Primary Hyperparathyroidism in Multiple Endocrine Neoplasia Type 2A in Denmark 1930­2021: A Nationwide Population-Based Retrospective Study

Studies of primary hyperparathyroidism (PHPT) in multiple endocrine neoplasia type 2A (MEN 2A) shows divergence in frequency, disease definition, reporting of clinical characteristics and traces of selection bias. This is a nationwide population-based retrospective study of PHPT in MEN 2A, suggesting a representative frequency, with complete reporting and a strict PHPT definition. The Danish MEN 2A cohort 1930-2021 was used. Of 204 MEN 2A cases, 16 had PHPT, resulting in a frequency of 8% (CI, 5-12). Age-related penetrance at 50 years was 8% (CI, 4-15). PHPT was seen in the American Thyroid Association moderate (ATA-MOD) and high (ATA-H) risk groups in 62% and 38% of carriers, respectively. Median age at PHPT diagnosis was 45 years (range, 21-79). A total of 75% were asymptomatic and 25% were symptomatic. Thirteen underwent parathyroid surgery, resulting in a cure of 69%, persistence in 8% and recurrence in 23%. In this first study with a clear PHPT definition and no selection bias, we found a lower frequency of PHPT and age-related penetrance, but a higher age at PHPT diagnosis than often cited. This might be affected by the Danish RET p.Cys611Tyr founder effect. Our study corroborates that PHPT in MEN 2A is often mild, asymptomatic and is associated with both ATA-MOD and ATA-H variants. Likelihood of cure is high, but recurrence is not infrequent and can occur decades after surgery.

Non-invasive three-dimensional electrical activation mapping to predict cardiac resynchronization therapy response: site of latest left ventricular activation relative to pacing site.

AIMS: Pacing remote from the latest electrically activated site (LEAS) in the left ventricle (LV) may diminish response to cardiac resynchronization therapy (CRT). We tested whether proximity of LV pacing site (LVPS) to LEAS, determined by non-invasive three-dimensional electrical activation mapping [electrocardiographic Imaging (ECGI)], increased likelihood of CRT response. METHODS AND RESULTS: Consecutive CRT patients underwent ECGI and chest/heart computed tomography 6-24 months of post-implant. Latest electrically activated site and the distance to LVPS (dp) were assessed. Left ventricular end-systolic volume (LVESV) reduction of ≥15% at clinical follow-up defined response. Logistic regression probabilistically modelled non-response; variables included demographics, heart failure classification, left bundle branch block (LBBB), ischaemic heart disease (IHD), atrial fibrillation, QRS duration, baseline ejection fraction (EF) and LVESV, comorbidities, use of CRT optimization algorithm, angiotensin-converting enzyme inhibitor(ACE)/angiotensin-receptor blocker (ARB), beta-blocker, diuretics, and dp. Of 111 studied patients [64 ± 11 years, EF 28 ± 6%, implant duration 12 ± 5 months (mean ± SD), 98% had LBBB, 38% IHD], 67% responded at 10 ± 3 months post CRT-implant. Latest electrically activated sites were outside the mid-to-basal lateral segments in 35% of the patients. dp was 42 ± 23 mm [31 ± 14 mm for responders vs. 63 ± 24 mm non-responders (P < 0.001)]. Longer dp and the lack of use of CRT optimization algorithm were the only independent predictors of non-response [area under the curve (AUC) 0.906]. dp of 47 mm delineated responders and non-responders (AUC 0.931). CONCLUSION: The distance between LV pacing site and latest electrical activation is a strong independent predictor for CRT response. Non-invasive electrical evaluation to characterize intrinsic activation and guide LV lead deployment may improve CRT efficacy.

Treatment History Characteristics Associated With Use of Isocarboxazid: A Nationwide Register-Based Study.

