defects caused by the excision of facial skin tumors, between January 2019 and January 2021. Demographic data, including sex, age, body mass index (BMI), pathology of the skin tumor, location and dimensions of the skin defect, dimensions of the skin graft, preoperative and postoperative photographs, and medical comorbidities, were collected.Fourteen patients were included in the study. We used the Manchester Scar Scale to evaluate postoperative esthetic outcomes . The ManInformed consent for the surgical procedures and photography was obtained from each patient, and the study was approved by the Institutional Review Board of Tri-Service General Hospital . This study was conducted in accordance with the principles of the Declaration of Helsinki.Following the excision of the facial skin lesions, we measured the size of the skin defects. In order to avoid the deformity of the donor site, we marked a larger skin area over the submental triangle. All patients demonstrated skin redundancy and laxity in the submental triangle. Full-thickness skin grafts were harvested from the donor site, and the underlying soft tissue and fat were trimmed off c,d. The 2 (ranging from 1.5 × 1.0 cm2 to 4.5 × 4.0 cm2). The frozen section of the excised skin lesion showed free margins in all cases, and full-thickness skin grafts were harvested from the submental triangle. The dimensions of the skin graft harvested from the submental triangle averaged 4.54 × 3.07 cm2 (ranged from 3.0 × 1.5 cm2 to 6.5 × 5.0 cm2). Details are presented in The study included seven women and seven men aged between 43 and 91 years, 70.6 years in average. Most cases (10 patients) were of basal cell carcinoma. There were other pre-cancerous skin tumors in three patients, and squamous cell carcinoma in one patient. The dimensions of the skin defects averaged 2.88 × 2.39 cmMedical comorbidities and lifestyle habits are listed in 2 in size (2 was harvested from submental triangle (Case 3: A 43-year-old woman had a poorly healed, crusted skin lesion over her left temporal region for 3–4 years a. She vi in size e, and a triangle b.At the 3-month follow-up, the skin graft was well matched in texture and contour g. Only a2 with a central ulcer approximately 0.7 × 0.5 cm2 in size, deep into the subcutaneous layer. The skin lesions were excised with a free margin of frozen section. The pathological examination revealed seborrheic keratosis with severe dysplasia. The skin defect over the left temporal scalp was 4.0 × 4.0 cm2 in size (2 was harvested from the submental triangle.Case 7: A 75-year-old man had a progressively enlarging skin lesion over his left temporal scalp for several years a. The ex in size b, and a At the 2-year follow-up, the skin graft had healed well with perfect matching of skin color, texture, and contour c. There Before the operation, the patient presented with a poor neckline and an obvious double chin a. At posThe management of patients with facial skin cancer is challenging, and esthetic outcomes should always be considered. The treatment options include surgical excision, cryotherapy, electrodesiccation, and curettage. Surgical excision is the standard treatment owing to a lower risk of recurrence than with other techniques ,7. UnforWe have different reconstruction options for managing skin defects in the reconstruction ladder. It is important to assess the characteristics of facial skin defects, including the size, location, and type of tissue lining, before choosing a suitable approach . Direct Skin grafts are classified as split-thickness and full-thickness skin grafts. Both types of skin grafts may result in primary and secondary contractures . In secoCommon local flaps include advancement, transposition, and rotation flaps, which provide similar skin color and texture . For larFrom a subjective point of view, Ebrahimi et al. compared the results between the local flap and the skin graft for the reconstruction of a cheek skin defect . There wFrom an objective point of view, Martinovic et al. assessed the skin quality in the facial region following reconstruction with local flaps and full-thickness skin grafts . There wWhile flaps theoretically result in better cosmetic effects than skin grafts, they also require longer skin incisions. In many cases, surgical wounds do not lie along the Langer’s lines of the face, which may result in noticeable scars . ConsideRegarding postoperative complications, Mamsen et al. discussed the differences between local flaps and full-thickness skin grafts in the reconstruction of facial defects . There w2 (In our study, all patients received full-thickness skin grafts for the reconstruction of facial skin defects. Considering the complexion, texture, and photo-damage, full-thickness skin grafts harvested from the retroauricular region and the frontal region at the hair border appear to be the best candidates. Unfortunately, the retroauricular region can only provide a limited amount of skin, and it can be challenging when confronted with larger skin defects. The frontal region is also an effective and fine donor site choice because the scar can be hidden at the hair border. However, if the facial skin defect is large, it may not be possible to provide a sufficiently sized and intact skin graft. On the other hand, care should be taken if we choose the frontal region at the hair border as the donor site choice for a full-thickness skin graft for male patients. Because there is a greater possibility of alopecia in males, unpleasant surgical scars might become visible. In case 3, the area of the skin defect over the left temporal region was 4.5 × 4.0 cm2 e, which 2 g. This wThere are only a few studies on the application of full-thickness skin grafts