Successful treatment of ceftazidime/avibactam combined with aztreonam in the NDM-producing Klebsiella pneumoniae bloodstream and intestinal infections in a NK/T lymphoma patient with agranulocytosis during autologous hematopoietic stem cell transplantation: a case report.

New Delhi metallo-beta-lactamase (NDM)-producing Klebsiella pneumoniae is increasingly reported worldwide. Clinicians face significant challenges in the treatment of this multidrug-resistant bacterium. The combination of ceftazidime/avibactam (CAZ/AVI) and aztreonam (ATM) is currently probably the most effective strategy for the treatment of such infection. We described a patient diagnosed with NK/T cell lymphoma who underwent autologous hematopoietic stem cell transplantation (ASCT) in the hematology department. The patient developed severe infection after ASCT. Blood and stool cultures showed carbapenem-resistant K. pneumoniae. Blood sample was detected as NDM-producing K. pneumoniae. We successfully treated this infection with CAZ/AVI and ATM.

Associations of preoperative serum high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels with the prognosis of ovarian cancer.

BACKGROUND: The effect of serum lipids on ovarian cancer is controversial. We conducted this study to evaluate the prognostic value of preoperative plasma lipid profile in patients with ovarian cancer. METHODS: The medical records of 156 epithelial ovarian cancer patients who underwent surgical resection in our department were retrospectively reviewed and analyzed. Serum lipids profiles, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), apolipoprotein A-I (apoA-I), apolipoprotein B (apoB) and clinicopathologic data, were analyzed. Cox proportional hazards regression analyses and Kaplan-Meier method were performed to evaluate the overall survival (OS) and progression-free survival (PFS). RESULTS: Multivariable Cox regression analysis found that preoperative higher LDL-C level was significantly associated with worse OS (HR 2.088, 95% CI 1.052-4.147, p = 0.035), whereas higher HDL-C level showed significant association with better PFS (HR 0.491, 95% CI 0.247-0.975, p = 0.042). Further Kaplan-Meier survival analysis demonstrated that OS was longer for patients with low levels of LDL-C (< 2.76 mmol/L) compared to those with high levels of LDL-C (≥ 2.76 mmol/L) (P = 0.028), and PFS was better for patients with high levels of HDL-C (≥ 1.19 mmol/L) compared to those with low levels of HDL-C (< 1.19 mmol/L) (P = 0.001). CONCLUSIONS: Preoperative HDL-C and LDL-C levels are significant predictors of clinical outcome in patients with epithelial ovarian cancer.

Rapid identification of pathogens by using surface-enhanced Raman spectroscopy and multi-scale convolutional neural network.

Salmonella is a prevalent pathogen causing serious morbidity and mortality worldwide. There are over 2600 serovars of Salmonella. Among them, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphi were reported to be the most common foodborne pathogenic serovars in the EU and China. In order to provide a more efficient approach to detect and distinguish these serovars, a new analytical method was developed by combining surface-enhanced Raman spectroscopy (SERS) with multi-scale convolutional neural network (CNN). We prepared 34-nm gold nanoparticles (AuNPs) as the label-free Raman substrate, measured 1854 SERS spectra of these three Salmonella serovars, and then proposed a multi-scale CNN model with three parallel CNNs to achieve multi-dimensional extraction of SERS spectral features. We observed the impact of the number of iterations and training samples on the recognition accuracy by changing the ratio of the number of the training and testing sets. By comparing the calculated data with experimental one, it was shown that our model could reach recognition accuracy more than 97%. These results indicate that it was not only feasible to combine SERS spectroscopy with multi-scale CNN for Salmonella serotype identification, but also for other pathogen species and serovar identifications.

An association of low high-density lipoprotein levels with recurrence of chronic subdural hematoma.