PURPOSE/BACKGROUND: The monoamine oxidase inhibitor isocarboxazid (Marplan) is occasionally used in the treatment of depression, but there is only little knowledge on the nature of the use of isocarboxazid in clinical practice. We aimed to identify treatment history characteristics associated with this use. METHODS/PROCEDURES: Via the nationwide Danish registers, we identified all adult incident users of isocarboxazid in the period from 2001 to 2018, as well as up to 5 matched controls using another antidepressant (matched on date of redeemed prescription, age, sex, and region of residence). The 5-year treatment history of the isocarboxazid users and the controls was assessed via the Danish registers. The association between treatment history characteristics and isocarboxazid use was examined by multivariate conditional logistic regression. FINDINGS/RESULTS: We identified 1455 isocarboxazid users and 7045 controls using another antidepressant. The following characteristics were associated with statistically significant increased likelihood of receiving isocarboxazid treatment: Prior treatment with a selective serotonin reuptake inhibitor (odds ratio [OR], 1.80 with 95% confidence interval [CI], 1.46-2.23), a serotonin-norepinephrine reuptake inhibitor (OR, 4.90; 95% CI, 4.08-5.89), a noradrenergic and specific serotonergic antidepressant (OR, 1.56; 95% CI, 1.30-1.88), a tricyclic antidepressant (OR, 5.05; 95% CI, 4.19-6.08), other antidepressants (OR, 4.74; 95% CI, 3.74-6.01), lithium (OR, 6.70; 95% CI, 5.08-8.83), an antipsychotic (OR, 1.43; 95% CI, 1.19-1.73), and each diagnosis of depression received in relation to psychiatric hospital treatment (OR, 1.31; 95% CI, 1.23-1.39). Forty percent of those initiating isocarboxazid had received treatment with drugs from 5 or more different psychopharmacological classes in the 5 preceding years. IMPLICATIONS/CONCLUSIONS: These findings suggest that isocarboxazid is typically used for treatment-resistant depression, consistent with guideline recommendations.

The B-Box Domain Protein BBX21 Promotes Photomorphogenesis.

B-box-containing (BBX) proteins play critical roles in a variety of cellular and developmental processes in plants. BBX21 (also known as SALT TOLERANCE HOMOLOG2), which contains two B-box domains in tandem at the N terminus, has been previously demonstrated as a key component involved in the COP1-HY5 signaling hub. However, the exact molecular and physiological roles of B-box domains in BBX21 are largely unclear. Here, we found that structurally disruption of the second B-box domain, but not the first one, in BBX21 completely abolishes its biological and physiological activity in conferring hyperphotomorphogenetic phenotype in Arabidopsis (Arabidopsis thaliana). Intact B-box domains in BBX21 are not required for interaction with COP1 and its degradation by COP1 via the 26S proteasome system. However, disruption of the second B-box of BBX21 nearly impairs its ability for binding of T/G-box within the HY5 promoter both in vitro and in vivo, as well as controlling HY5 and HY5-regulated gene expression in Arabidopsis seedlings. Taken together, this study provides a mechanistic framework in which BBX21 directly binds to the T/G-box present in the HY5 promoter possibly through its second B-box domain, which in turn controls HY5 and HY5-regulated gene expression to promote photomorphogenesis.

Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) plays crucial roles in various cellular processes via its E3 ubiquitin ligase activity in organisms, ranging from fungi to humans. As a key component in regulating various biological events, COP1 itself is precisely controlled at multiple layers. Here, we report a negative regulator of COP1, PINOID (PID), which positively mediates photomorphogenic development. Specifically, PID genetically and physically interacts with COP1 and directly phosphorylates COP1 at Ser20. As a result, this posttranslational modification serves to repress COP1 activity and promote photomorphogenesis. Our findings signify a key regulatory mechanism for precisely maintaining COP1 activity, thereby ensuring appropriate development in plants.

BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation.

BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub.

Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2's repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.

The RING-Finger E3 Ubiquitin Ligase COP1 SUPPRESSOR1 Negatively Regulates COP1 Abundance in Maintaining COP1 Homeostasis in Dark-Grown Arabidopsis Seedlings.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) functions as an E3 ubiquitin ligase in both plants and animals. In dark-grown Arabidopsis thaliana seedlings, COP1 targets photomorphogenesis-promoting factors for degradation to repress photomorphogenesis. Little is known, however, about how COP1 itself is regulated. Here, we identify COP1 SUPPRESSOR1 (CSU1), a RING-finger E3 ubiquitin ligase, as a regulator of COP1. Genetic evidence demonstrates that csu1 mutations suppress cop1-6 phenotypes completely in the dark. Furthermore, CSU1 colocalizes with COP1 in nuclear speckles and negatively regulates COP1 protein accumulation in darkness. CSU1 can ubiquitinate COP1 in vitro and is essential for COP1 ubiquitination in vivo. Therefore, we conclude that CSU1 plays a major role in maintaining COP1 homeostasis by targeting COP1 for ubiquitination and degradation in dark-grown seedlings.

Convergence of Light and ABA signaling on the ABI5 promoter.

Light is one of the most important environmental cues regulating multiple aspects of plant growth and development, and abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle and in plants' responses to various environmental stresses. How plants integrate the external light signal with endogenous ABA pathway for better adaptation and survival remains poorly understood. Here, we show that BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX) protein previously shown to positively regulate seedling photomorphogenesis, is also involved in ABA signaling. Our genetic data show that BBX21 may act upstream of several ABA INSENSITIVE (ABI) genes and ELONGATED HYPOCOTYL 5 (HY5) in ABA control of seed germination. Previous studies showed that HY5 acts as a direct activator of ABI5 expression, and that BBX21 interacts with HY5. We further demonstrate that BBX21 negatively regulates ABI5 expression by interfering with HY5 binding to the ABI5 promoter. In addition, ABI5 was shown to directly activate its own expression, whereas BBX21 negatively regulates this activity by directly interacting with ABI5. Together, our study indicates that BBX21 coordinates with HY5 and ABI5 on the ABI5 promoter and that these transcriptional regulators work in concert to integrate light and ABA signaling in Arabidopsis thaliana.

