A Split-Lung Ex Vivo Perfusion Model for Time- and Cost-Effective Evaluation of Therapeutic Interventions to the Human Donor Lung.

With the ongoing shortage of donor lungs, ex vivo lung perfusion (EVLP) offers the opportunity for objective assessment and potential therapeutic repair of marginal organs. There is a need for robust research on EVLP interventions to increase the number of transplantable organs. The use of human lungs, which have been declined for transplant, for these studies is preferable to animal organs and is indeed essential if clinical translation is to be achieved. However, experimental human EVLP is time-consuming and expensive, limiting the rate at which promising interventions can be assessed. A split-lung EVLP model, which allows stable perfusion and ventilation of two single lungs from the same donor, offers advantages scientifically, financially and in time to yield results. Identical parallel circuits allow one to receive an intervention and the other to act as a control, removing inter-donor variation between study groups. Continuous hemodynamic and airway parameters are recorded and blood gas, perfusate, and tissue sampling are facilitated. Pulmonary edema is assessed directly using ultrasound, and indirectly using the lung tissue wet:dry ratio. Evans blue dye leaks into the tissue and can quantify vascular endothelial permeability. The split-lung ex vivo perfusion model offers a cost-effective, reliable platform for testing therapeutic interventions with relatively small sample sizes.

Identification of a TRP channel-related risk model for predicting prognosis and therapeutic effects of patients with hepatocellular carcinoma.

PURPOSE: TRP channels have been implicated in cancer progression. Our study seeks to establish a prognostic model for hepatocellular carcinoma (HCC) by utilizing genes related to TRP channels. METHODS: We used the TCGA and ICGC databases as training and validation cohorts, respectively. We calculated the risk scores using Lasso-Cox regression analysis based on the expression levels of prognostic genes and performed survival analysis to compare overall survival between high- and low-risk groups. Then we compared the clinicopathologic characteristics and conducted biological functional analysis. We also explored immune cell infiltration and compared the drug sensitivity. RESULTS: Using bioinformatics algorithms, we identified 11 TRP-related genes and calculated the risk scores. Patients in the high-risk group demonstrated worse overall survival, as well as more advanced T stage and pathologic stage. The risk score showed a significant association with the cell cycle. The high-risk group had more ICI and RTK targets with elevated expression and showed better therapeutic effect to chemotherapy including 5-fluorouracil, camptothecin, docetaxel, doxorubicin, gemcitabine, and paclitaxel. Overall, an individualized nomogram was constructed by integrating the risk score and requisite clinicopathologic parameters to predict the overall survival of HCC patients. CONCLUSIONS: We successfully established a highly accurate prognostic model for predicting overall survival and therapeutic effects using TRP channel-related genes.

Andrographis modulates cisplatin resistance in lung cancer via miR-155-5p/SIRT1 axis.

Andrographis (Andro) has been identified as an anti-cancer herbal. This study was to explore its underlying regulatory routes regarding cisplatin (DDP) resistance in lung cancer. The impacts of Andro on cell viability in lung cancer cells and normal cells BEAS-2B were validated using CCK8 tests. Then, cell viability and apoptosis analysis was performed in the cells after DDP, Andro, or combined treatment. RT-qPCR was applied for evaluating miR-155-5p and SIRT1 mRNA expressions, while western blot was for evaluating SIRT1 protein expressions. Binding sites between SIRT1 and miR-155-5p were predicted on TargetScan and were confirmed using luciferase reporter assays. Xenograft animal models were established for in vivo validation of the regulatory function of Andro in lung cancer. Andro decreased the cell viability in lung cancer cells but not normal cells BEAS-2B. The combined treatment with DDP and Andro induced the lowest viability and highest apoptosis in both A549 and A549/DDP cells. MiR-155-5p expression was suppressed, and SIRT was promoted by the Andro treatment, while overexpression of miR-155-5p reversed effects of Andro in cells, which was further counteracted by SIRT1 activation. SIRT1 was verified to be a target of miR-155-5p in A549/DDP cells. Moreover, Andro synergized with DDP in mice with lung cancer via miR-155-5p/SIRT1. Andro modulates cisplatin resistance in lung cancer via miR-155-5p/SIRT1 axis.

