Progressive Calcification Research Articles

Progressive calcification of bioprosthetic mitral valve observed during pregnancy resulting from in vitro fertilization: a case report

BackgroundWomen with pre-existing cardiac conditions who undergo assisted reproductive technologies (ART) are believed to be at a heightened risk of cardiovascular events during both the treatment and pregnancy phases. An unresolved question within this context pertains to whether the ART procedure itself constitutes a risk factor for individuals with bioprosthetic heart valves (BHV). Additionally, there is ongoing controversy regarding whether pregnancies expedite the process of structural valve degeneration (SVD) in BHV. The purpose of this study is to present the developmental process of BHV calcification, which is considered the primary cause of SVD, during a pregnancy resulting from in vitro fertilization and embryo transfer (IVF-ET), an ART modality, and to elucidate the underlying mechanisms.Case presentationAt 7 + 3 weeks of gestation in a twin pregnancy resulting from IVF-ET, a 27-year-old woman with a bioprosthetic mitral valve manifesting severe mitral stenosis and moderate pulmonary arterial hypertension, was suspected of SVD. Despite undergoing fetal reduction, she experienced progressive calcification of the bioprosthetic valve, increasing pulmonary arterial pressure and ultimately deteriorated into heart failure. An elective cesarean section and redo valve replacement was subsequently administered to improve her cardiovascular condition. As a result, a healthy young boy was delivered and the dysfunctional BHV was replaced with a mechanical valve. She did not report any discomfort during the 3-month follow-up.ConclusionThe progressive calcification of the BHV was observed during IVF pregnancy, indicating a potential connection between fertility therapy, pregnancy and calcification of BHV. Pregnant women with pre-implanted BHV should be treated with caution, as any medical interventions during ART and pregnancy can have a significant impact on both maternal and fetal outcomes. Thus, involving a multidisciplinary team in decision-making early on, starting from the treatment of the original heart disease, throughout the entire process of ART and pregnancy, is crucial.

Global huntingtin knockout in adult mice leads to fatal neurodegeneration that spares the pancreas.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG tract in the huntingtin (HTT) gene, leading to toxic gains of function. HTT-lowering treatments are in clinical trials, but the risks imposed are unclear. Recent studies have reported on the consequences of widespread HTT loss in mice, where one group described early HTT loss leading to fatal pancreatitis, but later loss as benign. Another group reported no pancreatitis but found widespread neurological phenotypes including subcortical calcification. To better understand the liabilities of widespread HTT loss, we knocked out Htt with two separate tamoxifen-inducible Cre lines. We find that loss of HTT at 2 mo of age leads to progressive tremors and severe subcortical calcification at examination at 14 mo of age but does not result in acute pancreatitis or histological changes in the pancreas. We, in addition, report that HTT loss is followed by sustained induction of circulating neurofilament light chain. These results confirm that global loss of HTT in mice is associated with pronounced risks, including progressive subcortical calcification and neurodegeneration.

Antisense oligonucleotides enhance SLC20A2 expression and suppress brain calcification in a humanized mouse model

Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.

Identification of Circulating Inflammatory Proteins Associated with Calcific Aortic Valve Stenosis by Multiplex Analysis.

Calcific aortic valve stenosis (CAVS) is characterized by increasing inflammation and progressive calcification in the aortic valve leaflets and is a major cause of death in the aging population. This study aimed to identify the inflammatory proteins involved in CAVS and provide potential therapeutic targets. We investigated the observational and causal associations of 92 inflammatory proteins, which were measured using affinity-based proteomic assays. Firstly, the case-control cohort identified differential proteins associated with the occurrence and progression of CAVS. Subsequently, we delved into exploring the causal impacts of these associated proteins through Mendelian randomization. This involved utilizing genetic instruments derived from cis-protein quantitative loci identified in genome-wide association studies, encompassing a cohort of over 400,000 individuals. Finally, we investigated the gene transcription and protein expression levels of inflammatory proteins by single-cell and immunohistochemistry analysis. Multivariate logistic regression and spearman's correlation analysis showed that five proteins showed a significant positive correlation with disease severity. Mendelian randomization showed that elevated levels of two proteins, namely, matrix metallopeptidase-1 (MMP1) and sirtuin 2 (SIRT2), were associated with an increased risk of CAVS. Immunohistochemistry and single-cell transcriptomes showed that expression levels of MMP1 and SIRT2 at the tissue and cell levels were significantly higher in calcified valves than in non-calcified control valves. These findings indicate that MMP1 and SIRT2 are causally related to CAVS and open up the possibility for identifying novel therapeutic targets.

