Malignancy after transplantation is a leading cause of death among kidney transplant recipients. However, donor-derived malignancies are rare. We report a case of a high grade papillary urothelial carcinoma arising in a transplanted kidney.A 62-year-old female who received a kidney transplantation more than 30 years ago presented with urinary tract infection, acute renal failure, and hydronephrosis of the transplant kidney. Anterograde nephrostogram showed a large filling defect in the lower pole of the transplant kidney and in the proximal 3–4 cm of the ureter. A biopsy from the renal pelvic mass showed a high grade urothelial carcinoma. She underwent an anterior exenteration, resection of both transplant and native kidneys and bilateral pelvic lymph node dissection. Pathologic examination showed a high grade papillary urothelial carcinoma which appeared to arise in the pelvis of the graft kidney, involve the graft ureter and native urinary bladder. The tumor had metastasized to one left obturator lymph node but spared the two native kidneys and ureters. Short tandem repeat (STR) analysis confirmed the tumor to be of donor origin. Next-generation sequencing identified amplification of TERT and loss of CDKN2A/CDKN2B in the primary tumor.While it is known that transplant recipients have an increased risk of urothelial carcinoma compared to the general population, the lack of the well-documented risk factors, such as older age at transplantation, BK polyomavirus infection, and prolonged post-transplantation history and dissemination of the tumor in this case shed light on the de novo tumorigenesis of the graft kidney within the host microenvironment. Amplification of Telomerase reverse transcriptase (TERT) and loss of cyclin dependent kinase inhibitor 2A/2B (CDKN2A/CDKN2B) detected in the tumor by next gene sequencing suggests that they may play an important role in this case. Kidney transplantation has been established as the treatment of choice for patients with end-stage renal disease , 2. WithA 62-year-old female underwent living-related donor renal transplant in 1983 due to end stage kidney disease with primary renal hypertension. She was maintained on azathrothiozine and steroids. No episodes of rejection were reported post-transplant, and the transplant kidney functioned well. In 2017, the patient presented with a urinary tract infection. Medical evaluation revealed acute renal failure and hydronephrosis of the transplanted kidney. She was treated with Cefapime and urine cytology was positive for malignant cells. An anterograde nephrostogram showed a large filling defect in the lower pole of the transplant kidney as well as a filling defect in the proximal 3–4 cm of the ureter. At time of ureteroscopy, multiple biopsies from the renal pelvic mass demonstrated high grade urothelial carcinoma. Later on, the patient was found to have extensive bladder tumors; she underwent hysterectomy, bilateral salpingo-oophorectomy, nephrectomy of native kidneys and transplant kidney, cystectomy and bilateral pelvic lymph node dissections. Post operation follow-up, pelvic magnetic resonance imaging demonstrated a large liver mass which was confirmed by biopsy to be metastatic urothelial carcinoma. Immunotherapy was initiated and the liver metastasis responded well to the immunotherapy with dramatic reduction in size of liver mass. At last follow-up, the patient was well 44 months after her surgical resection.high grade papillary UC involving the graft kidney, graft ureter and the native bladder. The carcinoma was predominantly non-invasive, however, focal invasion into subepithelial connective tissue was identified in the renal pelvis. Metastatic tumor was identified in one of thirteen obturator lymph nodes. The two native kidneys and ureters were negative for carcinoma  is commonly seen in upper urinary tract UC , we perfTERT and loss of CDKN2A/CDKN2B, which are genomic alterations found in approximately 7 and 25% of bladder cancers respectively . These were not detected in the lymph node metastasis. The renal pelvic tumor also showed a missense variant (p.N596S) in MSH2 at a low VAF (3.5%). This same variant was detected in the lymph node metastasis, but at a VAF of 37%. The lymph node metastasis also showed two other missense variants in MSH6 and FANCA with VAFs of 34 and 41% respectively. Interpretation of these variants would suggest these are of uncertain significance (VUS), potentially of germline origin. Given the low donor contribution in this lymph node specimen from the STR analyses (8%), it is suggested that these may be of recipient (native) germline origin. The low VAF MSH2 detected in the donor kidney tumor confirms the 7% recipient (native) contribution detected in this specimen. Thus, for the lymph node, no apparent somatic alterations were detected, possibly due to the overall low tumor cellularity of the specimen.DNA extracted from the donor kidney tumor and the lymph node metastasis were submitted for mutation profiling of 147 genes. The results are provided in Table Malignancy is a leading cause of death in renal transplant recipients. Post-transplant malignancies most often develop within the first 5 years post transplantation. Papillary RCC is the most common tumor type, almost exclusively occurring within the native kidneys . These pOur case of a rare donor-derived UC arising in a graft kidney is even more unusual in having arisen 34 years after transplantation, lacking other risk factors for secondary malignancy post-transplant, such as older age at transplantation and BK virus infection –21, and There are three possibilities regarding the UC origin in our case. One is that the tumor originated in the native bladder, the most common site of post-transplant UC, and spread retrograde to the donor ureter and graft kidney. The second possibility is that the primary tumor originated from the renal pelvis, and spread to the donor ureter and native bladder. This could be supported by the larger size of the tumor in the renal pelvis compared to other sites. The final possibility is the concurrent development of UC in the renal pelvis and urinary bladder. To answer these questions, we performed STR analysis, a molecular analysis of 16-STR widely used in medicine for establishing paternity and quality control in pathology. This test confirmed that the tumor in all three sites was of donor origin, supporting a renal pelvic origin, with drop metastasis to the ureter and urinary bladder.Molecular analysis of the tumor showed a low VAF MSH2 mutation, which was not detected at the protein level by MSI IHC. This, suggests that MSI had a very limited role, if any, in this case. In contrast, amplification of telomerase reverse transcriptase (TERT) and loss of Cyclin Dependent Kinases Inhibitors (CDKN); CDKN2A/CDKN2B were detected in the tumor.Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase and is essential for telomerase activity. Upregulation of TERT expression and resulting telomerase activity occurs in the large majority of malignancies, including urothelial carcinoma , 23. ThiThe CDKN2A and CDKN2B genes, encoding p16 and p15 respectively, are located on chromosome 9p21. Homozygous and heterozygous deletions occur at this locus in many primary human tumors, including urothelial carcinoma. CDKN2A and CDKN2B inhibit cyclin dependent kinase 4 (CDK4) and CDK6 which regulate cellular proliferation by preventing entry into the S phase of the cell cycle. Their inactivation may contribute to uncontrolled growth in human cancer. Deletion of CDKN2A/CDKN2B genes in urothelial carcinoma were found to be a common event and have been shown to be a crucial event in the progression from normal urothelium to carcinoma , 25.Although the primary renal pelvic tumor only showed focal invasion into submucosal tissue, spread to the graft ureter, native bladder, lymph node and liver document the aggressive nature of the primary tumor. The aggressiveness in this case may be related to the patient’s immunosuppression. The role of TERT amplification and loss of CDKN2A/CDKN2B in such aggressive behavior is unknown and may require large cohort studies. Amplification of telomerase reverse transcriptase (TERT) and loss of CDKN2A/CDKN2B were not associated with UC grade and stage in tumors arising in the bladder. This may not be true in upper tract UC, as mutational differences exist between upper tract and bladder urothelial carcinomas .While it is known that transplant recipients have an increased risk of urothelial carcinoma compared to the general population, the lack of the well-documented risk factors, such as older age at transplantation and BK polyomavirus infection, provide an opportunity to identify possible mechanisms of tumorigenesis of the graft kidney within the host microenvironment. Amplification of TERT and loss of CDKN2A/CDKN2B detected in the tumor by next gene sequencing suggests that they may play an important role in this case.