Shikonin Inhibits Candida albicans Biofilms via the Ras1-cAMP-Efg1 Signalling Pathway.

Objective: To investigate the influence of shikonin (SK) on the formation of Candida albicans biofilms and discuss the possible mechanism. Methods: The inhibition of the formation of C. albicans biofilms by SK was observed by scanning electron microscopy. A silicone film method and a water-hydrocarbon two-phase assay were performed to investigate the effects of SK on cell adhesion. Real-time reverse-transcription polymerase chain reaction was used to analyse the expression of genes related to cell adhesion and Ras1-cyclic adenosine monophosphate (cAMP) - enhanced filamentous growth protein 1 (Efg1) signalling pathway. Finally, the level of cAMP in C. albicans was detected and exogenous cAMP rescue experiment was conducted. Results: The results showed that SK could destroy the typical three-dimensional structure of the biofilms, inhibit cell surface hydrophobicity and cell adhesion, downregulate the expression of Ras1-cAMP-Efg1 signalling pathway-related genes (ECE1, HWP1, ALS3, RAS1, CYR1, EFG1 and TEC1) and effectively reduce the production of key messenger cAMP in the Ras1-cAMP-Efg1 pathway. Meanwhile, exogenous cAMP reversed the inhibitory effect of SK on biofilms formation. Conclusion: Our results suggest that SK exhibits potential anti-C. albicans biofilms effects related to the inhibition of Ras1-cAMP-Efg1 pathway.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

In vitro antifungal activity of Shikonin against Candida albicans by inducing cellular apoptosis and necrosis.

BACKGROUND: Our previous studies showed that Shikonin (SK) had a strong anti-Candida albican (C. albicans) activity, especially against some fluconazole-resistant strains, which is probably due to the oxidative damage of SK to C. albicans. METHODS AND RESULTS: In this study, we expanded the antifungal spectrum and evaluate the toxicity of SK. The results indicated that SK also exhibited potent invitro antifungal activities against other pathogenic fungi such as other Candida, Aspergillus, Cryptococcus, and Dermatophytes, but did not display apparent toxicity to the mammalian cells, suggesting that SK is safe to be a potential antifungal drug. Furtherly, we analyze the exact mechanism of SK against C. albicans. We found that SK could induce a series of apoptosis characteristics, including phosphatidylserine externalization, chromatin condensation and fragmentation, decreased cytochrome c oxidase activity as well as caspase activation. CONCLUSIONS: In summary, this study highlighted the antifungal activity and mechanism of SK against C. albicans, providing a potential therapeutic strategy for C. albicans infection.

Characterization of the complete mitochondrial genome of Quasilineus sinicus Gibson, 1990 (Nemertea: Heteronemertea) and its phylogenetic implications.

In this study, we sequenced and characterized the complete mitochondrial genome (mitogenome) of Quasilineus sinicus Gibson, 1990 (Heteronemertea, Nemertea) using Illumina sequencing technology. The circular mitogenome was 16,358 bp in length and comprised 22 transfer RNA genes, 13 protein-coding genes, and two ribosomal RNA genes. Its overall base composition included 20.82% A, 41.06% T, 26.68% G, and 11.44% C; in fact, the mitogenome had a high A + T content of 61.88%. Furthermore, our phylogenetic analysis demonstrated that Paleonemertea, Pilidiophora, and Hoplonemertea were monophyletic groups, and Q. sinicus was most closely related to Iwatanemertes piperata.

Persistence of an epidemic cluster of Rhodotorula mucilaginosa in multiple geographic regions in China and the emergence of a 5-flucytosine resistant clone.

