Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Risk factors for mortality in 1528 Brazilian childhood-onset systemic lupus erythematosus patients.

OBJECTIVES: To identify associations between mortality in cSLE patients and their characteristics: clinical and laboratory features, disease activity and damage scores, and treatment; to evaluate risk factors associated with mortality in cSLE; and to determine the most frequent causes of death in this group of patients. METHODS: We performed a multicenter retrospective cohort using data from 1,528 cSLE patients followed in 27 pediatric rheumatology tertiary centers in Brazil. Patients' medical records were reviewed according to a standardized protocol, in which information regarding demographic and clinical features, disease activity and damage scores, and treatment were collected and compared between deceased cSLE patients and survivors. Univariate and multivariate analyses by Cox regression model were used to calculate risk factors for mortality, whereas survival rates were analyzed by Kaplan-Meier plots. RESULTS: A total of 63/1,528 (4.1%) patients deceased, 53/63 were female (84.1%), median age at death was 11.9 (9.4-13.1) years and median time interval between cSLE diagnosis and death was 3.2 (0.5-5.3) years. Sepsis was the main cause of death in 27/63 (42.8%) patients, followed by opportunistic infections in 7/63 (11.1%), and alveolar hemorrhage in 6/63 (9.5%) patients. The regression models resulted in neuropsychiatric lupus (NP-SLE) (HR = 2.56, 95% CI = 1.48-4.42) and chronic kidney disease (CKD) (HR = 4.33, 95% CI = 2.33-4.72), as risk factors significantly associated with mortality. Overall patient survival after cSLE diagnosis at 5, 10, and 15 years were 97%, 95.4%, and 93.8%, respectively. CONCLUSIONS: This study confirmed that the recent mortality rate in cSLE in Brazil is low, but still of concern. NP-SLE and CKD were the main risk factors for mortality, indicating that the magnitude of these manifestations was significantly high.

Chronic kidney disease in patients with childhood-onset systemic lupus erythematosus.

BACKGROUND: Lupus nephritis (LN) is a frequent manifestation of childhood-onset systemic lupus erythematosus (cSLE) with a potential risk for kidney failure and poor outcomes. This study aimed to evaluate stages III, IV, and V of chronic kidney disease (CKD) and investigate risk factors for CKD in cSLE patients. METHODS: We performed a nationwide observational cohort study in 27 pediatric rheumatology centers, including medical charts of 1528 cSLE patients. Data were collected at cSLE diagnosis, during follow-up, and at last visit or death, between September 2016 and May 2019. RESULTS: Of 1077 patients with LN, 59 (5.4%) presented with CKD, 36/59 (61%) needed dialysis, and 7/59 (11.8%) were submitted for kidney transplantation. After Bonferroni's correction for multiple comparisons (p < 0.0013), determinants associated with CKD were higher age at last visit, urinary biomarker abnormalities, neuropsychiatric involvement, higher scores of disease activity at last visit and damage index, and more frequent use of methylprednisolone, cyclosporine, cyclophosphamide, and rituximab. In the regression model analysis, arterial hypertension (HR = 15.42, 95% CI = 6.12-38.83, p ≤ 0.001) and biopsy-proven proliferative nephritis (HR = 2.83, 95%CI = 1.70-4.72, p ≤ 0.001) increased the risk of CKD, while children using antimalarials had 71.0% lower CKD risk ((1.00-0.29) × 100%) than children not using them. The Kaplan-Meier comparison showed lower survival in cSLE patients with biopsy-proven proliferative nephritis (p = 0.02) and CKD (p ≤ 0.001). CONCLUSIONS: A small number of patients manifested CKD; however, frequencies of dialysis and kidney transplantation were relevant. This study reveals that patients with cSLE with hypertension, proliferative nephritis, and absence of use of antimalarials exhibited higher hazard rates of progression to CKD. A higher resolution version of the Graphical abstract is available as Supplementary information.

Analysis of surgical and histopathological results of robot-assisted partial nephrectomy with use of three or four robotic arms: an early series results.

