Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.

Mapping the emergence of molecular vibrations mediating bond formation.

Fundamental studies of chemical reactions often consider the molecular dynamics along a reaction coordinate using a calculated or suggested potential energy surface1-5. But fully mapping such dynamics experimentally, by following all nuclear motions in a time-resolved manner-that is, the motions of wavepackets-is challenging and has not yet been realized even for the simple stereotypical bimolecular reaction6-8: A-B + C â†’ A + B-C. Here we track the trajectories of these vibrational wavepackets during photoinduced bond formation of the gold trimer complex [Au(CN)2-]3 in an aqueous monomer solution, using femtosecond X-ray liquidography9-12 with X-ray free-electron lasers13,14. In the complex, which forms when three monomers A, B and C cluster together through non-covalent interactions15,16, the distance between A and B is shorter than that between B and C. Tracking the wavepacket in three-dimensional nuclear coordinates reveals that within the first 60 femtoseconds after photoexcitation, a covalent bond forms between A and B to give A-B + C. The second covalent bond, between B and C, subsequently forms within 360 femtoseconds to give a linear and covalently bonded trimer complex A-B-C. The trimer exhibits harmonic vibrations that we map and unambiguously assign to specific normal modes using only the experimental data. In principle, more intense X-rays could visualize the motion not only of highly scattering atoms such as gold but also of lighter atoms such as carbon and nitrogen, which will open the door to the direct tracking of the atomic motions involved in many chemical reactions.

Endoscopic submucosal dissection for colorectal polyps: outcome determining factors.

BACKGROUND: Endoscopic submucosal dissection (ESD) has gained increasing popularity in the management of complicated colorectal polyps. However, clinical outcomes for ESD have remained highly inconsistent worldwide. This study investigated and analysed factors that significantly affect ESD outcomes. METHODS: We conducted a single-centred retrospective study on 220 colorectal polyps removed by ESD from 1st January 2016 to 31st December 2020. Data were collected and retrieved from clinical records. Variables studied included patient demographics, ESD technicalities and polyp characteristics. The primary outcome was completeness of resection based on en bloc and R0 resection rates. The secondary outcomes were recurrence, complications and hospital stay. Further analysis was performed for significant outcome determining factors. RESULTS: The en bloc resection and R0 resection rates were 97.3% and 65% respectively. Intraprocedural and delayed perforation rates were 3.2% and 0.5% respectively. Intraprocedural and delayed bleeding rates were both 1.8%. Post-polypectomy syndrome rate was 2.7%. The median hospital stay was 4 days. Submucosal fibrosis was a significant determining factor for lower en bloc resection (p = 0.004), lower R0 resection (p = 0.002), intraprocedural perforation (p = 0.001), intraprocedural bleeding (p = 0.025) and post-polypectomy syndrome (p = 0.039). Hybrid snaring was associated with lower en bloc resection (p < 0.001), while longer ESD time was associated with lower R0 resection (p = 0.003) and post-polypectomy syndrome (p = 0.025). Other significant factors for post-polypectomy syndrome included young age (p = 0.021) and large polyp size (p = 0.018). Secondary analysis showed that submucosal fibrosis was significantly associated with non-granular lesions (p < 0.001) and prior biopsy (p = 0.003). CONCLUSION: Submucosal fibrosis, hybrid snaring, ESD time, age and polyp size were significant outcome determining factors for ESD. By identifying these factors, strategies may be formulated to improve ESD outcomes.

Protein Hydrolysate from Spirulina platensis Prevents Dexamethasone-Induced Muscle Atrophy via Akt/Foxo3 Signaling in C2C12 Myotubes.

Loss of muscle mass is the primary symptom of sarcopenia. Protein intake is recommended to prevent muscle mass loss, and Spirulina platensis, a microalga with high protein content, is a potential protein supplement. Here, we evaluated the differentiation ability of C2C12 cells and the inhibitory effect of Spirulina hydrolysates (SPH) prepared by Collupulin on dexamethasone (DEX)-treated C2C12 cells. SPH contained 578.27 mg/g protein and 92.30 mg/g branched-chain amino acids. SPH increased C2C12 myotube length and diameter, likely owing to increased MyoD1 and Myf5 expression. Inhibition of increased Atrogin-1, MuRF-1, and FoxO3 expression by SPH in DEX-treated C2C12 cells suppressed DEX-induced muscle atrophy. Moreover, SPH inhibited the DEX-induced increase in cytosolic p-Akt protein expression and suppressed the increase in nuclear FoxO3a protein expression, thereby suppressing the increase in the protein expression of the ubiquitin-proteasome-related factors Atrogin-1 and MuRF-1, which are involved in muscle atrophy. SPH suppressed DEX-induced muscle atrophy by activating the Akt/FoxO3a pathway. SPH promoted C2C12 myoblast differentiation into myotubes and inhibited DEX-induced myotube atrophy by suppressing Atrogin-1 and MuRF-1 expression and regulating the FoxO3a transcription factor. Collectively, SPH can be used as a functional food to inhibit muscle atrophy and promote muscle regeneration.