Rhodotorula mucilaginosa, an environmental yeast widely used in industry and agriculture, is also an opportunistic pathogen resistant to multi-antifungals. During the national surveillance in China, R. mucilaginosa has been documented from various hospitals and regions. At present, the molecular epidemiology of invasive infections caused by R. mucilaginosa and their resistance profiles to antifungals were unknown. Here we collected 49 strains from four hospitals located in different geographic regions from 2009 to 2019 in China, determined their genotypes using different molecular markers and quantified susceptibilities to various antifungals. Sequencing of ITS and D1/D2 regions in rDNA indicated that 73.5% (36/49) of clinical strains belong to same sequence type (rDNA type 2). Microsatellite (MT) genotyping with 15 (recently developed) tandem repeat loci identified 5 epidemic MT types, which accounted for 44.9% (22/49) of clinical strains, as well as 27 sporadic MT types. Microsatellite data indicated that the presence of an epidemic cluster including 35 strains (71.4%) repeatedly isolated in four hospitals for eight years. Single nucleotide variants (SNVs) from the whole genome sequence data also supported the clustering of these epidemic strains due to low pairwise distance. In addition, phylogenetic analysis of SNVs from these clinical strains, together with environmental and animal strains showed that the closely related epidemic cluster strains may be opportunistic, zoonotic pathogens. Also, molecular data indicated a possible clonal transmission of pan echinocandins-azoles-5-flucytosine resistant R. mucilaginosa strains in hospital H01. Our study demonstrated that R. mucilaginosa is a multi-drug resistant pathogen with the ability to cause nosocomial infection.

Aquaporin-4 deletion attenuates opioid-induced addictive behaviours associated with dopamine levels in nucleus accumbens.

There is a lack of safe and effective non-opioid medications for the treatment of opioid addiction. Aquaporin-4 (AQP4), a water channel protein expressed in astrocytes, regulates the progression of neurological diseases. Our previous work demonstrated that AQP4 deficiency in mice attenuated morphine-induced physiological dependence. However, the role of AQP4 in the neurobiology of behaviours related to opioid addiction in mice remains unclear. Here, we report that Aqp4-knockout mice exhibited attenuated heroin consumption and heroin-seeking behaviours. Furthermore, Aqp4-knockout mice displayed diminished hyperactivity induced by morphine and heroin and subsequently showed dramatically inhibited morphine-induced behavioural sensitization. This attenuated hyperlocomotion to opioids was accompanied by a decreased dopamine response to the opioid-induced increase in the levels of extracellular dopamine in the NAc. In addition, Aqp4-knockout mice displayed upregulation of dopamine transporters in the striatum, suggesting a probable neurobiological mechanism for uptake of the extracellular dopamine. The present findings suggest that deficiency of AQP4 decreases opiate-induced drug seeking and taking behaviours, and AQP4 may be involved in the treatment of addiction. Therefore, the development of a pharmacological antagonist to AQP4 may be valuable to investigate as opioid addiction therapy.

MiR-27a-3p/Hoxa10 Axis Regulates Angiotensin II-Induced Cardiomyocyte Hypertrophy by Targeting Kv4.3 Expression.

Cardiac hypertrophy is a common pathological process of various cardiovascular diseases, which is often accompanied with structural and electrical remodeling, and can even lead to sudden cardiac death. However, its molecular mechanism still remains largely unknown. Here, we induced cardiomyocyte hypertrophy by angiotensin II (Ang II), and found that miR-27a-3p and hypertrophy-related genes were up-regulated. Further studies showed that miR-27a-3p-inhibitor can alleviate myocardial hypertrophy and electrical remodeling. Moreover, luciferase assay confirmed that miR-27a-3p could regulate the expression of downstream Hoxa10 at the transcriptional level by targeting at its 3'UTR. At the same time, the protein expression of Hoxa10 was significantly reduced in Ang II-treated cardiomyocytes. Furthermore, overexpression of Hoxa10 can reverse myocardial hypertrophy and electrical remodeling induced by Ang II in cardiomyocytes. Finally, we found that Hoxa10 positively regulated the expression of potassium channel protein Kv4.3 which was down-regulated in hypertrophic cardiomyocytes. Taken together, our results revealed miR-27a-3p/Hoxa10/Kv4.3 axis as a new mechanism of Ang II-induced cardiomyocyte hypertrophy, which provided a new target for clinical prevention and treatment of cardiac hypertrophy and heart failure.

C/EBPß enhances efficacy of sorafenib in hepatoblastoma.