BACKGROUND: Chronic subdural hematoma (CSDH) is a common illness in neurosurgical practice with a substantial recurrence rate. Previous studies found that serum lipids were associated with the risk of stroke and subarachnoid hemorrhage. In the current study, we aimed to identify the relationship between serum lipids and CSDH recurrence. METHODS: The medical records of 274 consecutive surgical patients with CSDH in our department were reviewed and analyzed. Patients were separated into recurrence and non-recurrence groups. Univariable and multivariable Cox proportional hazards regression analyses were performed to identify serum lipids (triglycerides, total cholesterol, LDL, HDL) and other potential predictors associated with CSDH recurrence, and the performance of predictors was assessed with receiver operating characteristic (ROC) curve. RESULTS: Of the 274 patients included in the study, 42 (15.3%) experienced at least 1 recurrence of CSDH. Univariate analysis showed that age, hypertension, diabetes mellitus, anticoagulant use, triglycerides, HDL, and midline shift were all significantly associated with CSDH recurrence. Multivariable Cox regression analysis found that only age, diabetes mellitus, midline shift, and HDL level were independent risk factors for CSDH recurrence. A higher HDL level (HR = 0.929, 95% CI 0.905-0.953) was significantly associated with a lower risk of recurrence, and ROC curve analysis revealed that the optimal HDL cut-off value as a predictor was 37.45 mg/dl. CONCLUSIONS: Low level of high-density lipoprotein is significantly associated with recurrence of chronic subdural hematoma.

Nurses' Emotional Stress Levels When Caring for COVID-19 Patients in an Intensive Care Unit.

CONTEXT: The COVID-19 pandemic has had an effect on many communities' physical and mental well-being, especially that of healthcare workers. During the pandemic, health workers have shown signs of depression and anxiety and have experienced sleep disturbances. Few studies have examined health workers' resilience during the pandemic. OBJECTIVE: The current study intended to examine the job stress and mental well-being of nurses who have supported, worked with, and cared for COVID-19 patients in an intensive care unit. DESIGN: The research team performed a narrative review by searching the Mendeley, ScienceDirect, Medline, PubMed, Google Scholar, and Springer databases. The search used many keywords, both alone and in combination, such as COVID-19, pandemic, nurses, healthcare professionals, stress, and frontline workers. The review considered only English journals. SETTING: This study was take place in Second Affiliated Hospital of Hainan Medical University, Hainan Province, China. RESULTS: During the current pandemic, COVID-19 prevention in social settings, governmental regulation during the pandemic, and provision of frontline care have faced notable challenges. In general, nurses who have assisted during the COVID-19 pandemic have been under severe strain. The key factors that influenced nurses' stress were being only children, their working time per week, and their levels of anxiety. CONCLUSIONS: COVID-19 has posed a vast threat to public health worldwide. The psychological stress of nurses should be managed in public-health emergencies.

Knockdown of long non-coding RNA small nucleolar RNA host gene 9 or hexokinase 2 both suppress endometrial cancer cell proliferation and glycolysis.

AIM: Endometrial cancer (EC) is a common type of malignant gynecological cancer. Small nucleolar RNA host gene 9 (SNHG9) has been discovered to serve a role in several types of cancer; however, the role of SNHG9 in EC remains unclear. The present study aimed to investigate the effects of lncRNA SNHG9 on cell proliferation and glycolysis in EC cells. METHODS: SNHG9 and hexokinase 2 (HK2) mRNA expression levels were measured by reverse transcription-quantitative PCR. Glucose consumption and lactate production were detected by the glycolysis cell-based assay kit. Cell Counting Kit-8 and colony formation assays were conducted to detect cell proliferation. The knockdown experiments of SNHG9 and HK2 were carried out by transfection of corresponding small interference RNAs (siRNA). The SNHG9-overexpressed plasmid was transfected into the cells to upregulate SNHG9. HK2 protein levels were analyzed by western blotting assay. RESULTS: SNHG9 expression levels were significantly upregulated in EC tissues and cells. The knockdown of SNHG9 subsequently effectively attenuated cell proliferation and glycolysis in vitro, while SNHG9 overexpression reported the opposite effects. Notably, the transfection of 2-DG partially reversed the promoting effect of SNHG9 on glycolysis. Downregulation of HK2 markedly decreased cell proliferation and glycolysis in EC cells antagonized SNHG9. CONCLUSION: Either downregulation of SNHG9 or HK2 inhibits EC cell proliferation and glycolysis via repressing EC cell proliferation and glycolysis.

[Determination of seven ingredients of different grades Alismatis Rhizoma by QAMS method].

