Regulation of ferroptosis by nanotechnology for enhanced cancer immunotherapy.

INTRODUCTION: This review explores the innovative intersection of ferroptosis, a form of iron-dependent cell death, with cancer immunotherapy. Traditional cancer treatments face limitations in efficacy and specificity. Ferroptosis as a new paradigm in cancer biology, targets metabolic peculiarities of cancer cells and may potentially overcome such limitations, enhancing immunotherapy. AREA COVERED: This review centers on the regulation of ferroptosis by nanotechnology to augment immunotherapy. It explores how nanoparticle-modulated ferroptotic cancer cells impact the TME and immune responses. The dual role of nanoparticles in modulating immune response through ferroptosis are also discussed. Additionally, it investigates how nanoparticles can be integrated with various immunotherapeutic strategies, to optimize ferroptosis induction and cancer treatment efficacy. The literature search was conducted using PubMed and Google Scholar, covering articles published up to March 2024. EXPERT OPINION: The manuscript underscores the promising yet intricate landscape of ferroptosis in immunotherapy. It emphasizes the need for a nuanced understanding of ferroptosis' impact on immune cells and the TME to develop more effective cancer treatments, highlighting the potential of nanoparticles in enhancing the efficacy of ferroptosis and immunotherapy. It calls for deeper exploration into the molecular mechanisms and clinical potential of ferroptosis to fully harness its therapeutic benefits in immunotherapy.

Study on the Anti-Aging Performance of Different Nano-Modified Natural Ester Insulating Oils Based on Molecular Dynamics.

In order to investigate the anti-aging performance of nano-modified natural ester insulating oils, in this paper, two different types of nanoparticles are selected to modify insulating oils. We studied the microscopic mechanism of nano-modified models using molecular simulation techniques. Three models were established: an oil-water model without the addition of nanoparticles and two which contained nano-Fe3O4 and nano-Al2O3 particles, where the concentration of water was 1 wt.%. The research found that the diffusion of water molecules in the nano-modified model was slow, and the water molecules generated from transformer insulation aging were adsorbed around the nanoparticles, which inhibited the diffusion of water molecules, reduced the hydrolysis of ester molecules, and effectively enhanced the anti-aging performance of natural ester insulating oil. Compared with two different types of nano-modified models, the interface compatibility between nano-Fe3O4 and natural ester insulating oil is better, the composite model is stable, the change rate of the diffusion coefficient with temperature is small, there are more hydrogen bonds generated by nano-Fe3O4 and water molecules, and the anti-aging performance of the nano-Fe3O4-modified oil model is better.

The exploration of surgical outcomes in patients with giant cell tumor of the tendon sheath in spine: An epidemiological, radiological, and follow-up investigation.

PURPOSE: This study aimed to characterize giant cell tumor of the tendon sheath (GCTTS) in spine more fully and further validate the therapeutical effect of surgcial resection for treating this neoplasm. METHODS: Patients diagnosed with spinal GCTTS and received surgical resection in our hospital between January 2009 and September 2021 were identified retrospectively. The clinical data and radiological images were summarized and the clinical outcomes of patients with a follow-up period of more than 12 months were analyzed. RESULTS: Thirty patients with benign GCTTS and one with malignant GCTTS were included. Preoperative radiological images were available in 28 of 30 benign cases. Benign lesions were revealed as soft tissue masses centering on the facet joint with osteolytic bone destruction in 26 patients on CT, and as prevertebral or intramuscular masses without bone erosion in 2. MRI showed the signal of isointensity or hypointensity on T1 weighted images (T1WI) in 25 patients and slightly hyperintense in three. On T2 weighted images (T2WI), 17 lesions displayed homogeneous hypointense signal, and eight lesions possessed heterogeneous signals. The remaining three lesions featured slightly hyperintense signal on T2WI. Follow-up data were available in 23 of 30 benign cases treated with gross-total resection, and two patients experienced recurrence. CONCLUSIONS: Spinal GCTTS should be suspected in cases with features such as the mass mainly involving the posterior bone elements, the lack of intralesional calcification, T2-weighted dark signals, and free of any cancer. Gross-total resection is an effective means for treating spinal GCTTS.

Peripheral and intestinal mucosal-associated invariant T cells in premature infants with necrotizing enterocolitis.