Acute promyelocytic leukemia (APL) is characterized by frequent complications due to a distinct coagulopathy. While advances in treatments have improved long-term survival, hemorrhagic and thrombotic complications remain the most common causes of death and morbidity. Improved understanding of the mechanisms of the coagulopathy associated with APL may lead to therapeutic interventions to mitigate the risk of hemorrhage and thrombosis. RARα) on chromosome 17, most commonly with the promyelocytic leukemia gene (PML) on chromosome 15, which leads to clonal proliferation of promyeloblasts  criteria as a distinct entity apart from rare variants of promyelocytic leukemia (Acute promyelocytic leukemia (APL) is caused by a translocation of the retinoic acid receptor alpha  and arsenic trioxide (ATO) , 4. For At steady state, blood flow is maintained in the face of vascular injury via complex interactions that balance primary and secondary hemostasis (mitigating blood loss) with antithrombotic mechanisms (preventing obstruction of flow and loss of coagulation factors) . Our undThis review highlights the unique pathophysiology of hemostasis and thrombosis in APL and the major clinical reports on the frequency and nature of hemorrhagic and thrombotic events. We will emphasize the commonly used interventions in the management of APL with an eye toward their potential impact on the mechanisms responsible for the coagulopathy present in this disease. Lastly, this review will not only present the current interventions used to mitigate the hemorrhagic risks but also emphasize potential treatment strategies and novel agents that merit clinical investigation to decrease coagulopathy-related death in these patients.2-antiplasmin-an inhibitor of the fibrinolytic pathway  . Most ofHigher white blood cell (WBC) count and decreased fibrinogen at presentation is associated with increased risk of severe or fatal bleeding , 41–43. In addition to typical coagulopathy assessments, other factors are associated with increased incidence of hemorrhagic events in APL. The PETHEMA Group report found a strong association on multivariate analysis of abnormal renal function and fatal hemorrhage, a metric not universally analyzed in other reports . While oMore so, while the first 7 days of therapy are associated with the highest incidence of bleeding events and early death, hemorrhagic risk and fatal bleeding events persist through the first month of therapy , 11, 42.Malignant cells in APL, like those from solid cancers, induce a procoagulant state. This thrombotic tendency is mostly mediated by expression of cancer procoagulant and tissue factor, though additional mechanisms are also involved. For instance, microparticles generated by various cells are elevated in the plasma of patients with APL compared with normal individuals , 49. Thein vivo study demonstrating >80% reduction in thrombomodulin levels after treatment with TNF-α , interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) . IL-1β ath TNF-α , 56. Thuth TNF-α , 58. Theth TNF-α .While bleeding is the most feared complication of APL, thrombosis is a common but under-recognized presentation. Multiple reports reveal a 5–20% risk of thrombotic events during induction therapy for APL, with the largest PETHEMA report finding 39/759 (5.1%) with a thrombotic event , 60–63. Reports of the ongoing risk of thrombosis and hemorrhage during the initial 30 days of treatment for APL underscore the pathophysiologic importance of the interaction of therapies with the promyeloblasts and resulting alterations in coagulopathy .Promyeloblasts secrete high amounts of proteases which degrade collagen and thereby activate enzymes of the coagulation cascade . ConventATRA's impact on coagulopathy in APL is more thoroughly studied and better understood compared with other therapies. ATRA-based regimens lead to less expression of tissue factor and annexin II by APL promyeloblasts, with a subsequent decrease of the dynamic coagulopathy after treatment initiation , 69. ATRNevertheless, the effects of ATRA on the coagulopathy of APL are complex and not fully elucidated. For instance, by inducing differentiation of malignant promyelocytes, ATRA could enhance ETosis in APL. This newly recognized form of cell death allows the nuclear chromatin to come into direct contact with intracellular enzymes and be released outside the cells . Outside9/L, cryoprecipitate to maintain fibrinogen >100–150 mg/dL, and fresh frozen plasma to maintain INR <1.5 (9/L and plasma to maintain fibrinogen >150 mg/dl in the 2013 ATRA/ATO trial published by Lo-Coco et al. (Standard supportive care to mitigate the hemorrhagic complications of APL includes platelet transfusion to goal >30–50 × 10INR <1.5 . Limitedo et al. .Given the predominant role of hyperfibrinolysis in APL, antifibrinolytics were evaluated to decrease bleeding risk. A retrospective study of 268 patients in the pre-ATRA era showed no significant difference in bleeding between those treated with antifibrinolytics or supportive care alone . AdditioHeparin's ability to decrease intravascular fibrin formation and coagulation factor consumption due to disseminated intravascular coagulation has been the subject of much speculation and some investigation. Low doses of unfractionated heparin or low molecular weight heparin act on endothelial cells to protect them from unwanted interactions with malignant promyeloblasts . By inteSome centers have used recombinant factor VII for APL patients with an intracranial hemorrhage, but there is limited literature to support standard utilization in life-threatening hemorrhage at this time , 81. TheCoagulopathy in APL shares some characteristics with DIC in sepsis, but the quantitative and qualitative platelet defects in APL are more profound . Agents APL, more than any other acute leukemia, is marked by a characteristic coagulopathy at diagnosis. Hemorrhagic syndromes vary from easy bruising or purpura to hematomas and even intracranial hemorrhage, the most serious complication and the main cause of early death.Since 1990, an immense body of basic science research has improved our understanding of the pathophysiology of coagulation in APL. Despite these advances in knowledge, interventional studies to prevent or manage hemorrhagic and thrombotic complications are infrequent and not powered to drive clinical practice forward. These failures to advance our management of coagulopathy in APL are underscored by the persistently high rates of early hemorrhagic and thrombotic death. While long-term outcomes have improved with the use of ATRA and ATO, the last major hurdle in APL is to prevent early hemorrhagic and thrombotic deaths.Multiple questions face clinicians aiming to decrease hemorrhagic and thrombotic risk in APL. Are there markers which can reliably and reproducibly identify degree of endothelial damage in an individual patient? Can we identify patients more prone to hemorrhage vs. thrombosis, and tailor risk mitigation strategies based on individual coagulopathy profile? Are there any biomarkers or risk factors that would be particularly helpful in making these decisions? Moreover, as the risk for bleeding and clotting events is dynamic during induction therapy, how can we assess these changes in individual patients? The dynamic nature of coagulopathy in APL suggests that risk mitigation strategies should vary depending on time in therapy. Such an approach may transfuse platelets earlier in induction, shifting to more aggressive use of cryoprecipitate for a higher fibrinogen goal during the second week of therapy.In addition to its role in APL, ATRA is able to differentiate non-APL myeloblasts. The relative contributions of annexin II, tissue factor, and cancer procoagulant to coagulopathy in non-APL acute leukemias are less well-understood, and further studies are needed to define if ATRA is able to mitigate the coagulopathy in other acute leukemias.Coagulation remains profoundly altered despite the administration of ATRA, even if much improved in comparison with the pre-ATRA era. Interventions such as recombinant human soluble thrombomodulin to mitigate the risk of coagulopathy remain understudied. Moreover, the role of recombinant factor VII in life-threatening hemorrhage requires additional research. Likewise, the approach to anticoagulation after confirmed thrombosis remains a clinical challenge with little data to guide bedside clinicians. To adequately study the aforementioned interventions, many centers will have to collaborate prospectively and/or pool their retrospective experience in this relatively rare malignancy.Such an effort will pose significant logistical challenges, but may finally leverage our pathophysiologic understanding into improved outcomes for patients with APL.All authors contributed to the design, writing, and critical review of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The development of safe, effective, affordable vaccines against COVID-19 remains the cornerstone to mitigating this pandemic. Early in December 2020, multiple research groups had designed potential vaccines. From 11 March 2021, several European countries temporarily suspended the use of the Oxford–AstraZeneca vaccine amid reports of blood clot events and the death of a vaccinated person, despite the European Medicines Agency (EMA) and the World Health Organization’s assurance that there was no indication that vaccination was linked. This study aimed to identify and analyse the thrombotic adverse reactions associated with the Oxford–AstraZeneca vaccine. This was a retrospective descriptive study using spontaneous reports submitted to the EudraVigilance database in the period from 17 February to 12 March 2021. There were 54,571 adverse reaction reports, of which 28 were associated with thrombotic adverse reactions. Three fatalities were related to pulmonary embolism; one fatality to thrombosis. With 17 million people having had the AstraZeneca vaccine, these are extremely rare events The EMA’s Pharmacovigilance Risk Assessment Committee (18 March 2021) concluded that the vaccine was safe, effective and the benefits outweighed the risks. Conducting further analyses based on more detailed thrombotic adverse event reports, including patients’ characteristics and comorbidities, may enable assessment of the causality with higher specificity. The pandemic of SARS-CoV 2, the causative agent of COVID-19, poses an unprecedented challenge to world economies and community health. The development of safe, effective, affordable vaccines against COVID-19 remains the cornerstone to mitigating new viral strains in this pandemic crisis and re-establishing normality going forward. Several pharmacological therapeutics were suggested early in the pandemic for the treatment of this disease such as remdesivir, favipiravir, hydroxychloroquine, tocilizumab, lopinavir–ritonavir (LPV/RTV) and convalescent plasma ,4. By eaChimpanzee adenovirus encoding the SARS-CoV-2 spike