Optical Kerr Effect of Liquid Acetonitrile Probed by Femtosecond Time-Resolved X-ray Liquidography.

Optical Kerr effect (OKE) spectroscopy is a method that measures the time-dependent change of the birefringence induced by an optical laser pulse using another optical laser pulse and has been used often to study the ultrafast dynamics of molecular liquids. Here we demonstrate an alternative method, femtosecond time-resolved X-ray liquidography (fs-TRXL), where the microscopic structural motions related to the OKE response can be monitored using a different type of probe, i.e., X-ray solution scattering. By applying fs-TRXL to acetonitrile and a dye solution in acetonitrile, we demonstrate that different types of molecular motions around photoaligned molecules can be resolved selectively, even without any theoretical modeling, based on the anisotropy of two-dimensional scattering patterns and extra structural information contained in the q-space scattering data. Specifically, the dynamics of reorientational (libration and orientational diffusion) and translational (interaction-induced motion) motions are captured separately by anisotropic and isotropic scattering signals, respectively. Furthermore, the two different types of reorientational motions are distinguished from each other by their own characteristic scattering patterns and time scales. The measured time-resolved scattering signals are in excellent agreement with the simulated scattering signals based on a molecular dynamics simulation for plausible molecular configurations, providing the detailed structural description of the OKE response in liquid acetonitrile.

Synthesis and biological evaluation of novel purinyl quinazolinone derivatives as PI3Kδ-specific inhibitors for the treatment of hematologic malignancies.

Phosphatidylinositol 3-kinases (PI3Ks) mediate intracellular signal transduction. Aberrant PI3K signaling is associated with oncogenesis and disease progression in solid tumors and hematologic malignancies. Idelalisib (1), a first-in-class PI3Kδ inhibitor for the treatment of hematologic malignancies, was developed, but its sales were limited by black box warnings due to unexpected adverse effects. Therefore, to overcome these adverse events, various quinazolinone derivatives were synthesized and evaluated in vitro based on their inhibitory activity against the PI3K enzyme and the viability of cell lines such as MOLT and SUDHL. Among them, 6f (IC50 = 0.39 nM) and 6m (IC50 = 0.09 nM) showed excellent enzyme activity, and 6m displayed an approximately four-fold higher selectivity for PI3Kγ/δ compared with Idelalisib (1). Furthermore, in vivo PK experiments with 6f and 6m revealed that 6f (AUClast = 81.04 h*ng/mL, Cmax = 18.34 ng/mL, Tmax = 0.5 h, t1/2 = 10.2 h in 1 mpk dose) had improved PK compared with 1. Finally, further experiments will be conducted with 6f selected as a candidate, and the potential for it to be developed as a treatment with good efficacy for hematologic malignancies will be determined.

Correlations of Fat Content in Human Milk with Fat Droplet Size and Phospholipid Species.

Fat globule size and phospholipid (PL) content in human milk (HM) were investigated. HM was classified into three groups depending on fat content (A < B < C). PL content (mg/100 g HM) was significantly higher in the C group (p < 0.05), indicating its positive relationship with HM fat content. When the PL content was normalized (mg/g fat), that of group A was significantly higher (p < 0.05) and fat droplet size in group C was slightly larger, suggesting that HM fat content is affected by fat droplet numbers to a larger extent than by fat droplet size. A correlation between PC and SM content in HM was observed regardless of fat content, while correlation between PE and either PC or SM increased in the order of C > B > A, hence the composition and content of PL species in HM varied according to its fat content.

Estimating signal and noise of time-resolved X-ray solution scattering data at synchrotrons and XFELs.

