NF-κB Decoy Oligodeoxynucleotide-Loaded Poly Lactic-co-glycolic Acid Nanospheres Facilitate Socket Healing in Orthodontic Tooth Movement.

Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides loaded with polylactic-co-glycolic acid nanospheres (PLGA-NfDs) to mitigate alveolar bone loss during orthodontic tooth movement (OTM) following the bilateral extraction of maxillary first molars in a controlled experiment involving forty rats of OTM model with ethics approved. The decreased tendency of the OTM distance and inclination angle with increased bone volume and improved trabecular bone structure indicated minimized alveolar bone destruction. Reverse transcription-quantitative polymerase chain reaction and histomorphometric analysis demonstrated the suppression of inflammation and bone resorption by downregulating the expression of tartrate-resistant acid phosphatase, tumor necrosis factor-α, interleukin-1ß, cathepsin K, NF-κB p65, and receptor activator of NF-κB ligand while provoking periodontal regeneration by upregulating the expression of alkaline phosphatase, transforming growth factor-ß1, osteopontin, and fibroblast growth factor-2. Importantly, relative gene expression over the maxillary second molar compression side in proximity to the alveolus highlighted the pharmacological effect of intra-socket PLGA-NfD administration, as evidenced by elevated osteocalcin expression, indicative of enhanced osteocytogenesis. These findings emphasize that locally administered PLGA-NfD serves as an effective inflammatory suppressor and yields periodontal regenerative responses following tooth extraction.

SDF-1 involvement in orthodontic tooth movement after tooth extraction.

The stromal cell-derived factor 1 (SDF-1)/chemokine receptor type 4 (CXCR4) axis plays a key role in alveolar bone metabolism during orthodontic tooth movement (OTM). Herein, the effects of the SDF-1/CXCR4 axis on the regional acceleratory phenomenon (RAP) in OTM velocity and on changes in the surrounding periodontium after adjacent tooth extraction in rats were investigated. Six-week-old male Wistar/ST rats underwent left maxillary first molar (M1) extraction and mesial OTM of the left maxillary second molar (M2) with a 10-g force closed-coil spring. Phosphate-buffered saline, immunoglobulin G (IgG) isotype control antibody, or anti-SDF-1 neutralizing monoclonal antibody were injected at the M1 and M2 interproximal areas (10 µg/0.1 mL) for the first three days. Analyses were performed after 1, 3, and 7 days (n = 7). The results demonstrated a significant increase in SDF-1 expression from day 1, which was effectively blocked via anti-SDF-1 neutralizing monoclonal antibody injection. On day 3, the M2 OTM distance and the number of positively stained osteoclasts significantly reduced alongside a reduction in inflammatory markers in the experimental group. Our results demonstrated that serial local injection of the anti-SDF-1 neutralizing monoclonal antibody reduces M2 OTM, osteoclast accumulation, and localized inflammatory responses in an OTM model with tooth extraction-induced RAP.

Molecular genetic diagnostics for inherited retinal dystrophies in the clinical setting.

OBJECTIVE: To evaluate the success of diagnostic genetic testing in inherited retinal dystrophy (IRD) patients in the clinical setting. DESIGN: Retrospective cohort analysis. PARTICIPANTS: A total of 446 consecutive participants from diverse ethnic backgrounds living in western Canada. METHODS: Clinical information was collected from participants, including family history, and they underwent a full ophthalmic examination with chart review. Those with a suspected IRD were offered panel-based genetic testing of 351 genes between March 1, 2019, and February 28, 2022. The main outcome measure was effect of the genetic testing results on clinical diagnosis. RESULTS: Genetic testing established a conclusive molecular diagnosis in 249 of 446 cases (55.8%), a clearly negative result in 90 of 446 cases (20.1%), and an inconclusive diagnosis in 108 of 446 cases (24.2%). Conclusive disease-causing variants were identified in 69 genes, and the most commonly affected genes were ABCA4 (31 variants), USH2A (25 variants), and RPGR (19 variants). The inconclusive group included likely novel autosomal dominant variants or a pathogenic variant with a variant of uncertain significance in the same gene for a recessive phenotype. Notably, an inconclusive molecular genetic diagnosis was seen in as many as 47.3% of East Asian participants with an outer retinal dystrophy. CONCLUSIONS: This study represents the largest review of molecular genetic testing in IRDs in Canada. That negative or inconclusive results obtained in approximately 45% of cases demonstrates that there is an important need for new research into molecular genetic causes of IRDs. This is particularly true in addressing the problem of interpreting a variant of uncertain significance in ethnic minorities.