glycoprotein (ChAdOx1-S) ,8 is theSince 11 March 2021, several European countries  temporarily suspended the use of the Oxford–AstraZeneca COVID-19 vaccine amid reports of blood clot events and the death of a vaccinated person ,13. WithTraditionally, the monitoring of vaccine safety after licensure is subject to a combination of passive and active surveillance. The passive surveillance system is the basis of pharmacovigilance, which comprises the databases into which spontaneous reports of suspected adverse drug reactions (ADRs) and adverse events following immunisation (AEFI) from physicians and patients are collected . EudraViThe monitoring of safety reports of drugs and vaccines is critical because clinical trials are designed to assess primarily the efficacy; safety is typically a secondary objective. These trials are able to identify only common adverse events such as local and systemic reactions related to the immunogenicity of the vaccine that occurs shortly after administration. The trials are unlikely to detect rare adverse events that occur with trials in larger populations or delayed reactions that occur a long time after use, therefore pharmacovigilance and post-marketing surveillance is of great importance to detect unusual adverse events ,16,17.This study aimed to identify and analyse the thrombotic adverse reactions associated with the Oxford–AstraZeneca vaccine following its temporary suspension in several EU countries using the European EV database ,13.This was a retrospective descriptive study using spontaneous reports submitted to the EV database in the period from 17 February to 12 March 2021. Data were extracted from the line listing section of reports submitted to the EV in relation to COVID-19 Vaccine AstraZeneca (ChAdOx1-S). The following search terms were applied regardless of age group, gender, geographical area, reporter or level of seriousness: thrombosis; embolism; thromboembolism; embolic and thrombotic . Data weEthical consideration: Open access data were used, therefore no access authorisation was requested. The access policy of EMA states that ”No authorisation for accessing the ICSR (Level 1) data set by means of the adrreports.eu portal is required i.e., all academic researchers can access adverse reaction data of interest.” n = 16; 57%) of the reports pertained to people aged over 85 years; 13 (47%) reports were from within the EU  were submitted by healthcare professionals. More than half (n the EU .n = 19) and n = 9) illustrate the type of reported thromboembolic reaction along with the clinical outcome of cases by age group  and deep vein thrombosis (DVT) have usually been associated with multiple causative factors. These can be either hereditary or naturally occurring causes including cancer, advanced age, trauma, smoking, inherited or acquired thrombophilic states, previous thromboembolism, and hospitalisation for congestive heart failure or acute exacerbation chronic obstructive pulmonary disease (COPD). PE and DVT are typically associated with morbidity and mortality. Their natural incidence ranges from 56 events per 100,000 persons to 182 events per 100,000 persons which should be considered against the three deaths from PE in 17 million following vaccination ,17,18. AIn Europe, a rare disease (AE—adverse event—in this scenario) is defined when it affects five in 10,000 people . Ultra or very rare AE is defined as when it affects one in 50,000 people or 20/million. With a simple calculation of 28 thrombotic events reported in relation to the AstraZeneca vaccine for 17 million people who had the vaccine these would be cautiously labelled as extremely rare events [Notably, it would be challenging and difficult to determine the causality and link the thrombotic events to AstraZeneca vaccine using spontaneous reports. Additionally, to apply statistical calculations could be misleading because of the very limited safety and clinical data on the use of vaccine in the people affected, together with under-reporting and the low numbers of exposed individuals. This situation is further complicated by different confounding factors such as the indications and the wide spectrum of unknown comorbidities of patients ,22,23.While the AstraZeneca vaccine has been the focus, other vaccines have also been reporting adverse events. Searching the EMA EV database for Moderna vaccine reports for deep vein thrombosis and pulmonary embolism identified 29 DVTs, of which 12 also had pulmonary embolism; no reported deaths or fatal events . Similarn = 19) than males (n = 9). Thrombotic events in younger females are typically associated with ovarian hyperstimulation syndrome (OHSS) following reproductive treatment [This study also identified approximately double the occurrence of potential thrombotic events reported in females  advised that vaccination in pregnancy should be considered in women who are frontline health or social care workers or have underlying conditions that put them at very high risk of being infected with, transmitting or experiencing serious complications of COVID-19, however, no details of this group were available for analysis in this study .The vaccine components itself include active immunising antigens, conjugating agents, preservatives, stabilizers, adjuvants, and culture media used in the preparation of the vaccine, which can be considered as potential triggers for an allergic reaction. Many delayed reactions are classified as Type III hypersensitivity reactions which are attributed primarily to the formation of immune complexes that including T cell-mediated processes. The most common signs of delayed-type reactions include rashes, which may include urticaria, erythema and angioedema ,29.The limitations of this study include the small number of potential thrombotic adverse events and the limited safety data currently available for the vaccine. There may be under-reporting by healthcare professionals and patients; the quality of reports may play a significant role that may cause false negative or false positive safety signals. Many confounders and incomplete data together with reporting biases may also limit the generalisability of the findings .However, this study has provided valuable information about this current topical area from a trusted database. It has identified a very low number of investigated adverse reactions linked to the AstraZeneca vaccine that could be attributed to multiple causative factors and not merely the vaccines. The EMA’s Pharmacovigilance Risk Assessment Committee (PRAC) met on 18 March 2021. It concluded that the AstraZeneca vaccine was safe, effective and the benefits outweighed the risks and so urged people to accept the vaccination when offered .It has been suggested that administration of the COVID-19 vaccine AstraZeneca should be considered carefully and only when the potential benefit outweighs any potential risks, particularly in patients with a history of cerebral venous sinus thrombosis, acquired or hereditary thrombophilia, heparin-induced thrombocytopenia ,30. HealConducting further analyses based on more detailed thrombotic adverse event reports, including patients’ characteristics and comorbidities, may enable the assessment of the causality with higher specificity. It must be undertaken by experienced personnel to identify any real risks associated with any and all COVID-19 vaccines.This study has identified only 28 thrombotic events linked to the AstraZeneca vaccine out of 54,571 adverse reactions reported on the EV database. This finding should be interpreted cautiously, as underreporting together with reporting biases may limit the generalisability of the findings.It is difficult to determine a causal effect of the vaccine on the number of thromboembolic diseases reported. No clear causal effect can be confirmed, and multiple causative factors for thrombotic events were untested and undetermined. Further research is required to help national vaccine advisory boards and vaccine resistant people to make better informed, evidence-based decisions.


An initial diagnosis of cancer is usually based on symptoms, abnormal physical examination and imaging tests. Ovarian cancer is difficult to be diagnosed timely due to the nonspecific symptoms, thus resulting in the high-risk mortality. Despite of the various diagnostic methods, there is still no reliable diagnostic test. Clinically, carbohydrate antigen 125(CA125) is widely recognized as a diagnosis biomarker of ovary cancer. However, CA125 is not sensitive to detect the ovary cancer at the early stage. It is essential to explore other potential biomarkers. Human epididymis protein 4 (HE4) in the whey/four-disulfide core (WFDC) proteins family shows satisfactory sensitivity in the early diagnosis of ovary cancer. In this present review, we summarized the important effects of WFDC family proteins on the proliferation, apoptosis and migration of ovary cancer and intended to provide more evidence to explore the possibility of WFDC protein as a diagnosis biomarker. Although ovarian cancer accounts for only a third of gynecologic cancers, it results in 55% of deaths from gynecologic malignancies and 6% of all cancer deaths in women. The 50~70-year-old group is a group with the highest incidence of disease et al. confirmed that whey acidic protein was the main protein in the milk of rats and mice. The whey-acidic-protein motif in this protein is the prototypic member of a subfamily of the whey/four-disulfide core proteins (WFDC) In 1980, 'four-disulfide core' (FDC) structure and overall folding patterns have been initially found between snake venom postsynaptic neurotoxin and wheat germ agglutinin domain. This structure is composed of four similar disulfide bonds with different size ring With the emergence of selective molecular targeted therapies, biomarkers play an increasingly crucial role in the clinical management of cancer patients Figure 1). Among these, HE4 has been repeatedly confirmed to have significant expression changes in ovarian cancer tumor. Related basic experiments have proved that HE4 has an impact on tumor progression, such as proliferation, apoptosis invasion and migration Figure 2), the same as HE4, indicating the crucial role of WFDC family proteins as biomarkers.The attention of researches on cancer has gradually focused on genes coding WFDC family proteins. Most notably, the genes coding for WAP four disulfide core domain protein 14 , WAP four disulfide core domain protein 4 , WAP four disulphide core domain protein 1  and WAP four disulphide core domain protein 2 , have been studied on ovarian cancer. In the past ten years, SLPI and HE4 were the most studied molecules of the WFDC family in pathogenesis and development of ovarian cancer HE4 is currently most studied in ovarian cancer. It has been currently evaluated for diagnosing ovarian malignant tumor as a novel biomarker It is interesting that HE4 has an essential effect on tumorigenesis and development without directly exerting its serine protease inhibitory function + T cells in tissue of epithelial ovarian cancer patients Meanwhile, HE4 contributes to occurrence of inflammation in tumor environment. Recently, it was the first time to confirm that HE4 suppresses STAT3 to upregulate IL8 and HIF1A gene expression. This progress affects microenvironment and angiogenesis of ovarian cancer. Expression of HE4 was associated with reduced CD8SLPI is reported to overexpress in ovarian cancer while it has low expression levels in normal organs Applying to SLPI as tissue-specific promoter is a potentially useful method for ovarian cancer and allows targeted options for better gene therapy of ovarian cancer. In the ovarian tumor, gene expression regulated by the SLPI promoter was shown to be higher than in normal tissues such as the liver, lung, heart, kidney, and so on. Under the expression control of the SLPI promoter, HSVtk/GCV-mediated cell killing for ovarian cancer considerably improved survival in mice model to a comparable extent as the CMV promoter The first report connecting Elafin to ovarian cancer was presented on 2010. Elafin expression correlated with poor overall survival in late-stage, high-grade serous ovarian carcinomas. Importantly, its expression can be transcriptionally upregulated by immune cytokines via the activation of NF-κB pathway in primary tumors Prostate stromal 20 (ps20) is increasingly recognized as an important regulator of tumor growth, mostly in progressive prostate cancer Table 1).In comparison to other cancers associated with women, 77% of them are diagnosed as endometrial cancers stage I, 55% of breast cancers and 83% of cervical cancers respectively. While only 23% of ovarian malignancies are detected at an early stage CA125 is originally used to monitor the chemotherapy response of ovarian cancer and detect the relapse after surgery In a randomized controlled trial, annual multimodal screening with serum CA125 and transvaginal ultrasound sonography as a second-line diagnostic demonstrated minimal effectiveness Secretory HE4 showed significant difference as urinary biomarker in the differential diagnosis between ovarian cancer and benign tumors, followed by creatinine, carcinoembryonic antigen, vascular cell adhesion molecule (VCAM) and transthyretin (TTR) Table . The serum CA125 and computed tomography (CT) imaging are currently used for evaluating primary therapy response evaluation in epithelial ovarian cancer 72 Table . Several clinical trials have noted that HE4 levels had equal sensitivity but higher specificity in detecting ovarian cancer when compared to CA125 levels Diagnostic comparisons of potential single biomarker, including HE4, CA125, carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), transthyretin (TTR) and apolipoprotein A-1 (ApoA-1), also indicate HE4 has important roles in diagnosing Table 2). It is an initial enthusiasm to estimate the possibility of malignancy as a triage to ovarian masses and diagnostic aid, although they are not ideal diagnostic tests for early-stage detection. It presents a trend of variations that exploring biomarkers alternatives to CA125 alone and complements diagnostic performance in early-stage. A new insight of combination of markers to detect the occurrence and development of early ovarian cancer may improve the sensitivity and accuracy of detection (Table 2). Multiple biomarker panels, including HE4 protein, are being studied in depth Advances in biomarker discovery have led to several FDA-approved tests superior to CA125 in preoperative evaluation of women with a pelvic mass, including ROMA, Overa and OVA1 test (WFDC family proteins are involved in several aspects of the occurrence and development of ovarian cancer, including proliferation, apoptosis, invasion and metastasis. HE4 Ovarian Cancer Monitoring Trial set up to measure the HE4 protein level in blood for the diagnosis. Basic researches and clinical trials are expected to investigate the efficacy of other molecules of WFDC family compounds as biomarker in monitoring, diagnosis and treatment of ovarian cancer, especially in early-stage. We anticipate that further studies on the sensitivity and specificity of molecules other than HE4 in clinical trials will be undertaken to aid in the diagnosis, treatment and prognostic monitoring of early-stage ovarian cancer.Chen Zhang wrote the original draft. Haoyue Hu revised the manuscript. Xiaoyan Wang summarized the literature. Yajuan Zhu provided an overall idea. Ming Jiang modified the framework and re-designed pictures and tables.


Tooth decay and gum disease are two of the most common chronic health conditions in the United States, are reversible and preventable, and impact approximately 68% of older adults nationwide . While the Affordable Care Act added provisions to health prevention services, oral health prevention coverage was only included for children, leaving many adults and older adults without coverage . The research team used a rapid review process using 17 key search term combinations to identify literature in three medical databases  to identify system and policy level barriers and opportunities to address oral health equity issues for older adults in the United States. 40 articles met inclusion criteria for thematic analysis. Findings revealed three barrier categories: 1) poor oral health literacy of patients and health care providers, 2) reimbursement variability contributing to access and utilization barriers, 3) workforce and scope of practice variability. In addition, four opportunity categories were identified: 1) community-based oral health programming for older adults, 2) new reimbursement models, 3) medical-dental collaborations, and 4) policy and practice act updates. The COVID-19 public health crisis has impacted the implementation of some system and policy level opportunities. However, new health care initiatives specific to Medicare in discussion at the national level provide an opportunity to make some headway on the policy updates needed to address the oral health of older Americans. Findings and implications will be shared with the audience.


The KCNMA1 variant p.(Gly356Arg) abolishes the function of BKC and blocks the generation of K+ current. The phenotype of this variant includes developmental delay, and visceral and connective tissue malformations. So far, only three cases of LWAS have been described, one of which also had neonatal diabetes (ND). We present the case of a newborn affected by LIWAS carrying the p.(Gly375Arg) variant who manifested diabetes in the first week of life. The description of our case strongly increases the frequency of ND in LIWAS patients and suggests a role of BK inactivation in human insulin secretion. The knowledge on the role of BKC in insulin secretion is very poor. Analyzing the possible mechanisms that could explain the association of LIWAS with ND, we speculate that BK inactivation might impair insulin secretion through the alteration of ion-dependent membrane activities and mitochondrial functions in β-cells, as well as the impaired intra-islet vessel reactivity. Liang-Wang syndrome (LIWAS) is a polymalformative syndrome first described in 2019 caused by heterozygous mutation of the KCNMA1 gene encoding the Ca They have a complex regulation pattern that includes the modulatory β-subunits, subunit phosphorylation , pH, and endogenous messengers  and modulators  I in β-cells, the loss of their plasma membrane polarizing activity might leave more VDCC in the inactivated state, thereby affecting insulin exocytosis I, and consequently promotes cell contraction, while, under conditions favoring vasodilation, the BKC activation by hyperpolarizing the membrane leads to an inhibition of LTCC [On the other hand, BCKs might prevent Ca in vivo   could impair insulin secretion. We suggest considering the KCNMA1 p.(Gly375Arg) variant as a mutation potentially associated with ND in LIWAS and propose including baseline endocrine evaluation at the time of diagnosis and in the ongoing monitoring of patients affected by this rare syndrome. New studies focused on the role of BKCs in human insulin secretion are warranted.


Social distancing and shelter-in-place orders designed to curb the spread of COVID-19 increased isolation among persons with memory concerns (PWMC) and increased the burden on individuals providing their care. Technology, such as smartphones or tablets, has demonstrated potential to improve the social connections and mental health of older adults; however, older adults historically have been reluctant to adopt new technology. We aimed to understand why some PWMC and their caregivers used new technology to adapt to lifestyle changes during the COVID-19 pandemic while others did not. In this study, we used data collected in 20 qualitative interviews from June to August, 2020 with PWMC and their family caregivers to assess changes in and barriers to technology use. Qualitative thematic analysis identified three themes which explained motivations for using new technology during a pandemic: 1) seeking relief from caregiver burden, 2) alleviating boredom, and 3) maintaining social connection. Results further revealed lingering barriers to PWMC and caregiver adoption of technologies, including: 1) PWMC dependence upon caregivers, 2) a lack of familiarity with technology, and 3) difficulties using technology. This in-depth investigation suggests that technology has the ability to provide caregivers relief from caregiving duties and provide PWMC with more independence during periods of pronounced isolation


The membrane trafficking system establishes and maintains the correct distribution and compartmentalisation of proteins within the cell. Broadly speaking, membrane trafficking processes encompass the selection of protein cargoes, their packaging into transport intermediates, and the delivery and fusion of these transport intermediates with the proper acceptor organelle. As such, this machinery functions constantly and ubiquitously within almost every cell of the body. Perhaps counterintuitively then, given the ubiquity of these processes, a large and increasing number of pathogenic mutations affecting the membrane trafficking machinery have been identified with relatively restricted pathologies . Notablevia dynamic translocation of receptors and signalling partners. As such, genetic mutations affecting proteins of the membrane trafficking machinery underlie diverse disorders with immunological components, including familial haemophagocytic lymphohistiocytosis   . Characterisation of these mutants has given great insight into secretory granule transport, docking, priming and fusion . However, the potential role of heterozygous mutations is still debated. N. Benavides and A. Spessott et al. investigate the effects of a heterozygous mutation (R190C) in syntaxin-binding protein-2 (STXBP2) identified accompanied by a hemizygous glucose 6-phosphate dehydrogenase  mutation. They show that the heterozygous mutation does not interfere with protein or mRNA stability, nor interaction with syntaxin-11 (STX11). Since this protein region is conserved, they hypothesize binding to an unknown interaction partner. Overexpression in healthy control CD8+ T cells reduced secretory granule exocytosis dramatically, suggesting a dominant-negative effect of this mutation. This supports the growing evidence that heterozygous mutations in synergy with other mutations could lead to fHLH manifestation. Importantly, family members carrying the heterozygous R190C mutation in STXPB2 should be excluded as hematopoietic stem cell donors.Impaired cytotoxic function due to reduced secretory granule exocytosis by NK and CD8s (fHLH) . NumerouOhishi et al investigate an undescribed homozygous mutation, RAB27A p., identified in a patient with HLH without any associated albinism. They demonstrate that RAB27A Val143Ala does not affect the RAB27A–melanophilin (MLPH)/SLAC2-A interaction, essential for hair and skin pigmentation. However, in line with some previously characterised RAB27A mutations, CD8+ T cell function is impaired and reduced binding activity towards MUNC13-4 and SLP2-A is suggested. The authors summarize other RAB27A mutations sine albinism to demonstrate the importance of RAB27A sequencing in patients with suspected HLH without pigmentary dilution.Most homozygous mutation of RAB27A described to date predispose to HLH with albinism. + T cells. Sleiman et al. suggest a role for Synaptotagmin7 (SYT7), a high affinity calcium sensor, in the transport of secretory granules to the immunological synapse (IS), especially during the second phase of prolonged secretory granule fusion. They demonstrate that SYT7 KO CD8+ T cells display a conditional granule fusion defect under high 10mM [Ca2+] conditions, rescued by transduction of SYT7 wild type constructs.Calcium plays a regulatory role in serial secretory granule exocytosis and killing function of CD8Mastio and Saeed et al. provide a review highlighting the links between membrane trafficking defects, their consequent immunological deficiencies, and the subsequent development of B-cell malignancies. They detail the processes leading to malignancies and explore the involvement of actin regulators and immune synapse defects in transformation and tumor surveillance.Whilst the primary immunological defect is often the best understood in terms of mechanism and pathogenesis it is important to remember that downstream sequalae may contribute significantly to a patient’s clinical needs. via their dynamic translocation between subcellar compartments. Taguchi and Mukai et al. give an overview of current knowledge concerning STING translocation between the ER and Golgi and highlight a recently described autoinflammatory disorder (COPA syndrome) arising from mutations in the α-COP subunit of the COP-I coat. In a complementary review Amadio et al. build a case for more detailed exploration of the role of the actin machinery in regulating STING and TLR9 distribution and function. They discuss recent evidence of aberrant DNA sensing associated with the actin-related primary immuno-deficiency Wiskott-Aldrich syndrome and draw links to the roles of actin both upstream, and downstream of DNA sensing by STING and TLR9.A focus of much attention in recent years has been the role of subcellular compartmentalisation in the innate immune response to so-called pathogen associated molecular patterns (PAMPs). Two reviews in the current research focus examine the critical regulation of DNA-sensing pattern recognition receptors Together, the articles contained in this special Research Topic give insight into two of the many important roles that membrane trafficking plays in maintaining the proper function of the immune system. Further exploration of this subject will be greatly aided by continuing advances in both the medical genetics of immunodeficiency as well as experimental approaches to the study of membrane trafficking and the manipulation of immune cells. As such we believe that this will be a growing and important field of investigation in the coming years.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.Funding SFB 1160/IMPATH for SA and YTB is supported by the Swedish Research Council, Cancer Foundation, Childhood Cancer Foundation, the Knut and Alice Wallenberg Foundation, the Center for Innovative Medicine and Stockholm Region.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite the projected rise in the diversity of caregivers and caregiving in the US, the health system is not prepared to accommodate this growth. Interventions and supports often are not adequately tailored to meet the cultural needs of older adults. Additionally, the limited interventions available for racial/ethnic minority populations frequently fail to capture and report culturally tailored perspectives. Therefore, the purpose of this presentation is to describe how culture influences caregiving in the US. Specifically, it will: (1) provide a contemporary definition of culture; (2) identify cultural domains that impact caregiving; (3) offer examples of how caregiving is influenced by different cultural/demographic backgrounds; (4) provide examples of culturally tailored caregiving programs, and (5) discuss how to approach cultural needs that may not be addressed by current interventions.


Recent fecal microbiome transplant (FMT) studies demonstrated the modulatory effects of FMT on the composition and diversity of the gut microbiome in patients with refractory cancers and the potential to improve the efficacy of ICIs.The current review assessed the effects of the gut microbiome on clinical outcomes of immunotherapy and related adverse events (AEs) in cancer patients. Studies , hepatocellular carcinoma (HCC) (n = 2), non-small cell lung carcinoma (NSCLC) (n = 1) and one study examined combination both NSCLC and renal cell carcinoma (RCC) (n = 1). These studies consistently reported that the gut microbiome profile prior to administering immune checkpoint inhibitors (ICIs) was related to clinical response as measured by progression-free survival (PFS) and overall survival (OS). Two studies reported that a low abundance of Bacteroidetes was associated with colitis. Two studies showed that patients with anti-PD-1 refractory metastatic melanoma experienced improved response rates and no added toxicity when receiving fecal microbiota transplant (FMT) from patients with anti-PD-1 responsive disease. Conclusions: Overall, significant differences in the diversity and composition of the gut microbiome were identified in ICIs responders and non-responders. Our findings provide new insights into the value of assessing the gut microbiome in immunotherapy. Further robust randomized controlled trials (RCTs) examining the modulatory effects of the gut microbiome and FMT on ICIs in patients not responding to immunotherapy are warranted. Cancer immunotherapy, a novel paradigm for cancer treatment, has demonstrated significantly increased survival rates in patients with metastatic cancer who were diagnosed with melanoma, non-small cell lung cancer (NSCLC) and renal cell cancer (RCC) when compared with standard care. In addition, immunotherapy has been approved as treatment for several other cancers, including head and neck squamous cell cancer (HNSCC), refractory classical Hodgkin lymphoma, urothelial carcinoma, microsatellite instability-high cancer, recurrent or metastatic gastric and cervical cancer, refractory or relapsed primary mediastinal B cell lymphoma, advanced hepatocellular and Merkel cell carcinoma, and colorectal cancer (CRC) ,2.The two main types of immunotherapies with well-established efficacy are immune checkpoint inhibitor monoclonal antibodies (ICIs), including cytotoxic T-lymphocyte associated protein 4 (CTLA4) and programmed death cell protein 1 (PD1)/ligands (PDL1) . ICIs spDespite these immunotherapies demonstrating increased survival in cancer patients at various advanced stages of cancer, they have benefited only a subset of patients with varied individual therapeutic responses ,8. For eRecently, an increasing number of studies have suggested that the dysbiosis of the gut microbiome is a risk factor for chronic disease, including cancer ,13,14. NTo date, few studies have examined the impact of the gut microbiome on the clinical response of ICIs, namely progression-free survival (PFS), overall survival (OS) and irAEs, although several studies have suggested a critical role for the gut microbiome in mediating responses to immunotherapies. Previous reviews have included both animal models and clinical studies and have attempted to elucidate the underlying mechanisms of dysbiosis of gut microbiota in immune responses ,17. Our An extensive search of the electronic databases PubMed, Medline, and ScienceDirect was conducted from inception to March 2021. Studies were searched with terms “cancer” and “immunotherapy/immune checkpoint inhibitor” and “microbiome/microbiota”. Inclusion criteria were studies conducted with adults (>18 years), published with full texts in English, and published in peer-reviewed journals. In addition, references contained in the included studies were manually searched to identify relevant papers that may have been missed by electronic searches.Ten studies were identified from the three electronic databases  and included in this review . Of the α-DiversityNumber and Evenness of Distribution of Taxa within a Given Sampleβ-diversityThe difference in diversity of taxa from one sample to another, i.e., the number of taxa that are not the same (or not similarly distributed) in two different samples.16S rRNA gene sequencingSequencing of the 16S rRNA marker geneMetagenomic sequencingSequencing of the entire metagenome , also allowing analysis of the functional capacity of the microbiomen ≥ 65), whereas seven studies were conducted with small sample sizes (n ≤ 42). Six studies assessed patients diagnosed with melanoma, two with HCC, and one each study with lung cancer and with mixed lung and RCC. Five studies were conducted in the USA, three in China and two in France. Total study populations for ICIs  and type of ICI interventions  varied across studies. Study designs included in this review were diverse, but the main primary outcome of individual studies were clinical outcomes including, response to ICI (n = 9) and irAEs (n = 1). Four studies collected fecal samples once prior to ICIs interventions, whereas six studies collected these multiple times . Three studies analysed the gut microbiome profile with the 16S ribosomal RNA (16S rRNA) gene sequencing method, three with shotgun metagenomic sequencing, and four with both the 16S rRNA and shotgun metagenomics sequencing. Interestingly, an analysis of gene sequencing regions of 16S rRNA varied across studies: V3–V4 (n = 2), V4 (n = 4), and V4–V5 (n = 1).The ten selected studies included a total of 467 patients with a range of 8–89 in each of the studies. Only three studies were conducted with moderate sample sizes  (n = 24) compared to those who did not (non-responders) (n = 15)  that consistently reported that diversity and composition of the gut microbiome prior to ICIs was related to clinical response, although, the relationship between significant enriched taxa and clinical response in cancer patients varied across the nine studies  on recurrent Clostridioides difficile infection and showed strong effects in favour of the efficacy of FMT resulting in its successful introduction into recent clinical practice guidelines [These response rates are comparable to other studies that examined the effect of FMT on the recurrence of idelines ,38,39.These compelling novel studies illuminate new opportunities for treatment in advanced cancer patients who are refractory to immunotherapy and/or have limited further treatment options. A major challenge remains to identify the precise mechanisms and components of FMT that contribute to a favourable response to immunotherapy in patients with refractory cancer. Hence, additional well-controlled studies are required to determine specific features of donors and recipients that leads to the transformation from unfavourable to favourable responses in immunotherapy.The current review is significant in that it included only clinical studies in order to provide updated meaningful evidence for clinicians, cancer patients and carers, whereas previous reviews, examining both animal models and clinical studies in order to elucidate the role of gut microbiota in ICIs, resulted in gaps in their application to real-world clinical practice ,40.For instance, the composition of the human gut microbiota can be affected by various factors including diet, lifestyle, stress, environment and genetics which not only complicates comparisons with animal model studies, but also needs to be controlled as possible contributors towards populations’ differences in identified differential microbial taxa. In this review, we also assessed the relationship between the gut microbiome and irAEs in addition to clinical response. The importance of managing cancer treatment-related symptoms in cancer care is well recognized and several studies have identified irAEs during ICIs . FurtherBacteroidetes phylum bacteria are associated with colitis. Given that the management of irAEs is critically important in cancer care, the relationship between the gut microbiome and irAEs and clinical response is worth considering in future studies. In recognition of the weak association between the dysbiosis of gut microbiota and the efficacy of ICIs, we further reviewed the modulatory effects of the gut microbiome and FMT to examine causal factors and found that FMT has the potential to improve the efficacy of ICIs in advanced cancer patients.Despite this, only two studies have examined and identified that decreases in While the main strength of the current review is that it was conducted with clinical studies, our review has several limitations. Firstly, we found that a number of studies were conducted with heterogeneous samples of cancer patients diagnosed with advanced metastatic melanoma, HCC, NSCLC and mixed NSCLC and RCC. Despite these issues with heterogeneity, our review shows that clinical response to ICIs was consistently found to be related to alpha diversity in the composition of the gut microbiome. However, heterogeneity in the biasing of results, does have the advantage of increasing the generalizability of those results and more readily allow their application to patients diagnosed with various stages of cancers in real-world clinical practice settings. Furthermore, most studies included in this review analysed fecal samples using a 16S rRNA sequencing method that can measure the composition of gut bacteria from phylum to genus level, and occasionally species level, while limited studies conducted analyses using metagenomics sequencing methods that measure from phylum to species level. Thus, there are wide variances in the composition of gut microbiota reported among studies included in this review . In ordeIn conclusion, our findings from this clinical review demonstrates the potential benefits of utilising the gut microbiome to predict clinical response in advanced cancer patients undergoing ICIs and provides further insight into the gut microbiome in immunotherapy. Moreover, it revealed that gut microbiota can play a crucial role in augmenting the therapeutic effects of immunotherapy in advanced cancer patients who may have limited treatment options. However, several challenges remain to be answered before translating microbiome therapy into routine clinical practice.


PLT transcription and thereby, a finite meristem size. We furthermore demonstrate how this constrained PLT transcriptional domain enables independent control of meristem size and division rates, further supporting a division of labor between auxin and PLT. We subsequently reveal how the weaker auxin antagonism of the earlier active Arabidopsis response regulator 12 (ARR12) may arise from the absence of a DELLA protein interaction domain. Our model indicates that this reduced strength is essential to prevent collapse in the early stages of meristem expansion while at later stages the enhanced strength of Arabidopsis response regulator 1 (ARR1) is required for sufficient meristem size control. Summarizing, our work indicates that functional constraints significantly contribute to shaping the auxin–cytokinin–PLT regulatory network.After germination, the meristem of the embryonic plant root becomes activated, expands in size and subsequently stabilizes to support post-embryonic root growth. The plant hormones auxin and cytokinin, together with master transcription factors of the PLETHORA (PLT) family have been shown to form a regulatory network that governs the patterning of this root meristem. Still, which functional constraints contributed to shaping the dynamics and architecture of this network, has largely remained unanswered. Using a combination of modeling approaches we reveal how the interplay between auxin and PLTs enables meristem activation in response to above-threshold stimulation, while its embedding in a PIN-mediated auxin reflux loop ensures localized  The plant hormones auxin and cytokinin, along with the PLETHORA (PLT) and Arabidopsis response regulators 1 and 12 (ARR1 and ARR12) transcription factors, all play critical roles in controlling root growth and zonation. PLT transcription factors, which form a gradient emanating from the root quiescent center, have been shown to support division and prevent differentiation . A complPreviously, we showed that the auxin-induced PLT transcription factors form a gradient that controls the location of the stem cell region as well as the size of the meristem in a dosage-dependent manner . This PLPLT transcription, while PLTs enhance auxin production and transport. While such positive feedback may be instrumental in meristem activation, how it can be simultaneously consistent with a long-term finite meristem size even in absence of cytokinin antagonisms remains an open question. Indeed, PLT transcription is in fact largely limited to the root’s SCN domain and its direct surroundings. Considering its key importance for SCN and meristem establishment and maintenance, at first sight, a larger transcription domain would seem developmentally more robust. Likely, thus far unidentified selective advantages or functional constraints have contributed to molding this highly constrained PLT transcriptional domain. On a similar note, we discerned separate auxin–PLT–ARR12 and auxin–PLT–ARR1 subnetworks involved in controlling early and late phases of meristem development , an, anPLT tion 4 in A [21,22,ion 4 in A throughImportantly, while cytokinin biosynthesis appears to occur predominantly in the root tip meristem, the downstream cytokinin signalling mediated by ARR-Bs 1 and 12 involved in controlling root tip meristem size is focused in the EZ ,7,25,26.The thus generated model enabled us to reproduce auxin, PLT and ARR-B patterns as well as developmental meristem growth dynamics in wild type  and mutaarr1arr12 double mutant settings. This enabled us to first focus on the auxin and PLT feedback dynamics. Simulations were initialized with a small, germination stage root meristem of 8 rows of cells, subjected to a variable level of shoot-derived auxin influx entering the top of the vasculature , bistability ensures the inheritance of the high auxin high PLT state after each division event F,G. As aPLT transcription domain remained largely constrained to the SCN area. Thus we can exclude PLT dilution as the main determinant of limited meristem expansion in the Root tip model.We first reasoned that actual meristem expansion in planta and in the Root tip model could, at least in part, be constrained by growth-induced PLT dilution. PLT dynamics are governed by slow, high auxin levels requiring expression combined with slow turnover. As a consequence, upon cell growth and division, cytoplasmic and nuclear volume increases cause PLT levels to become diluted. For cells that are displaced through growth to lie outside the high auxin-dependent PLT expression domain centered on the QC, these diluted PLT levels do not become automatically restored through high PLT transcription. Instead, in these cells, the diluted PLT levels result in limited induction of auxin production which in turn only slowly and to a limited extent induces PLT expression. This causes a net decrease of PLT levels after each new round of division, thereby limiting division-mediated growth of the high PLT domain. To investigate this hypothesis we removed division-induced PLT dilution from our model, something that is impossible in planta, observing substantially enhanced, but still limited and auxin sensitive meristem size expansion . StrikinBased on our observation that auxin localization restrained the PLT transcription domain even in absence of PLT dilution, we reasoned that the PIN mediated auxin reflux loop, which focuses the largest fraction of root tip auxin around the quiescent center (QC), might be the necessary factor to limiting meristem expansion. We tested this hypothesis by reducing the strong basal polarity of vascular PINS normally occurring in planta. Specifically, we replaced all PINs across all cell files with a universal but reduced apolar PIN . Next, we re-introduced a weak polarity by increasing the basal PIN level in the vasculature by 10% (to ensure the high auxin domain starts at the tip end of the meristem). In these minimally polar PIN settings, we now observe fully bistable meristem growth dynamics, with, depending on parameter settings, either meristem collapse D,E or unWe thus show the polar transport to be necessary to pin down the auxin PLT positive feedback loop. To also show that it is sufficient, we extend the simplified, Single compartment ODE model to two connected cell compartments, one representing cells in theSCN and one representing cells in the transit-amplifying (TA) domain A. The twRaising the polar transport rate resulted in Having established that the expansion of the meristem in the Root tip model is limited through strongly polarized auxin transport, we next turn to how this finite meristem size is further controlled by cytokinin signalling through ARR 1 and ARR12.Our previous study revealed that in order for our model to reproduce experimentally measured meristem size dynamics, we had to assume that ARR1 not only activates later but also more strongly antagonizes auxin levels than ARR12 . Thus, aThe delayed activity of ARR1 arises from its DELLA-dependent transcription, with DELLA becoming derepressed only at 5dpg when the levels of its repressor giberellin have sufficiently decreased . InteresTo investigate this we set out to locate and identify the binding motif for DELLA on ARR1. All members of the type-B ARR family  previously shown to interact with DELLA do so with the tail end of the protein . We perfWith the molecular basis for the difference between ARR1 and ARR12 auxin antagonism strength established, we set out to investigate the functional significance of having type-B ARRs that not only differ in timing but also affect strength. To investigate this we first quantified the effects of varying ARR12 and ARR1 strengths over a 4-fold range, while keeping their timing dynamics as in the original model C. As expNext, we ran a series of alternative model simulations. In one hypothetical scenario, we let ARR1 which normally becomes active from 5dpg activate already at 36hpg. In another scenario, we let ARR12 which normally activates at 36hpg become active only at 5dpg. That is, in these scenarios we gave the ARRs the timing properties of one another. We compared the outcomes to those of simulations with normal ARR12 and ARR1 timing dynamics. For the wild type and early acting ARR1, simulations were run for two conditions, differing in the amount of shoot-derived auxin influx D. We seeAbove, we established how the auxin–PLT feedback loop enables meristem activation, while its interaction with the PIN reflux loop constrains the PLT transcriptional domain to the SCN thereby limiting meristem expansion. A remaining open question is what the functional consequences of two separate PLT domains, the SCN serving as a PLT producing “source” and the TA “sink” merely inheriting and receiving PLT protein, are.In our earlier work, using a simplified, rectangular root topology and without cytokinin signalling we observed that meristem size increases as a function of higher PLT cell-to-cell movement rates . In our Interestingly, when only increasing cell-to-cell PLT movement in the stem cell region, we observe a larger increase in meristem size and extension of the PLT gradient than when we increased PLT mobility in both the SCN and in the TA E–H,M. InRoot growth rate ultimately depends on the size of the meristem, which sets the number of dividing cells, and the rate of division of these meristem cells. Together these determine the cellular output rate of the meristem. In this context, the division rate of rapid transit-amplifying divisions is seen as a key determinant of meristematic output, while the importance of the rate of stem cell divisions is rarely considered. Based on the observed importance of the SCN domain as a PLT source, with PLT mobility in the SCN having a strong impact on PLT gradient and meristem size, we decided to reconsider these assumptions.We see that increasing SCN division rate by lowering cell cycle duration from 20 h to 14 h increases the number of transit-amplifying meristem cells  and therThe importance of the plant hormones auxin and cytokinin, as well as the PLT transcription factors in controlling root developmental zonation as well as root growth rate, has long been appreciated ,3,4,5,35In a recent study, we demonstrated that the auxin dependence of ARR1 and ARR12, combined with their reciprocal antagonism with auxin and PLTs, play a key role in patterning the meristematic PLT and EZ cytokinin signalling domain . In the Through combining our Root tip model with a simplified Single-compartment model we demonstrated that the positive feedback between auxin and PLT can give rise to bistability. This bistability enables the switching of the root meristem from an inactive low auxin–low PLT state, to an active, division supporting high auxin–high PLT state when stimulated beyond a certain threshold, as occurs during seed germination.Still, despite dividing meristem cells inheriting the maternal high auxin–high PLT state, both experimental data and our Root tip model simulations indicate that even in absence of antagonistic cytokinin signalling, after an initial growth phase meristem size stabilizes. Expanding our Single compartment to a Two-compartment model incorporating auxin transport we subsequently demonstrated that this stabilization of meristem size arises from the PIN mediated focusing of auxin around the QC. Due to this focusing of auxin, the division induced displacement of cells away from the SCN results in a substantial drop of auxin levels and a subsequent slower decrease in PLT levels that prevent the unlimited maintenance of the initially inherited high auxin–high PLT stateThus, locally, for an initial small meristem, the feedback between auxin and PLT enables an all-or-none bistable switch, while globally, for a larger meristem the presence of the auxin reflux loop prevents limitless meristem expansion and instead allows for a graded, auxin-dependent finite meristem size. This combination of all-or-nothing and graded behavior, with the all-or-nothing behavior relying on positive feedback and the gradedness on the spatially or temporally graded nature of recruiting localized positive feedback, has been observed in multiple biological systems. Examples are the calcium-induced calcium release in cardiac muscle cells which amplitude governs muscle contraction strength ,37 and tMutual repression is a frequently encountered motif in developmental patterning systems in which a boundary between two alternative cell fates or behaviors is being imposed, with the Drosophila segmental patterning systems being one of the most well-studied examples ,41,42,43Combined, these ARRs provide for the regulation of root tip meristem size, in addition to the reflux loop-mediated constraint. Importantly, ARR12 and ARR1 do not merely differ in auxin antagonism strength but also the time of action, with ARR12 becoming active from 36 hpg, while ARR1 is active only from 5dpg. Here, we used our model to demonstrate that in absence of normal, early ARR12 activity, meristem size initially overshoots final stable meristem size, whereas non-normal, early ARR1 activity causes the meristem to become vulnerable to collapse. This indicates that early in development when the auxin PLT feedback loop is gearing up ARR activity is needed to control meristem growth yet can not antagonize auxin too strongly. Later, when the high auxin high PLT state is firmly established, a stronger antagonism is needed to sufficiently control meristem size.Previously, we established the importance of a limited, prolonged high-auxin dependent PLT transcriptional domain combined with a longer range, division and cell-to-cell motion dependent PLT protein gradient . We demoHere we further probed the relevance of the PLT transcribing SCN and non-transcribing proximal meristem subdivision by independently altering the division rate or plasmodesmatal aperture in either of these two domains. Model outcomes show that the SCN is in control of the precise translation of auxin signals into a PLT gradient. Through affecting SCN PLT transcription level, plasmodesmatal aperture or division speed, meristem size is affected, while changes in division or PLT transport in the rest of the meristem hardly affect meristem size. This enhances the previously established division of labor between auxin and PLTs , showingA potential limitation of our model is that it does not incorporate plasmodesmatal auxin transport , and actInterestingly, under phosphate starvation, enhanced uptake of iron results in reduced plasmodesmatal aperture and subsequent reduction of main root meristem size . Thus faSummarizing, we uncovered how the auxin-PLT positive feedback loop embedded in the root tip auxin reflux loop enables the robust and reproducible activation of finite meristems, with additional size control by ARR12 and ARR1 tuned to early and late phases of meristem development. Additionally, we discovered a hitherto underappreciated role for the SCN in controlling meristem size and output through variations in local division rates and plasmodesmatal aperture, enabled through the confinement of PLT transcription to this niche.In this paper, we use 3 distinct but related models to better understand how the feedbacks between Auxin, PLT and the ARR-Bs control root tip growth dynamics.The first model is a 2D anatomically detailed Root tip model based on the Salvi et al. 2020 model, enhanced and altered to better fit experimental observations. In this model, cells can grow and divide into daughter cells, undergo vacuolar expansion and differentiation. Cells make these developmental decisions based on the state of their hormonal-gene regulatory network. For a more detailed overview of the model and its parameters see the Auxp is the auxin production rate, Plts,AuxKM is the PLT concentration for which auxin production is half maximum, Auxd is the auxin degradation rate, influx is the auxin influx rate, Pltsp is the PLT production rate, Auxin,PltsKM is the Auxin concentration for which PLT production is half maximum, and Pltsd is the PLT degradation rate. For the derivation of these equations see the The second model is a single compartment ODE system that captures in a simplified manner the essence of the Auxin–PLT positive feedback loop dynamics The model on the one hand serves as a minimal model enabling detailed phase plane and bifurcation analysis, while on the other hand allows us to identify which properties of the multi-scale Root tip model are of a more complex, emergent nature. The Single-compartment model is described by the following set of equations:ApolarTrans is the baseline apolar auxin transport rate between cell compartments and PolarTrans is the rate at which auxin is rapidly transported from the TA to the SCN, SCNdil is the effective growth dilution rate in the SCN (assuming a 22 h division rate) and TAdil is the effective growth dilution rate in the TA (assuming an 11 h dilution rate). The default parameters of the Two-compartment model used in this paper are given in The Two-compartment model is an extension of the above described Single-compartment model, generated through coupling two compartments to model an SCN compartment and a TA compartment connected through auxin transport. To model the effect of cell growth and division, we incorporate a PLT protein level division rate-dependent dilution term. In addition, we add PIN-mediated transport between the two compartments of which the polarity/directionality can be varied, and shoot-derived auxin influx into TA compartment. Combined this results in the following system of equations:https://tbb.bio.uu.nl/rdb/grindC.html, accessed on 12 March 2021) developed by Prof. Rob de Boer  [For the Single and Two-compartment models, we used GRIND (erlands)  to run tWe aimed to compare the known DELLA binding ARR-Bs ARR1, ARR2, and ARR14 to the two ARR-Bs known to be present and active earlier in development when Gibberellin levels are still high and therefore DELLA levels are low ,26, name


Nonalcoholic fatty liver disease (NAFLD) has emerged as a leading cause of chronic liver disease worldwide in the past few decades as a consequence of the global obesity epidemic and is associated with significant morbidity and mortality. NAFLD is closely associated with components of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, suggesting a plausible metabolic mechanistic basis. Metabolic inflexibility is considered a nidus for NAFLD pathogenesis, causing lipotoxicity, mitochondrial dysfunction and cellular stress leading to inflammation, apoptosis and fibrogenesis, thus mediating disease progression into nonalcoholic steatohepatitis (NASH) and ultimately cirrhosis. In this review, we describe they key metabolic drivers that contribute to development of NAFLD and NASH, and we explain how NASH is a metabolic disease. Understanding the metabolic basis of NASH is crucial for the prevention and treatment of this disease. Non-alcoholic fatty liver disease (NAFLD) has become one of the most common causes of chronic liver disease worldwide and is rapidly becoming the most common indication for liver transplantation , 2. It iStrong evidence has shown that the metabolic basis of NAFLD is a part of a metabolic disease cluster due to its association with obesity, insulin resistance, type 2 diabetes mellitus (T2DM), hyperlipidemia and cardiovascular disease (CVD) . IndividIn contrast, the Mediterranean diet plays a beneficial role in reducing liver fat and improving cardiovascular risk in patients with NAFLD . This diMetabolic flexibility refers to the body’s ability to adequately handle substrates and maintain energy homeostasis Figure 1The resultant high influx of FFAs to the liver combined with increased de novo lipogenesis leads to lipotoxic stress. The process of conversion of FFAs to TG within hepatocytes inhibits peroxisome proliferator‐activated receptor‐α (PPARα) signalling  and thusThere is growing body of evidence supporting the role of gut microbiome in the development and progression of NAFLD Table 1 Recently, there has been an increased interest in investigating the role of skeletal muscles in the pathogenesis of NAFLD . SarcopeIt is now recognized that the muscle is a key metabolic organ and buffers the functions of the liver. While its role as an ammonia buffer is well known with the hepatology community, its role in maintaining body composition is less well appreciated. Muscles primarily utilize free fatty acids in the fasted state and are critical for mobilization of fat stores with diet and exercise. In the metabolically inflexible sarcopenic state, it depends on glucose as its principal substrate for energy and switches to protein breakdown when glucose is not available further contributing to sarcopenia. A host of myokines have been described of which myostatin is the best known –75. TheyFrom a pathophysiological perspective, striated muscles play a key role in metabolic homeostasis –79. UndeKey pathogenic mechanisms driving the progression from hepatic steatosis to NASH include aberrant lipid metabolism, oxidative stress, mitochondrial dysfunction, inflammatory cytokines, immune response and alterations in gut microbiome . Lipotox(PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7), glucokinase regulator (GCKR), hydroxysteroid 17-beta dehydrogenase-13 (HSD17B13) have been identified as key modifiers of NAFLD development and progression  secretion and lipoprotein metabolism. The rs58542926 C>T polymorphism results in loss of this gene’s function or reduction of its hepatic expression, leading to impaired secretion of TG‐rich lipoproteins and increased hepatocyte TG content  and aspartate aminotransferase (AST) levels , are more likely to develop steatohepatitis and fibrosis  levels , 119. Pafibrosis , 121. Stfibrosis , 123.PNPLA3 I148M variant led to NASH and fibrosis focused on its function as a triglyceride lipase and the loss of function with this mutation . In addition, PNPLA3-I148M significantly worsened the severity of histological features of NASH including steatosis, hepatocellular ballooning and lobular inflammation (p< 0.001). The principal observation in this study is that the hallmark feature linked to PNPLA3-I148M-induced NASH acceleration is ‘metabolic reprogramming’ of the liver with increased TGs and diglycerides, n3 polyunsaturated fatty acids depletion and increased ceramides. PNPLA3-I148M also had a significant impact on several cellular processes including proteosomal, phagosomal, and lysosomal function, amino-acyl T RNA synthesis, circadian rhythm and ER function. PNPLA3-I148M expression promoted cell death and increased oxidative stress and ER stress. Multiple inflammatory pathways were activated with PNPLA3-I148M, such as JAK-STAT system. Specifically, the STAT3 system was activated significantly in the presence of the I148M variant. Integrated pathway analysis revealed a strong signature of ceramide related inflammatory activation including h-ras and downstream PLA2 activation, suggesting a key role for sphingolipids in the inflammatory response to PNPLA3-I148M. Furthermore, PNPLA3-I148M was associated with activation of several fibrogenic pathways such as those for Procollagen I, III and α-smooth muscle actin mRNA, as well as increased transforming growth factor beta (TGF-β1) protein levels  and its progression to cirrhosis represents a complex set of gene-environment interactions. While environmental factors such as changes in diet play a central role, the role of other factors such as environmental pollutants are also becoming apparent and worthy of further investigation.148M variant of PNPLA3 and progressive NASH, an increasing number of genetic variants have also been linked to the disease. However, for several of these the specific mechanisms by which they affect NASH have not been clarified. Particularly the HSD17B13 splice variant which has a protective role is an important new discovery and future studies will be needed to define how it contributes to the disease and even potentially negates some of the deleterious effects of the PNPLA3 mutation.The role of genetics in modulating the metabolic drivers of NASH will be a major area of research over the next decade. Following the landmark studies identifying a linkage between the IIt is further clear that individual mutations can not only have an impact based on their biological effect but multiple mutations in different genes in various metabolic pathways can interact to modulate the metabolic response to the lipotoxic load being delivered to the liver. Development of a rigorously validated polygenic risk score remains an important unmet need in the field.A key focus of this manuscript has been the growing body of literature on the role of the extrahepatic milieu as a cause of the liver disease. Much more work needs to be done on the relationship between changes in diet and the changes in intestinal epithelium and how these alter the meal stimulated gut hormonal response. This is likely to be a key modulator of the state of metabolism and the development of both the metabolic syndrome and fatty liver disease. Early data from the metabolic benefits of duodenal mucosal resurfacing provide a strong rationale to continue to pursue this line of research. These are also likely to yield potential therapeutic approaches beyond duodenal mucosal surfacing.The role of the intestinal microbiome continues to be elucidated as well. There is already a plethora of literature demonstrating changes in specific taxa that are linked to the NASH and even advanced fibrosis. While some data on metagenomics are available, they are not tightly concordant with the metabolomic signatures in stool and direct interrogation of the microbial transcriptome will be needed to better understand the role of altered microbial taxa in NASH. Further functional analyses of changes in the microbiome will provide mechanistic insights into the nature of microbial contribution to the disease and its progression. It will also set the stage for phase-based therapeutics for NASH.Additional intestinal factors such as secondary bile acids are likely to also be important. Many bacteria are susceptible to the antibiotic functions of secondary bile acids and decrease when secondary bile acids increase whereas bacteria that require such bile acids for growth increase. These may provide a potential explanation for the changes in microbial composition. Further systemic uptake of secondary bile acids may have direct effects on metabolism via FXR, TGR5 and affect processes such as senescence to cause hepatocellular cancer.Finally, the importance of striated muscle and adipose tissue in driving NASH is also becoming apparent and direct modification of muscle function by specifically engineered diets designed to break metabolic inflexibility to improve the systemic metabolic state are likely to improve not only NAFLD but the systemic state of dysmetabolism.In summary, while much progress has been made in understanding the metabolic drivers of NASH, much additional work remains to be done. These are likely to have a major impact on the understanding of the role of the systemic changes in metabolic syndrome and how these lead to NASH. Gene-environmental interactions and studies to define subpopulations based on clustering of specific genetic and environmental factors may provide insights on the heterogeneity of NAFLD and new approaches to treat individual patients.Both authors contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.AJS is President of Sanyal Biotechnology and has stock options in Genfit, Akarna, Tiziana, Indalo, Durect, Exhalenz and Hemoshear. He has served as a consultant to Astra Zeneca, Conatus, Coherus, Bristol Myers Squibb, Blade, Tobira, Takeda, Siemens, Merck, Genentech, Tern, Gilead, Lilly, Poxel, Artham, Boehringer Ingelhiem, Novo Nordisk, NGM Bio, Birdrock, Novartis, Pfizer, and Genfit. He has been an unpaid consultant to Intercept, Echosens, Perspectum, Immuron, Galectin, Fractyl, Affimune, Chemomab, Nordic Bioscience. His institution has received grant support from Gilead, Salix, Tobira, Intercept, Bristol Myers, Shire, Merck, Astra Zeneca, Malinckrodt, Cumberland and Novartis. He receives royalties from Elsevier and UptoDate.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.