Embracing the (r)evolution of social media and digital scholarship in pediatric nephrology education.

Free Open-Access Medical Education (FOAMed) has transformed medical education in the past decade by complementing and substituting for traditional medical education when needed. The attractiveness of FOAMed resources is due to their inexpensive nature, wide availability, and user ability to access on demand across a variety of devices, making it easy to create, share, and participate. The subject of nephrology is complex, fascinating, and challenging. Traditional didactic lectures can be passive and ineffective in uncovering these difficult concepts and may need frequent revisions. Active teaching methods like flipped classrooms have shown some benefits, and these benefits can only be multifold with current social media tools. Social media will inspire the involvement of students and allow them to create and share educational content in a "trendy way," encouraging the participation of their peers and thus building an educational environment more conducive to them while promoting revision and retainment. FOAMed also promotes asynchronous learning, spaced learning, microlearning, and multimodal presentation with a meaningful variation. This article discusses the evolution of digital education, social media platforms, tools for creating and developing FOAMed resources, and digital scholarship.

Establishing content validity for the migraine Global Impression Item (mGI-I) assessment: a modified single-item migraine symptom severity questionnaire.

OBJECTIVE: To establish content validity of a single-item, migraine-specific symptom severity questionnaire for completion by migraine patients, key family members (KFMs) of migraine patients, and Healthcare Professionals (HCPs) who treat migraine patients. BACKGROUND: Migraine is a common disabling primary headache disorder with high prevalence and significant socioeconomic burden and personal impacts. There is a need for a global assessment of migraine symptom severity to evaluate potential new therapies from multiple perspectives. METHODS: The migraine Global Impression Item (mGI-I) was drafted and tested in a non-interventional, qualitative study comprising telephone interviews with 15 migraine patients, 15 KFMs of migraine patients, and 15 migraine treating HCPs. The mGI-I was drafted with two different item stem options and two different response scale options to ask about the patient's migraine from the perspective of each respondent. Cognitive interviews were conducted to test comprehensiveness, clarity and ease of completion of the different versions of the mGI-I iteratively in three sequential waves of respondents. RESULTS: Revisions were made to the draft mGI-I after Wave 1 and Wave 2 of the interviews. Changes were made to simplify the item stem (removing unnecessary text), make language more patient-friendly (e.g. use of "migraine attack"), and add clarity to the item stem for consistent interpretation (include descriptive language of migraine attacks). Across both waves there was a preference for a 5-point response scale compared to a 7-point scale. In Wave 3, all respondents found the revised instructions, item stem, and 5-point response scale comprehensive, easy to understand and to answer. No further changes to the mGI-I were made after Wave 3. CONCLUSIONS: This qualitative study of 45 total respondents across 3 subpopulations, established the content validity and appropriateness of the mGI-I in migraine patients, KFMs, and migraine-treating HCPs. The study specifically confirmed that the mGI-I is comprehensive, easily understood and answered for each respondent population.

Reducing the physical, social, and emotional impact of episodic migraine: Results from erenumab STRIVE and ARISE phase III randomized trials.