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X-ray free-electron laser (XFEL) to provide intense and short X-ray pulses. Considering the limited opportunities to use these facilities, it is necessary to verify the plausibility of a target experiment prior to the actual experiment. For this purpose, a program has been developed, referred to as S-cube, which is short for a Solution Scattering Simulator. This code allows the routine estimation of the shape and signal-to-noise ratio (SNR) of TRXL data from known experimental parameters. Specifically, S-cube calculates the difference scattering curve and the associated quantum noise on the basis of the molecular structure of the target reactant and product, the target solvent, the energy of the pump laser pulse and the specifications of the beamline to be used. Employing a simplified form for the pair-distribution function required to calculate the solute-solvent cross term greatly increases the calculation speed as compared with a typical TRXL data analysis. Demonstrative applications of S-cube are presented, including the estimation of the expected TRXL data and SNR level for the future LCLS-II HE beamlines.

The effects of zinc on radiation-induced dysgeusia: a systematic review and meta-analysis.

PURPOSE: Many head and neck cancer patients who receive radiation therapy experience radiation-induced dysgeusia (RID), which has no standard treatment. The only supplement controlled clinical trials have evaluated for the treatment of RID is zinc. However, the results of these and other studies investigating the use of zinc for RID have been inconsistent. To assess the validity of zinc as a treatment for RID, we conducted a systematic literature search and performed a meta-analysis to determine the extent to which zinc affects RID incidence and the degree to which ongoing RID responds to zinc. METHODS: We searched the Ovid MEDLINE, Ovid Embase, PubMed, and Cochrane Library databases to identify studies investigating the use of zinc-based therapy for RID in head and neck cancer patients treated with radiation that were published between January 1, 2003, and November 9, 2017. Using American Society of Clinical Oncology criteria, we selected studies with a high level of evidence for inclusion in the meta-analysis. RESULTS: Of the 32 full-text articles eligible for inclusion, three were included in the final review and meta-analysis. The meta-analysis showed that, compared with placebo, zinc reduces the incidence of RID (risk ratio, 0.72; 95% confidence interval, 0.67-0.92) but does not improve taste acuity more rapidly following radiation therapy (risk ratio, 2.58; 95% confidence interval, 0.97-6.88). CONCLUSION: Our findings indicate that zinc-based therapy reduces the incidence of RID but has a minimal effect on ongoing RID. Our findings also highlight the need for additional evidence-based research on this topic.

Review on colorectal endoscopic submucosal dissection focusing on the technical aspect.

BACKGROUND: Endoscopic submucosal dissection (ESD) enables removal of broad-based colorectal polyps or lateral spreading tumors with a higher rate of en bloc resection and a lower risk of local recurrence. However, it is technically demanding. Over the past 20 years, various equipment and innovative techniques have been developed to reduce the difficulty of ESD. The information in the literature is scattered. Our aim is to provide a comprehensive review on the setup and technical aspects of colorectal ESD. METHODS: We searched the PubMed database and systemically reviewed all original and review articles related to colorectal ESD. Further manual search according to reference lists of identified articles were done. The selected articles were categorized and reviewed. Original figures were created to help readers understand some of the ESD techniques. RESULTS: A total of 216 articles were identified, in which 25 of them were review articles and 191 of them were original articles. They were categorized and reviewed. An in-depth appraisal of the setting, equipment, and technical aspects of colorectal ESD was performed. CONCLUSIONS: Although ESD is a technically demanding procedure that requires expert endoscopic skills, it can be mastered. With good peri-procedural preparation, sufficient knowledge of the equipment, and thorough understanding of the useful endoscopic tricks and maneuvers, colorectal ESD can be performed smoothly and safely.

Transoral Low-Level Laser Therapy Via a Cylindrical Device to Treat Oral Ulcers in a Rodent Model.

BACKGROUND AND OBJECTIVE: Various clinical and animal studies have applied low-level laser therapy (LLLT) to treat oral ulcers. However, most previous studies applied lasers with small pinpoint irradiation, which required multiple laser irradiations to cover the complete extent of the ulcer. The objective of this study was to evaluate the effect of LLLT using a 635 nm diode laser via a transoral device to cover the whole lesion on oral ulcers in an animal model. STUDY DESIGN/MATERIALS AND METHODS: An animal model of oral ulcers was developed with a 6 mm skin punch in the right buccal mucosa of Wistar rats (males, body weight 200-250 g). Three days after the mucosal injury, LLLT (spot size 2 cm2 ) was conducted once a day for 5 days. Twenty-eight rats were randomly assigned into four groups according to energy density (control group, 5, 20, 75 J/cm2 ). The size of the ulcers was measured and histologic analysis were performed ten days after the initial mucosal injury. RESULTS: The mean size of the oral ulcers was significantly smaller in rats treated with an energy density of 20 J/cm2 than that of any other group (control group or energy densities of 5 or 75 J/cm2 ). The irradiation of oral ulcers with an energy density of 20 J/cm2 accelerated the oral mucosa wound healing process and decreased inflammation and granulation tissue, resulting in good reepithelization. However, the histologic outcomes of rats irradiated with energy densities of 5 or 75 J/cm2 were comparable with those of the control group. CONCLUSION: LLLT using a 635 nm diode laser for oral ulcers with a transoral cylindrical device for wide light distribution may accelerate the wound healing process. LLLT with large-surface irradiation may be a substitute for previous LLLT for oral mucosal lesions conducted in a punctuate manner. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Gold Nanoparticle Formation via X-ray Radiolysis Investigated with Time-Resolved X-ray Liquidography.

