Important Tools for Use by Pediatric Endocrinologists in the Assessment of Short Stature

Assessment and management of children with growth failure has improved greatly over recent years. However, there remains a strong potential for further improvements by using novel digital techniques. A panel of experts discussed developments in digitalization of a number of important tools used by pediatric endocrinologists at the third 360° European Meeting on Growth and Endocrine Disorders, funded by Merck KGaA, Germany, and this review is based on those discussions. It was reported that electronic monitoring and new algorithms have been devised that are providing more sensitive referral for short stature. In addition, computer programs have improved ways in which diagnoses are coded for use by various groups including healthcare providers and government health systems. Innovative cranial imaging techniques have been devised that are considered safer than using gadolinium contrast agents and are also more sensitive and accurate. Deep-learning neural networks are changing the way that bone age and bone health are assessed, which are more objective than standard methodologies. Models for prediction of growth response to growth hormone (GH) treatment are being improved by applying novel artificial intelligence methods that can identify non-linear and linear factors that relate to response, providing more accurate predictions. Determination and interpretation of insulin-like growth factor-1 (IGF-1) levels are becoming more standardized and consistent, for evaluation across different patient groups, and computer-learning models indicate that baseline IGF-1 standard deviation score is among the most important indicators of GH therapy response. While physicians involved in child growth and treatment of disorders resulting in growth failure need to be aware of, and keep abreast of, these latest developments, treatment decisions and management should continue to be based on clinical decisions. New digital technologies and advancements in the field should be aimed at improving clinical decisions, making greater standardization of assessment and facilitating patient-centered approaches.     

Takotsubo Syndrome: Clinical Manifestations,Etiology and Pathogenesis

The purpose of the review is the analysis of clinical and experimental data on the etiology and pathogenesis of takotsubo syndrome (TS). TS is characterized by contractile dysfunction, which usually affects the apical region of the heart without obstruction of coronary artery, moderate increase in myocardial necrosis markers, prolonged QTc interval (in 50% of patients), sometimes elevation of ST segment (in 19% of patients), increase N-Terminal Pro-B-Type Natriuretic Peptide level, microvascular dysfunction, sometimes spasm of the epicardial coronary arteries (in 10% of patients), myocardial edema, and life-threatening ventricular arrhythmias (in 11% of patients). Stress cardiomyopathy is a rare disease, it is observed in 0.6 - 2.5% of patients with acute coronary syndrome. The occurrence of takotsubo syndrome is 9 times higher in women, who are aged 60-70 years old, than in men. The hospital mortality among patients with TS corresponds to 3.5% - 12%. Physical or emotional stress do not precede disease in all patients with TS. Most of patients with TS have neurological or mental illnesses. The level of catecholamines is increased in patients with TS, therefore, the occurrence of TS is associated with excessive activation of the adrenergic system. The negative inotropic effect of catecholamines is associated with the activation of β₂ adrenergic receptors. An important role of the adrenergic system in the pathogenesis of TS is confirmed by studies which were performed using ¹²⁵I-metaiodobenzylguanidine (¹²⁵I -MIBG). TS causes edema and inflammation of the myocardium. The inflammatory response in TS is systemic. TS causes impaired coronary microcirculation and reduces coronary reserve. There is a reason to believe that an increase in blood viscosity may play an important role in the pathogenesis of microcirculatory dysfunction in patients with TS. Epicardial coronary artery spasm is not obligatory for the occurrence of TS. Cortisol, endothelin-1 and microRNAs are challengers for the role of TS triggers. A decrease in estrogen levels is a factor contributing to the onset of TS. The central nervous system appears to play an important role in the pathogenesis of TS.     