Hepatoblastoma (HB) is the predominant hepatic neoplasm in infants and young children. Sorafenib has been used to treat adult and pediatric hepatocellular carcinoma. However, efficacy of monotherapy of sorafenib in HB is not sustained. In this study, we tested a possible combinatory therapy of sorafenib with the CCAAT/enhancer-binding proteins (C/EBP) overexpression in HB cell line. Firstly, we evaluated the expression level of C/EBPß in the patients with HB by analyzing The Cancer Genome Atlas data. Lower level of C/EBPß was observed in tumor tissues in comparison with matched normal tissues. Next, we observed that combination of sorafenib and C/EBPß overexpression led to dramatic growth and migration inhibition of live tumor cells which implied promising probability for clinical trial. Mechanistically, C/EBPß which can be downregulated by Ras v12, augmented messenger RNA and protein levels of p53. These data suggested that a combination of sorafenib and C/EBPß overexpression inhibited tumor growth synergistically and provided a promising approach to treat HB.

A case of primary hepatic extranodal marginal zone B-cell mucosa-associated lymphoid tissue (MALT) lymphoma treated by radiofrequency ablation (RFA), and a literature review.

Primary hepatic mucosa-associated lymphoid tissue (MALT) lymphoma is an extremely rare liver malignancy that usually lacks characteristic imaging findings and which is often misdiagnosed. We report a 63-year-old woman diagnosed with primary hepatic extranodal marginal zone B-cell lymphoma, MALT type. The patient underwent needle biopsy and radiofrequency ablation (RFA), and showed no signs of relapse during the 12-month postoperative follow-up. This case stresses the rarity of primary hepatic MALT-type lymphoma and the unique and effective treatment for this patient. Our patient received RFA, which showed good efficacy and which provides a new option for the treatment of hepatic MALT lymphoma. We also present our findings from a systematic review to improve the current understanding of this disease.

Tumor Differentiation and EGFR Mutation Associated with Disease-Free Survival in Stage IA Lung Adenocarcinoma Patients with Curative Surgery.

BACKGROUND: Nearly 30% of stage IA non-small-cell lung cancer patients eventually die of recurrence or metastasis. This study aimed to predict stage IA lung adenocarcinoma (LADC) patients who underwent radical resection with a high risk of recurrence or metastasis. METHODS: Information on clinicopathological, genetic and therapeutic features and recurrence status was collected in this retrospective and two-center study. A nomogram based on multivariate analysis was established to predict disease-free survival. Further stratification was performed to identify populations with a high risk of relapse. RESULTS: A total of 1584 patients with pathological stage IA LADC who underwent radical surgery between 2011 and 2015 were enrolled from two medical institutions in this study. The nomogram including tumor differentiation and EGFR mutation had a higher C-index of 0.880 (95% CI 0.833-0.926) compared to 0.598 (95% CI 0.486-0.711) for the AJCC 8th TNM staging system. Furthermore, the C-index for the validation cohort was 0.798 (95% CI 0.738-0.857). In addition, the 3-year cumulative nonrecurrence rate in the high-risk group stratified by this model was 21.8% compared to 98.1% in the low-risk group. CONCLUSION: This study proposed a new nomogram including tumor differentiation and EGFR mutation to predict recurrence or metastatic probability in stage IA LADC patients who underwent radical surgery. This nomogram could identify patients in the high-risk group and help guide adjuvant treatment in the future.

Low HOXC10 expression in liver cancer regulates proliferation via a mechanism involving miR-221 and the MAPK signaling pathway.