harvested from the submental triangle. Its first clinical use was reported in 2002 by Field et al. . A full-Owing to the thick subcutaneous fat in the submental triangle, it is relatively safe to harvest skin from this region. The donor site is primarily closed after a full-thickness skin graft is harvested from the submental triangle, as the redundant skin is removed. It presents effects similar to those of a neck lift. In case 7, the cervicomental angle improved immediately after the operation and at the 2-year follow-up b. Care sPossible complications of the recipient site include infection, hematoma, wound dehiscence, and graft necrosis, which make careful hemostasis and rigid fixation of skin grafts essential. On the other hand, wound dehiscence, seroma, hematoma, and fat necrosis might happen as complications of the donor site, so attentive hemostasis and meticulous suture with layers by layers during operation is very important. Luckily, infection and unacceptable scars of the donor site rarely occur due to the easy maintenance of skin cleanliness of the neck and skin laxity of the submental region. Although the number of cases enrolled in our study was small and there was a relatively high proportion of comorbidities that could impair wound healing, no complications occurred at either the donor or recipient sites in our study.Based on the results of our study, we believe that full-thickness skin grafts harvested from the submental triangle are a good option for the reconstruction of facial skin defects. The limitations of our study include the small sample size and a lack of long-term follow-ups of outcomes and recurrence. Furthermore, this is a single institutional, retrospective, case series study without a comparison group, such as a reconstruction with full-thickness skin grafts harvested from other donor sites, or with local flaps, which increases the odds of bias. Future prospective trials, with a larger sample size and a long-term follow-up of at least a year, are needed to validate these results. Recurrence also needs to be assessed in future studies.In conclusion, the skin of the submental triangle provides a good possible option for good full-thickness skin grafts, especially for the reconstruction of skin defects in the facial region. It is particularly useful in elderly patients who have redundant neck skin or double chins. Using this method, both donor and recipient sites can achieve satisfactory esthetic outcomes.


Genetic and molecular knowledge of a species is crucial to its gene discovery and enhanced breeding. Here, we report the genetic and molecular dissection of ginseng, an important herb for healthy food and medicine. A mini-core collection consisting of 344 cultivars and landraces was developed for ginseng that represents the genetic variation of ginseng existing in its origin and diversity center. We sequenced the transcriptomes of all 344 cultivars and landraces; identified over 1.5 million genic SNPs, thereby revealing the genic diversity of ginseng; and analyzed them with 26,600 high-quality genic SNPs or a selection of them. Ginseng had a wide molecular diversity and was clustered into three subpopulations. Analysis of 16 ginsenosides, the major bioactive components for healthy food and medicine, showed that ginseng had a wide variation in the contents of all 16 ginsenosides and an extensive correlation of their contents, suggesting that they are synthesized through a single or multiple correlated pathways. Furthermore, we pair-wisely examined the relationships between the cultivars and landraces, revealing their relationships in gene expression, gene variation, and ginsenoside biosynthesis. These results provide new knowledge and new genetic and genic resources for advanced research and breeding of ginseng and related species. Panax ginseng Mey.) and American ginseng (P. quinquefolius L.), has long been used for healthy food and medicine  (CAS) (DS) (FPS) (SE) (SS) (UGT71A27) (UGT74AE2) (CYP716A53v2) (CYP716A52v2) (CYP716A47)  , cycloarS) (CAS) , dammareAS) (DS) , farnesyS) (FPS) , squalenPS) (SE) , squalenSE) (SS) , UDP-glyGT71A27) , UDP-glyGT74AE2) , cytochr16A53v2) , cytochr16A52v2) , and cytP716A47) . MoreoveP716A47) , differeP716A47) , differeP716A47) , and difP716A47)  to identThe germplasm collection of a species is the gene pool of the species containing most, if not all, of the genes necessary for its genetic improvement and adaptation to climate change. Therefore, genetic and molecular evaluation and dissection of a species’ germplasm collection is paramount to its efficient utilization for continued genetic improvement, gene discovery, and favorable allele mining and for deciphering the species’ speciation, genetic variation, and evolution . SeveralThe aims of the present study were to construct a mini-core germplasm collection representative for Jilin ginseng and characterize the collection at genetic and molecular levels. We first collected 1,168 germplasm lines from the origin and diversity center of Jilin ginseng, developed the mini-core germplasm collection, and analyzed its genetic variation using 16 ginsenosides. Then, we sequenced the 4-year-old plant root transcriptomes of the mini-core collection, identified the SNPs contained in functional genes, quantified the expressions of the genes and transcripts, and characterized it using high-density genic SNP markers and gene expression profiles to facilitate gene discovery, favorable allele mining, and application of the research result for enhanced breeding. Analysis of the genic SNPs and gene expressions provided a comprehensive insight into molecular diversity, population structure, and evolution of the ginseng species. In addition, a relationship