Towards sustainable diagnostics: replacing unstable H2O2 by photoactive TiO2 in testing systems for visible and tangible diagnostics for use by blind people

Blind and color blind people cannot use colorimetric diagnostics; the problem is especially severe in rural areas where high temperatures and the absence of electricity challenge modern diagnostics. Here we propose to replace the unstable component of a diagnostic test, H₂O₂, with stable TiO₂. Under UV irradiation, TiO₂ forms reactive oxygen species that initiate polymerization of acrylamide causing liquid-to-gel transition in an analyte-dependent manner. We demonstrate that specific DNA sequences can be detected using this approach. This development may enable the detection of biological molecules by users with limited resources, for example in developing countries or for travelers in remote areas.

Blind and color blind people cannot afford colorimetric diagnostics; the problem is especially severe in rural areas where high temperatures and the absence of electricity challenge modern diagnostics.

An ideal diagnosis, including diagnosis for infectious diseases, should meet the ASSURED criteria: (i) affordable by those at risk of infection; (ii) sensitive; (iii) specific; (iv) user-friendly; (v) rapid, and robust, for example not requiring refrigerated storage; (vi) equipment-free; (vii) delivered to those who need it.¹Analytic methods with visually detectable outputs (e.g. color change) satisfy criteria (iv) and (vi), and are therefore among the most common. Indeed, pregnancy tests, test strips for measuring acetone and glucose in urine for diabetic people, and pH strips are known to be the best to make the analysis easiest in data output. However, such methods cannot be used by the visually impaired.Recently, we described an alternative output signal that cannot only be detected by sight, but also by touch and applied it in the detection of adenosine triphosphate (ATP) and deoxyribonucleic acid (DNA).² The method is based on the analyte-dependent radical polymerization of acrylamide into polyacrylamide in the presence of hydrogen peroxide (H₂O₂). H₂O₂ serves as a source of radicals. The test uses affordable reagents and does not require any instrumentation for signal readout. Such test systems can be adopted for detection of a wide variety of biological analytes. Unfortunately, H₂O₂ is subject to light decomposition and should be refrigerated for long term storage; it is also prone to exploding at high concentrations. Therefore, substituting H₂O₂ with a more stable ingredient would increase the shelf life of the test system and make it usable in those environments with limited access to refrigeration. This work is devoted to addressing the ASSURED criterion (v): we substituted perishable H₂O₂ with stable titania (TiO₂) as an alternative source of initiators of radical polymerization.Recently we demonstrated the possibility of converting electromagnetic energy into pH gradients³ or generating reactive oxygen species (ROS) using TiO₂,^3,4 which makes this approach attractive for controlling interactions between chemical networks. Moreover, combining several functional chemical networks can result in a network with new functions. We have also shown that the pH gradient on titania can be used for regulating enzymatic reaction networks.⁵ In this work, we were interested in using photogenerated ROS (such as superoxide anion O₂˙⁻, hydroxyl radical ˙OH, and hydrogen peroxide H₂O₂) (Fig. 1) to initiate radical polymerization with the aim of making a visual and tactile portable sensor for the detection of biologically important molecules (DNA, RNA, ATP). Under ultraviolet (UV) irradiation, TiO₂ splits water, which results in the generation of a high concentration of ROS and free radicals.⁶Open in a separate windowFig. 1(i) A portable sensor based on a light-induced liquid-to-gel transition for polymer by radical polymerization on TiO₂ particles. The details of the sensor design are shown in Fig. 5; (ii) ROS formation on titania via reactions with photogenerated photohole (h⁺) and photoelectron (e⁻).The sensing system is based on deoxyribozyme sensor that produces ROS in the presence of a specific analyte, thereby triggering the radical polymerization of acrylamide into polyacrylamide. The buffer contained acetylacetone, H₂O₂, 40% acrylamide/bisacrylamide, hemin and a split deoxyribozyme sensor with peroxidase-like activity (PxR).