Background: Necrotizing enterocolitis (NEC) is a potentially fatal inflammatory gastrointestinal disease in preterm infants with unknown pathogenesis. Mucosal-associated invariant T (MAIT) cells primarily accumulate at sites where exposure to microbes is ubiquitous and regulate immunological responses. As the implications of these cells in NEC development in premature infants remain unknown, we investigated the role and characteristics of MAIT cells in NEC pathogenesis. Methods: The percentage of different MAIT cell subsets in peripheral blood samples of 30 preterm infants with NEC and 22 control subjects was estimated using flow cytometry. The frequency of MAIT cells in the intestinal tissues of five NEC patients and five control subjects was also examined. The level of serum cytokines was estimated using cytometric bead array. Potential associations between the different measurements were analyzed using the Spearman's correlation test. Results: Compared with controls, the NEC patients were found to have significantly reduced percentages of circulating CD161+ CD3+ CD8αα+ T cells and CD161+ CD3+ TCRγδ-TCRVa7.2+ MAIT cells. In the intestinal tissues, the percentage of MAIT cells was significantly higher in samples from the NEC patients than the controls. Furthermore, the percentage of circulating MAIT cells in the peripheral blood samples was inversely correlated with that in the intestinal tissues of the NEC patients. The percentage of CD8αα+ MAIT cells was found to be significantly reduced in both peripheral blood and intestinal tissues of NEC patients. Following treatment, the frequency of circulating MAIT cells significantly increased in NEC patients and reached a level similar to that in the control subjects. However, there was no difference in the percentage of circulating CD8αα+ MAIT cells before and after treatment in the NEC patients. Conclusion: Our results suggested that during the development of NEC MAIT cells accumulate in the inflammatory intestinal tissues, while the percentage of CD8aa+ MAIT cells is significantly decreased, which may lead to the dysfunction of MAIT cells in gut immunity.

Systematic Identification of Genomic Markers for Guiding Iron Oxide Nanoparticles in Cervical Cancer Based on Translational Bioinformatics.

Purpose: Magnetic iron oxide nanoparticle (MNP) drug delivery system is a novel promising therapeutic option for cancer treatment. Material issues such as fabrication and functionalized modification have been investigated; however, pharmacologic mechanisms of bare MNPs inside cancer cells remain obscure. This study aimed to explore a systems pharmacology approach to understand the reaction of the whole cell to MNPs and suggest drug selection in MNP delivery systems to exert synergetic or additive anti-cancer effects. Methods: HeLa and SiHa cell lines were used to estimate the properties of bare MNPs in cervical cancer through 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and enzyme activity assays and cellular fluorescence imaging. A systems pharmacology approach was utilized by combining bioinformatics data mining with clinical data analysis and without a predefined hypothesis. Key genes of the MNP onco-pharmacologic mechanism in cervical cancer were identified and further validated through transcriptome analysis with quantitative reverse transcription PCR (qRT-PCR). Results: Low cytotoxic activity and cell internalization of MNP in HeLa and SiHa cells were observed. Lysosomal function was found to be impaired after MNP treatment. Protein tyrosine kinase 2 beta (PTK2B), liprin-alpha-4 (PPFIA4), mothers against decapentaplegic homolog 7 (SMAD7), and interleukin (IL) 1B were identified as key genes relevant for MNP pharmacology, clinical features, somatic mutation, and immune infiltration. The four key genes also exhibited significant correlations with the lysosome gene set. The qRT-PCR results showed significant alterations in the expression of the four key genes after MNP treatment in HeLa and SiHa cells. Conclusion: Our research suggests that treatment of bare MNPs in HeLa and SiHa cells induced significant expression changes in PTK2B, PPFIA4, SMAD7, and IL1B, which play crucial roles in cervical cancer development and progression. Interactions of the key genes with specific anti-cancer drugs must be considered in the rational design of MNP drug delivery systems.

Effects of low back pain on balance performance in elderly people: a systematic review and meta-analysis.