of the ginsenosides was proposed in the biosynthesis of ginsenosides. These resources and findings promote ginseng research such as genome-wide identification of genes involved in ginsenoside biosynthesis and enhance ginseng breeding such as marker-assisted selection and gene-based breeding .A mini-core germplasm collection that is representative for the genetic variation and diversity of traits and genes in a species is necessary for its deeper research in genetics, genomics, and breeding. Therefore, to develop a mini-core germplasm collection for Jilin ginseng, we previously collected a total of 1,168 cultivars and landraces from 16 major ginseng-producing counties of Baishan, Jilin, Tonghua, and Yanbian, Jilin Province (43°42'N 126°12'E), China, in 2009 Figure 1During plant growth and development from 2010 to 2014, the cultivars and landraces were recorded for main quality and yield potential traits and phylogenetically analyzed with 50 SSR markers . The quar = 0.90 to 0.98 (P < 0.0001) between plants collected from a field trial replicate, different field trial replicates, and across years. Therefore, the mRNA of one plant root was sequenced for this study. Total RNA was extracted using the RNeasy Plant Mini Kit , following the manufacturer’s protocol. The integrity and quantity of the RNAs were estimated by Agilent 2100 Bioanalyzer . Poly(A) mRNA was purified from total RNA by NEBNext Oligo(dT)25 beads . cDNA libraries were constructed from the mRNAs using the NEBNext Ultra RNA Library Prep Kit for Illumina , as described by the manufacturer. Library sequencing was accomplished using the Illumina HiSeq 4000 platform with a 150-base paired-end (150PE) module at Gene Denovo Biotechnology Co . Raw reads of low quality were removed, including those with adapters, containing more than 10% unknown nucleotides, and containing more than 50% low-quality bases (<Q10).The root sample collected above from one root per cultivar or landrace was used for transcriptome sequencing. Single-nucleotide polymorphisms (SNPs) and nucleotide insertions/deletions (InDels) were called and genotyped among the mini-core collection cultivars and landraces by the SAMtools software with the default parameters  using thWe constructed the population structure of the mini-core collection using STRUCTURE 2.3.4 . BecauseFurthermore, the population structure of the mini-core collection was confirmed by phylogenetic analysis, followed by principal component analysis (PCA) using all high-quality genic SNPs. The phylogenetic tree was constructed using the TASSEL 5.2.79 software  with theThe molecular diversity of the mini-core collection was estimated using allele frequency, gene diversity, heterozygosity, and polymorphism information content (PIC). The allele frequency, gene diversity, heterozygosity, and PIC were determined with the PowerMarker software  using thBecause ginsenosides are the main bioactive components for healthy food and medicine, we assayed the contents of ginsenosides in the roots of the cultivars and landraces of the mini-core collection to further characterize it. The roots of the cultivars and landraces collected above, with two roots per cultivar or landrace and one root from each field replicate, were used for ginsenoside content quantification. The roots were dried in an oven at 35°C to consistent weight , and theWe performed a correlation analysis between the 16 ginsenosides using their contents in the 4-year-old plant roots of the entire mini-core collection, each subpopulation, and the cultivars and landraces collected from each geographical region. The correlation analyses between ginsenosides were based on the content variations of the ginsenosides in the cultivars and landraces of the entire mini-core collection, each subpopulation, or those collected from each geographical region. The results were then used to deduce their relationships in ginsenoside biosynthesis. The relationships of the ginsenosides by content were determined by Person’s correlation coefficient calculated using the SPSS package (IBM SPSS Statistics 23) and by network analysis based on ginsenoside contents using the BioLayout Express3D (Version 3.3) . Their pThe ginsenoside contents of the cultivars and landraces clustered into different subpopulations and admixture group or collected from different geographical regions were compared by ANOVA  with a single factor, followed by Tukey’s HSD (honestly significant difference) test using Excel. A two-tailed significance level was used for the analysis.As different transcripts spliced from a gene may have different biological functions , the expMoreover, we examined the expressions and interactions of the 11 genes previously cloned that encode the key enzymes involved in ginseng ginsenoside biosynthesis to further characterize the mini-core collection . The expressions of 15 transcripts of the 11 ginsenoside biosynthesis genes were extracted from the expression dataset of the mini-core collection. Expression heatmaps of the 15 gene transcripts were constructed and visualized for the cultivars and landraces collected from different geographical regions and for different subpopulations and the admixture group using an R language package . The co-Pearson’s correlation coefficients were calculated pair-wisely between cultivars and landraces of the mini-core collection to estimate their relationships using an R language package . The corA mini-core collection was constructed for Jilin ginseng from 344 cultivars and landraces Table S1A total of 16.3 billion 150-nucleotide clean reads or over 244 Gb transcriptome sequences were obtained for the transcriptomes of the 344 cultivars and landraces of the mini-core collection, with a range from 40.1 