We report the generation of gold nanoparticles (AuNPs) from the aqueous solution of chloro(2,2',2″-terpyridine)gold(III) ion ([Au(tpy)Cl]2+) through X-ray radiolysis and optical excitation at a synchrotron. The original purpose of the experiment was to investigate the photoinduced structural changes of [Au(tpy)Cl]2+ upon 400 nm excitation using time-resolved X-ray liquidography (TRXL). Initially, the TRXL data did not show any signal that would suggest structural changes of the solute molecule, but after an induction time, the TRXL data started to show sharp peaks and valleys. In the early phase, AuNPs with two types of morphology, dendrites, and spheres, were formed by the reducing action of hydrated electrons generated by the X-ray radiolysis of water, thereby allowing the detection of TRXL data due to the laser-induced lattice expansion and relaxation of AuNPs. Along with the lattice expansion, the dendritic and spherical AuNPs were transformed into smaller, raspberry-shaped AuNPs of a relatively uniform size via ablation by the optical femtosecond laser pulse used for the TRXL experiment. Density functional theory calculations confirm that the reduction potential of the metal complex relative to the hydration potential of X-ray-generated electrons determines the facile AuNP formation observed for [Au(tpy)Cl]2+.

Prosthodontic treatment of a patient with Ewing sarcoma of the left maxillary sinus: A clinical report.

This clinical report describes the prosthodontic rehabilitation of a 22-year-old woman with a substantial treatment history of Ewing sarcoma of the left maxillary sinus. The patient was diagnosed with Ewing sarcoma at the age of 7 years and went through chemotherapy, radiation, surgical resection, and free-flap reconstruction, initially without prosthodontic rehabilitation. The patient aged 22 years was referred to the oral oncology clinic at The University of Texas MD Anderson Cancer Center for prosthodontic treatment. The patient's prosthetic rehabilitation with dental implants and a definitive maxillary obturator prosthesis is presented in detail.

Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline.

Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss.

Earlier-born secondary hair follicles exhibit phenotypic plasticity.

The mouse pelage is composed of four distinct hair types. The fact that the follicles that generate these hair types form in successive waves during late embryonic development suggested the model that distinct epigenetic states of the inductive mesenchyme fixed when the follicles are formed specify the distinctive hair morphologies. This model is inconsistent with the observation that many follicles produce different hair types in successive hair cycles. In this study, the characteristics of the hair follicles that switch between the production of different hair types were examined. These follicles were born earlier than those that do not switch between hair types and made longer hairs. They also expressed a higher level of Sox2 in the dermal papilla and had more DP cells per follicle. These observations are consistent with the hypothesis that different birthdates specify the potential of different follicles. However, rather than directly specifying hair type, birthdate correlates with three types: guard hairs, a plastic population that can make awl, auchene or zigzag hairs, and a population that normally makes only zigzag hairs. Although Sox2 expression levels in the DP identify this subset during the morphogenetic cycle, Sox2 expression is not a fixed epigenetic state specified when the follicle is first formed.

The first report of Bathynellacea in the subterranean water of Mongolia: A new species of Altainella Camacho, 2020 (Crustacea, Bathynellidae).

Background: We report the first finding of Bathynellacea, discovered in Mongolia. We also report a new species of the genus Altainella Camacho, 2020, which was previously recorded only in the western edge of Russia. New information: Altainellamongoliensis sp. nov. was found in the interstitial hyporheic region of the Onon River Basin, Mongolia, by inserting a core approximately 1.2 m deep and extracting the underground interstitial water. The new species exhibited sexual dimorphism in thoracopod VII, uniquely observed within the genus Altainella. We provide a morphological description and remarks on the new species with molecular information based on the 18S rDNA and partial CO1 gene sequences. We emphasise the need for continued research on the subterranean biodiversity in previously neglected regions by reporting the first discovery of macro-stygobionts in Mongolia.