OBJECTIVE: The purpose of this study was to examine changes in the functional impact of migraine following treatment with erenumab, as measured by the Migraine Functional Impact Questionnaire (MFIQ). BACKGROUND: The MFIQ, a novel patient-reported outcome (PRO) measuring the impact of migraine on four domains (physical function, social function, and emotional function [PF, SF, and EF]; usual activities [UAs]) and a single item assessing overall impact on UA, was included in phase III trials evaluating erenumab 70 and 140 mg monthly for migraine prevention among people with episodic migraine (EM). METHODS: In the ARISE study, 577 patients with EM were randomized to erenumab 70 mg or placebo. In the STRIVE study, 955 patients with EM were randomized to erenumab, 70 mg or 140 mg or placebo. Pairwise comparisons of least-squares mean (LSM) change from baseline in MFIQ scores (with associated 95% confidence interval [CI]) were assessed for each active treatment versus placebo. RESULTS: In ARISE, greater reductions from baseline to month 3 were observed for 70 mg versus placebo for PF (LSM [95% CI]: -3.2 [-6.4 to -0.1]; p = 0.046) and EF (-4.0 [-7.3 to -0.7]; p = 0.019) domain scores. In STRIVE, between-group differences also reflected reductions from baseline to the average of months 4-6 that favored erenumab on all four MFIQ domain scores. Reductions in impact for 70 mg compared to placebo were -4.3 (95% CI: -6.8 to -1.7; p < 0.001) for PF, -4.0 (-6.3 to -1.7; p < 0.001) for UA, -3.7 (-6.1 to -1.2; p = 0.003) for SF, and -5.3 (-7.9 to -2.6; p < 0.001) for EF domain scores. Improvements were also observed for 140 mg versus placebo with between-group differences of -5.7 (95% CI: -8.2 to -3.2; p < 0.001) in PF, -5.1 (-7.5 to -2.8; p < 0.001) in UA, -5.0 (-7.4 to -2.6; p < 0.001) in SF, and -7.2 (-9.9 to -4.5; p < 0.001) in EF domain scores. There were also greater improvements in the overall impact on UA score for 70 mg (LSM [95% CI]: -4.3 [-7.0 to -1.7]; p = 0.001) and 140 mg (-5.3 [-8.5 to -3.2]; p < 0.001) versus placebo. CONCLUSIONS: The MFIQ measures the frequency of impacts and level of difficulty on multiple functional domains that provide a more complete picture of the effects of migraine. MFIQ scores showed that in comparison with placebo, patients treated with erenumab had greater reductions in the functional impact of migraine, providing insight into treatment benefits that extend beyond improvements in clinical status and health-related quality of life previously reported based on clinical end points and other PROs.

Role of therapeutic apheresis in the treatment of pediatric kidney diseases.

Therapeutic apheresis utilizes apheresis procedures in the treatment of a variety of conditions including kidney disease. Therapeutic plasma exchange (TPE) is the most common modality employed with the rationale of rapid reduction of a pathogenic substance distributed primarily in the intravascular compartment; however other techniques which adsorb such pathogenic substances or alter the immune profile have been utilized in diseases affecting native and transplanted kidneys. This article discusses the modalities and technical details of therapeutic apheresis and summarizes its role in individual diseases affecting the kidney. Complications related to pediatric apheresis procedures and specifically related to apheresis in kidney disease are also discussed. Though therapeutic apheresis modalities are employed frequently in children with kidney disease, most experiences are extrapolated from adult studies. International and national registries need to be established to elucidate the role of apheresis modalities in children with kidney disease.

Erenumab patient characteristics, medication adherence, and treatment patterns in the United States.

OBJECTIVE: To describe patient characteristics, adherence, and treatment patterns, among adult migraine patients in the United States prescribed erenumab. BACKGROUND: Migraine is a highly prevalent and debilitating disease characterized by recurrent attacks of moderate to severe headache accompanied by non-headache symptoms. Erenumab is a first-in-class calcitonin gene-related peptide receptor (CGRP-R) antagonist indicated for migraine prophylaxis in adults. METHODS: This retrospective longitudinal cohort study used IQVIA's open-source longitudinal pharmacy (LRx) and medical (Dx) claims databases to identify adult migraine patients with an initial claim (index date) for erenumab between May 1, 2018 and April 30, 2019. Patients were required to have ≥180 days of follow-up. Erenumab dosing patterns, persistence, and adherence (using medication possession ratio [MPR] and proportion of days covered [PDC]), and discontinuation of other commonly prescribed acute and prophylactic anti-migraine therapies were assessed. Dose changes in acute therapies after initiation of erenumab were assessed in a subset of patients with an adequate trial of erenumab (≥2 additional erenumab claims within the 80 days following the index claim). RESULTS: A total of 64,174 patients met the study criteria. Mean (SD) age was 48 (13) years and 85.2% (n = 54,656) were female. The initial erenumab dose was 70 mg for the majority of patients (65.1%; n = 41,790); most (81.4%; n = 34,019) maintained their index dose during follow-up. Overall, 30.8% (n = 19,797) of patients had a PDC ≥ 0.80 and 41.7% (n = 26,769) had a MPR ≥ 0.80. Discontinuation rates of acute and other prophylactic migraine therapies after initiation of erenumab (among users of the respective therapies) were 48.7% (22,965/47,190) and 36.1% (16,602/46,006), respectively. Dose decreases among triptan, ergot compound, opioid, and barbiturate users were observed after initiation of erenumab. CONCLUSIONS: Almost all patients had prior use of acute or preventive therapy. Adherence to erenumab was higher than traditional oral prophylactic migraine therapies; however, overall adherence was still suboptimal. The decrease in use of acute and preventive prescription medications following initiation of erenumab suggests effectiveness in the real-world setting.