million to 51.5 million clean reads and an average of 47.2 million clean reads per cultivar or landrace. From the transcriptome sequences, 1,532,337 genic SNPs were identified, with an average of 25.6 SNPs per gene, suggesting a high variation of ginseng germplasm. These SNPs spanned 2,761.8 Mb (92.5%) of the 2,984.9 Mb ginseng genome assembly , with a The population structure of the mini-core collection was evaluated based on 6,232 SNPs that were chosen from the 26,600 high-quality SNPs, with one SNP per approximately 500 kb along the ginseng genome. The structure of the mini-core collection was estimated using the LnP(K) and ΔK values, respectively, at a number (K) of subpopulations varying from 2 to 10 Table S1Furthermore, we evaluated the molecular relationships among the 344 cultivars and landraces of the mini-core collection by phylogenic analysis using all 26,600 high-quality SNPs  based on all 26,600 high-quality SNPs also verified the population structure of the mini-core collection determined with the 6,232 SNPs  using 6,232 of the high-quality SNPs selected with one SNP in approximately 500 kb of the ginseng genome. Pairwise genetic distance analysis excluding the admixture group showed that the mini-core collection had a pairwise genetic distance varying from 0.0167 to 0.0707, with a variation of 4.4-fold and an average genetic distance of 0.0488. The pairwise mean genetic distance of the mini-core collection was larger than the within-sub1, within-sub2, and within-sub3 mean genetic distances of 0.0436, 0.0398, and 0.0296, respectively. It was apparent that the pairwise genetic distances between subpopulations were much larger than the within-subpopulation genetic distances , but their within-subpopulation or admixture group variations were dramatic among cultivars and landraces (P < 0.05). R0 had the highest content in sub2, followed by admixture group, sub1, and sub3. Rf was the most abundant in sub3, and then admixture group, sub1, and sub2 in descending order. The content of Rb2 was the highest in the admixture group, followed by sub2, sub1, and sub3. Rd was also the highest in content in the admixture group, but followed by sub2, sub3, and sub1  of the 120 possible pairwise correlations of the 16 ginsenosides were significant, 44 of which were significant at rrelated Table S5P ≤ 0.05, with each ginsenoside strongly correlated with one or more other ginsenosides. This result suggested that they were synthesized through a single complicated pathway or multiple pathways that are correlated in ginsenoside biosynthesis , including six PPD-typed ginsenosides, one PPT-typed ginsenoside, and one oleanane-typed ginsenoside, but with a lower correlation coefficient varying from 0.120 to 0.380, suggesting that these eight ginsenosides are likely in a more distant position or existence of one or more other ginsenosides between them in the ginsenoside biosynthesis pathway , thus distinguishing the origin of the cultivars and landraces among geographical regions. Among the four geographical regions, the cultivars and landraces from Jilin had the network with the largest number of gene nodes, the largest number of gene interaction edges, and the highest connectivity (P < 0.01), differing the cultivars and landraces among subpopulations and admixture group. Subpopulation 3 (sub3) had the largest number of gene interaction edges and the highest connectivity . To have a glimpse into variation of the expression activities and interactions of these genes in the mini-core collection, we constructed their expression heatmaps and co-expression networks in 4-year-old plant roots of different subpopulations and admixture group and cultivars and landraces collected from different geographical regions. Comparative analysis of the heatmaps showed that the genes expressed in low activity in almost all cultivars and landraces analyzed, regardless of their geographical regions and there was no tendency that the cultivars and landraces collected from a similar geographical region were grouped into a similar cluster , with an average of 0.78 and a CV of 12.66% (P < 0.01) , with an average of 0.76 and a CV of 31.29% (P < 0.01) , with an average of 0.73 and a CV of 25.66% (P < 0.01) , with an average of 0.56 and a CV of 18.13% (P < 0.01)  (P < 0.05) (P < 0.05) (P < 0.05)  of the mini-core collection using the genic SNPs. The mini-core collection has been clustered into three subpopulations and an admixture group using a selection of 6,232 SNPs, which is in consistency with the results of phylogenetic analysis and PCA determined with all 26,600 high-quality SNPs. This indicates that the population structure obtained in this study is highly reliable. Although the population structure of the mini-core collection was weaker with a low ΔK value, relative to other plant species such as rice , similarThe cultivars and landraces of the mini-core collection were collected from four geographical regions of the origin and diversity center of Jilin ginseng, but only a limited relationship was observed between the subpopulations and geographical regions from which they were collected. This result was previously observed by This study has examined the genetic diversity of the mini-core collection by major allele frequency, heterozygosity, gene diversity, and polymorphism information content (PIC) based on 6,232 genic SNPs. Ginsenosides are the most important bioactive components in ginseng for healthy food and medicine; therefore, they are widely used to evaluate ginseng quality. However, variation of ginsenoside contents across genotypes, environments, ages, seasons, and tissues  makes itThe present study has, for the first time, revealed that extensive correlations exist in content