NF-κB Decoy ODN-Loaded Poly(Lactic-co-glycolic Acid) Nanospheres Inhibit Alveolar Ridge Resorption.

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides (ODNs) are double-stranded DNA sequences capable of downregulating the expression of downstream genes of the NF-κB pathway, which is recognized for regulating prototypical proinflammatory signals, physiological bone metabolism, pathologic bone destruction, and bone regeneration. The aim of this study was to investigate the therapeutic effect of NF-κB decoy ODNs on the extraction sockets of Wistar/ST rats when delivered by poly(lactic-co-glycolic acid) (PLGA) nanospheres. Microcomputed tomography and trabecular bone analysis following treatment with NF-κB decoy ODN-loaded PLGA nanospheres (PLGA-NfDs) demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1ß, tumor necrosis factor-α, receptor activator of NF-κB ligand, turnover rate, and increased transforming growth factor-ß1 immunopositive reactions and relative gene expression. These data demonstrate that local NF-κB decoy ODN transfection via PLGA-NfD can be used to effectively suppress inflammation in a tooth-extraction socket during the healing process, with the potential to accelerate new bone formation.

Novel pathogenic variants in Tubulin Tyrosine Like 5 (TTLL5) associated with cone-dominant retinal dystrophies and an abnormal optical coherence tomography phenotype.

Purpose: Autosomal recessive cone and cone-rod dystrophies (CD/CRD) are inherited forms of vison loss. Here, we report on and correlate the clinical phenotypes with the underlying genetic mutations. Methods: Clinical information was collected from subjects, including a family history with a chart review. They underwent a full ophthalmic examination, including best-corrected visual acuity, direct and indirect ophthalmoscopy, color vision testing, color fundus photography, contrast sensitivity, autofluorescence, and spectral domain-optical coherence tomography (SD-OCT), and full-field electroretinography. Next-generation panel-based genetic testing was used to identify DNA variants in subject buccal swab samples. Results: Genetic testing in two patients revealed three novel variants in the TTLL5 gene associated with CD/CRD: two missense variants (c.1433G>A;p.(Arg478Gln), c.241C>G;p.(Leu81Val), and one loss-of-function variant (c.2384_2387del;p.(Ala795Valfs*9). Based on in-silico analysis, structural modeling, and comparison to previously reported mutations, these novel variants are very likely to be disease-causing mutations. Combining retinal imaging with SD-OCT analysis, we observed an unusual sheen in the CD/CRD phenotypes. Conclusion: Based on the protein domain location of novel TTLL5 variants and the localization of TTLL5 to the connecting cilium, we conclude that the CD/CRD disease phenotype is characterized as a ciliopathy caused by protein tracking dysfunction. This initially affects cone photoreceptors, where photoreceptor cilia express a high level of TTLL5, but extends to rod photoreceptors over time. Fundus photography correlated with SD-OCT imaging suggests that the macular sheen characteristically seen with TTLL5 mutations derives from the photoreceptor's outer segments at the posterior pole.

Microelectrode Arrays, Electrocatalysis, and the Need for Proper Characterization.

Indirect electrochemical methods are a powerful tool for synthetic chemistry because they allow for the optimization of chemical selectivity in a reaction while maintaining the advantages of electrochemistry in terms of sustainability. Recently, we have found that such methods provide a handle for not only the synthesis of complex molecules, but also the construction of complex, addressable molecular surfaces. In this effort, the indirect electrochemical methods enable the placement or synthesis of molecules by any electrode or set of electrodes in a microelectrode array. The success of these surface-based reactions are typically evaluated with the use of fluorescence labelling studies. However, these fluorescence-based evaluations can be misleading. While they are excellent for determining that a reaction has occurred in a site-selective fashion on an array, they do not provide information on whether that reaction is the one desired or how well it worked. We describe here how the use of a "safety-catch" linker strategy allows for a more accurate assessment of reaction quality on an array, and then use that capability to illustrate how the use of transition metal mediated cross-coupling reactions on an array prevent unwanted background reactions that can occur on a polymer-coated electrode surface. The method enables a unique level of quality control for array-based transformations.

RNA modifications in brain tumorigenesis.

