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At least 523 records · Page 29Linked to original sources
PubMed2003

Estimation of postmortem interval using the protein marker cardiac Troponin I.

The importance of determining the time since death is crucial to criminal, civil and forensic cases. A technique exploiting the degradation of a protein, cardiac Troponin I (cTnI), to estimate the postmortem interval (PMI) was investigated. Cardiac Troponin I is a basic regulatory protein found as part of a ternary complex responsible for calcium dependent muscle contraction. An efficient extraction protocol to analyze the banding pattern of cTnI in postmortem tissue was developed. The analysis involves extraction of the protein, separation by denaturing gel electrophoresis (SDS-PAGE) and visualization by Western blot using cTnI specific monoclonal antibodies. A bovine model was used to develop and optimize the protocol. The homology of cTnI amongst mammalian species allows for the cross-reaction of human anti-cTnI antibodies with bovine cTnI. The results indicate a characteristic banding pattern amongst human cadavers (n=6), a pseudo-linear relationship between percent cTnI degraded and the log of the time since death (r>0.95), and a qualitative degradation band pattern that in a simple comparative analysis with a standard human heart (known time since death) can be used to estimate the postmortem interval. The degradation-banding pattern of tissue cTnI is useful in the determination of the early postmortem (pm) interval (0-5 days). Overall, this technique offers advantages such a wide postmortem interval, measurable degradation pattern, a temporal semiquantitative relationship and manageable temperature dependence over direct temperature methods.

Animals↗
PubMedDate not supplied

Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes.

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.

Antibodies↗
PubMed1985

Theoretical considerations for optimizing intensity differences between primary musculoskeletal tumors and normal tissue with spin-echo magnetic resonance imaging.

In the radiographic assessment of primary musculoskeletal tumors, it is important for therapy planning to accurately define the extent of a tumor. Using a double spin-echo pulse sequence, the T1 and T2 relaxation times and relative hydrogen densities of several neoplastic tissues and of several normal tissues in four patients were measured. Neoplasms measured included one fibrosarcoma, two osteosarcomas, and one giant cell tumor. Normal tissues measured included normal muscle, fat, and bone marrow. Using a mathematical model of the double spin-echo pulse sequence, the intensity difference between each tumor and each normal tissue for multiple values of TR and TE was calculated. These calculated intensity differences were then used to plot isodifference contour curves for each tissue pair. These plots enabled us to pick combinations of TR and TE that optimized the signal difference between tumor and normal tissue. When comparing tumor with predominantly fatty tissue such as marrow or subcutaneous fat, optimal signal difference in our imager occurred at a TR of 600 to 800 msec and a very short TE. When comparing tumor with muscle, optimal signal difference occurred with very long TR times, and TE times ranging from 30 to 90 msec. These preliminary results suggest that an optimal scanning protocol for primary musculoskeletal tumors should contain at least two different pulse sequences with widely separated TR values (500 and 2000 msec in our instrument), and short to intermediate values of TE (28 and 56 msec in our instrument). It is believed that analysis of isodifference contour plots is a useful method for optimizing intensity differences between any two tissue types.

Bone Neoplasms↗
PubMed1994

Management of patients with acute asthma: what do we know? What do we need to know?

Management of patients with acute, severe asthma mandates the use of comprehensive monitoring and aggressive therapeutic drug regimens. Global clinical and objective assessment criteria can help determine the severity of the acute episode and will also help determine optimal management protocols. The response to aggressive therapy, rather than the initial severity of the attack, predicts outcome. Beta 2 sympathomimetic agents are the treatment of choice for acute asthmatic episodes. Aminophylline and parasympatholytic agents may be useful adjuvants, but more data are needed before firm conclusions can be reached regarding risk-benefit ratios. The optimal timing of steroid therapy is not yet known, but even this form of treatment is slow in producing results.

Acute Disease↗
PubMed2000

Outpatient parenteral inotropic therapy for advanced heart failure.