among the 16 ginsenosides in the mini-core collection, and when they were subjected to network analysis, they have formed only a single network consisting of two clusters and with each being correlated with at least one of the other ginsenosides. These results suggest that the ginsenosides are synthesized through a single or multiple related biological pathways and reflect their relationships in the pathway. However, variation of the correlations has been observed among the cultivars and landraces collected from different geographical regions. Because the samples used for the ginsenoside assay were collected from the cultivars and landraces grown from the same trials, the variation of the ginsenoside content correlations might result from genetic difference of the cultivars and landraces collected from different geographical regions. The correlations of the ginsenosides provide a clue useful for construction of the pathway of ginsenoside biosynthesis and information necessary for breeding varieties of high quality in multiple ginsenosides. Therefore, we have inferred the relationships of the ginsenosides in the pathway of ginsenoside biosynthesis, which is helpful for comprehensively deciphering the molecular mechanisms underlying ginsenoside biosynthesis.β-AS, CAS, DS, FPS, SE, SS, UGT71A27, UGT74AE2, CYP716A53v2, CYP716A52v2, and CYP716A47 , even though they had a wide variation among the cultivar and landrace pairs. These results indicate that the cultivars and landraces of Jilin ginseng currently used in production and breeding are substantially related with each other in both gene expression and mutations. Nevertheless, the patterns of their relationships revealed were not consistent between the gene transcript expressions and genic SNPs. Although the inconsistency could be partly attributed to the facts that gene expressions are consequences of the interactions of multiple factors, such as genotype-by-environment, gene-by-non gene elements, nucleotide methylation, chromatin modification, and small RNA regulation, whereas SNPs are relatively stable across these factors, it would be rather more likely to reflect different aspects of relationships between the cultivars and landraces. The relationships of 83%–89% of the cultivar and landrace pairs revealed by expressions of 15 ginsenoside biosynthesis gene transcripts or the contents of 16 ginsenosides were significant (P < 0.05), thus confirming the general conclusion that the cultivars and landraces of Jilin ginseng tended to be related. These results indicate that the expressions of the ginsenoside biosynthesis genes as well as the biosynthesis of the ginsenosides in which the genes are involved somehow are related or involved in a single or related processes. Moreover, it should be pointed out that the relationships do not seem to dent the robustness and applications of the mini-core collection for advanced ginseng research as this study did not identify significant kinships among the cultivars and landraces. Finally, the relationships of the cultivars and landraces revealed by any of genome-wide gene transcript expressions, ginsenoside biosynthesis gene transcript expressions, ginsenoside contents, and genic SNPs were significantly different between subpopulations of the mini-core collection, or the cultivars and landraces collected from different geographical regions.Knowledge of the relationships between cultivars, landraces, and germplasm lines is crucial to understanding and use of the germplasm collection for advanced ginseng research and breeding. The present study has, for the first time, examined the relationships between ginseng cultivars and landraces using genome-wide gene transcript expressions, ginsenoside biosynthesis gene transcript expressions, ginsenoside contents, and genic SNPs. Of these characters, the relationships of all pairs of the cultivars and landraces revealed by either expressions of 10,000 random gene transcripts or 6,232 high-quality genic SNPs were significant  can be found below: MPZ and YW conceived and designed the project, and MPZ revised and edited the manuscript. SL and YFW wrote the manuscript draft. HH, PC, JL, and YJ extracted and quantified ginsenosides. SL, LL, KW, and MZZ extracted, purified, and quantified the RNA. SL, MC, YH, and LZ conducted data analysis. All authors contributed to the article and approved the submitted version.


Krüppel-like transcriptional factor is important in maintaining cellular functions. Deletion of Krüppel-like transcriptional factor usually causes abnormal embryonic development and even embryonic death. KLF4 is a prominent member of this family, and embryonic deletion of KLF4 leads to alterations in skin permeability and postnatal death. In addition to its important role in embryo development, it also plays a critical role in inflammation and malignancy. It has been investigated that KLF4 has a regulatory role in a variety of cancers, including lung, breast, prostate, colorectal, pancreatic, hepatocellular, ovarian, esophageal, bladder and brain cancer. However, the role of KLF4 in tumorigenesis is complex, which may link to its unique structure with both transcriptional activation and transcriptional repression domains, and to the regulation of its upstream and downstream signaling molecules. In this review, we will summarize the structural and functional aspects of KLF4, with a focus on KLF4 as a clinical biomarker and therapeutic target in different types of tumors. Transcription factors regulate the expression of numerous genes in the organism and play a crucial role in embryonic development, physiological and pathologic processes. The transcription factor Krüppel-like factor 4 is important in the differentiation of