RNA modifications are emerging as critical regulators in cancer biology, thanks to their ability to influence gene expression and the predominant protein isoforms expressed during cell proliferation, migration, and other pro-oncogenic properties. The reversibility and dynamic nature of post-transcriptional RNA modifications allow cells to quickly adapt to microenvironmental changes. Recent literature has revealed that the deregulation of RNA modifications can promote a plethora of developmental diseases, including tumorigenesis. In this review, we will focus on four key post-transcriptional RNA modifications which have been identified as contributors to the pathogenesis of brain tumors: m6A, alternative polyadenylation, alternative splicing and adenosine to inosine modifications. In addition to the role of RNA modifications in brain tumor progression, we will also discuss potential opportunities to target these processes to improve the dismal prognosis for brain tumors.

A method for going from 2D laparoscope to 3D acquisition of surface landmarks by a novel computer vision approach.

PURPOSE: This paper presents a method to use the Smart Trocars-our new surgical instrument recognition system-or any accurate localization system of surgical instrument for acquiring intraoperative surface data. Complex laparoscopic surgeries need a proper guidance system which requires registering the preoperative data from a CT or MRI scan to the intraoperative patient state. The Smart Trocar can be used to localize the instruments when it comes to contact with the soft tissue surface. METHOD: Two successive views through the laparoscope at different angles with the 3D localization of a fixed tool at one single location using the Smart Trocars can point out visible features during the surgery and acquire their location in 3D to provide a depth map in the region of interest. In other words, our method transforms a standard laparoscope system into a system with three-dimensional registration capability. RESULT: This method was initially tested on a simulation for uncertainty assessment and then on a rigid model for verification with an accuracy within 2 mm distance. In addition, an in vivo experiment on pig model was also conducted to investigate how the method might be used during a physiologic respiratory cycle. CONCLUSION: This method can be applied in a large number of surgical applications as a guidance system on its own or in conjunction with other navigation techniques. Our work encourages further testing with realistic surgical applications in the near future.

The SmartOR: a distributed sensor network to improve operating room efficiency.

BACKGROUND: Despite the significant expense of OR time, best practice achieves only 70% efficiency. Compounding this problem is a lack of real-time data. Most current OR utilization programs require manual data entry. Automated systems require installation and maintenance of expensive tracking hardware throughout the institution. This study developed an inexpensive, automated OR utilization system and analyzed data from multiple operating rooms. STUDY DESIGN: OR activity was deconstructed into four room states. A sensor network was then developed to automatically capture these states using only three sensors, a local wireless network, and a data capture computer. Two systems were then installed into two ORs, recordings captured 24/7. The SmartOR recorded the following events: any room activity, patient entry/exit time, anesthesia time, laparoscopy time, room turnover time, and time of preoperative patient identification by the surgeon. RESULTS: From November 2014 to December 2015, data on 1003 cases were collected. The mean turnover time was 36 min, and 38% of cases met the institutional goal of ≤30 min. Data analysis also identified outlier cases (>1 SD from mean) in the domains of time from patient entry into the OR to intubation (11% of cases) and time from extubation to patient exiting the OR (11% of cases). Time from surgeon identification of patient to scheduled procedure start time was 11 min (institution bylaws require 20 min before scheduled start time), yet OR teams required 22 min on average to bring a patient into the room after surgeon identification. CONCLUSION: The SmartOR automatically and reliably captures data on OR room state and, in real time, identifies outlier cases that may be examined closer to improve efficiency. As no manual entry is required, the data are indisputable and allow OR teams to maintain a patient-centric focus.

Image-guided simulation of tissue deformation using a mechanical model on a surgical application.

This paper presents a method for localizing the position of a liver and a tumor within the tissue during a minimally invasive liver operation. From pre-operative CT scans, the liver volume and its internal structures are segmented using image-processing techniques. Based on these segmentations, a three-dimensional mechanical model is built to compute the liver volume and internal structure displacement under boundary conditions such as external forces from the surgical instrument. This can help the surgeon understand the motion of internal structures when manipulating the liver. To validate our method, an experiment on a porcine liver explant was performed to assess the difference between actual tissue motion and the mechanical model.

A robust and non-obtrusive automatic event tracking system for operating room management to improve patient care.