BACKGROUND: Patients with advanced heart failure generally have hemodynamic perturbation characterized by low cardiac output and high ventricular filling pressures. This creates a clinical milieu with profound symptomatology that includes weakness, fatigue, and fluid-retention states causing peripheral edema, mesenteric congestion, and dyspnea syndromes. Great morbidity including hospital admissions and readmissions as well as high mortality rates ensue. Though medication and/or surgical intervention often attenuate heart failure symptomatology, morbidity, and mortality, some patients reach more advanced stages despite aggressive maneuvers. Indeed, patients presenting with acute decompensation of chronic congestive heart failure frequently receive parenteral inotropic drugs during their hospitalization with clinical improvement. Because these agents generally increase cardiac output and reduce pre-load and afterload, they can be lifesaving. Some patients, however, have symptomatic and hemodynamic rebound to worsened heart failure states during or shortly after inotrope weaning. METHODS: It was, then, a logical step to segue from acute inpatient inotrope infusion to long-term administration of these drugs in the outpatient setting when patients were dependent on these agents. Dopamine, dobutamine, and milrinone are all generally available inotropes that have been used singly or in combination in a chronic outpatient infusion setting. CONCLUSIONS: Data from a few small clinical trials and anecdotal case experience suggest that these drugs result in both hemodynamic and clinical improvement that is generally sustained during chronic administration, and even noted long after discontinuation of infusions in some patients. Some reports have suggested that intermittent infusion therapy in outpatients (so-called pulsed therapy) is effective in attenuating congestive heart failure symptoms long term, with more data supporting chronic infusion of these agents. Though questions regarding safety of these agents have been raised, a reasonable compendium of data published to date supports the contention that inotropic drugs used in this fashion ameliorate symptoms. Legitimate concern may be raised regarding exacerbation of arrhythmias with subsequent sudden cardiac death syndrome; however, in severely symptomatic heart failure patients, the trade-off between symptomatic amelioration and the chance of sudden cardiac death may be worthwhile. Unfortunately, precise guidance regarding the best drug, dose, optimal administration technique, weaning protocol, and actual risk/benefit ratio are not well characterized. Practice as been guided, in large part, by anecdotal experience. However, it appears that chronic or pulsed outpatient parenteral inotropic infusion therapy is frequently prescribed and that this treatment option is an effective alternative for carefully selected patients with severely symptomatic and advanced heart failure. Formulating optimal protocols for home inotropic drug infusion therapy by conducting properly designed clinical trials will be an essential endeavor.

Adrenergic beta-Agonists↗
PubMed2003

Cryoloop vitrification in assisted reproduction: analysis of survival rates in > 1000 human oocytes after ultra-rapid cooling with polymer augmented cryoprotectants.

While human oocytes have been successfully cryopreserved using traditional slow-rate freezing protocols, inconsistent results post-thaw have limited the routine clinical application of oocyte cryopreservation. Despite interest in the potential benefits of vitrification as an alternative laboratory approach to long-term oocyte preservation in assisted reproduction, there is little agreement on how best to configure such cryopreservation protocols to optimize oocyte viability. To comparepost-thaw oocyte survival rates,we performed cryoloop vitrification of human oocytes utilizing two different cryoprotectant mixtures that included polymer macromolecules. Human oocytes (n = 1120) were obtained from consenting patients undergoing in vitro fertilization, but only failed-matured (uninseminated) or failed-fertilized (inseminated but without 2pn development) were included in this investigation. Protocol A consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.4 M sucrose and 20% synthetic serum substitute. Protocol B consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.65 M sucrose, 1 mg/ml polyethylene glycol, 10 mg/ml Ficoll and 20% synthetic serum substitute. Following cryostorage for 10-14 d at -196 degrees C, the survival rate for oocytes vitrified with protocol A was 80.9%, whereas the post-thaw viability among protocol B oocytes was 80.6% (p > 0.005). Our results indicate that an ethylene glycol + dimethyl sulphoxide mixture (with or without polymer macromolecules) can be an effective cryoprotectant strategy for human oocyte vitrification; either approach can be employed without any observed compromise in post-warming survival and/or morphology.

Cell Survival↗
PubMed1987

Cryopreservation of in vitro cultured mouse preimplantation embryos.

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.

Animals↗
PubMed1982

Determination of half-dose depth in skin for soft x-rays.