² In the presence of the specific analyte sequence (A1 in this study), the sensor hybridized with the analyte and formed PxR, which bound hemin and decomposed H₂O₂ to ROS. The latter oxidized acetylacetone to the acetylacetone radical, which initiated the polymerization of acrylamide, resulting in liquid-to-gel conversion. Here, the aim of the research is to increase the system sustainability by changing H₂O₂ to TiO₂ particles. Noteworthy is that the system is externally controlled by UV light, since TiO₂ produces ROS only upon irradiation.Firstly, we quantified the TiO₂-derived ROS with the aim of finding the optimal conditions for the generation of the minimum amount of ROS needed for the polymerization. We used luminol chemiluminescence (CL) calibrated with hydrogen peroxide. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a widely used CL reagent and has CL emission at different wavelengths depending on the conditions.⁶ Traditionally, luminol CL is observed in the presence of H₂O₂ in alkaline solutions (Fig. 2), which is catalyzed by metal ions, metal complexes or vitamins.^7–10Open in a separate windowFig. 2Mechanism of chemiluminescence of luminol and O₂⁻˙.The luminol CL signaling is conveniently used for the detection of ROS (particularly superoxide anion, hydrogen peroxide, hydroxyl radical) in biological systems.^11–16Irradiation of TiO₂ suspensions of different concentrations (0.03 M and 0.06 M) at two wavelengths (365 nm and 254 nm) revealed more ROS being formed upon irradiation at 254 nm – as was expected (Fig. 3).Open in a separate windowFig. 3Log concentration of ROS generated in TiO₂ suspensions of different concentration (0.03 M and 0.06 M) under irradiation by UV light with different wavelengths vs. irradiation time.According to the data obtained, the minimum concentration of TiO₂ needed to trigger polymerization after 5 min of irradiation at 365 nm was 1.25 mM (Fig. 4). We therefore used these conditions in the following experiments, since short irradiation time and longer wavelength of UV-A irradiation are less damaging to the DNA-based biosensor component than UV-C light with wavelength of 254 nm. It should be noted that control experiments were made without TiO₂ particles and polymerization did not occur. Next, we optimized the conditions for the analyte-dependent activation of PxR resulting in acrylamide polymerization. If polymerization occurs, the gel will stick to the bottom of the inverted tubes (Fig. 5).² To demonstrate the general applicability of the approach, we designed a sensor for an analyte of biomedical significance, nucleic acids, as an example. A sequence of 16S rRNA, which was represented in this study by synthetic A1 sequence (5′-CAT TAC TCA CCC GTC CGC CAC TCG TCA GCG AAG CAG CAA GCT GCT TCC TGT TAC CGT TCG), of pathogenic E. coli O157:H7 was chosen as the target analyte,. The binding of A1 to PxR1 and PxR2 stabilized the G-quadruplex structure, which then binds to hemin and catalyzes the polymerization (Fig. 5A).Open in a separate windowFig. 4The dependence of the log concentration of ROS generated on TiO₂ particles on TiO₂ suspension concentration. Inserts show the minimum concentration of TiO₂ particles in a suspension (therefore minimum concentration of ROS generated on TiO₂ particles), needed for the gel formation.Open in a separate windowFig. 5Polymerization-based visual and tactile detection of A1 analyte. (A) Sensor design: RNA strands PxR1 and PxR2 bind A1 sequence and form a G-quadruplex structure, which then binds to hemin (green oval) and catalyzes the radical polymerization of the acrylamide solution. The dotted lines represent triethylene glycol linkers. (B and C) Inverted test-tubes with radical polymerization of acrylamide initiated by reactive oxygen species generated on TiO₂ particles under UV light 365 nm. (B) Systems with different concentrations of hemin (

Functional reclassification of variants of uncertain significance in the HCN4 gene identified in sudden unexpected death

The HCN4 gene encodes a subunit of the hyperpolarization‐activated cyclic nucleotide‐gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N‐terminus, p.D546N in the C‐linker domain, and both p.S935Y and p.R1044Q in the C‐terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild‐type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C‐linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild‐type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time‐consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C‐linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.     