OBJECTIVES: The aim of this study was to evaluate whether criteria exist to guide election between the use the three- or four-arm technique in robotic partial nephrectomy (RPN) instead of just the surgeon's preference. MATERIAL AND METHODS: We performed a retrospective review of 80 patients submitted to RPN from May 2016 to February 2020. The patients were divided into two groups of 40, the first submitted to the surgical procedure with use of three robotic arms and the second with four arms. The group division was performed independently of the complexity of the cases, age or gender of the patients and laterality of the renal lesions. Peri- and postoperative data were analyzed for comparison between the two groups. RESULTS: Both techniques had similar oncological outcomes (positive tumor margins), renal function preservation (warm ischemia time) and hemorrhagic complications (estimated blood loss and renal artery pseudoaneurysm), with a small difference in the need for blood transfusion, favoring the technique with three arms. CONCLUSIONS: The two robotic partial nephrectomy techniques had similar oncological and postoperative outcomes, with minimal perioperative complications. The three-arm technique is safe and feasible regardless of the complexity and size of the tumor. Additionally, the use of the three-arm technique reduced surgery costs by US$ 413.00 per patient.

The new 2019-EULAR/ACR classification criteria specific domains at diagnosis can predict damage accrual in 670 childhood-onset systemic lupus erythematosus patients.

OBJECTIVE: To evaluate if the 2019-European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) criteria at diagnosis of childhood-onset systemic lupus erythematosus (cSLE) are associated with higher rates of early damage scored by Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI). METHODS: This retrospective multicenter study included 670 cSLE patients with ≤5 years of disease duration. All patients fulfilled both 2019-EULAR/ACR and 1997-ACR classification criteria. Total score of 2019-EULAR/ACR criteria and each of its specific domains were assessed at diagnosis as predictors of damage accrual at the last visit, according to the presence of any organ damage (defined by SDI ≥ 1). RESULTS: Median disease duration was 2.8 (IQR 1.8-3.8) years and 200 (29.9%) patients had at least one organ damage (SDI ≥ 1). The most frequent domains were neuropsychiatric (12%), renal (7%), and musculoskeletal (6%). There was a higher frequency of renal (58% vs 43%, p = 0.0004) and neuropsychiatric domain (21% vs 7%, p < 0.0001) of 2019-EULAR/ACR criteria in patients with damage (SDI ≥ 1) compared to those without damage (SDI = 0). Patients scoring renal or neuropsychiatric domains of the 2019-EULAR/ACR criteria at diagnosis were associated with renal damage (odds ratio 9.701, 95% confidence interval 3.773-24.941, p < 0.001) or neuropsychiatric damage (OR 9.480, 95% CI 5.481-16.399, p<0.0001) at latest visit, respectively. cSLE patients with positive anti-dsDNA at diagnosis were also associated with renal damage by the latest visit (OR 2.438, 95% CI 1.114-5.3381, p = 0.021). Constitutional, hematologic, mucocutaneous, serosal, and musculoskeletal domains and specific criteria as well as other immunologic criteria were not associated with damage accrual. Median of SLEDAI-2K was significantly higher in patients with global damage (19.5 (2-51) vs 14 (0-51), p<0.001). 2019-EULAR/ACR score >25 was associated with more overall (SDI ≥ 1) (38% vs 25%, p = 0.0002) and renal damage (11% vs 5%, p = 0.023). CONCLUSIONS: The 2019-EULAR/ACR criteria at diagnosis were associated with a higher rate of early damage in cSLE patients, especially for renal and neuropsychiatric damage. Of note, damage was particularly associated with high disease activity at diagnosis and 2019-EULAR/ACR score >25.

Findings regarding non-sexual penile fracture in a referral emergency hospital.