Unlike superficial x-rays, the soft x-rays normally used in dermatologic practice spare unaffected underlying organs during treatment of cutaneous malignancies. However, since the dose with depth from soft x-rays varies markedly, it is important to know this relationship for optimal therapeutic results. The peak kilovoltage, and thus the energy of the beam, is generally selected so that the dose to the base of the lesion is one-half the surface dose. An absorbed dose of 3,400 rads to the surface and a dose of about one-half this amount to the base of most malignant lesions is one standard protocol for optimal therapeutic results. An accurate value of half-depth dose in skin is therefore necessary and is readily obtained from ordinary half-value layer measurements using the technic described.

Aluminum↗
PubMedDate not supplied

Detection of etiological agent for cholera by PCR protocol.

BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report. We report the detection of Vibrio species in drinking water samples in a duplex PCR reaction. The target loci used in the study were ctxA and tcpA. The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, wherein monitoring of target organisms is very crucial. MATERIAL AND METHODS: Single step thermocycling programs have been reported for amplification of specific target loci. We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR. The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue. This was further treated with proteinase K and heat to yield DNA compatible for PCR. The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples. RESULTS: The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction. The sensitivity of detection achieved for the pathogen was 100 cells per reaction. The specificity of the primers was demonstrated by spiking the reaction with non-specific template. The developed protocol was successfully extended to environmental samples. CONCLUSIONS: The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples. This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.

Bacterial Outer Membrane Proteins↗
PubMed2002

[Stress and stress tolerance in chronic heart failure].

BACKGROUND: Exercise intolerance in patients with chronic heart failure (CHF) shows no correlation to the degree of left ventricular dysfunction. This surprising finding has directed attention to peripheral changes in CHF. During the last years several different peripheral factors as determinants of exercise intolerance have been defined, i.e. abnormalities in ventilation, reduced endothelium-dependent vasodilatation of peripheral conduit and resistance vessels, and altered skeletal muscle metabolism. Skeletal muscle alterations are characterized by a reduced oxidative capacity, a catabolic state with reduced local IGF-I expression and muscle atrophy, chronic inflammation with local expression of the inducible isoform of nitric oxide synthase (iNOS) and an accelerated rate of programmed cell death (apoptosis). EFFECTS OF PHYSICAL EXERCISE: Physical exercise training has evolved as an important therapeutic approach to influence these non-cardiac causes of exercise intolerance. After the first studies documenting the effect of aerobic training on the peripheral causes of exercise intolerance in CHF the question was asked: Should we treat the heart or the periphery to improve exercise intolerance in CHF? Today, we have come closer to the answer: It is now clear that these two systems are not mutually exclusive. Exercise training in CHF has been shown to improve skeletal muscle metabolism and function, to avert muscle catabolism, to reduce neurohumoral overactivation, to reverse endothelial dysfunction and to contribute to the prevention of pathologic left ventricular remodeling. After 6 months of regular exercise training oxidative capacity of the working skeletal muscle increases by approximately 40%. Regular exercise training leads to a significant improvement of endothelium-dependent vasodilatory capacity of peripheral resistance vessels, thereby reducting peripheral resistance in particular during exercise. These beneficial training effects result in a small, but significant improvement of stroke volume and reduction in cardiomegaly. CONCLUSION: Although several questions regarding patient selection, optimal training protocol and training intensity remain unanswered, exercise training can been seen as an established adjunct to pharmacotherapy in CHF. We may soon reach the conclusion that by treating the periphery with exercise programs we are in fact treating the heart, as well. All exercise-induced adaptations converge to increase peak oxygen uptake by up to 2 ml/kg.min. For patients in stable CHF on optimal cardiac medication a combination of in-hospital and home-based aerobic endurance training in combination with local muscle strength training seems most promising. Although exercise training offers no causal treatment of CHF, it has great potentials as an adjunct therapy directed at improving exercise tolerance and expanding the physical limits of CHF patients.

Chronic Disease↗
PubMed1990

Optimization of multidimensional high-performance liquid chromatography for the determination of drugs in plasma by direct injection, micellar cleanup and photodiode array detection.