Understanding the migraine treatment landscape prior to the introduction of calcitonin gene-related peptide inhibitors: Results from the Assessment of TolerabiliTy and Effectiveness in MigrAINe Patients using Preventive Treatment (ATTAIN) study.

BACKGROUND: Calcitonin gene-related peptide (CGRP) inhibitors were introduced in the United States (US) in 2018. To understand the changing patterns of preventive treatment following the introduction of these new agents, we must first characterize the patterns which preceded their introduction. OBJECTIVE: To characterize the burden, unmet need, and treatment patterns in patients with migraine initiating preventive migraine medications before the introduction of CGRP inhibitors in the US. METHODS: Between March 2016 and October 2017, we enrolled episodic (EM) and chronic migraine (CM) patients initiating or changing preventive treatment at primary care or neurology clinic visits in the US, in a real-world observational study using a prospective cohort design. At baseline and monthly thereafter for 6 months, we collected data from study sites and patients on migraine frequency, treatment modifications, migraine impact on functioning, and work productivity for a descriptive analysis of migraine patient experience and treatment patterns. RESULTS: From the sample of 234 completers, 118 had EM (50.4%) and 116 had CM (49.6%). Mean age at enrollment was 41 years (SD = 12) and mean age at first migraine diagnosis was 22 years (SD = 11). Most participants were females (n = 204/234; 87.2%) and white (n = 178/234; 76.1%). The majority (n = 164/234; 70.1%) had not used preventive migraine treatment in the 5 years prior to enrollment (treatment naïve). At baseline, mean monthly migraine days were 9.6 days (SD = 5.0) for the preventive treatment naïve group and 12.4 days (SD = 7.0) for treatment experienced patients. The majority had severe Migraine Disability Assessment (Grade IV, total score ≥21), including 67.1% (n = 110/164) of the preventive treatment naïve and 77.1% (n = 54/70) of the preventive treatment experienced patients. Headache Impact Test total scores indicating severe impairment (score >59) occurred in 88.4% (n = 145/164) of the treatment naïve and 88.6% (n = 62/70) of treatment experienced patients. Mean work productivity loss as measured by the Work Productivity and Activity Impairment questionnaire in the subsample of employed patients was 53.3% loss. The most used acute medications at baseline were nonsteroidal anti-inflammatory agents (n = 124/234; 53.0%), acetaminophen-based products (n = 112/234; 47.9%), and triptans (n = 105/234; 44.9%). The most commonly initiated preventive treatments were topiramate (n = 100/234; 42.7%), tricyclic antidepressants (n = 39/234; 16.7%), beta-blockers (n = 26/234; 11.1%), and onabotulinumtoxinA (n = 24/234; 10.3%). Over the 6-month follow-up period, almost half of patients (n = 116/234, 49.6%) modified their preventive treatment and discontinued treatment (n = 88/312 total modifications; 28.2%) or modified their pattern of use by increasing, decreasing, or skipping doses (n = 224/312 total modifications; 71.8%), often without seeking medical advice. Avoiding side effects was the main reason reported among patients who discontinued (n = 52/88; 59.1%), decreased frequency or dose (n = 37/89; 41.6%), and skipped doses (n = 29/86; 33.7%). Perceived lack of efficacy was another frequent reason reported among those who discontinued (n = 20/88; 22.7%), decreased frequency or dose (n = 15/89; 16.9%), and skipped doses (n = 18/86; 20.9%). Despite initiation of preventive treatment and improvements observed in number of headache and migraine days, migraine patients continued to experience substantial disability, headache impact, and reduced productivity throughout the 6-month follow-up period. CONCLUSIONS: Prior to 2018, the burden of migraine was high for patients initiating preventive treatments. Despite having more than 9 days of migraine per month on average, the majority (70.1%) of patients initiating prevention had been treatment naïve, indicating underuse of preventive treatments. The preventive treatments used in this study were poorly tolerated and were reported by patients to lack efficacy, resulting in suboptimal adherence. The high discontinuation rates suggest that the preventive medications being offered during the period of the study did not meet the treatment needs of patients. In addition, the decisions by about half of patients to alter their prescribed treatment plan without consulting their provider can pose substantial health risks. These findings pertain to the broad set of preventive treatments initiated in this study and do not support inferences about individual preventive treatments, due to limitations in sample size. These findings suggest the need for more effective and better tolerated preventive treatment options.