PURPOSE: To describe penile fracture (PF) findings with non-sexual etiology in a referral emergency hospital, with emphasis on demographic data, clinical and intraoperative findings and long-term outcomes. MATERIALS AND METHODS: Patients with PF of non-sexual cause operated at our institution from January 2014 to January 2019 were submitted to surgical treatment and monitored for at least three months after surgery. Etiology of trauma, epidemiological and clinical presentation data, time to intervention and operative findings were reviewed retrospectively. The evaluation of postoperative erectile function was carried out by filling out the International Index of Erection Function - 5 (IIEF-5). The tool used to assess urinary function was the International Prostate Symptom Score (IPSS) questionnaire. RESULTS: Of a total of 149 patients submitted to surgical treatment for PF, 18 (12%) reported non-sexual etiology. Twelve (66.6%) cases were due to penile manipulation through the act of bending the penis during morning erection, three (16.6%) when rolling over in bed with erect penis, one (5.5%) when embracing the wife during erection, one (5.5%) to laying on the partner with erect penis and the other (5.5%) when sitting on the toilet with an erection. Operative findings were unilateral corpus cavernosum injury in all cases. Only one (5.5%) patient had a partial urethral lesion. Follow-up time varied from 3 to 18 months (mean, 10.1 months). Three (16.6%) patients developed erectile dysfunction six months after surgery. However, all of them responded to treatment with IPDE-5 and reported improvement of erection, with no need for medication, on reevaluation after 18 months. One (5.5%) patient developed penile curvature < 30 degrees. Thirteen (72.2%) patients developed penile nodules. No patient presented voiding complaints during follow-up. CONCLUSIONS: PF is a rare urologic emergency, especially with the non-sexual etiology. However, PF should always be considered when the clinical presentation is suggestive, regardless of the etiology. Penile manipulation and roll over in bed were the most common non-sexual causes. These cases are related to low-energy traumas, usually leading to unilateral rupture of corpus cavernosum. Urethral involvement is uncommon but may be present. Early treatment has good long-term clinical outcome, especially when performed in specialized centers with extensive experience in FP.

Lessons learned after 20 years' experience with penile fracture.

OBJECTIVE: To report our experience over the past 20 years in the diagnosis and surgical treatment of penile fracture (PF). MATERIALS AND METHODS: Between January 1997 and January 2017, patients with clinical diagnosis of PF were admitted to our facility and retrospectively assessed. Medical records were reviewed for clinical presentation, etiology and operative findings. Postoperative complications, sexual and urinary function were evaluated. RESULTS: Sexual trauma was the main etiological factor, responsible for 255 cases (88.5%): 110 (43.1%) occurred with the "doggy style" position, 103 (40.3%) with "man on top" position, 31 (12.1%) with the "woman on top" position and 11 (4.3%) in other sexual positions. The most common findings in the clinical presentation were hematoma, in all cases and detumescence in 238 (82.6%). Unilateral corpus cavernosum injuries were found in 199 (69%) patients and bilateral in 89 (31%) patients. Urethral injuries were observed in 54 (18.7%) cases. Nine (14.7%) patients developed erectile dysfunction and eight (13.1%) had penile curvature. Only two (3.7%) patients had complications after urethral reconstruction. CONCLUSIONS: PF has typical clinical presentation and no need for additional tests in most cases. Hematoma and immediate penile detumescence are the most common clinical findings. Sexual activity was the most common cause. The 'doggy style' and 'man-on-top' was the most common positions and generally associated with more severe lesions. Concomitant urethral injury should be considered in cases of high-energy trauma. Surgical reconstruction produces satisfactory results, however, it can lead to complications, such as erectile dysfunction and penile curvature.

First Latin American clinical practice guidelines for the treatment of systemic lupus erythematosus: Latin American Group for the Study of Lupus (GLADEL, Grupo Latino Americano de Estudio del Lupus)-Pan-American League of Associations of Rheumatology (PANLAR).