epithelial cells and deletion of KLF4 leads to impaired barrier function of skin and death of the newborn.KLF4 is one of the most reported transcription factors among KLF family members, and nearly half of the reports are associated with tumors. Evidently, KLF4 plays an important role in the vast majority of tumors, including lung, breast, prostate, colorectal, pancreatic, liver, ovarian, esophageal, bladder, and brain cancers.Studies over the past three decades have shown that KLF4 inhibits tumor progression in most types of tumors. Therefore, it is considered as an “tumor suppressor”. However, in a few types of tumors, such as breast cancer and glioblastoma, KLF4 appears to be a “oncogene”. Also, there is increasing evidence that KLF4 can promote the precancerous lesions, such as the early lesions of pancreatic cancer, ADM and PanIN.The exact molecular mechanisms how KLF4 executes its function in tumors remains unclear, although the most important molecules downstream KLF4 include P21, P27, P53 and Cyclin-D. Whether KLF4 promotes precancerous lesions in other types of tumors has not been reported.Does KLF4, which promotes precancerous lesions of pancreatic cancer, similarly promote the precancerous process in other types of tumors?KLF4 inhibits tumor growth in the advanced stage of pancreatic cancer, while it plays the opposite role in the precancerous lesions. Is this a function of KLF4 itself, involving in completely opposite regulatory mechanisms?Can KLF4 be used as a marker for specific types of tumors or precancerous lesions and be used for early diagnosis and detection through exosomes and other modalities?Can KLF4 be used as a drug target to inhibit tumorigenesis and progression by targeting the transcriptional activation and repression domains of KLF4 with small molecule inhibitors, or altering its transcription and translation?Krüppel-like Factor 4 (KLF4) is a member of evolutionarily conserved family of zinc finger transcription factors, which was first discovered in the differentiated epithelium, colon and small intestine of newborn mice by Garrett-Sinha and Shields in 1996 . KLF4 isKLF4 is a transcription factor regulating not only cellular physiological processes such as proliferation, differentiation and apoptosis, but also pathogenesis of inflammation and tumorigenesis. However, the mechanism of its numerous physiological and pathological functions remain unclear , and theThe KLF family is composed of at least 18 transcription factors. They are widely distributed in eukaryotes and involved in embryonic development, tissue and organ maturation, stem cell generation, the process of inflammation and tumorigenesis, to name a few . To exer2-4-Cys-X12-His-X3-4-His, coordinates zinc ions and mediates the binding of the CACCC motifs of target DNA [Since zinc finger proteins are highly homologous to KLF in the DNA-binding region, KLF4 is classified into the KLFs family . Structurget DNA , 22. Intrget DNA . Howeverrget DNA . Differerget DNA , suggestrget DNA . Mutatiorget DNA . Similarrget DNA . Whetherrget DNA . FinallyAs one of the Yamanaka factors , KLF4 is best known for its role in maintaining the stemness of embryonic stem cells and inducing the formation of induced pluripotent stem cell (iPSC) , 28. iPSPhysiologically, KLF4 critically regulates the epithelial cell differentiation and the development of bone and kidney –34. EmbrPathologically, KLF4 acts as a context-dependent anti- and pro-inflammatory factor. KLF4 played a protective role in the kidney and antifibrosis role by inhibiting inflammation . The antKLF4 plays an important role in the development and progression of various types of cancers, including lung, breast, colorectal and pancreatic cancers. However, whether KLF4 is cancer-promoting or cancer-suppressing remains a mystery. In general, the dual effect of KLF4 on cancers depend on the type/subtype of cancer and the molecular regulation in signaling pathways. KLF4 acted as a tumor suppressor in gastrointestinal tumors  and cutai.e., wild-type KLF4 is expressed in the nucleus, whereas the isomer KLF4α is localized in the cytoplasm, and they indeed exhibit the opposite effects [Lung cancer is a lethal human disease with the second highest number of new cases . KLF4 ne effects .Breast cancer is the second lethal cancer in women, with the highest number of new cases . The effi.e., KLF4 was usually expressed in the cytoplasm of highly malignant prostate cancers [KLF4 generally inhibits the development of prostate cancer. For instance, the expression of KLF4 is decreased in primary and metastatic prostate cancer tissues. Preclinical studies have also verified that KLF4 inhibits the growth and migration of prostate cancer cells . Further cancers .The antitumor role of KLF4 in colorectal cancer is evidently clear. KLF4 was downregulated in human colorectal cancer tissues as compared with normal mucosa . KLF4 acAlthough the incidence of PDA is not as high as that of breast, lung and colorectal cancers, the mortality rate is almost as high as the morbidity rate due to the lack of effective treatment. The current measures for PDA management have shifted to early diagnosis and treatment. In PDA, KLF4 promoted the formation of precancerous lesions in mouse models, including ADM and PanIN. Human PDA was associated with chronic pancreatitis, in which ADM was observed , and ADMHowever, KLF4 inhibits cancer cell proliferation, EMT, invasion and metastasis in the advanced stage. PDA had a significantly reduced KLF4 expression as compared to normal pancreatic tissues and benign pancreatic lesions . KLF4 inKLF4 plays a role in inhibiting the development of hepatocellular carcinoma (HCC). KLF4 expression is lower in HCC tissues than in adjacent