Therapeutic plasma exchange: single-center experience in children with kidney disorders.

BACKGROUND: Therapeutic plasma exchange (TPE) is used in kidney diseases as an adjunct treatment. Little has been described as to its effectiveness in kidney disorders in children. This study aimed to assess the safety, efficacy, and outcomes of patients who underwent TPE for kidney indications. METHODS: Retrospective chart review of patients receiving TPE from 2010 to 2018 for kidney indications, such as antibody-mediated rejection, bone marrow transplant-associated thrombotic microangiopathy (TA-TMA), atypical hemolytic uremic syndrome, transplant recurrence of focal segmental glomerulosclerosis, and glomerulonephritis. Outcomes assessed were trends in kidney function, mortality, and progression to stage 5 chronic kidney disease (CKD 5). Significant hypocalcemia was defined as ionized calcium < 1 mmol/L. RESULTS: A total of 641 TPE procedures were performed on 47 patients (25 male). Average age was 12.8 ± 5.9 years. Median glomerular filtration rate (GFR) improved from baseline to end of TPE treatments (pre 44.9 (19.8, 79), end 56.1 (23, 98) [p = 0.02]). Ten out of 47 children developed CKD 5. Seven out of 47 patients died; 5 had TA-TMA. Initial 7 consecutive sessions were reviewed for complications. Among 335 procedures, 41 episodes of significant hypocalcemia were noted (12.2%); only 1 was symptomatic. Of the 26 episodes (7.7%) of allergic reactions, all were associated with the use of FFP; 5 were anaphylactic. No TPE-associated mortality was noted. CONCLUSIONS: TPE is a relatively well-tolerated useful adjunct therapy in children with kidney indications. The benefit of TPE has to be balanced with risks such as hypocalcemia and allergic reactions which can occur more frequently with FFP. Graphical abstract.

United States Patients' Perspective of Living With Migraine: Country-Specific Results From the Global "My Migraine Voice" Survey.