Improvements in a multidimensional liquid chromatography system for the direct determination of drug substances in blood plasma are reported. The system employs an on-line micellar chromatographic cleanup followed by a reversed-phase analytical separation. The limit of detection of propranolol is improved by a factor 10 compared to previously reported work. The technique is applied towards the determination of a multicomponent mixture of tricyclic anti-depressants in blood plasma. A protocol for optimization is described.

Chromatography, High Pressure Liquid↗
PubMed1996

Macrophage targeting with technetium-99m labelled J001 acylated poly-galactoside for scintigraphy of inflammation: optimization and assessment of imaging specificity in experimental arthritis.

J001, an acylated poly-(1,3)-galactoside purified from the membrane of Klebsiella pneumoniae, associates selectively with macrophages via the binding to CD11b and CD14 molecules. Inflammatory foci known to recruit macrophages could thus be imaged with technetium-99m labelled J001. This study aims to define the optimal scintigraphic protocol for 99mTc-J001 imaging and to evaluate the specificity of J001 scans. A dose range study was conducted in rabbits with immunological arthritis using six different specific activities ranging from 370 to 11840 MBq·mg-1 while the intravenously injected activity was constant (37 MBq) Radiochemical purity for each preparation was documented together with the in vivo stability of the 99mTc-J001 complex using exclusion-diffusion radioHPLC of serum collected 1 h after radiopharmaceutical administration. Scintigraphic images were recorded at 2, 3 and 4h and analysed using indexes calculated from regions of interest. Specificity of the macrophage imaging was assessed by comparison with scans obtained after administration of 99mTcO4(- )or 99mTc-albumin nanocolloids. A protocol of plasma transfusion was also used to inject 99mTc-J001 after complete removal of radioactive colloids likely to be generated during the labelling. For the higher specific activities (5920 and 11840 MBq.mg-1), radiochemical purity degradation and in vitro 99mTc transchelation were noted. To prevent transchelation and 99mTc bond hydrolysis likely to impair imaging specificity, 1480 MBq.mg-1 corresponding to 25microg injected J001 was found to be the optimal usable specific activity. Results obtained with the various tracers support the hypothesis that macrophage targeting is the main factor involved in the J001 imaging of arthritis.

Animals↗
PubMed2001

Tailoring immunosuppressive therapy for renal transplant recipients.

During the past decade several new potent immunosuppressive agents with different modes of action and different side-effect profiles have become available. Nowadays immunosuppression after renal transplantation is no longer one single regimen applicable to all patients. In the selection of the optimal immunosuppressive protocol, individual drug-related toxicity, recipient-related risk factors as well as donor organ characteristics have to be taken into account. This article will give an overview of the most recently developed immunosuppressive agents available for clinical use. Their individual mode of action and their different efficacy and safety profile will be described as basis for selection of each of these drugs in an attempt to tailor the optimal therapeutic regimen for the individual patient both in terms of short-term and long-term outcome.

Antibodies, Monoclonal↗
PubMed2002

Clinical implications of thromboprophylaxis in the management of total hip and knee arthroplasty.

Thrombosis is the most common cause of mortality in the United States, resulting in more than 2 million deaths per year. Almost an equal number of individuals are affected each year by nonfatal thrombosis, including deep vein thrombosis and nonfatal pulmonary embolism. A large proportion of thrombotic episodes can be prevented by the appropriate selection of prophylactic therapy--a clinical decision that figures greatly in numerous clinical conditions associated with an increased risk of thrombosis, including major orthopedic surgery. Orthopedic surgeons are well aware of the risks for complications inherent in total hip arthroplasty and total knee arthroplasty in particular. However, determining which protocols are optimal for thromboprophylaxis remains a matter of contention, and the choice of prophylactic therapy is a critical factor in the successful completion of any major orthopedic surgical procedure. Although there are key differences between total hip and knee arthroplasty in terms of the measures available for thromboprophylaxis and the data documenting their relative degree of effectiveness, the two procedures share many similarities in these respects as well as in their surgical protocols. By reviewing the data and practice guidelines on thromboprophylaxis in total hip and knee arthroplasty together, orthopedic surgeons can more clearly see the implications for clinical success that the choice of prophylactic therapy has on their management of these two vitally important procedures.