The present study is to establish a quantitative analysis of multi-components by single marker(QAMS) for determining contents of seven compositions in Alismatis Rhizoma, alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B, alisol B 23-acetate and 11-deoxy-alisol B. Six relative correction factors(RCFs) of alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B and 11-deoxy-alisol B were established in the UPLC method with alisol B 23-acetate as the internal standard, which was to calculate the mass fraction of each. The mass fraction of seven effective constituents in Alismatis Rhizoma was calculated by the external standard method(ESM) at the same time. Compared with the content results determined by the ESM and QAMS, the feasibility and accuracy of QAMS method were verified. Within the linear range, the RCFs of alismoxide, alisol C 23-acetate, alisol A, alismol, alisol B, 11-deoxy-alisol B were 0.946, 4.183, 0.915, 1.039, 0.923 and 1.244, respectively, with good repeatability in different experimental conditions. There was no significant difference between the QAMS method and ESM method. Then, QAMS method was applied to determination of the different degree Alismatis Rhizoma from different areas. As a result, the concentrations of 7 components have differences in different areas, but no significant differences in different grades. The QAMS method is feasible and accurate for the determination of the seven chemical compositions, and which can be used for quality control of Alismatis Rhizoma.

Ordered raft domains induced by outer leaflet sphingomyelin in cholesterol-rich asymmetric vesicles.

Sphingolipid- and cholesterol-rich liquid-ordered (Lo) lipid domains (rafts) are thought to be important organizing elements in eukaryotic plasma membranes. How they form in the sphingolipid-poor cytosolic (inner) membrane leaflet is unclear. Here, we characterize how outer-leaflet Lo domains induce inner-leaflet-ordered domains, i.e., interleaflet coupling. Asymmetric vesicles studied contained physiologically relevant cholesterol levels (∼ 37 mol %), a mixture of SM (sphingomyelin) and DOPC (dioleoylphosphatidylcholine) in their outer leaflets, and DOPC in their inner leaflets. Lo domains were observed in both leaflets, and were in register, indicative of coupling between SM-rich outer-leaflet-ordered domains and inner-leaflet-ordered domains. For asymmetric vesicles with outer-leaflet egg SM or milk SM, a fluorescent lipid with unsaturated acyl chains (NBD-DOPE) was depleted in both the outer- and inner-leaflet-ordered domains. This suggests the inner-leaflet-ordered domains were depleted in unsaturated lipid (i.e., DOPC) and thus rich in cholesterol. For asymmetric vesicles containing egg SM, outer-leaflet Lo domains were also depleted in a saturated fluorescent lipid (NBD-DPPE), while inner-leaflet Lo domains were not. This indicates that inner- and outer-leaflet Lo domains can have significantly different physical properties. In contrast, in asymmetric vesicles containing outer-leaflet milk SM, which has long acyl chains capable of interdigitating into the inner leaflet, both outer- and inner-leaflet Lo domains were depleted, to a similar extent, in NBD-DPPE. This is indicative of interdigitation-enhanced coupling resulting in inner- and outer-leaflet Lo domains with similar physical properties.

The influence of natural lipid asymmetry upon the conformation of a membrane-inserted protein (perfringolysin O).

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.

Decreasing Transmembrane Segment Length Greatly Decreases Perfringolysin O Pore Size.

Perfringolysin O (PFO) is a transmembrane (TM) ß-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM ß-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

Altering hydrophobic sequence lengths shows that hydrophobic mismatch controls affinity for ordered lipid domains (rafts) in the multitransmembrane strand protein perfringolysin O.