BACKGROUND: Migraine is associated with debilitating symptoms that can affect daily functioning. "My Migraine Voice" was a large, cross-sectional, multi-country online survey aimed at understanding disease burden directly from people with migraine. OBJECTIVE: This study reports on the social and economic impacts of migraine, specifically the impact on activities of daily living and the costs of migraine, from the point of view of people with migraine in the United States. METHODS: The online survey was administered to adults with a self-reported diagnosis of migraine who experienced 4 or more monthly migraine days each month for the previous 3 months. Prespecified screening quotas were used so that 90% of respondents reported current or past use of preventive migraine medication, 80% of whom switched treatment (ie, changed their prescribed preventive medication at least once). The remaining 10% were preventive treatment naïve (ie, never used any prescribed preventive medication). Burden of migraine on activities of daily living and caregivers (eg, functional limitations, fear of next migraine attack, sleep problems) and economic burden (eg, out-of-pocket costs, impact on work productivity using the validated work productivity and activity impairment questionnaire) reported by respondents from the United States are presented. Results are stratified by employment status, migraine frequency (chronic vs episodic migraine), and history of preventive treatment. RESULTS: Thousand hundred and one individuals with migraine from the United States responded to the survey. Respondents reported limitations completing daily activities during all migraine phases, including during the premonitory/aura and postdrome phases. Most (761/1101 (69%)) relied on family, friends, or others for help with daily tasks and reported being helped a median of 9 days (25th percentile 5 days, 75th percentile 15 days) within the last 3 months. Respondents with chronic migraine reported being helped for more days (median 10 days, 25th percentile 5 days, 75th percentile 23 days) in the last 3 months. Almost all (962/1101 (87%)) experienced sleep difficulties and 41% (448/1101) (48% (336/697) of those with 2 or more preventive treatment failures) were very or extremely fearful of a next migraine attack. Median (25th percentile, 75th percentile) monthly out-of-pocket costs of $90.00 ($30.00, $144.00) in doctor's fees (n = 504), $124.00 ($60.00, $234.00) in health insurance (n = 450), $40.00 ($20.00, $100.00) for prescriptions (n = 630), and $50.00 ($0.00, $100.00) for complementary therapies (n = 255) were reported. Those with 2 or more preventive treatment failures reported higher monthly out-of-pocket doctor fees (median $99.00 ($30.00, $150.00), n = 388). Among employed respondents (n = 661), migraine resulted in 22% absenteeism, 60% presenteeism, 65% work productivity loss, and 64% activity impairment. CONCLUSIONS: Migraine impacts individuals' activities of daily living, work-life, and financial status, especially individuals with high needs, namely those with 4 or more monthly migraine days and prior treatment failures. People with migraine are impaired during all migraine phases, experience fear of their next migraine attack and sleep difficulties, and pay substantial monthly out-of-pocket costs for migraine. Burden is even greater among those who have had 2 or more preventive treatment failures. Impacts of migraine extend beyond probands to caregivers who help people with migraine with daily tasks, employers who are affected by employee absenteeism, presenteeism, and reduced productivity, and society which is burdened by lost and reduced economic productivity and healthcare costs.

Ambulatory blood pressures and central blood pressures are associated with cardiovascular morbidity in adolescent and young adult patients receiving chronic hemodialysis.

BACKGROUND: Left ventricular hypertrophy (LVH) and arterial stiffness measured by pulse wave velocity (PWV) are independent predictors of cardiovascular (CV) mortality in adults receiving chronic dialysis. Hypertension strongly associates with elevated left ventricular mass index (LVMI) and PWV, with ambulatory blood pressure monitoring (ABPM), and central blood pressure (CBP) superior to office blood pressures (BP) in predicting CV morbidity. Few studies have described associations of office BP, ABPM, and CBP with LVMI and PWV in adolescent and young adult patients receiving hemodialysis (HD). METHODS: Cross-sectional study of 22 adolescents and young adults receiving chronic HD. Pre- and post-dialysis office BP and CBP using applanation tonometry were obtained. Twenty-four-hour ABPM was obtained midweek post-dialysis. Pre- and post-dialysis carotid-brachial PWV were obtained same day as BP measurements. Annual echocardiograms for standard care were reviewed for LVH. RESULTS: Pre-dialysis CBP index correlated with LVMI (r = 0.3, p = 0.04) and PWV (r = 0.48, p = 0.02). Hypertensive patients identified by ABPM had worse LVMI; daytime ABPM systolic BP index correlated with LVMI (r = 0.5, p = 0.02). Office BP was not associated with LVMI; only office diastolic BP was associated with PWV (r = 0.46, p = 0.02). There was no correlation of LVMI or PWV with bone health parameters, anemia, interdialytic weight gain, or residual renal function. CONCLUSIONS: Ambulatory blood pressure monitoring is superior to casual office BP obtained at time of dialysis in delineating cardiovascular morbidity in adolescent and young adult HD patients. CBP is easily performed and correlates with LVMI and PWV in adolescent and young adult HD patients; however, large-scale normative data is needed.