Systemic lupus erythematosus (SLE), a complex and heterogeneous autoimmune disease, represents a significant challenge for both diagnosis and treatment. Patients with SLE in Latin America face special problems that should be considered when therapeutic guidelines are developed. The objective of the study is to develop clinical practice guidelines for Latin American patients with lupus. Two independent teams (rheumatologists with experience in lupus management and methodologists) had an initial meeting in Panama City, Panama, in April 2016. They selected a list of questions for the clinical problems most commonly seen in Latin American patients with SLE. These were addressed with the best available evidence and summarised in a standardised format following the Grading of Recommendations Assessment, Development and Evaluation approach. All preliminary findings were discussed in a second face-to-face meeting in Washington, DC, in November 2016. As a result, nine organ/system sections are presented with the main findings; an 'overarching' treatment approach was added. Special emphasis was made on regional implementation issues. Best pharmacologic options were examined for musculoskeletal, mucocutaneous, kidney, cardiac, pulmonary, neuropsychiatric, haematological manifestations and the antiphospholipid syndrome. The roles of main therapeutic options (ie, glucocorticoids, antimalarials, immunosuppressant agents, therapeutic plasma exchange, belimumab, rituximab, abatacept, low-dose aspirin and anticoagulants) were summarised in each section. In all cases, benefits and harms, certainty of the evidence, values and preferences, feasibility, acceptability and equity issues were considered to produce a recommendation with special focus on ethnic and socioeconomic aspects. Guidelines for Latin American patients with lupus have been developed and could be used in similar settings.

Takayasu arteritis in childhood: misdiagnoses at disease onset and associated diseases.

Juvenile-Takayasu arteritis (j-TA) is a difficult diagnosis and some patients develop uncommon manifestations and associated diseases that may contribute to the delayed diagnosis. Our aim was to identify the misdiagnoses, the associated diseases and the atypical manifestations observed in a j-TA Brazilian multicentre study. 71 children and adolescents who met the classification criteria for j-TA were included. The misdiagnoses, the associated diseases and the atypical manifestations were evaluated. 19 (26.8%) patients had misdiagnoses. The most common of them was aortic coarctation in six (8.4%) patients, followed by rheumatic fever in five (7.0%) and one patient presented with both former diagnoses. Limb pain (two patients), spondyloarthropathy, juvenile idiopathic arthritis (JIA), spinal arteriovenous malformation, polyarteritis nodosa (PAN) and fever of unknown origin (FUO) were other misdiagnoses. Patients who had misdiagnoses previously to j-TA diagnosis presented a trend to have a longer diagnosis delay. 11 (15.5%) patients had 14 TA-associated diseases, such as pulmonary tuberculosis (5 patients), rheumatic fever (2 patients), spondyloarthropathy, polyarticular JIA, Crohn's disease, Prader-Willi disease, diabetes mellitus, Moyamoya and primary immunodeficiency. 7 (9.9%) patients presented 10 atypical manifestations, such as pyoderma gangrenosum, erythema nodosum, myositis, chorea, enthesitis, episcleritis, uveitis, hepatomegaly, splenomegaly and necrosis of extremities. Our study emphasizes the main misdiagnoses, associated diseases and atypical manifestations that occur in patients with j-TA and warns of the features that may alert paediatricians to this diagnosis, such as constitutional symptoms and elevated inflammatory markers.

What Is the Best Way to Prepare A Buccal Mucosa Graft for Urethroplasty? A Histology-Based Preliminary Report.

PURPOSE: To evaluate the histological characteristics of buccal mucosa samples prepared using different dissection techniques. METHODS: The buccal mucosae harvested from 9 men submitted to a substitution urethroplasty were used in this study. Biopsy specimens of buccal mucosae were grouped according to the macroscopic appearance after dissection: Group 1 - yellowish appearance (minimal dissection), Group 2 - whitish appearance (more aggressive dissection), and Group 3 - translucent appearance (ultra-aggressive dissection). The biopsy specimens were evaluated using histomorphometric techniques. The thicknesses of the graft, epithelium, and sub-epithelial connective tissue were compared. RESULTS: The samples from Group 3 and Groups 2 and 3 presented lower total and lower sub-epithelial connective tissue thickness, respectively, when compared with Group 1. The epithelial thickness was not affected by the dissection method. Muscular and adipose tissues were present in 77 and 55% of samples from Group 1, respectively, whereas these were observed in 44 and 22% of samples from Group 2 respectively. The muscular tissue was observed in 11% of the cases, and the adipose tissue was not observed in samples prepared with ultra-aggressive dissection. CONCLUSION: We conclude that an intermediate graft dissection (whitish appearance, as performed in Group 2) presents the best balance between subepithelial connective tissue preservation and adipose and muscle tissue removal.