Photomodulative effects of low-level laser therapy on tracheal fenestration developed in in vivo model.

The effect of low-level laser therapy (LLLT) on variable mucosal lesions in the upper aerodigestive tract has been reported. However, the effect of LLLT on tracheostomy sites or tracheal fenestration is rarely reported. In this study, we evaluate the effect of LLLT performed using 635 nm laser light based on a cylindrical diffuser and an animal model with tracheal fenestration. An animal model of tracheal fenestration is developed by suturing the trachea to the skin after performing a vertical tracheostomy from the second to the fifth tracheal ring of Wistar rats (male, body weight 200-250 g). LLLT (spot size: 2 cm2) is conducted once daily for five days using a handheld cylindrical device. Twenty-four rats are randomly assigned to a no-therapy or LLLT group with an energy density of 20 J/cm2. Histological analysis is performed at 7 and 14 days after tracheal fenestration. Irradiation at the tracheal fenestration site with an energy density of 20 J/cm2 improves the wound healing, as shown at 2 weeks after tracheostomy. Histological analysis shows significantly decreased acute inflammation and granulation tissue, as well as better cartilage regeneration and less tracheal wall thickening. Therefore, LLLT demonstrates therapeutic potential for preventing tracheal stenosis and granuloma after tracheostomy.

Cellular and molecular imaging of CAR-T cell-based immunotherapy.

Chimeric Antigen Receptor T cell (CAR-T) therapy has emerged as a transformative therapeutic strategy for hematological malignancies. However, its efficacy in treating solid tumors remains limited. An in-depth and comprehensive understanding of CAR-T cell signaling pathways and the ability to track CAR-T cell biodistribution and activation in real-time within the tumor microenvironment will be instrumental in designing the next generation of CAR-T cells for solid tumor therapy. This review summarizes the signaling network and the cellular and molecular imaging tools and platforms that are utilized in CAR-T cell-based immune therapies, covering both in vitro and in vivo studies. Firstly, we provide an overview of the existing understanding of the activation and cytotoxic mechanisms of CAR-T cells, compared to the mechanism of T cell receptor (TCR) signaling pathways. We further describe the commonly employed tools for live cell imaging, coupled with recent research progress, with a focus on genetically encoded fluorescent proteins (FPs) and biosensors. We then discuss the utility of diverse in vivo imaging modalities, including fluorescence and bioluminescence imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and photoacoustic (PA) imaging, for noninvasive monitoring of CAR-T cell dynamics within tumor tissues, thereby providing critical insights into therapy's strengths and weaknesses. Lastly, we discuss the current challenges and future directions of CAR-T cell therapy from the imaging perspective. We foresee that a comprehensive and integrative approach to CAR-T cell imaging will enable the development of more effective treatments for solid tumors in the future.

Extracorporeal shock wave therapy is not useful after arthroscopic rotator cuff repair.

PURPOSE: Extracorporeal shock wave therapy (ESWT) is known to accelerate the healing of musculoskeletal tissue. The purpose of this study was to test the hypothesis that ESWT stimulates rotator cuff healing after arthroscopic repair. METHODS: Seventy-one consecutive patients with a small- to large-sized rotator cuff tear underwent arthroscopic rotator cuff repair. The patients were randomized into two groups: 35 patients underwent ESWT at 6 weeks after surgery (ESWT group) and 36 patients did not (control group). Cuff integrity was evaluated with computed tomographic arthrography at 6 months after surgery. Constant and UCLA scores were measurable outcomes. RESULTS: All patients were available for a minimum one-year follow-up. The mean age of the ESWT and control groups was 59.4 (SD: 7.7) and 58.6 years (SD: 7.8) (n.s.). There were no significant differences in tear size and repair method between the two groups (n.s.). The mean Constant and UCLA scores, respectively, increased from 54.6 to 90.6 (P < 0.001) and from 18.5 to 27.4 (P < 0.001) in the ESWT group, and from 58.9 to 89.3 (P < 0.001) and 18.5 to 27.4 in the control group. Computed tomographic arthrography was performed in 26 patients from the ESWT group and 24 from the control group, and cuff integrity was maintained in 46 out of 50 patients. Definite re-tear was observed in two patients of the ESWT group and four of the controls. There were no complications associated with ESWT. CONCLUSION: This study failed to prove that ESWT stimulates rotator cuff healing after arthroscopic rotator cuff repair. Additional ESWT after rotator cuff repair could theoretically be advantageous, and it was proven to be safe in this study. LEVEL OF EVIDENCE: II.