Cinacalcet for secondary hyperparathyroidism in end-stage renal disease patients below age 5 years
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BACKGROUND: Management of chronic kidney disease mineral bone disorder (CKD-MBD) in pediatric patients with end-stage renal disease (ESRD) is challenging. While the use of calcimimetics is well-studied in adults on dialysis, few studies have been performed in pediatrics. Little is known about the use of cinacalcet in young children with ESRD. The aim of this study was to report the efficacy and safety of cinacalcet for treatment of secondary hyperparathyroidism in chronic dialysis patients younger than 5 years. MATERIALS AND METHODS: We included children aged < 5 years on chronic dialysis, either hemodialysis (HD) or peritoneal dialysis (PD), who were prescribed cinacalcet for more than 1 month. Retrospective chart review was performed to gather demographics, dialysis prescription, relevant mineral imbalance laboratory parameters, and medications. Data was collected for 6 consecutive months. RESULT: 18 patients (9 male), mean age at initiation of cinacalcet was 2.3 years; 13 PD and 5 HD. Average starting dose of cinacalcet: 6.2 mg daily, 0.55 mg/kg/day. Average time on dialysis was 14.4 months. Parathyroid hormone significantly decreased over the 1st month following initiation of cinacalcet from 929 (IQR 572 - 1,056) to 427 (IQR 256 - 778) pg/mL, p = 0.009. Three patients developed asymptomatic hypocalcemia (Ca < 9.4 mg/dL). Serum phosphorous decreased after initiation, and this was persistent at 6 months. Significant improvement in linear growth was observed while on cinacalcet and growth hormone (GH). CONCLUSION: Cinacalcet can be effectively used in young children on dialysis with minimal side effects. Good linear growth was seen in patients on cinacalcet and GH therapy. Long-term large scale data is necessary to confirm. Institution-based management algorithm incorporating cinacalcet would be helpful to maintain uniformity in role of cinacalcet for management of CKD-MBD.

Faster rate of blood volume change in pediatric hemodialysis patients impairs cardiac index.

BACKGROUND: Intradialytic hypotension and myocardial stunning are proposed as contributing to the pathogenesis of increased cardiovascular disease burden and death in patients receiving maintenance hemodialysis (HD). Noninvasive cardiac output measurements provide a dynamic, real-time assessment of hemodynamic parameters. We investigated intradialytic changes in hemodynamic parameters in pediatric outpatients receiving chronic HD and determined patient and treatment risk factors associated with such intradialytic changes. METHODS: Hemodialysis was performed using linear fluid removal over 4 h with polysulfone dialyzers. Continuous wave Doppler ultrasound was used to measure hemodynamic parameters prior, 2 h into, and after the mid-week HD treatment session. Pulse wave tonometry was performed at the same time. The percentage change in blood volume was measured by noninvasive hematocrit monitoring during HD. RESULTS: Twenty-two patients fit the inclusion criteria, of whom 16 (73 %) were male. The mean age of the patients was 17 ± 3.8 years, and the dialysis vintage was 47.8 ± 33.7 months. The cardiac index decreased significantly midway through the HD treatment session and remained low until the end of treatment. A significant decline in cardiac index without hypotension occurred in 12 (54 %) patients. Expected increase in systemic vascular resistance index to preserve the cardiac index was not observed. Weight, percentage fluid overload, dialysis vintage, and adequacy did not correlate with the observed decline in the cardiac index. The decrease in blood volume at the 2 h (R = 0.43, p = 0.045) and 4 h (R = 0.56, p = 0.007) time points was the only factor associated with cardiac index decline. CONCLUSION: The cardiac index and stroke volume decreased significantly during the HD session. Patients with larger blood volume changes during the first 2 h of HD and at 4 h showed a significant decrease in cardiac index that did not recover at the completion of the HD treatment. Rate of fluid removal was the only significant risk factor for compromised cardiac index during HD. Conventional methods currently used for assisting fluid removal in HD are inadequate to assess hemodynamic changes.