tissues, and high KLF4 expression is considered as a marker of benign prognosis after surgical resection . In contOvarian cancer can be divided into epithelial ovarian cancer (EOC) and non-epithelial ovarian cancer, with the former accounting for more than 95% of ovarian malignancies . KLF4 haMore than 90% of esophageal cancers are squamous cell cancers (SCC) . The antBladder cancer (BC) can be divided into uroepithelial carcinoma of the bladder, squamous carcinoma of the bladder, adenocarcinoma of the bladder and bladder sarcoma (UBC). UBC is the most common type with an incidence of over 90%. Limited data indicated that KLF4 inhibited the development of BC, while KLF4 was downregulated in a variety of UBC tissues and cells as compared to normal bladder epithelial tissues and cells. Overexpression of KLF4 inhibited BC cells growth and induced apoptosis . MechaniThe incidence of brain cancer accounts for approximately 1–3% of all human tumors. The most common benign brain cancers are pituitary tumors and meningiomas, while the most common malignant brain cancer is glioma. Clinical meningioma is frequently associated with KLF4 mutations and low expression of KLF4 RNA and protein , 125. Ine.g., p53, p21 and cyclin-D. The inconsistent roles of KLF4 in tumor development and progression may be due to multiple factors, including the types of tumors, stage of tumor progression, isomers and their regulation. The different roles of KLF4 are best exemplified in PDA, where KLF4 plays opposing role at different stages of PDA development and progression. Tumor subtypes clearly dictate the regulatory roles for KLF4 as exemplified by the evidence that KLF4 is more likely to promote tumor development from endothelial and squamous cells, while the opposite role in others. Therefore, the function of KLF4 in tumorigenesis is highly complex, given the context of different tumor subtypes, diverse molecular regulation and malignancy of tumor cells. Therefore, the signaling pathways of KLF4 are summarized next in an attempt to understand the dual regulatory role of KLF4 on tumors.In summary, as a transcription factor, KLF4 has a broad regulatory role in tumors and its signaling pathways are mainly involved in cell cycle and proliferation, KLF4 regulates a large number of genes and biological factors involved in proliferation, differentiation and apoptosis, and crucially regulates the development and progression of various types of tumors . In PDA,e.g., p21, p27Kip1, CD44, MSI2, Caveolin-1, and FOXM1 [Generally, KLF4 inhibits PDA progression by binding to various promoters of genes related to proliferation, nd FOXM1 , 95, 141nd FOXM1 . Pharmocnd FOXM1 . In addind FOXM1 . Conversnd FOXM1 .Finally, KLF4 also regulates the development and progression of several other cancers by directly regulating cell proliferation and differentiation factors, including CyclinD1, p53, Keratin4, and Keratin19 . KLF4 alNumerous studies have demonstrated that KLF4 inhibits the progression of colorectal, gastric, and hepatocellular carcinomas, and low KLF4 expression is clearly related to poor overall survival. Importantly, the higher the malignancy of tumor, the lower the expression of KLF4 . This phAlthough KLF4 inhibits the tumor proliferation and metastasis in most cases of SCLC and NSCL, KLF4 expression is higher in some NSCL than in normal tissue, which implies a positive association between KLF4 and NSCL . In moste.g., KRAS and TP53 [PDA cells can originate from a variety of cells in the pancreas, such as acinar cells, ductal cells, central acinar cells, endocrine cells and their progenitor cells, but not from a single cell type –153. Tisand TP53 . Susceptand TP53 , 156. Inand TP53 . Downregand TP53 . During and TP53 , whereasKRAS mutations, caerulein treatment and pancreatic duct ligation [ADM is an important precancerous lesion of PDA, in which KLF4 is upregulated with ligation . KLF4 inligation , 158–161ligation . ADM canligation , 162. ThIn other tumors, KLF4 remains “complex” as a molecular marker for diagnosis and prognosis. There are many KLF4 downstream molecules, certain of which can play a dominant role in certain types or subtypes of tumors . TherefoKLF4 acts as a context-dependent antitumor or tumor-promoting factor, holding limited potential as a therapeutic target. However, few compounds have been found to act directly on KLF4. Sulforaphane, a natural compound derived from broccoli, inhibited the progression of colon cancer by inducing KLF4 and enhancing the KLF4-p21 signaling . KenpaulCertain drugs also regulate KLF4 expression through epigenetic modifications. Decitabine, the most potent inhibitor of gene methylation, upregulated KLF4 in renal fibrosis by inhibiting the methylation of KLF4 promoter, which contributed to the inhibition of EMT . Small mCurrently, a few compounds have been designed to target the tumor-regulating effects of KLF4, and are available for clinical trials. APTO-253, a targeted inducer of KLF4, have been found in a clinical trial to have successfully halted the progression of some advanced tumors, especially leukemia . HoweverKLF4 expression is finely orchestrated during embryonic development and physiology . UnderstKLF4 regulates a large number of genes related to cell proliferation and differentiation. Expression of KLF4 has a significant impact on the formation of precancerous lesions, such as ADM and PanIN . In conjSupplementary references for Table 1