Hearts of stone: Rapidly progressive left ventricular myocardial calcifications in severe sepsis: Case series.

Rapidly progressive left ventricular myocardial calcification (RPLVMC) is a rare phenomenon of severe sepsis which is associated with long-term complications like irreversible cardiomyopathy and arrhythmias. To date, only 19 cases have been reported in literature. We present a case series of two patients with RPLVMC which manifested within a period of days in the setting of severe sepsis. Unique to previous case reports, the patients in the current case series had no pre-existing systemic risk factors such as end-stage kidney disease or endocrinological dysfunction. This case series aims to increase awareness of RPLVMC in severe sepsis, improve its opportunistic detection on routine medical imaging (namely chest x-ray and computed tomography), and spur future research to develop potential prevention strategies.

Biomechanical Evaluation of Aortic Valve Stenosis by Means of a Virtual Stress Test: A Fluid-Structure Interaction Study.

The impact of aortic valve stenosis (AS) extends beyond the vicinity of the narrowed leaflets into the left ventricle (LV) and into the systemic vasculature because of highly unpredictable valve behavior and complex blood flow in the ascending aorta that can be attributed to the strong interaction between the narrowed cusps and the ejected blood. These effects can become exacerbated during exercise and may have implications for disease progression, accurate diagnosis, and timing of intervention. In this 3-D patient-specific study, we employ strongly coupled fluid-structure interaction (FSI) modeling to perform a comprehensive biomechanical evaluation of systolic ejection dynamics in a stenosed aortic valve (AV) during increasing LV contraction. Our model predictions reveal that the heterogeneous ∆P vs. Q relationship that was observed in our previous clinical study can be attributed to a non-linear increase (by ~ 1.5-fold) in aortic valve area as LV heart rate increases from 70 to 115bpm. Furthermore, our results show that even for a moderately stenotic valve, increased LV contraction during exercise can lead to high-velocity flow turbulence (Re = 11,700) in the aorta similar to that encountered with a severely stenotic valve (Re ~ 10,000), with concomitant greater viscous loss (~3-fold increase) and elevated wall stress in the ascending aorta. Our FSI predictions also reveal that individual valve cusps undergo distinct and highly non-linear increases (>100%) in stress during exercise, potentially contributing to progressive calcification. Such quantitative biomechanical evaluations from realistic FSI workflows provide insights into disease progression and can be integrated with current stress testing for AS patients to comprehensively predict hemodynamics and valve function under both baseline and exercise conditions.

Calciphylaxis in kidney graft recipient with type 1 diabetes: A case report

Calcifylaxis calcifying uremic arteriolopathy is a rare disease that occurs in patients with chronic kidney disease and is characterized by progressive calcification of small-caliber skin vessels and thrombosis of small (arterioles), accompanied by necrosis of the skin, subcutaneous tissue and soft tissues with severe pain syndrome. The article describes a patient with a long-term, severe course of diabetes mellitus, the development of diabetic nephropathy with an outcome in terminal renal failure, which required renal replacement therapy with peritoneal dialysis and kidney transplantation. In the posttransplantation period, the patient registered the occurrence of calcifylaxis, which is a rare complication.
 The lack of treatment protocols for calcifylaxis in patients with chronic kidney disease requires further research. It is important to constantly monitor patients with risk factors for the development of this serious disease and timely treatment, which will improve their condition and slow down the progression of the process.