Anticoagulants↗
PubMedDate not supplied

Childhood non-Hodgkin's lymphoma--results of treatment with ALL high-risk protocol.

Between August 1984 and September 1990, 13 children with non-Hodgkin's lymphoma (NHL) were treated with ALL high-risk protocol (5 with TCLSG 842 regimen and 8 with TPOG 882B protocol) at Taichung Veterans General Hospital. Diagnosis of NHL was confirmed by histology. Nine had lymphoblastic lymphoma, three had histiocytic lymphoma and one had small non-cleaved cell lymphoma, according to the Rappaport classification. After thorough clinical evaluation eight children were NHL stage IV; four were stage III; and one was stage II. All these children had received chemotherapy as acute lymphocytic leukemia (ALL) high-risk protocol for a total of three years, including central nervous system (CNS) prophylaxis. Of the 13 patients, 9 (69.2%) gained complete remission. Over-all survival rate was 46.2%, with a median interval of 36 months. The complete remission rate and survival rate for the nine children with lymphoblastic lymphoma was better at 77.7% & 66.6% respectively. Within the nonresponsive group, two failed to remit because of early withdrawal from the protocol; the other two patients were refractory to treatment. Relapse was noted in one patient. As to the side effects, neutropenic fever was the most common problem encountered (9 occurrences in 13 patients). Other major complications included severe mucositis, massive GI bleeding, intracranial thrombosis induced by l-asparaginase and tumor lysis syndrome. Childhood NHL is often of diffuse types in pathology, and most affected children have advanced diseases at diagnosis. Choosing an optimal treatment protocol is important for good treatment results.

Adolescent↗
PubMedDate not supplied

Extending the applicability of carboxyfluorescein in solid-phase synthesis.

Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound collections, coupling protocols were developed reducing the amount of coupling reagent and fluorophore. To enable further chemical derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanalytical applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compound collections with uniform labeling yields.

Fluoresceins↗
PubMed2001

Kidney morcellation in laparoscopic nephrectomy for tumor: recommendations for specimen sampling and pathologic tumor staging.

Laparoscopic nephrectomy is a novel approach for small renal tumors in selected patients; however, removal of the kidney through the small laparoscopic abdominal wall incision site requires the kidney to be morcellated into small fragments while still in situ. Morcellation presents two problems for the pathologist. First, guidelines for optimal sampling of morcellated fragments have not been described. Second, morcellation precludes complete pTNM tumor staging, in particular, tumor size, margins, and renal vein involvement. Based on our initial experience with 23 laparoscopic nephrectomies/nephroureterectomies (13 clinically suspected neoplasms, confirmed pathologically as renal cell carcinoma [RCC, n = 7], urothelial carcinoma of the renal pelvis [n = 3], angiomyolipoma [n = 1], and cystic nephroma [n = 1], and 10 clinically benign entities) and a conservative statistical model, we present a decision analysis model of various specimen sampling protocols that optimize cost, labor, or time to diagnosis (single vs sequential sampling). Using the tumor-to-kidney volume ratio (TKR), calculated from preoperative radiologic imaging and specimen gross weight, several specimen sampling algorithms were compared. For the average situation in which TKR is > or =0.15, the algorithm that most significantly optimizes cost and labor is one that initially samples 5% of the morcellated specimen. However, additional sampling may be required in one fourth of the cases. The optimal amount of sampled tissue may indeed be less than 5% because this assumes no suspicious tissue is grossly visible and in all our cases of RCC grossly visible tumor was identified. Additional nomograms for a spectrum of TKR, sampling success, and cost are presented to allow pathologists their own discretion in determining optimal sampling of the morcellated kidney. Tumor staging is severely limited by morcellation. Tumor size, renal capsule involvement, and renal vein involvement cannot be fully pathologically evaluated for RCC, whereas invasion cannot be definitively assessed for urothelial carcinoma of the renal pelvis. Knowledge of the radiologic features (lesion size, capsule, and vein involvement) is important in sampling and staging morcellated kidneys removed laparoscopically.

Algorithms↗
PubMed1997

An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis.

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.

Adolescent↗