BACKGROUND: Research suggests that individuals with low back pain (LBP) may have poorer motor control compared to their healthy counterparts. However, the sample population of almost 90% of related articles are young and middle-aged people. There is still a lack of a systematic review about the balance performance of elderly people with low back pain. This study aimed to conduct a systematic review and meta-analysis to understand the effects of LBP on balance performance in elderly people. METHODS: This systematic review and meta-analysis included a comprehensive search of PubMed, Embase, and Cochrane Library databases for full-text articles published before January 2020. We included the articles that 1) investigated the elderly people with LBP; 2) assessed balance performance with any quantifiable clinical assessment or measurement tool and during static or dynamic activity; 3) were original research. Two independent reviewers screened the relevant articles, and disagreements were resolved by a third reviewer. RESULTS: Thirteen case-control studies comparing balance performance parameters between LBP and healthy subjects were included. The experimental group (LBP group) was associated with significantly larger area of centre of pressure movement (P < 0.001), higher velocity of centre of pressure sway in the anteroposterior and mediolateral directions (P = 0.01 and P = 0.02, respectively), longer path length in the anteroposterior direction (P < 0.001), slower walking speed (P = 0.05), and longer timed up and go test time (P = 0.004) than the control group. CONCLUSION: The results showed that balance performance was impaired in elderly people with LBP. We should pay more attention to the balance control of elderly people with LBP.

Identification of key genes for guiding chemotherapeutic management in ovarian cancer using translational bioinformatics.

The emergence of resistance to chemotherapy drugs in patients with ovarian cancer is still the main cause of low survival rates. The present study aimed to identify key genes that may provide treatment guidance to reduce the incidence of drug resistance in patients with ovarian cancer. Original data of chemotherapy sensitivity and chemoresistance of ovarian cancer were obtained from the Gene Expression Omnibus dataset GSE73935. Differentially expressed genes (DEGs) between sensitive and resistant ovarian cancer cell lines were screened by Empirical Bayes methods. Overlapping DEGs between four chemoresistant groups were identified by Venn map analysis. Protein-protein interaction networks were also constructed, and hub genes were identified. The hub genes were verified by in vitro experiments as well as The Cancer Genome Atlas data. Results from the present study identified eight important genes that may guide treatment decisions regarding chemotherapy regimens for ovarian cancer, including epidermal growth factor-like repeats and discoidin I-like domains 3, NRAS proto-oncogene, hyaluronan and proteoglycan link protein 1, activated protein C receptor, CD53, cyclin-dependent kinase inhibitor 2A, insulin-like growth factor 1 receptor and roundabout guidance receptor 2 genes. Their expressions were found to have an impact on the prognosis of different treatment groups (cisplatin, paclitaxel, cisplatin + paclitaxel, cisplatin + doxorubicin and cisplatin + topotecan). The results indicated that these genes may minimise the occurrence of ovarian cancer drug resistance and may provide effective treatment options for patients with ovarian cancer.

Loss of high-temperature requirement protein A2 protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia.

Cellular homeostasis requires tight coordination between nucleus and mitochondria, organelles that each possesses their own genomes. Disrupted mitonuclear communication has been found to be implicated in many aging processes. However, little is known about mitonuclear signaling regulator in sarcopenia which is a major contributor to the risk of poor health-related quality of life, disability, and premature death in older people. High-temperature requirement protein A2 (HtrA2/Omi) is a mitochondrial protease and plays an important role in mitochondrial proteostasis. HtrA2mnd2(-/-) mice harboring protease-deficient HtrA2/Omi Ser276Cys missense mutants exhibit premature aging phenotype. Additionally, HtrA2/Omi has been established as a signaling regulator in nervous system and tumors. We therefore asked whether HtrA2/Omi participates in mitonuclear signaling regulation in muscle degeneration. Using motor functional, histological, and molecular biological methods, we characterized the phenotype of HtrA2mnd2(-/-) muscle. Furthermore, we isolated the gastrocnemius muscle of HtrA2mnd2(-/-) mice and determined expression of genes in mitochondrial unfolded protein response (UPRmt ), mitohormesis, electron transport chain (ETC), and mitochondrial biogenesis. Here, we showed that HtrA2/Omi protease deficiency induced denervation-independent skeletal muscle degeneration with sarcopenia phenotypes. Despite mitochondrial hypofunction, upregulation of UPRmt and mitohormesis-related genes and elevated total reactive oxygen species (ROS) production were not observed in HtrA2mnd2(-/-) mice, contrary to previous assumptions that loss of protease activity of HtrA2/Omi would lead to mitochondrial dysfunction as a result of proteostasis disturbance and ROS burst. Instead, we showed that HtrA2/Omi protease deficiency results in different changes between the expression of nuclear DNA- and mitochondrial DNA-encoded ETC subunits, which is in consistent with their transcription factors, nuclear respiratory factors 1 and 2, and coactivator peroxisome proliferator-activated receptor γ coactivator 1α. These results reveal that loss of HtrA2/Omi protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia. The novel mechanistic insights may be of importance in developing new therapeutic strategies for sarcopenia.