BACKGROUND: Optimization of OR management is a complex problem as each OR has different procedures throughout the day inevitably resulting in scheduling delays, variations in time durations and overall suboptimal performance. There exists a need for a system that automatically tracks procedural progress in real time in the OR. This would allow for efficient monitoring of operating room states and target sources of inefficiency and points of improvement. STUDY DESIGN: We placed three wireless sensors (floor-mounted pressure sensor, ventilator-mounted bellows motion sensor and ambient light detector, and a general room motion detector) in two ORs at our institution and tracked cases 24 h a day for over 4 months. RESULTS: We collected data on 238 total cases (107 laparoscopic cases). A total of 176 turnover times were also captured, and we found that the average turnover time between cases was 35 min while the institutional goal was 30 min. Deeper examination showed that 38 % of laparoscopic cases had some aspect of suboptimal activity with the time between extubation and patient exiting the OR being the biggest contributor (16 %). CONCLUSION: Our automated system allows for robust, wireless real-time OR monitoring as well as data collection and retrospective data analyses. We plan to continue expanding our system and to project the data in real time for all OR personnel to see. At the same time, we plan on adding key pieces of technology such as RFID and other radio-frequency systems to track patients and physicians to further increase efficiency and patient safety.

Interferon-λ restricts West Nile virus neuroinvasion by tightening the blood-brain barrier.

Although interferon-λ [also known as type III interferon or interleukin-28 (IL-28)/IL-29] restricts infection by several viruses, its inhibitory mechanism has remained uncertain. We used recombinant interferon-λ and mice lacking the interferon-λ receptor (IFNLR1) to evaluate the effect of interferon-λ on infection with West Nile virus, an encephalitic flavivirus. Cell culture studies in mouse keratinocytes and dendritic cells showed no direct antiviral effect of exogenous interferon-λ, even though expression of interferon-stimulated genes was induced. We observed no differences in West Nile virus burden between wild-type and Ifnlr1(-/-) mice in the draining lymph nodes, spleen, or blood. We detected increased West Nile virus infection in the brain and spinal cord of Ifnlr1(-/-) mice, yet this was not associated with a direct antiviral effect in mouse neurons. Instead, we observed an increase in blood-brain barrier permeability in Ifnlr1(-/-) mice. Treatment of mice with pegylated interferon-λ2 resulted in decreased blood-brain barrier permeability, reduced West Nile virus infection in the brain without affecting viremia, and improved survival against lethal virus challenge. An in vitro model of the blood-brain barrier showed that interferon-λ signaling in mouse brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and signal transducer and activator of transcription 1 (STAT1)-independent manner. Our data establish an indirect antiviral function of interferon-λ in which noncanonical signaling through IFNLR1 tightens the blood-brain barrier and restricts viral neuroinvasion and pathogenesis.

Endoscopic suture fixation of self-expanding metallic stents with and without submucosal injection.

BACKGROUND: Self-expanding metallic stents (SEMS) are useful for treating leaks after bariatric procedures but stent migration (12 to 40 %) remains a problem. Suture fixation has been used to minimize migration but has attendant risks of transmural penetration. We hypothesized that submucosal injection would decrease the risk of full thickness suture penetration while still providing greater pullout forces than endoscopic clipping. METHODS: Porcine explant models and 155 mm SEMS were used with a force meter to measure the pullout forces required to dislodge the stent. Stents were first deployed without fixation and this pullout force acted as a control. The explants were then randomized to receive fixation with clips, endoscopic suturing with submucosal elevation, or endoscopic suturing without submucosal elevation. The pullout force was again measured and the ratio of the experimental to control pullout forces was used as a measure of the efficacy of the fixation. RESULTS: Endoscopic suture fixation after submucosal injection resulted in statistically significant increased pullout force compared to clip fixation (n = 5; mean force ratio 462 %; 95 % confidence interval [CI] 281-643 %; p < 0.01). Endoscopic suturing fixation without submucosal injection also resulted in statistically significant increased pullout force (n = 5; mean force ratio 765 %; 95 % CI 258-632 %; p < 0.01). Fixation with clips did not result in significantly increased pullout force compared to no fixation (n = 5; mean force ratio 108 %; 95 % CI 56-159 %; p < 0.01). Submucosal injection also eliminated full thickness suture penetration (0/10 submucosal injection; 7/10 no injection). CONCLUSIONS: Endoscopic suture fixation of SEMS resulted in a statistically significant increase in pullout force necessary to displace the stents. Submucosal injection prior to suture fixation preserves the increased pullout force while minimizing the risk of transmural penetration with the potential for less risk of injury to mediastinal structures.