Alpha1- and beta1-adrenoceptor signaling fully compensates for beta3-adrenoceptor deficiency in brown adipocyte norepinephrine-stimulated glucose uptake

To assess the relative roles and potential contribution of adrenergic receptor subtypes other than the beta3-adrenergic receptor in norepinephrine-mediated glucose uptake in brown adipocytes, we have here analyzed adrenergic activation of glucose uptake in primary cultures of brown adipocytes from wild-type and beta3-adrenergic receptor knockout (KO) mice. In control cells in addition to high levels of beta3-adrenergic receptor mRNA, there were relatively low alpha1A-, alpha1D-, and moderate beta1-adrenergic receptor mRNA levels with no apparent expression of other adrenergic receptors. The levels of alpha1A-, alpha1D-, and beta1-adrenergic receptor mRNA were not changed in the beta3-KO brown adipocytes, indicating that the beta3-adrenergic receptor ablation does not influence adrenergic gene expression in brown adipocytes in culture. As expected, the beta3-adrenergic receptor agonists BRL-37344 and CL-316 243 did not induce 2-deoxy-d-glucose uptake in beta3-KO brown adipocytes. Surprisingly, the endogenous adrenergic neurotransmitter norepinephrine induced the same concentration-dependent 2-deoxy-D-glucose uptake in wild-type and beta3-KO brown adipocytes. This study demonstrates that beta1-adrenergic receptors, and to a smaller degree alpha1-adrenergic receptors, functionally compensate for the lack of beta3-adrenergic receptors in glucose uptake. Beta1-adrenergic receptors activate glucose uptake through a cAMP/protein kinase A/phosphatidylinositol 3-kinase pathway, stimulating conventional and novel protein kinase Cs. The alpha1-adrenergic receptor component (that is not evident in wild-type cells) stimulates glucose uptake through a phosphatidylinositol 3-kinase and protein kinase C pathway in the beta3-KO cells.     

FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

The large family of fibroblast growth factors (FGFs) has been known for its important roles in regulating critical cellular processes during embryonic development and homeostasis of normal tissues (1–3). While most FGFs act as cytokines or hormonelike proteins that mediate their pleiotropic cellular processes by binding to cell surface receptors endowed with intrinsic tyrosine kinase activity (FGFRs), a subfamily of FGFs (FGF 11–14) was shown to be uniquely expressed intracellularly. The mechanism of action and physiological roles of intracellular FGFs are poorly understood (4–6).In contrast to most receptor tyrosine kinases (RTKs) that are activated by a single ligand molecule that binds with high affinity to the extracellular domain of its cognate RTK with a dissociation constant in the subnanomolar range, the binding affinities of FGFs to FGFRs are, at least, 1,000–10,000 fold weaker with dissociation constants in the submicromolar range (7–9). The weak binding affinities toward FGFRs of the largest subfamily of FGF molecules designated canonical FGFs are offset by interactions with cell surface heparan sulfate proteoglycans (HSPGs). Both biochemical and structural studies revealed how multiple interactions between heparin or HSPG with both FGF and FGFR mediate tight association enabling robust receptor dimerization and tyrosine kinase activation (10, 11).The three endocrine FGFs, FGF19, 21, and 23 are part of an additional subfamily of FGF molecules. Endocrine FGFs function as circulating hormones that play essential roles in the control of various metabolic processes (12). In addition to the conserved FGF-domain found in all FGF ligands, endocrine FGFs contain unique C-terminal tails (CTs) composed of 46 (FGF19), 34 (FGF21), or 89 (FGF23) amino acids that serve as specific and high-affinity ligands for the two members of the Klotho family of surface receptors. It was shown that KLA serves as a high-affinity receptor for FGF23 while β-Klotho (KLB) functions as a high-affinity surface receptor for both FGF19 and FGF21 (13–16). Structural analyses of free and ligand-occupied Klotho proteins revealed the molecular basis underlying the specificity and high affinity of KLA and KLB toward endocrine FGFs. It also showed that Klotho proteins function as the primary receptors for endocrine FGFs whereas FGFR functions as a catalytic subunit that mediates cell signaling via its tyrosine kinase domain (8, 17, 18). Accordingly, endocrine FGFs stimulate their cellular responses by forming a ternary complex with Klotho proteins and FGFRs to induce receptor dimerization, tyrosine kinase activation, and cell signaling. Unlike FGFRs that are ubiquitously expressed, the expressions of KLA and KLB are restricted to specific tissues and organs to enable precise targeting of endocrine FGFs to stimulate their physiological responses in specific cells and tissues (19–22). The ability of endocrine FGFs to circulate is attributed to the loss of conserved heparin binding sites that are essential for the function of canonical FGFs (23).FGF23 is a 32-kDa glycoprotein, mainly produced in the bone by osteoblasts and osteocytes, that serve as a key hormone in regulating phosphate homeostasis, vitamin D, and calcium metabolism (24, 25). Circulating levels of physiologically active FGF23 are regulated by proteolytic cleavage to produce a FGF23 molecule lacking its unique CT (26, 27). The cleavage resulting in FGF23 inactivation prevents assembly and stimulation of the FGF23/FGFR/KLA complex. Additionally, the processing of FGF23 includes several posttranslational modifications which affect its stability and susceptibility toward proteolysis. Secreted FGF23 was shown to be O-glycosylated in its C-terminal cleavage site (28, 29) to protect the protein from C-terminal cleavage. In order for the cleavage site to be exposed, FGF23 has to be first phosphorylated in this region (30). Phosphorylation prevents glycosylation and exposes the cleavage site to proteolysis.In this paper, we demonstrate that the CT of FGF23 contains two tandem repeats and that each repeat binds with high affinity to KLA. This contrasts with FGF19 and FGF21, whose CTs contain a single binding site to KLB. Engineered FGF23 variants containing each of the two repeats individually or both repeats bind specifically to KLA and stimulate cell signaling to a similar extent. We also demonstrate that two cysteine residues flanking the second repeat form a disulfide bridge in FGF23 secreted by mammalian cells. However, both oxidized or unbridged forms of FGF23 exhibit similar KLA binding characteristics and similar cellular stimulatory activities. We also show that FGF23-WT induces mitogen-activated protein kinase (MAPK) activation in cells expressing chimeric KLA-FGFR proteins and use TIRFM imaging of individual KLA molecules on the cell surface to demonstrate that FGF23 has the capacity for simultaneous binding to two KLA molecules. These insights reveal the complexity of FGF23 regulation and its role in assembling the FGF23/FGFR/KLA signaling complex.     

Dead cells in melanoma tumors provide abundant antigen for targeted delivery of ionizing radiation by a mAb to melanin

Melanoma is a cancer with a rising incidence, and metastatic disease is almost always lethal. We investigated the feasibility of targeting melanin, an intracellular melanocyte pigment, to deliver cytotoxic radiation to human melanoma cells in vivo by using a melanin-binding mAb (6D2). Nude mice bearing MNT1 pigmented human melanoma tumors were treated with mAb 6D2 labeled with 1.5 mCi (1 Ci = 37 GBq) of the beta-emitter 188-Rhenium (188Re) and manifested inhibition of tumor growth and prolonged survival. mAb 6D2 bound tumor melanin and demonstrated no crossreactivity with normal melanized tissues in black mice. The mechanism of melanin targeting involved Ab binding to extracellular melanin released during tumor cell turnover or to dying cells with permeable membranes. In this approach, the cytotoxic radiation emanating from labeled Ab bound to melanin is presumably delivered by "crossfire" effect to the adjacent viable tumor cells. Our results establish the feasibility of targeting melanin released from dead melanoma cells in tumors with radiolabeled Abs to achieve a therapeutic effect. In contrast to conventional tumor antigens, melanin is insoluble, resistant to degradation, and can be expected to accumulate in targeted tissues, suggesting that the efficacy of therapy could increase with each subsequent treatment cycle.