Homeodomain-containing gene 10 (HOXC10) is associated with the progression of a variety of different types of human cancer; however, the role of HOXC10 in liver cancer is not completely understood. The present study aimed to investigate the mechanisms underlying the effects of HOXC10 on liver cancer tumorigenesis. Quantitative PCR and western blotting were used to detect the expression patterns of HOXC10 in cancer and adjacent healthy tissues. EdU, Cell Counting Kit-8 and colony formation assays were used to determine the functions of HOXC10 in liver cancer cell lines. ENCORI, TargetScan and miRTarBase were used to identify microRNAs that target HOXC10. The verification of the interaction between HOXC10 and microRNA-221 was determined by a luciferase assay. Compared with adjacent non-cancerous tissues, the expression of HOXC10 was markedly decreased in liver cancer tissues. A HOXC10 small interfering (si)RNA significantly attenuated HOXC10 expression at the mRNA and protein levels, and enhanced cell proliferation compared with the siRNA-negative control group. In addition, the luciferase reporter assay indicated that microRNA-221 directly bound to the 3'-untranslated region of HOXC10, and interfered with the inhibitory effect of HOXC10 on proliferation. In addition, HOXC10 knockdown elevated the expression levels of mitogen-activated protein kinase signaling pathway markers compared with the siRNA-negative control group. Therefore, the results of the present study may aid with the development of novel therapeutic regimens and diagnostic markers of liver cancer.

Genomic characteristics in Chinese non-small cell lung cancer patients and its value in prediction of postoperative prognosis.

BACKGROUND: The genomic profile of non-small cell lung cancer (NSCLC) in Asians is distinct from that of Caucasians, but comprehensive genetic profiling reports have been limited for Asian patients. We aimed to elucidate genomic characteristics of Chinese NSCLC patients and develop potential model including genomic characteristics to predict postoperative prognosis. METHODS: Resected tumor samples from 511 patients with stage I-IV lung cancer were subjected to targeted sequencing using a panel of 295 cancer-related genes. Based on the molecular profiles and clinical features, we established nomogram models with predictors consisting of integrated clinical and genomic characteristics to provide post-operative risk stratification. RESULTS: Compared to the TCGA population (mainly Caucasians), there was a significantly higher frequency of EGFR (53.7% vs. 14.4%) and NOTCH3 (8.4% vs. 1.3%) mutations and less mutated KRAS (11.0% vs. 32.6%), KEAP1 (4.4% vs. 17.4%) and LRP1B (16.3% vs. 29.6%) in Chinese lung adenocarcinomas (LUAD). Distinct patterns of mutually exclusive and co-occurring mutations were identified between LUAD and lung squamous cell carcinoma (LUSC), indicating the unique histology-specific tumorigenesis mechanism of each subtype. We observed alterations in pathways correlated with clinical characteristics. Additionally, we constructed nomogram model with predictors consisting of clinical and genomic characteristics, which were more accurate than models with clinical characteristics or TNM staging only both in stage I-IIIA patients and T1-2N0M0 sub-cohort. CONCLUSIONS: This study revealed Chinese NSCLC patients have unique genomic profile. Furthermore, the nomogram model combining clinical features with genomic characteristics could improve risk stratification in early-stage NSCLC.

Aquaporin 4 is involved in chronic pain but not acute pain.

Accumulating evidence has revealed that spinal glia plays an important role in the processing of pain, particularly chronic pain. Aquaporin 4 (AQP4), the predominant water channel exists in astrocytes, has been proved to modulate astrocytic function and thus participate in many diseases of the central nervous system. However, there is still controversy over whether AQP4 is involved in pain modulation. In the present study, we investigated the effects of AQP4 on pain by examining chronic inflammatory pain, neuropathic pain, and thermal, chemical, and mechanical stimuli-induced acute pain in AQP4 knockout mice. In Complete Freund's adjuvant-induced chronic inflammatory pain and spared nerve injury-induced neuropathic pain models, AQP4-/- mice attenuated pain-related behavioral responses compared with AQP4+/+ mice, demonstrating that AQP4 deficiency relieved chronic inflammatory pain and neuropathic pain. In the tail-flick and hot-plate tests, two acute pain models of thermal stimuli, no differences in pain-related behaviors were detected between AQP4+/+ and AQP4-/- mice. In the formalin and capsaicin tests, two models of chemical stimuli-induced acute pain, no differences in the durations of licking the injected hindpaw were found between AQP4+/+ and AQP4-/- mice. In the von Frey hair test, a model of mechanical stimuli-induced acute pain, no significant differences in withdrawal thresholds were found between these two genotypes mice as well. These results indicated that AQP4 deficiency did not affect acute pain induced by thermal, chemical, and mechanical stimuli. Taken together, our findings suggested that AQP4 contributes to chronic pain, but not acute pain.

Pediatric Epistaxis and Its Correlation Between Air Pollutants in Beijing From 2014 to 2017.

BACKGROUND: Epistaxis is a common symptom in children. The effect of air pollution on epistaxis is not yet clear. OBJECTIVES: To explore the characteristics of pediatric epistaxis in Beijing and its correlation with air pollutants. MATERIAL AND METHODS: Data were collected from 2014 to 2017 in Otolaryngology Department of Capital Institute of Pediatrics. Children diagnosed with epistaxis with relevant information with the same period of municipal air pollutants' concentration were compared. RESULTS: The annual visits of epistaxis showed a bimodal trend. The incidence of epistaxis in infants was low, increased with age, reached the peak between the ages of 4 to 5, and then gradually decreased with age. In different age groups, male patients were more than females. From 2014 to 2017 in Beijing, particulate matter less than 2.5 µm in diameter (PM2.5), particulate matter less than 10 µm in diameter (PM10), sulfur dioxide (SO2), nitrogen dioxide (NO2), and carbon monoxide (CO) showed a downtrend, lower in summer than in the other 3 seasons. Ozone (O3) was significantly higher in 2016 and 2017, showed an increase trend in summer. The incidence of epistaxis was negatively correlated with PM2.5, PM10, SO2, NO2 and CO, which was positively correlated with O3 (P < .05). CONCLUSIONS: Pediatric epistaxis in Beijing changes with age and has obvious seasonal variation. There are some correlations between air pollutants and the incidence of epistaxis in children.

Synthesis and Kinetics of the N-(2-Methyl-6-ethyl phenyl)-1-methoxypropyl-2-imine Schiff Base Catalyzed by NKC-9 Cation Exchange Resin.

The kinetics of condensation reaction of methoxyacetone with 2-methyl-6-ethyl aniline catalyzed by NKC-9 cation exchange resin was studied for the first time. The reaction temperature of Schiff base synthesis was determined in the range of 367.15 to 401.15 K by the batch experiments, and influences of reactant molar ratio, temperature, catalyst dosage, and particle size on the ultimate conversion were also studied. The dynamic data were used to be relevant with PH, ER(1), ER(2), and Langmuir Hinshelwood Hougen Watson homogeneity models. Model parameters, including reaction equilibrium constants, activation energy, enthalpy change, entropy change, and rate constants, were solved. The accuracy of the model was validated by means of both experimental proofs and standard deviation between the predicted and experimental data. Finally, a series of characterization tests such as Fourier transform infrared spectroscopy, X-ray diffraction, and polarizing microscopy were performed to investigate the structure and properties of NKC-9.

[Treatment of obstructive sleep apnea-hypopnea syndrome for children refractory asthma].

OBJECTIVE: The aim of this study was to understand the effect of different treatment of obstructive sleep apnea-hypopnea syndrome (OSAHS) for refractory asthma in children. METHODS: Fifty two children (32 in surgical group, 20 in conservative group) with refractory asthma and OSAHS were included in the study. All children received asthma condition assessment and polysomnography (PSG) examination before and after treatment, and were followed up for 6 months. RESULTS: All children got improved in PSG values 3 months after treatment, more significant improvement was achieved in surgical group than in conservative group (P < 0.05). While compared of OSAHS treatment, there were 2 cure, 6 notable effective, 9 effective, 3 in vain cases in conservative group, 8 cure, 16 notable effective, 8 effective, 0 in vain cases in surgery group. There was significant difference between the two groups (χ² = 8.91, P = 0.031). All children got improved in asthma condition evaluation parameters and decreased the use number of short acting ß2 agonists after 6 months treatment. More significant improvement was achieved in surgical group than in conservative group. The differences of all the items had statistical significance (P < 0.05). There was statistical correlation between days mutation rate of peak expiratory flow (PEF) and apnea hypopnea index (r = 0.712, P < 0.01), and between days mutation rate of PEF and lowest oxygen saturation (r = 0.726, P < 0.01). CONCLUSIONS: Active treatment of OSAHS can improve asthma symptoms and reduce asthma medication effectively. The curative effect of surgical treatment is superior to conservative treatment.