Ti-24Nb-4Zr-8Sn Alloy Pedicle Screw Improves Internal Vertebral Fixation by Reducing Stress-Shielding Effects in a Porcine Model.

To ensure the biomechanical properties of Ti-24Nb-4Zr-8Sn, stress-shielding effects were compared between Ti-24Nb-4Zr-8Sn and Ti-6Al-4V fixation by using a porcine model. Twelve thoracolumbar spines (T12-L5) of 12-month-old male pigs were randomly divided into two groups: Ti-24Nb-4Zr-8Sn (EG, n = 6) and Ti-6Al-4V (RG, n = 6) fixation. Pedicle screw was fixed at the outer edge of L4-5 vertebral holes. Fourteen measuring points were selected on the front of transverse process and middle and posterior of L4-5 vertebra. Electronic universal testing machine was used to measure the strain resistance of measuring points after forward and backward flexion loading of 150 N. Meanwhile, stress resistance was compared between both groups. The strain and stress resistance of measurement points 1, 2, 5, 6, 9, and 10-14 in Ti-24Nb-4Zr-8Sn fixation was lower than that of Ti-6Al-4V fixation after forward and backward flexion loading (P < 0.05). The strain and stress resistance of measurement points 3, 4, 7, and 8 was higher in Ti-24Nb-4Zr-8Sn fixation than that of Ti-6Al-4V fixation (P < 0.05). Stress-shielding effects of Ti-24Nb-4Zr-8Sn internal fixation were less than that of Ti-6Al-4V internal fixation. These results suggest that Ti-24Nb-4Zr-8Sn elastic fixation has more biomechanical goals than conventional Ti-6Al-4V internal fixation by reducing stress-shielding effects.

Knockdown of long non-coding RNA HOXD-AS1 inhibits the progression of osteosarcoma.

Long non-coding RNA HOXD-AS1 (HOXD-AS1) has recently been shown to be involved in the development and progression of multiple cancers. However, the expression, significance, and biological function of HOXD-AS1 in osteosarcoma (OS) remain unknown. Here, we found that the expression level of HOXD-AS1 was significantly upregulated in OS tissues and cells. Furthermore, high expression of HOXD-AS1 was positively associated with the clinical and pathological characteristics of OS, including tumor stage and lymph node metastasis, and negatively correlated with overall survival rate. in vitro assays confirmed that knockdown of HOXD-AS1 suppressed cell proliferation, colony formation, migration, and invasion, and promoted cell cycle arrest at G1 stage and apoptosis in OS cells. in vivo assays confirmed that knockdown of HOXD-AS1 significantly decreased tumor growth in xenograft mice, and decreased tumor size and weight. Importantly, we also showed that knockdown of HOXD-AS1 significantly reduced signal transducer and activator of transcription 3 and its target protein (CyclinD1, Bcl-2, and MMP-2) expression in vitro and in vivo. Moreover, overexpression of STAT3 could reverse the suppression of proliferation ability induced by sh-HOXD-AS1 in U2OS cells. Collectively, our data indicated that HOXD-AS1 might be an oncogenic long non-coding RNA (lncRNA) and might be a potential attractive therapeutic target for OS.

K-rod dynamic internal fixation versus microendoscopic discectomy for the treatment of single-segment lumbar disc herniation.