Psychotria viridis: Chemical constituents from leaves and biological properties.

The phytochemical study of hexane, chloroform and methanol extracts from leaves of Psychotria viridis resulted in the identification of: the pentacyclic triterpenes, ursolic and oleanolic acid; the steroids, 24-methylene-cycloartanol, stigmasterol and ß-sitosterol; the glycosylated steroids 3-O-ß-D-glucosyl-ß-sitosterol and 3-O-ß-D-glucosyl-stigmasterol; a polyunsaturated triterpene, squalene; the esters of glycerol, 1-palmitoylglycerol and triacylglycerol; a mixture of long chain hydrocarbons; the aldehyde nonacosanal; the long chain fat acids hentriacontanoic, hexadecanoic and heptadenoic acid; the ester methyl heptadecanoate; the 4-methyl-epi-quinate and two indole alkaloids, N,N-dimethyltryptamine (DMT) and N-methyltryptamine. The chemical structures were determined by means of spectroscopic (IR, 1H and 13C NMR, HSQC, HMBC and NOESY) and spectrometric (CG-MS and LCMS-ESI-ITTOF) methods. The study of biologic properties of P. viridis consisted in the evaluation of the acetylcholinesterase inhibition and cytotoxic activities. The hexane, chloroform, ethyl acetate and methanol extracts, the substances 24-methylene-cycloartanol, DMT and a mixture of 3-O-ß-D-glucosyl-ß-sitosterol and 3-O-ß-D-glucosyl-stigmasterol showed cholinesterase inhibiting activity. This activity induced by chloroform and ethyl acetate extracts was higher than 90%. The methanol and ethyl acetate extracts inhibit the growth and/or induce the death of the tumor cells strains B16F10 and 4T1, without damaging the integrity of the normal cells BHK and CHO. DMT also demonstrated a marked activity against tumor cell strains B16F10 and 4T1.

Novel population genetics in ciliates due to life cycle and nuclear dimorphism.

Our understanding of population genetics comes primarily from studies of organisms with canonical life cycles and nuclear organization, either haploid or diploid, sexual, or asexual. Although this template yields satisfactory results for the study of animals and plants, the wide variety of genomic organizations and life cycles of unicellular eukaryotes can make these organisms behave differently in response to mutation, selection, and drift than predicted by traditional population genetic models. In this study, we show how each of these unique features of ciliates affects their evolutionary parameters in mutation-selection, selection-drift, and mutation-selection-drift situations. In general, ciliates are less efficient in eliminating deleterious mutations-these mutations linger longer and at higher frequencies in ciliate populations than in sexual populations--and more efficient in selecting beneficial mutations. Approaching this problem via analytical techniques and simulation allows us to make specific predictions about the nature of ciliate evolution, and we discuss the implications of these results with respect to the high levels of polymorphism and high rate of protein evolution reported for ciliates.

Takayasu arteritis in a Brazilian multicenter study: children with a longer diagnosis delay than adolescents.

OBJECTIVES: To evaluate and compare demographic, clinical, laboratory and angiographic data of Brazilian children and adolescents with Takayasu's arteritis. METHODS: In this Brazilian multicentre, retrospective study which included 10 paediatric rheumatology centres, we identified 71 children and adolescents with Takayasu's arteritis which were diagnosed before their 19th birthday. The patients' demographic, clinical, laboratorial and angiographic data were recorded. The participants were divided into two groups: children, defined by the WHO as younger than 10 years old (group 1: 36 patients) and adolescents, defined as individuals aged 10 to 19 years old (group 2: 35 patients). Features of both groups concerning disease manifestations were compared. RESULTS: A total of 21 (58.3%) patients in group 1 and 30 (85.7%) patients in group 2 were girls (p=0.01). The mean age at disease onset, the mean time to diagnosis, and the mean follow-up time were 5.7 and 12.7, 1.8 and 0.7, 7.2 and 3.6 years, respectively, in groups 1 and 2 (p<0.001, 0.001 and <0.001). At initial evaluation, constitutional symptoms (77.5%) were the most predominant symptoms and decreased peripheral pulses (85.9%) was the most predominant clinical sign without differences between groups. The main laboratory findings were increased erythrocyte sedimentation rate followed by leukocytosis. Anaemia, thrombocytosis and higher platelet levels were significantly more frequent in group 1 (p=0.031, 0.001 and 0.018). Angiographic data were similar in both groups. CONCLUSIONS: Children presented more laboratory abnormalities but clinical and angiographic characteristics were similar to those presented by the adolescents. Diagnosis delay is longer in younger patients.