The B-box family gene STO (BBX24) in Arabidopsis thaliana regulates flowering time in different pathways.

Flowering at the appropriate time is crucial for reproductive success and is strongly influenced by various pathways such as photoperiod, circadian clock, FRIGIDA and vernalization. Although each separate pathway has been extensively studied, much less is known about the interactions between them. In this study we have investigated the relationship between the photoperiod/circadian clock gene and FRIGIDA/FLC by characterizing the function of the B-box STO gene family. STO has two B-box Zn-finger domains but lacks the CCT domain. Its expression is controlled by circadian rhythm and is affected by environmental factors and phytohormones. Loss and gain of function mutants show diversiform phenotypes from seed germination to flowering. The sto-1 mutant flowers later than the wild type (WT) under short day growth conditions, while over-expression of STO causes early flowering both in long and short days. STO over-expression not only reduces FLC expression level but it also activates FT and SOC1 expression. It also does not rely on the other B-box gene CO or change the circadian clock system to activate FT and SOC1. Furthermore, the STO activation of FT and SOC1 expression is independent of the repression of FLC; rather STO and FLC compete with each other to regulate downstream genes. Our results indicate that photoperiod and the circadian clock pathway gene STO can affect the key flowering time genes FLC and FT/SOC1 separately, and reveals a novel perspective to the mechanism of flowering regulation.

Molecular interactions of BBX24 and BBX25 with HYH, HY5 HOMOLOG, to modulate Arabidopsis seedling development.

BBX24 and BBX25 are two important transcriptional regulators, which regulate seedling photomorphogenesis in Arabidopsis. Very recently, we have shown that BBX24 and BBX25 negatively regulate the expression of BBX22, reducing the function of HY5, by physically interacting with its bZIP domain. (1) Furthermore, HY5 HOMOLOG, HYH, has been reported to heterodimerize with HY5 and enhances its photomorphogenic function in seedling de-etiolation by serving as coactivator. (8) Here, we further report that BBX24 and BBX25 physically interact with HYH. The physical interactions of BBX24 and BBX25 with HYH could lead to depletion of HYH molecules from the active pool and, thus indirectly, reduce the function of HY5 in promoting photomorphogenesis. Hence, our results suggest another mode of regulation by which BBX24 and BBX25 exert their negative effects on HY5 indirectly through HYH for the fine-tuning of seedling photomorphogenesis.

The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis.

ELONGATED HYPOCOTYL5 (HY5) is a basic domain/leucine zipper (bZIP) transcription factor, central for the regulation of seedling photomorphogenesis. Here, we identified a B-BOX (BBX)-containing protein, BBX25/SALT TOLERANCE HOMOLOG, as an interacting partner of HY5, which has been previously found to physically interact with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). BBX25 physically interacts with HY5 both in vitro and in vivo. By physiological and genetic approaches, we showed that BBX25 is a negative regulator of seedling photomorphogenesis. BBX25 and its homolog BBX24 regulate deetiolation processes and hypocotyl shade avoidance response in an additive manner. Moreover, genetic relationships of bbx25 and bbx24 with hy5 and cop1 revealed that BBX25 and BBX24 additively enhance COP1 and suppress HY5 functions. BBX25 accumulates in a light-dependent manner and undergoes COP1-mediated degradation in dark and light conditions. Furthermore, a protoplast cotransfection assay showed that BBX24 and BBX25 repress BBX22 expression by interfering with HY5 transcriptional activity. As HY5 binds to the BBX22 promoter and promotes its expression, our results identify a direct mechanism through which the expression of BBX22 is regulated. We suggest that BBX25 and BBX24 function as transcriptional corepressors, probably by forming inactive heterodimers with HY5, downregulating BBX22 expression for the fine-tuning of light-mediated seedling development.

Function of B-BOX under shade.