PURPOSE: This study compared the clinical outcome of K-rod dynamic internal fixation versus microendoscopic discectomy (MED) for the treatment of single-segment lumbar disc herniation. METHODS: This retrospective study included 34 patients with L4-L5 single-segment lumbar disc herniation who underwent K-rod dynamic internal fixation ( n = 18) or MED ( n = 16). The pain was evaluated by the Oswestry disability index (ODI) and visual analogue scale (VAS). The neurological function was assessed by the Japanese Orthopaedic Association (JOA) scores. The height of intervertebral space was calculated using X-ray images, and the disc degeneration was evaluated based on Pfirrmann scores. The mean follow-up time was 31 months (range, 18-46 months). RESULTS: In both groups, the ODI scores, VAS scores and JOA scores were significantly improved at the last follow-up compared with the preoperative values ( p < 0.05). There was no significant difference in the improvement of ODI, VAS and JOA scores between the two groups ( p > 0.05). The height of intervertebral space in the surgical segments, proximal segments and distal segments, was significantly greater in the K-rod group than in the MED group. The disc degeneration occurred more in the MED group than in the K-rod group ( p < 0.05). CONCLUSION: K-rod internal fixation and MED produce satisfactory outcomes in the treatment of single-segment lumbar disc herniation. K-rod internal fixation is superior to MED in preventing adjacent segment degeneration.

Oligosaccharide nanomedicine of alginate sodium improves therapeutic results of posterior lumbar interbody fusion with cages for degenerative lumbar disease in osteoporosis patients by downregulating serum miR-155.

Degenerative lumbar disease (DLD) is a significant issue for public health. Posterior lumbar intervertebral fusion with cages (PLIFC) has high-level fusion rate and realignment on DLD. However, there are some complications following the surgery. Alginate oligosaccharides (AOS) have antioxidant and anti-inflammatory activities and may be suitable for infection therapy. MiR-155 is a biomarker associated with inflammatory and oxidative stress. AOS may promote PLIFC therapy by regulating miR-155. Pluronic nanoparticles and oligosaccharide nanomedicine of alginate sodium (ONAS) were prepared with ampicillin at size <200 nm. Ninety-six DLD osteoporosis patients received PLIFC and were evenly assigned into ONAS group (OG, oral administration of 100 mg ONAS daily) and control group (PG, 100 mg pluronic nanoparticles). Serum miR-155 level was measured by real-time quantitative PCR. The levels of superoxide dismutase (SOD), glutathione (GSH), aspartate aminotransaminase (AST), alanine aminotransferase (ALT), interleukin-1ß (IL-1ß), and interleukin-1 receptor antagonist (IL-1ra) were measured. Weighted mean difference (WMD), relative risk (RR), complications, surgery infection rate, fusion rate, and Japanese Orthopaedic Association (JOA) scores were used to evaluate therapeutic efficacy. After 1-month therapy, infection rates and side effects were lower in OG than those in PG (RR =0.64, 95% confidence interval [CI] [0.48, 0.84], P=0.001). The fusion rates were higher in OG than in PG (WMD =21.96, 95% CI [-0.24, 37.62], P=0.021). The JOA scores were higher in OG than in PG (RR =0.52, 95% CI [0.33, 0.84], P=0.007), and no significant difference was found for the visual analog scale and Oswestry Disability Index. Serum levels of miR-155, ALT, AST, and IL-1ß were lower while SOD, GSH, and IL-1ra were higher in OG than in PG. MiR-155 mimic increased the levels of ALT, AST, and IL-1ß and reduced the levels of SOD, GSH, and IL-1ra. In contrast, miR-155 inhibitor had reverse results. Therefore, ONAS has better improvement in complications and therapeutic effects on DLD by regulating serum miR-155.

Chitin Oligosaccharide (COS) Reduces Antibiotics Dose and Prevents Antibiotics-Caused Side Effects in Adolescent Idiopathic Scoliosis (AIS) Patients with Spinal Fusion Surgery.