Abstract 14286: Rapid Involution of a Bioprosthetic Transcatheter Aortic Valve Replacement Presenting as Late Bioprosthetic Stenosis

A 65 year old male with a bioprosthetic 29 mm Edwards Sapien Ultra S3 TAVR implant for symptomatic severe aortic stenosis presented for evaluation after a 30-day transthoracic echocardiogram demonstrated bioprosthetic valve stenosis. Following TAVR deployment, TEE showed normal bioprosthetic valve function without evidence of significant paravalvular regurgitation. There were no procedural complications. Echocardiogram performed 1-day post-implantation demonstrated expected transvalvular gradients and valve area. The patient reported immediate symptomatic improvement following TAVR. At 30 days post-implant, TTE demonstrated a peak velocity of 3.37 m/s, mean gradient of 29 mmHg, dimensionless index of 0.29, and effective valve orifice area of 0.9 cm2. This was associated with a progressive decline in exercise capacity. At 57 days post-implant, TEE showed tissue growth of the aortic root anterior to the valve stent, causing deformation and under-expansion of the valve architecture from a circular to an ovoid shape. Notably, the bioprosthetic leaflets did not appear thickened or calcified, but exhibited restricted motion as a result of the abnormal valve architecture. Peak velocity had further increased to 4.6 m/s, with a mean gradient of 57 mmHg, dimensionless index of 0.21, and calculated effective orifice area of 0.52 cm2. Bioprosthetic aortic valve valve stenosis is an expected complication of valve replacement, though typically characterized by progressive leaflet calcification and pannus formation over several years or by valve thrombosis. Here, we present a rare case of rapidly progressive bioprosthetic TAVR stenosis identified at 30 days post-implantation after normal morphology and gradients on immediate and 1-day imaging. Stenosis was characterized by a process of tissue growth anterior to the stent with involution and underexpansion of the stent architecture and morphologically normal leaflet tissue.

Risk Factors and Outcomes With Progressive Mitral Annular Calcification.

Background Mitral annular calcification (MAC) is a chronic degenerative process that may progress. This study aimed to investigate associating factors and clinical implications of MAC progression. Methods and Results Among 560 patients with MAC identified by transthoracic echocardiography between January 2012 and June 2016, 138 patients (mean±SD age 72.7±10.2 years, 73 women) with mild or moderate MAC who received follow-up examination within 18 to 36 months were retrospectively analyzed. Progressive MAC was defined as hemodynamic or structural profiles that had worsened by more than 1 grade. Hemodynamic features were assessed by the transmitral mean diastolic pressure gradient (MDPG), and structural features were assessed by the MAC angle in the parasternal short-axis view. The clinical outcome was defined as a composite of all-cause mortality, hospitalization for heart failure, and occurrence of ischemic stroke. Forty-three patients (31.2%) showed progressive MAC. Patients with progressive MAC had higher systolic blood pressure, pulse pressure, MAC angle, and MDPG than those with stable MAC. Patients with progressive MAC had smaller left ventricular (LV) end-systolic dimensions and higher LV ejection fractions compared with those with stable MAC. In multivariate analysis, pulse pressure, LV ejection fraction, MAC angle, and MDPG at baseline were significantly associated with MAC progression. During a median of 39.2 months' follow-up, patients with progressive MAC showed poorer clinical outcomes than those with stable MAC (log-rank P=0.015). Conclusions MAC progression is not rare and is associated with structural substrate and hemodynamic loads that result in mechanical stress. Patients with progressive MAC have poor outcomes.

Rapidly Progressive Pediatric Intervertebral Disc Calcification

Rapidly Progressive Pediatric Intervertebral Disc Calcification

Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression.

In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.

Progressive Calcification of Supraspinatus Tendon in Patients With Calcific Tendinitis: Two Case Reports

Background. Calcific tendinitis (CT) is a common disease characterized by the presence of calcific deposits in the tendons of the rotator cuff. CT has a wave-like course, and the formed calcification tends to be resorbed. The lysis of the calcium deposits is characterized by a strong pain syndrome, and the site of the calcification is replaced by collagen.
 The aim to demonstrate clinical observations in which the deposited calcification in the rotator cuff did not resorb but increased over time.
 Cases presentation. We report on two rare clinical cases of an increase in calcification of the rotator cuff in patients aged 51 and 50 years old. Calcific tendinitis occurred with periods of remission and exacerbation. During exacerbations, conservative treatment was carried out, including courses of massage, physiotherapy, and pain relief therapy. In one case, a single injection of a hormonal drug was performed. Control X-rays and MRI during one of the exacerbations showed an increase in the size of the calcifications. Due to the lack of effect from conservative treatment and the detected increase in calcifications, a decision was made to perform surgical treatment. Arthroscopic removal of calcifications with re-fixation of the supraspinatus tendon and elimination of internal damage was performed on both patients. Good results were noted on the follow-up examination according to the ASES orthopedic score, as well as clinical examination data.
 Conclusion. The presented case reports illustrate the possible increase in calcification, unlike the standard course of the disease, in which the calcification is resorbed. Further study of this pathology is necessary to establish the causes and mechanisms of calcification increase over time and its dependence on the phase of the disease.

Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis

BackgroundDiffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 (ENT1−/−), a preclinical model of DISH, and behavioral indicators of pain.MethodsA longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1−/− mice at 2, 4, and 6 months. At endpoint, spinal cords were isolated for immunohistochemical analysis of astrocytes (GFAP), microglia (IBA1), and nociceptive innervation (CGRP).ResultsIncreased spine calcification in ENT1−/− mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1−/− mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1−/− mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1−/− mice compared to wild-type, suggesting an increase in nociceptive innervation.ConclusionThese data suggest that ENT1−/− mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.

The clinical and genetic spectrum of primary familial brain calcification

Primary familial brain calcification (PFBC), formerly known as Fahr’s disease, is a rare neurodegenerative disease characterized by bilateral progressive calcification of the microvessels of the basal ganglia and other cerebral and cerebellar structures. PFBC is thought to be due to an altered function of the Neurovascular Unit (NVU), where abnormal calcium-phosphorus metabolism, functional and microanatomical alterations of pericytes and mitochondrial alterations cause a dysfunction of the blood–brain barrier (BBB) and the generation of an osteogenic environment with surrounding astrocyte activation and progressive neurodegeneration. Seven causative genes have been discovered so far, of which four with dominant (SLC20A2, PDGFB, PDGFRB, XPR1) and three with recessive inheritance (MYORG, JAM2, CMPK2). Clinical presentation ranges from asymptomatic subjects to movement disorders, cognitive decline and psychiatric disturbances alone or in various combinations. Radiological patterns of calcium deposition are similar in all known genetic forms, but central pontine calcification and cerebellar atrophy are highly suggestive of MYORG mutations and extensive cortical calcification has been associated with JAM2 mutations. Currently, no disease-modifying drugs or calcium-chelating agents are available and only symptomatic treatments can be offered.

Effect of vitamin K supplementation on serum calcification propensity and arterial stiffness in vitamin K-deficient kidney transplant recipients: A double-blind, randomized, placebo-controlled clinical trial

Vitamin K deficiency is common among kidney transplant recipients (KTRs) and likely contributes to progressive vascular calcification and stiffness. In this single-center, randomized, double-blind, placebo-controlled trial, we aimed to investigate the effects of vitamin K supplementation on the primary end point, serum calcification propensity (calciprotein particle maturation time, T50), and secondary end points arterial stiffness (pulse wave velocity [PWV]) and vitamin K status in 40 vitamin K-deficient KTRs (plasma dephosphorylated uncarboxylated matrix Gla protein [dp-ucMGP] ≥500 pmol/L). Participants (35% female; age, 57 ± 13 years) were randomized 1:1 to vitamin K2 (menaquinone-7, 360 μg/day) or placebo for 12 weeks. Vitamin K supplementation had no effect on calcification propensity (change in T50 vs baseline +2.3 ± 27.4 minutes) compared with placebo (+0.8 ± 34.4 minutes; Pbetween group = .88) but prevented progression of PWV (change vs baseline −0.06 ± 0.26 m/s) compared with placebo (+0.27 ± 0.43 m/s; Pbetween group = .010). Vitamin K supplementation strongly improved vitamin K status (change in dp-ucMGP vs baseline −385 [−631 to −269] pmol/L) compared with placebo (+39 [−188 to +183] pmol/L; Pbetween group < .001), although most patients remained vitamin K-deficient. In conclusion, vitamin K supplementation did not alter serum calcification propensity but prevented progression of arterial stiffness, suggesting that vitamin K has vascular effects independent of calciprotein particles. These results set the stage for longer-term intervention studies with vitamin K supplementation in KTRs. Trial registryEU Clinical Trials Register (EudraCT Number: 2019-004906-88) and the Dutch Trial Register (NTR number: NL7687).