In vitro and in vivo characterization of a West Nile virus MAD78 infectious clone.

The viral determinants governing the varied neuropathogenicity of different West Nile virus (WNV) strains are poorly understood. Here, we generated an infectious clone (WNV-MAD(IC)) of the non-pathogenic strain WNV-MAD78 and compared its replication to that of parental WNV-MAD78 and a WNV-MAD78 infectious clone (WNV-MAD(TX-UTRs)) containing the 5' and 3' untranslated regions (UTRs) of the pathogenic strain WNV-TX. All three viruses replicated at similar rates and caused similar lethality in mice. Thus, the infectious clone is indistinguishable from parental virus in replication and neurovirulence, and the UTRs alone do not account for the increased virulence of WNV-TX compared to WNV-MAD78.

IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling.

Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-ß after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-ß and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-ß or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.

A FOXO3-IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses.

Antiviral responses must be tightly regulated to defend rapidly against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses and their transcription is regulated by a variety of transcription factors; principal among these is the family of interferon regulatory factors (IRFs). The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.

The lineage-defining factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns.

The T-box transcription factor T-bet is important for the differentiation of naive CD4(+) T helper cells (Th cells) into the Th1 phenotype. Much is known about T-bet's role as a transcriptional activator, but less is known about the mechanisms by which T-bet functionally represses alternative Th cell genetic programs. In this study, we first identify Socs1, Socs3, and Tcf7 (TCF-1) as gene targets that are negatively regulated by T-bet. Significantly, T-bet's role in the repression of these genes is through a direct interaction with their promoters. Consistent with this, we identified two T-bet DNA-binding elements in the Socs1 promoter that are functionally used to down-regulate transcription in primary Th1 cells. Importantly, T-bet's novel role in transcriptional repression is because of its ability to physically associate with, and functionally recruit, the transcriptional repressor Bcl-6 to a subset of promoters. Furthermore, T-bet functionally recruits Bcl-6 to the Ifng locus in late stages of Th1 differentiation to repress its activity, possibly to prevent the overproduction of IFN-γ, which could result in autoimmunity. Collectively, these data establish a novel mechanism for T-bet-mediated gene repression in which two lineage-defining transcription factors, one a classical activator and one a repressor, collaborate to promote and properly regulate Th1 development.

A novel rotationally invariant region-based hidden Markov model for efficient 3-D image segmentation.

We present a novel 3-D region-based hidden Markov model (rbHMM) for efficient unsupervised 3-D image segmentation. Our contribution is twofold. First, rbHMM employs a more efficient representation of the image data than current state-of-the-art HMM-based approaches that are based on either voxels or rectangular lattices/grids, thus resulting in a faster optimization process. Second, our proposed novel tree-structured parameter estimation algorithm for the rbHMM provides a locally optimal data labeling that is invariant to object rotation, which is a highly valuable property in segmentation tasks, especially in medical imaging where the segmentation results need to be independent of patient positioning in scanners in order to minimize methodological variability in data analysis. We demonstrate the advantages of our proposed technique over grid-based HMMs by validating on synthetic images of geometric shapes as well as both simulated and clinical brain MRI scans. For the geometric shapes data, our method produced consistently accurate segmentation results that were also invariant to object rotation. For the brain MRI data, our white matter and gray matter segmentation resulted in substantially higher robustness and accuracy levels with improved Dice similarity indices of 4.60% (p=0.0022) and 7.71% , respectively.

A hybrid geometric-statistical deformable model for automated 3-D segmentation in brain MRI.

We present a novel 3-D deformable model-based approach for accurate, robust, and automated tissue segmentation of brain MRI data of single as well as multiple magnetic resonance sequences. The main contribution of this study is that we employ an edge-based geodesic active contour for the segmentation task by integrating both image edge geometry and voxel statistical homogeneity into a novel hybrid geometric-statistical feature to regularize contour convergence and extract complex anatomical structures. We validate the accuracy of the segmentation results on simulated brain MRI scans of both single T1-weighted and multiple T1/T2/PD-weighted sequences. We also demonstrate the robustness of the proposed method when applied to clinical brain MRI scans. When compared to a current state-of-the-art region-based level-set segmentation formulation, our white matter and gray matter segmentation resulted in significantly higher accuracy levels with a mean improvement in Dice similarity indexes of 8.55% ( p < 0.0001) and 10.18% ( p < 0.0001), respectively.