Urinary stones composed of calcium carbonate are extremely rare, accounting for 0.01%-1.4% of urolithiasis (UL) cases. Urolithiasis is an infrequent condition in the pediatric population worldwide and in Mexico; nevertheless, the incidence in the Yucatán Peninsula is higher than that reported in other areas of Mexico and the world. Urolithiasis is the second most common urinary disease among pediatrics in the Yucatán Peninsula, which makes it an endemic region for this disease. We describe the case of a five-year-old male from the southeast region of Mexico who presented with signs and symptoms of urinary tract infection (UTI) and was diagnosed with bilateral staghorn stones of calcium carbonate, successfully treated by mini endoscopic combined intra-renal surgery, and dietary adjustments to prevent recurrence. Urolithiasis (UL) is a common condition among adults, with a global prevalence of up to 20% within this demographic; conversely, its occurrence among pediatrics accounts for only 2%-3% of the total cases, rendering it rare -3. In MeA five-year-old male from the Yucatán Peninsula in Mexico presented with fever, malaise, and dysuria. There was a family history of UL on the patient’s father and paternal grandparents. The child's growth status was within normal/high limits according to his age, with a height of 113 centimeters, or 44.5 inches, which is between the 75 and 90 percentile; in terms of weight, the patient's BMI-for-age percentile was located between the 89 and 95 percentiles (17 kg/m²), classifying him as overweight. Dietary habits were characterized by a high-calorie intake, frequent consumption of hypersodic foods, and inadequate hydration.Due to the presenting symptoms, an initial urinalysis was conducted, which revealed pyuria, hematuria, proteinuria, and a pH of 6.7 . The patient's serum creatinine level was 0.61 mg/dL, but renal damage was detected because of the presence of proteinuria in the urinalysis. Because of the family history of UL and the endemicity of the disease in Yucatán, a suspicion of urolithiasis was raised, and therefore renal ultrasound was performed, where hyperechoic images with acoustic shadowing were observed in both kidneys, suggestive of UL. Given these findings, an abdominopelvic CT scan and 3D reconstruction were obtained Figures , 1B to cBilateral hyperdense renal images encompassing the entire collecting system were observed, confirming the diagnosis of bilateral staghorn calculi.A staged surgical management with bilateral mini-endoscopic combined intra-renal surgery (ECIRS) was successfully performed, and the stone analysis conducted through spectroscopy Figure  and X-raComplementary, a 24-hour urine metabolic analysis was conducted, and hypercalciuria, hypomagnesuria, and hypocitraturia were identified Table .These alterations were addressed by implementing an appropriate diet and administering oral potassium citrate, with good results after treatment.A case of a five-year-old male from the Yucatán Peninsula diagnosed with bilateral staghorn lithiasis composed of pure calcite was presented. Globally, the incidence of pediatric UL constitutes a small fraction of total UL cases , and calUrolithiasis in the pediatric population is a growing disease, with studies showing up to a five-fold increase . DespiteIn the Yucatán Peninsula, the presence of both modifiable and non-modifiable risk factors makes its inhabitants more susceptible to kidney stone formation . It has Regarding stone composition, according to the cohort study conducted by Degheili et al. in 2022, which analyzed the stone composition of 624 patients, it was found that calcium carbonate stones accounted for only nine (1.4%) cases, which makes it very rare . The patOver the past 25 years, in Mexico, pediatric lithiasis has exhibited a yearly upward trend of 6%-10%, particularly in areas where it is considered endemic. This underscores the significance of considering UL as a potential diagnosis in pediatric patients who frequently present with symptoms of urinary tract infection, abdominal pain radiating to the scrotum or labia, fever, microscopic or macroscopic hematuria, nausea, and vomiting . The firSome authors consider pediatric UL as a symptom rather than a disease; therefore, when addressing the diagnosis of lithiasis in pediatric patients, it is essential to conduct an early and comprehensive approach that combines metabolic studies, nutritional interviews, and, in feasible cases, analysis of the mineral composition of the calculi to determine what is causing the child to develop stones. Urinary stones developed during childhood have a risk of recurrence of up to 20%, and those with metabolic disturbances have a five-fold increased risk of developing another UL in the short- to mid-term, compared to those with adequate nutrition . A 24-hoDue to the recurrence of the disease, it is essential to introduce a prophylactic treatment after the removal of kidney stones . The maiAs for invasive treatment, mini-ECIRS has proven to be a novel technique of renal puncture and safe fragmentation, which increases surgical success while maintaining a high stone-free rate, reduces postoperative pain, and avoids postoperative complications such as renal bleeding, ureteral injury, and renal dysfunction, so it could be considered the best therapeutic option to treat large renal stones in pediatric patients ,16.There is a pressing need to direct research efforts toward comprehending the risk factors for calcite stone formation and recurrence, as well as its impact on the risk of acute kidney injury and chronic kidney disease in the pediatric population.Calcium carbonate calculi are exceedingly rare, even more so in pediatric patients. Stone composition and metabolic studies should be conducted routinely to identify abnormalities and prevent lithiasis recurrence. Currently, there are no studies about the correlation between risk factors and calcite stone formation and recurrence, making this a promising area for pediatric and urological research.