Ambushing the Ambush Hypothesis: predicting and evaluating off-frame codon frequencies in prokaryotic genomes.

BACKGROUND: In this paper, we address the evidence for the Ambush Hypothesis. Proposed by Seligmann and Pollock, this hypothesis posits that there exists a selection for off-frame stop codons (OSCs) to counteract the possible deleterious effects of translational frameshifts, including the waste of resources and potential cytotoxicity. Two main types of study have been used to support the hypothesis. Some studies analyzed codon usage and showed that codons with more potential to create OSCs seem to be favored over codons with lower potential; they used this finding to support the Ambush Hypothesis. Another study used 342 bacterial genomes to evaluate the hypothesis directly, finding significant excesses of OSCs in these genomes. RESULTS: We repeated both analyses with newer datasets and searched for other factors that could explain the observed trends. In the first case, the relative frequency of codons with the potential to create OSCs is directly correlated with the GC content of organisms, as stop codons are GC-poor. When evaluating the frequency of OSCs directly in 1,976 bacterial genomes we also detected a significant excess. However, when comparing the excess of OSCs with similarly obtained results for the frequency of out-of-frame sense codons, some sense codons have a more significant excess than stop codons. CONCLUSIONS: Two avenues of study have been used to support the Ambush Hypothesis. Using the same methods as these previous studies, we demonstrate that the evidence in support of the Ambush Hypothesis does not hold up against more rigorous testing.

An alternative look at code evolution: using non-canonical codes to evaluate adaptive and historic models for the origin of the genetic code.

The canonical code has been shown many times to be highly robust against point mutations; that is, mutations that change a single nucleotide tend to result in similar amino acids more often than expected by chance. There are two major types of models for the origin of the code, which explain how this sophisticated structure evolved. Adaptive models state that the primitive code was specifically selected for error minimization, while historic models hypothesize that the robustness of the code is an artifact or by-product of the mechanism of code evolution. In this paper, we evaluated the levels of robustness in existing non-canonical codes as well as codes that differ in only one codon assignment from the standard code. We found that the level of robustness of many of these codes is comparable or better than that of the standard code. Although these results do not preclude an adaptive origin of the genetic code, they suggest that the code was not selected for minimizing the effects of point mutations.

Imaging in urethral stricture disease: an educational review of current techniques with a focus on MRI.

Urethral stricture disease refers to narrowing of the urethral lumen obstructing the flow of urine. Urethral strictures can significantly impact daily life due to incontinence, incomplete emptying, hesitancy, and increased risk of urinary tract infections. Imaging is central to the evaluation of suspected urethral stricture, as assessment of stricture length and severity is crucial for guidance of surgical management. The currently employed modalities include radiography, chiefly retrograde urethrography (RUG) and voiding cystourethrography (VCUG); magnetic resonance urethrography (MRU); and sonourethrography (SUG). MRU has become a recent focus of research as it provides high spatial resolution, multiplanar capacity, and soft tissue type differentiation for evaluation of periurethral compartments and concurrent soft tissue defects. The protocol for MRU has evolved over the years to now include dynamic micturition imaging and image reconstruction options. In this review, we discuss each of the imaging modalities used in the diagnosis and evaluation of urethral stricture and provide an overview of literature on MRU over the last decade, including suggested indications that have not yet been incorporated into current guidelines. We delineate scenarios where special diagnostic imaging beyond radiography is beneficial, providing examples from our practice and description of our techniques for each modality.