Plants are capable of perceiving changes in the light environment and finely adjust their growth and development. Reductions of red to far-red ratio (R:FR) generated by an increase of the plant canopy above the plant are sensed by the phytochrome system triggering the shade-avoidance syndrome (SAS) that includes elongation of vegetative structures, reduction of branching and acceleration of flowering. Albeit the SAS is a strategy of major adaptative significance in plant communities, involving massive changes in gene expression, our knowledge of the SAS signaling network is still fragmented. By a selection and characterization of a T-DNA mutant with a long hypocotyl under shade, we identified BBX21, a protein with two B-box domains involved in the SAS. BBX21 belongs to a small eight member family of B-box containing proteins with both opposite and additive functions in the SAS signaling. BBX21 down-regulates the gene expression of auxin, brassinosteroid and ethylene signaling pathway components under shade. Furthermore BBX21 is a transcription factor that interacts genetically with COP1. We propose a model in which a dynamic balance of positive and negative B-box transcriptional regulators acts as a gas-and-brake mechanism into the COP1 signaling to regulate the expression of SAS.

AtBBX21 and COP1 genetically interact in the regulation of shade avoidance.

Plants grown at high densities perceive the reduction in the ratio of red (R) to far-red (FR) light as a warning of competition. This light signal triggers morphological responses such as hypocotyl and stem elongation, and acceleration of flowering, which are known collectively as the shade-avoidance syndrome (SAS). Mutations in the photomorphogenic repressor COP1 suppress the SAS, but how COP1 modulates these responses is uncertain. We identified a new mutant with altered responses to natural shade, named lhus (long hypocotyl under shade). lhus seedlings have longer hypocotyls than wild-type under a low R:FR ratio, but not under sunlight or darkness. The lhus phenotype is due to a mutation affecting a B-box zinc finger transcription factor encoded by At1g75540, a gene previously reported as AtBBX21 that interacts with COP1 to control de-etiolation. Mutations in genes encoding other members of this protein family also result in impaired SAS regulation. Under short-term canopy shade, LHUS/BBX21 acts as positive regulator of SAS genes such as PAR1, HFR1, PIL1 and ATHB2. In contrast, global expression analysis of wild-type and lhus/bbx21 seedlings revealed that a large number of genes involved in hormonal signalling pathways are negatively regulated by LHUS/BBX21 in response to long-term canopy shade, and this observation fits well with the phenotype of lhus/bbx21 seedlings grown under a low R:FR ratio. Moreover, the bbx21 bbx22 double mutation restored the SAS in the cop1 background. We propose that LHUS/BBX21 and other B-box-containing proteins, such as BBX22, act downstream of COP1, and play a central role in early and long-term adjustment of the SAS in natural environments.

LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-box protein involved in light-dependent development and gene expression, undergoes COP1-mediated ubiquitination.

B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination.

STH2 has 2 B there: An insight into the role of B-box containing proteins in Arabidopsis.

Recently we reported the identification of a novel B-box transcription factor SALT TOLERANCE HOMOLOG 2 (STH2) that interacts genetically with two key regulators of the light-signaling pathway, HY5 and COP1.1 We also provided phenotypic and genetic characterization of the sth2 mutant suggesting that STH2 plays a positive role in regulating photomorphogenesis both independently or together with HY5. Functional assays in protoplasts revealed that STH2 could act as a transcriptional activator. To our knowledge this is the first report of a B-box domain containing protein playing a direct role in activating transcription in plants. Here we discuss the possible position of STH2 in the transcriptional network and comment on the role of the B-box domain in plants.

SALT TOLERANCE HOMOLOG2, a B-box protein in Arabidopsis that activates transcription and positively regulates light-mediated development.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and ELONGATED HYPOCOTYL5 (HY5) are two major regulators of light signaling in plants. Here, we identify SALT TOLERANCE HOMOLOG2 (STH2) as a gene that interacts genetically with both of these key regulators. STH2 encodes a B-box-containing protein that interacts physically with HY5 in yeast and in plant cells. Whereas STH2 is uniformly nuclear by itself, it shows a COP1-dependent localization to speckles when coexpressed with COP1. We identified two independent T-DNA insertion lines in STH2. Both alleles are hyposensitive to blue, red, and far-red light. The sth2 mutant, like hy5, shows an enhanced number of lateral roots and accumulates less anthocyanin. Analysis of double mutants between sth2 and hy5 indicates that STH2 has both HY5-dependent and -independent functions. Furthermore, besides partially suppressing the hypocotyl phenotype of dark-grown cop1 alleles, sth2 also suppresses the reduced number of lateral roots and high anthocyanin levels in light-grown cop1 alleles. Interestingly, we found that STH2 can activate transcription. Transient transfection assays in protoplasts using a LUC reporter driven by the chalcone isomerase promoter show that the B-boxes in STH2 and a functional G-box element in the promoter are required for this activity. In conclusion, we have identified STH2, a B-box protein in Arabidopsis thaliana, as a positive regulator of photomorphogenesis and report that the B-box domain plays a direct role in activating transcription in plants.

Opposite root growth phenotypes of hy5 versus hy5 hyh mutants correlate with increased constitutive auxin signaling.

The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development.