Transcatheter Mitral Valve Replacement in Patients with Mitral Annular Calcification: A Review.

Mitral annular calcification (MAC) is a progressive degenerative calcification of the mitral valve (MV) that is associated with mitral stenosis, regurgitation or both. Patients with MAC are poor candidates for MV surgery because of technical challenges and high peri-operative mortality. Transcatheter MV replacement (TMVR) has emerged as an option for such high surgical risk patients. This has been described with the use of the SAPIEN transcatheter heart valve (valve-in-MAC) and dedicated TMVR devices. Careful anatomic assessment is important to avoid complications of TMVR, such as left ventricular outflow tract obstruction, valve migration, embolization and paravalvular mitral regurgitation. In this review, we discuss the pathology, importance of preprocedural multimodality imaging for optimal patient selection, clinical outcomes and complications associated with TMVR in patients with MAC.

ARACNOIDITE OSSIFICANTE NA COLUNA VERTEBRAL

ABSTRACT Introduction: Arachnoiditis ossificans (AO) in the spine is a rare entity characterized by progressive calcification of the arachnoid and dural sac, with consequent neurological involvement. Objective: Review the causes, clinical manifestations, and complementary studies for their correct diagnosis. Method: Systematic review under PRISMA guidelines, with search in Pubmed, Lilacs, and Embase. Patient demographics (sex and age), history reported as a cause of AO and time elapsed between cause and diagnosis of AO, clinical manifestations, and complementary studies used for diagnosis were collected. Results: 38 articles, of which we collected 46 patients (25 women, 21 men), mean age of 52 years. The most frequent cause was previous spine surgery and myelography with fat-soluble contrast. The most frequent symptoms were insufficient muscle strength (74%) and pain (69%). CT was used in 76%. The most frequent location was thoracic (35%). Conclusion: Its pathogenesis is unclear; described as the final cause of a chronic inflammatory process in the arachnoid with the consequent bone metaplasia. Diagnosis usually precedes a long period of pain and progressive neurological symptoms. The most sensitive and specific complementary study for the diagnosis is the tomography without contrast, which should be requested in case of clinical suspicion. Level of Evidence II; Systematic Review.

Radiation-induced Brain Calcification Leads to L-dopa-resistant Parkinsonism and Cerebellar Ataxia

We experienced a young patient who presented with progressive parkinsonism and cerebellar ataxia. Brain magnetic resonance imaging revealed progressive brain calcification, expanding from the bilateral basal ganglia to the central pons, caused by a delayed reaction to the radiation therapy that she had received to treat craniopharyngioma 14 years earlier. Heterogeneous clinical symptoms due to radiation-induced brain calcification have been described, but parkinsonism has never been reported. While dopamine transporter-single photon emission computed tomography revealed only slight damage to the dopaminergic striatal pathway, the extension of calcification to the periventricular white matter was likely responsible for her parkinsonism.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

New Percutaneous Approaches for the Treatment of Heavily Calcified Mitral Valve Stenosis.

Important breakthroughs have considerably improved the outcomes of the percutaneous treatment of valvular heart diseases during the last decades. However, calcium deposition and progressive calcification of the left-sided heart valves present a challenge with prognostic implications that have not been addressed until recently. In the case of native mitral stenosis with no surgical options, a compelling need for tackling heavily calcified valves has led to the development of novel debulking techniques and to the use of aortic balloon-expandable bioprosthesis in the mitral position. In this section of the special issue "Mitral Valve Disease: State of the Art", we will review standard approaches and indications for the treatment of native mitral stenosis; summarize these two innovative solutions and their evidence, describing both procedures in a "step-by-step" fashion; and briefly comment on future directions in this field.