The hypothesis that mismatch between transmembrane (TM) length and bilayer width controls TM protein affinity for ordered lipid domains (rafts) was tested using perfringolysin O (PFO), a pore-forming cholesterol-dependent cytolysin. PFO forms a multimeric barrel with many TM segments. The properties of PFO mutants with lengthened or shortened TM segments were compared with that of PFO with wild type TM sequences. Both mutant and wild type length PFO exhibited cholesterol-dependent membrane insertion. Maximal PFO-induced pore formation occurred in vesicles with wider bilayers for lengthened TM segments and in thinner bilayers for shortened TM segments. In diC(18:0) phosphatidylcholine (PC)/diC(14:1) PC/cholesterol vesicles, which form ordered domains with a relatively thick bilayer and disordered domains with a relatively thin bilayer, affinity for ordered domains was greatest with lengthened TM segments and least with shortened TM segments as judged by FRET. Similar results were observed by microscopy in giant vesicles containing sphingomyelin in place of diC(18:0) PC. In contrast, in diC(16:0) PC/diC(14:0) PC/diC(20:1) PC/cholesterol vesicles, which should form ordered domains with a relatively thin bilayer and disordered domains with a relatively thick bilayer, relative affinity for ordered domains was greatest with shortened TM segments and least with lengthened TM segments. The inability of multi-TM segment proteins (unlike single TM segment proteins) to adapt to mismatch by tilting may explain the sensitivity of raft affinity to mismatch. The difference in width sensitivity for single and multi-TM helix proteins may link raft affinity to multimeric state and thus control the assembly of multimeric TM complexes in rafts.

National tertiary public hospital performance appraisal: Using FOCUS-PDCA to improve external quality assessment.

PURPOSE: To innovatively use the FOCUS-PDCA quality improvement strategy to establish an external quality assessment (EQA) working group to continuously improve EQA performance, an important indicator of the national tertiary public hospital performance appraisal. METHODS: The project was carried out at the National Center for Clinical Laboratories. Using FOCUS-PDCA, which combines problem-focused steps (FOCUS) and improvement steps (PDCA), a project team was established to carry out improvement work. Root cause analysis was carried out to analyze the problems in quality control from EQA project application to results analysis and an improvement plan was implemented according to the steps of FOCUS-PDCA. The project was executed in three cycles from 2019 to 2021 to obtain more satisfactory results. RESULTS: After implementing three cycles of FOCUS-PDCA, the EQA participation rate increased from 66.5% in 2018 to 100% in 2021, and the EQA pass rate increased from 94.9% in 2018 to 99.3% in 2021. Consequently, the hospital moved into the top 50 in performance assessment for the first time in 2020 and ranked 27th in 2021. CONCLUSION: The use of the FOCUS-PDCA quality improvement strategy can improve the EQA performance of national tertiary public hospitals and help them achieve satisfactory results in the national examination.

Dual-mode upconversion sensors for detecting differently charged biotargets based on the oxidase-mimicking activity of Ce4+ and electrostatic control.

Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets.

Association of Antenatal Corticosteroids with Neonatal Outcomes among Very Preterm Infants Born to Mothers with Clinical Chorioamnionitis: A Multicenter Cohort Study.

The objective of this study was to assess the relationship of ACS with neonatal outcomes among very preterm infants born to mothers with clinical chorioamnionitis in China. This was a multicenter retrospective cohort study. Study participants included infants born at <32 weeks' gestation with clinical chorioamnionitis and registered in the Chinese Neonatal Network from 1 January 2019 to 31 December 2020. Infants were divided into two groups: any amount of ACS or no administration of ACS. Multivariable generalized linear models using generalized estimating equations were used to assess the association between ACS and neonatal outcomes among the study population. We identified 2193 infants eligible for this study; 1966 (89.6%) infants had received ACS therapy, and 227 (10.4%) had not received any ACS therapy. Among very preterm infants born to mothers with clinical chorioamnionitis, any ACS usage was significantly associated with decreased risks of early death (aRR 0.56, 95% CI 0.32, 0.99) and severe ROP (aRR 0.51, 95% CI 0.28, 0.93) after adjustment for maternal hypertension, gestational age at birth, Caesarean section, being inborn, and administration of systemic antibiotics to the mother within 24 h before birth. In addition, out of the 2193 infants, the placentas of 1931 infants underwent pathological examination with recorded results. Subsequently, 1490 of these cases (77.2%) were diagnosed with histological chorioamnionitis. In 1490 cases of histologic chorioamnionitis, any ACS usage was significantly related to decreased risks of overall mortality (aRR 0.52, 95% CI 0.31, 0.87), severe ROP (aRR 0.47, 95% CI 0.25, 0.97), and respiratory distress syndrome (aRR 0.52, 95% CI 0.31, 0.87). We concluded that any ACS was associated with reduced risks for neonatal early death and severe ROP among very preterm infants born to mothers with clinical chorioamnionitis.