Antibiotics are always considered for surgical site infection (SSI) in adolescent idiopathic scoliosis (AIS) surgery. However, the use of antibiotics often causes the antibiotic resistance of pathogens and side effects. Thus, it is necessary to explore natural products as drug candidates. Chitin Oligosaccharide (COS) has anti-inflammation and anti-bacteria functions. The effects of COS on surgical infection in AIS surgery were investigated. A total of 312 AIS patients were evenly and randomly assigned into control group (CG, each patient took one-gram alternative Azithromycin/Erythromycin/Cloxacillin/Aztreonam/Ceftazidime or combined daily), experiment group (EG, each patient took 20 mg COS and half-dose antibiotics daily), and placebo group (PG, each patient took 20 mg placebo and half-dose antibiotics daily). The average follow-up was one month, and infection severity and side effects were analyzed. The effects of COS on isolated pathogens were analyzed. SSI rates were 2%, 3% and 8% for spine wounds and 1%, 2% and 7% for iliac wound in CG, EG and PG (p < 0.05), respectively. COS reduces the side effects caused by antibiotics (p < 0.05). COS improved biochemical indexes and reduced the levels of interleukin (IL)-6 and tumor necrosis factor (TNF) alpha. COS reduced the antibiotics dose and antibiotics-caused side effects in AIS patients with spinal fusion surgery by improving antioxidant and anti-inflammatory activities. COS should be developed as potential adjuvant for antibiotics therapies.

Association of single nucleotide polymorphisms of IL23R and IL17 with necrotizing enterocolitis in premature infants.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal inflammatory disease in neonates, particularly in preterm infants. The interleukin (IL) 23/IL17 axis has been shown to play an important role in the gastrointestinal inflammation. However, the association of gene polymorphisms in the IL23/IL17 axis and the development of NEC remains unknown. In this study, we aimed to explore a possible genetic role of IL23R and IL17 in the development of NEC. We identified single nucleotide polymorphisms (SNPs) in IL23R (rs10889677), IL17A (rs2275913), and IL17F (rs763780) by polymerase chain reaction and Sanger sequencing. A total of 102 NEC patients (stage II, n = 75; and stage III, n = 27) and 120 control subjects were recruited for the study. All of the participants were premature (gestational age < 37 weeks). Our results revealed that the combination of the IL17F rs763780 (TC + CC) genotype and the C allele both significantly increased the risk of NEC [odds ratio (OR) 1.89, 95% confidence interval (CI) 1.04-3.43, P = 0.035; OR 1.82, 95% CI 1.06-3.13, P = 0.028, respectively]. Furthermore, the rs763780 (TC + CC) genotype was associated with increased severity of NEC and the incidence of NEC-related perforation [OR 2.80, 95% CI 1.10-7.12, P = 0.031; OR 3.86, 95% CI 1.10-13.53, P = 0.035, respectively]. However, IL23R rs10889677 and IL17A rs2275913 were not associated with the susceptibility to NEC. In conclusion, our data suggest that a variant of IL17F (rs763780) may contribute to the development of NEC.

Does heterotopic ossification affect the outcomes of cervical total disc replacement? A meta-analysis.

STUDY DESIGN: Meta-analysis. OBJECTIVE: The purpose of this study was to answer the following questions: (1) Does heterotopic ossification (HO) negatively influence clinical outcomes after cervical total disc replacement (CTDR)? (2) Should patients be classified into HO and non-HO groups? (3) Is there a more rational classification? SUMMARY OF BACKGROUND DATA: Heterotopic ossification has emerged as a common complication after CTDR and has been an important reason for reoperation, thus limiting the use of the surgery. However, the influence of HO on clinical outcomes after CTDR has not been well established. METHODS: A meta-analysis was conducted with studies identified by searches of MEDLINE, EMBASE, and the Cochrane Library. We calculated the weighted mean differences of the visual analogue scale pain score, Neck Disability Index, and range of motion (ROM). Patients were classified into 2 groups under 3 classifications on the basis of the grade of HO. Results were pooled using a fixed effect model or a random effects model, according to the heterogeneity. RESULTS: There were significant differences in ROM under all 3 classifications. The visual analogue scale pain score and the Neck Disability Index between the patients with and without HO showed no significant difference after CTDR. Significant differences in visual analogue scale pain score were observed when patients were classified into a "high-grade HO" group (McAfee grades 3 or 4 HO) and a "low-grade HO" group (McAfee grade 0, 1, or 2 HO). CONCLUSIONS: The presence of HO is not associated with clinical outcomes after CTDR. However, the severity of HO actually impacts clinical outcomes in an inverse manner, which needs further investigation. It is inappropriate to classify patients on the basis of the presence of HO; further studies of the classifications (ROM-affecting HO vs. ROM-preserving HO; high-grade HO vs. low-grade HO) and cervical stability after CTDR are needed. LEVEL OF EVIDENCE: 2.