The growing story of (ARABIDOPSIS) CRINKLY 4.

Receptor kinases play important roles in plant growth and development, but only few of them have been functionally characterized in depth. Over the past decade CRINKLY 4 (CR4)-related research has peaked as a result of a newly discovered role of ARABIDOPSIS CR4 (ACR4) in the root. Here, we comprehensively review the available (A)CR4 literature and describe its role in embryo, seed, shoot, and root development, but we also flag an unexpected role in plant defence. In addition, we discuss ACR4 domains and protein structure, describe known ACR4-interacting proteins and substrates, and elaborate on the transcriptional regulation of ACR4 Finally, we address the missing knowledge in our understanding of ACR4 signalling.

BINDING PROTEIN is a master regulator of the endoplasmic reticulum stress sensor/transducer bZIP28 in Arabidopsis.

BINDING PROTEIN (BiP) is a major chaperone in the endoplasmic reticulum (ER) lumen, and this study shows that BiP binds to the C-terminal tail of the stress sensor/transducer bZIP28, a membrane-associated transcription factor, retaining it in the ER under unstressed conditions. In response to ER stress, BiP dissociates from bZIP28, allowing it to be mobilized from the ER to the Golgi where it is proteolytically processed and released to enter the nucleus. Under unstressed conditions, BiP binds to bZIP28 as it binds to other client proteins, through its substrate binding domain. BiP dissociates from bZIP28 even when bZIP28's exit from the ER or its release from the Golgi is blocked. Both BiP1 and BiP3 bind bZIP28, and overexpression of either BiP detains bZIP28 in the ER under stress conditions. A C-terminally truncated mutant of bZIP28 eliminating most of the lumenal domain does not bind BiP and is not retained in the ER under unstressed conditions. BiP binding sites in the C-terminal tail of bZIP28 were identified in a phage display system. BiP was found to bind to intrinsically disordered regions on bZIP28's lumen-facing tail. Thus, the dissociation of BiP from the C-terminal tail of bZIP28 is a major switch that activates one arm of the unfolded protein response signaling pathway in plants.

Polypyrimidine tract-binding proteins of potato mediate tuberization through an interaction with StBEL5 RNA.

Polypyrimidine tract-binding (PTB) proteins are a family of RNA-binding proteins that function in a wide range of RNA metabolic processes by binding to motifs rich in uracils and cytosines. A PTB protein of pumpkin was identified as the core protein of an RNA-protein complex that trafficks RNA. The biological function of the PTB-RNA complex, however, has not been demonstrated. In potato, six PTB proteins have been identified, and two, designated StPTB1 and StPTB6, are similar to the phloem-mobile pumpkin type. RNA binding assays confirmed the interaction of StPTB1 and StPTB6 with discrete pyrimidine-rich sequences of the 3'-untranslated regions of the phloem-mobile mRNA, StBEL5. The promoter of StPTB1 was active in companion cells of phloem in both stem and petioles. Expression of both types was evident in phloem cells of roots and in stolons during tuber formation. RNA accumulation of both PTB proteins was induced by short days in leaves in correlation with enhanced accumulation of StBEL5 RNA. StPTB suppression lines exhibited reduced tuber yields and decreased StBEL5 RNA accumulation, whereas StPTB overexpression lines displayed an increase in tuber production correlated with the enhanced production in stolons of steady-state levels of StBEL5 transcripts and RNA of key tuber identity genes. In StPTB overexpression lines, both the stability and long-distance transport of StBEL5 transcripts were enhanced, whereas in suppression lines stability and transport decreased. Using a transgenic approach, it is shown that the StPTB family of RNA-binding proteins regulate specific stages of development through an interaction with phloem-mobile transcripts of StBEL5.

Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.

Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.

Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase.

Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser(475) and Thr(478) in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein-protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser(475) and Thr(478) in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.