Solamargine improves the therapeutic efficacy of anti-PD-L1 in lung adenocarcinoma by inhibiting STAT1 activation.

OBJECTIVE: The effect of solamargine on lung adenocarcinoma and its effect on STAT1 signaling pathway mediated immune escape were studied through network pharmacology and in vitro and in vivo experiments. METHODS: The solamargine targets were screened using the TCMSP and the LUAD targets were screened using the GeneCard, OMIM, PharmGkb, TTD and DrugBank databases. PPI network analysis and target prediction were performed using GO and KEGG. Colony formation assay, EDU staining, wound healing, transwell assay, Hoechst and flow cytometry were used to detect the effects of solamargine on the proliferation, migration and apoptosis of LUAD. Western blotting (WB) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect P-STAT1 and PD-L1 expression. And immunofluorescence was used to detect P-STAT1 expression. In vivo experiments, C57BL/6 mice were divided into control group, low concentration group, high concentration group, positive control group and combination group. Every other day, following seven consecutive doses, the size of the tumor was assessed. Finally, the expressions of P-STAT1, STAT1, PD-L1 and apoptosis index proteins were detected by WB. RESULTS: The anti-LUAD effect of solamargine was found by wound healing, colony formation assay, transwell assay, hoechst and EdU staining. The results of network pharmacological analysis showed that solamargine could suppress STAT1 expression level. Further enrichment assay of STAT1 showed that STAT1 was associated with immune-related pathways. In addition, molecular signal analysis by WB and RT-qPCR indicated that solamargine could reduce the expression levels of P-STAT1 and PD-L1 in a concentration-dependent manner. According to the results of in vivo assays, combination of solamargine and immune checkpoint inhibitors (ICIs) durvalumab could significantly inhibit the growth of Lewis transplanted tumors in C57BL/6 mice, and no toxic side effect was recoded. CONCLUSION: These results indicated that solamargine could inhibit the proliferation and promote the apoptosis of LUAD. It also could reduce the expression level of P-STAT1 protein and inhibit the expression level of PD-L1. At the same time, the combination with the ICIs can better block the expression of PD-L1 in cells, thereby inhibiting the immune escape pathway of tumor cells and achieving anti-tumor effects. This study proposed a novel combined therapeutic approach, involving the inhibition of STAT1 by solamargine in conjunction with ICIs.

Csn-miR156d-CsSPL1 regulates flowering and anthocyanin metabolism.

MiR156 play important roles in regulation of plant growth and development, secondary metabolite synthesis, and other biological processes by targeting the SQUAMOSA promoter binding protein-like (SPL) family. Our previous sequencing data analysis suggested that Csn-miR156d may regulate flowering and anthocyanin accumulation by cleavage and degradation of the expression of the SPL in tea plant, but it remains to be elucidated. In this study, 5'RLM-RACE experiment, tobacco transient transformation, qRT-PCR, and antisense oligonucleotide (asODN) were used to verify that CsSPL1 is the target gene of Csn-miR156d. Stable transformation of Arabidopsis revealed that Csn-miR156d could delay flowering by negatively regulating the transcript levels of FT, AP1, FUL, and SOC1, while overexpression of CsSPL1 showed an opposite effect. Additionally, overexpression of Csn-miR156d in Arabidopsis could enhance the transcription of the anthocyanin biosynthesis-related structural genes DFR, ANS, F3H, UGT78D2, and LDOX, as well as regulatory genes PAP1, MYB113, GL3, MYB11, and MYB12, leading to anthocyanin accumulation. Moreover, asODN experiment revealed that Csn-miR156d could increase the anthocyanin content in tea plant. These results suggest that Csn-miR156d regulates flowering and anthocyanin accumulation in tea plant by suppressing the expression of CsSPL1. Our study provides new insights into the development and anthocyanin accumulation in tea plant and lays a theoretical foundation for further research on the molecular mechanism of miRNAs in regulating tea plant growth and secondary metabolism.

Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol.

Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts.