Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae.

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

Evaluating clinical efficacy of hospital-based surveillance with mammography and ultrasonography for breast cancer.

Periodic mammography and/or sonography examinations are conducted across numerous hospitals nationalwidely, especially for antedees with a positive mammography screening. Despite the regular practice, clinical efficacy of hospital-based breast cancer surveillance remains unclear. Specifically, the impact of surveillance interval upon survival and prognostic surrogates stratified by menopausal status, as well as malignant transition rate should be deciphered. We retrieved cancer registry to ascertain 841 breast cancers with surveillance history through administration data. Healthy controls underwent breast surveillance and were concurrently free of cancer. More benign diseases rather than cancers were identified from premenopausal women (age ≤50 years) with sonography alone within one year, as well as older women (age >50) with both mammography and sonography one to two years before a cancer or benign diagnosis. Among breast cancers, mammography alone during the antecedent one to two years had a protective effect for diagnosing carcinoma in situ rather than invasive cancer (age-adjusted odds ratio: 0.048, P = 0.016). Three-state time homogeneous Markov model showed that hospital-based breast surveillance within 2 years of disease onset reduced the malignant transition rate by 65.16% (59.79-76.74%). The clinical efficacy of breast cancer surveillance was evidenced.

Clinical characteristics and influencing factors of chronic obstructive pulmonary disease in patients with non-small cell lung cancer in Hubei province / 公共卫生与预防医学

Objective To analyze the clinical characteristics and influencing factors of non-small cell lung cancer (NSCLC) patients with chronic obstructive pulmonary disease (COPD) in Hubei province, and to provide a theoretical basis for the diagnosis and treatment of NSCLC patients with COPD. Methods A total of 246 NSCLC patients admitted to our hospital from 2018 to 2020 were selected and divided into control group (without COPD, n=125) and observation group (with COPD, n=121) according to COPD. The clinical characteristics of chest pain, hemoptysis, emasculation, atelectasis and pleural effusion were compared between the two groups. The values of FEV1/FVC, RV/TLC and DLCO in the two groups were measured by pulmonary function detector. The age, gender, smoking, smoking history, proportion of lung squamous cell carcinoma, TNM stage and other clinical data of all subjects were analyzed by self-made survey scale of our hospital. Univariate analysis and logistic regression were used to analyze the risk factors of COPD in NSCLC patients. Results Among 246 NSCLC patients, 121 patients (49.19%) were complicated with COPD, including 76 males and 45 females, and there was a statistical difference between the two groups (χ2=4.891, P>0.05). The average age of the observation group (61.02±4.82) was significantly higher than that of the control group (59.76±4.73) (t=2.069, P0.05). Male (OR=2.982), smoking history (OR=2.623) and lung squamous cell carcinoma (OR=3.147) were risk factors for COPD in NSCLC patients (P<0.05). Conclusions NSCLC patients with COPD are more common in male smokers in Hubei Province, often accompanied by pleural effusion , severe hemoptysis and other symptoms , and their lung function is decreased. Early detection and standardized treatment of COPD in the treatment of NSCLC can improve the prognosis of patients.

Azide-free cyclization reaction access to 4-aryl-NH-1,2,3-triazoles: P-toluenesulfonyl hydrazide and sulfamic acid as nitrogen sources.

An iodine-mediated cyclization has been developed to 4-aryl-NH-1,2,3-triazoles, with p-toluenesulfonyl hydrazide and sulfamic acid used as nitrogen sources. Sulfamic acid plays a crucial role in this reaction by both acting as a substrate and providing an acidic environment. This reaction offers a metal- and azide-free strategy to access NH-1,2,3-triazoles.

Understanding the Connection Among Ikigai, Well-Being, and Home Robot Acceptance in Japanese Older Adults: Mixed Methods Study.

BACKGROUND: Ikigai (meaning or purpose in life) is a concept understood by most older adults in Japan. The term has also garnered international attention, with recent academic attempts to map it to concepts in the Western well-being literature. In addition, efforts to use social and home robots to increase well-being have grown; however, they have mostly focused on hedonic well-being (eg, increasing happiness and decreasing loneliness) rather than eudaimonic well-being (eg, fostering meaning or purpose in life). OBJECTIVE: First, we explored how Japanese older adults experience ikigai and relate these to concepts in the Western well-being literature. Second, we investigated how a home robot meant to promote ikigai is perceived by older adults. METHODS: We used a mixed methods research design-including 20 interviews with older adults, a survey of 50 older adults, and 10 interviews with family caregivers. For interviews, we asked questions about older adults' sources of ikigai, happiness, and social support, along with their perception of the robot (QT). For surveys, a number of well-being scales were used, including 2 ikigai scales-ikigai-9 and K-1-and 6 Patient-Reported Outcomes Measurement Information System scales, measuring meaning and purpose, positive affect, satisfaction with participation in social roles, satisfaction with participation in discretionary social activities, companionship, and emotional support. Questions related to the perception and desired adoption of the robot and older adults' health status were also included. RESULTS: Our results suggest that health is older adults' most common source of ikigai. Additionally, although self-rated health correlated moderately with ikigai and other well-being measures, reported physical limitation did not. As opposed to social roles (work and family), we found that ikigai is more strongly related to satisfaction with discretionary social activities (leisure, hobbies, and friends) for older adults. Moreover, we found that older adults' sources of ikigai included the eudaimonic aspects of vitality, positive relations with others, contribution, accomplishment, purpose, and personal growth, with the first 3 being most common, and the hedonic aspects of positive affect, life satisfaction, and lack of negative affect, with the first 2 being most common. However, the concept of ikigai was most related to eudaimonic well-being, specifically meaning in life, along the dimension of significance. Finally, we found that Japanese older adults have high expectations of a home robot for well-being, mentioning that it should support them in a multitude of ways before they would likely adopt it. However, we report that those with the highest levels of meaning, and satisfaction with their leisure life and friendships, may be most likely to adopt it. CONCLUSIONS: We outline several ways to improve the robot to increase its acceptance, such as improving its voice, adding functional features, and designing it to support multiple aspects of well-being.

Chronic Hepatitis C Virus Infection: An Ongoing Challenge in Screening and Treatment.

With the advent of direct-acting antiviral agents (DAA) in the recent few years, hepatitis C virus (HCV) infection has become a curable infectious disease. Successful clearance of HCV could lead to improvement of both hepatic and extrahepatic outcomes, such as complications of cirrhosis, hepatocellular carcinoma, cardiovascular diseases, and incident diabetes. However, challenges persist in reaching the HCV elimination goals of the World Health Organization by 2030. Among these challenges are identifying those already infected or undiagnosed subjects, re-linking to the care of known but untreated HCV-infected subjects, and developing strategies to enhance treatment rates and compliance in specific or high-risk populations. In addition, issues of post-DAA viral clearance, including avoiding or preventing reinfection in high-risk populations and surveillance of hepatocellular carcinoma, are important to consolidate the treatment's short- and long-term efficacies. In the current DAA era, treatment is the most effective prevention strategy not only in its excellent efficacy and safety but also in preventing HCV spread. All of the surveillance or measures should center on DAA treatment in clinical practice.

Photoconduction Properties in Tungsten Disulfide Nanostructures.

We reported the photoconduction properties of tungsten disulfide (WS2) nanoflakes obtained by the mechanical exfoliation method. The photocurrent measurements were carried out using a 532 nm laser source with different illumination powers. The results reveal a linear dependence of photocurrent on the excitation power, and the photoresponsivity shows an independent behavior at higher light intensities (400-4000 Wm-2). The WS2 photodetector exhibits superior performance with responsivity in the range of 36-73 AW-1 and a normalized gain in the range of 3.5-7.3 10-6 cm2V-1 at a lower bias voltage of 1 V. The admirable photoresponse at different light intensities suggests that WS2 nanostructures are of potential as a building block for novel optoelectronic device applications.

Tandem Ene/[4 + 2] Cycloaddition Reaction for the Synthesis of 9-Benzylphenanthrenes from Arynes and α-(Bromomethyl)styrenes.

A tandem reaction for the synthesis of phenanthrenes from arynes and α-(bromomethyl)styrenes is reported. The transformation proceeds via an ene reaction of α-(bromomethyl)styrenes with arynes, followed by a [4 + 2] cycloaddition reaction. The reaction generates 9-benzylphenanthrene derivatives in moderate to excellent yields.

Precise application of topical tranexamic acid to enhance endoscopic hemostasis for peptic ulcer bleeding: a randomized controlled study (with video).

BACKGROUND AND AIMS: Peptic ulcer recurrent bleeding occurs in 20% to 30% of patients after standard endoscopic hemostasis, particularly within 4 days after the procedure. The application of additional tranexamic acid (TXA) to the ulcer may enhance hemostasis. This study investigated the effectiveness of TXA powder application on bleeding ulcers during endoscopic hemostasis. METHODS: This study enrolled patients who had peptic ulcer bleeding between March 2022 and February 2023. After undergoing standard endoscopic therapy, the patients were randomly assigned to either the TXA group or the standard group. In the TXA group, an additional 1.25 g of TXA powder was sprayed endoscopically on the ulcer. Both groups then received 3 days of high-dose (8 mg/h) continuous infusion proton pump inhibitor therapy. Second-look endoscopy was conducted on days 3 to 4. The primary end point of early treatment failure was defined as ulcer recurrent bleeding within 4 days or major stigmata of recent hemorrhage on the second-look endoscopy. RESULTS: Sixty patients (30 in each group) with peptic ulcer bleeding and balanced baseline characteristics were randomly assigned to a treatment group. The early treatment failure rate was lower in the TXA group (6.7%) than in the standard group (30%) (P = .042). The freedom from treatment failure periods for 4 and 28 days was significantly longer in the TXA group than in the standard group (P = .023). No adverse events from TXA were recorded. CONCLUSIONS: The precise delivery of topical TXA alongside standard endoscopic hemostasis reduced the early treatment failure rate in patients with bleeding peptic ulcers. (Clinical trial registration number: NCT05248321.).

Generation and characterization of avian single chain variable fragment against human Alpha-Enolase.

Overexpression of human alpha-enolase (hEno1)has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.

Experience and acceptability for HPV self-sampling among women in Jiangsu province, China: a cross-sectional survey.

This aim of this study was to investigate women's knowledge about HPV along with their experience and acceptability of self-sampling in Jiangsu province, China. A total of 862 women aged 25-63 years old from Jiangsu province who purchased an HPV self-sampling test kit were invited to complete a questionnaire designed by the authors. Participants had high acceptability for HPV self-sampling with a mean score of 4.2 (95% [CI], 4.1-4.22) out of 5 points. 27% of participants preferred clinician-sampling, 33% preferred self-sampling, other 40% expressed no preference. Women with good knowledge about HPV and with a good experience with HPV self-sampling were more acceptable for self-sampling (P < 0.05). The biggest concern about HPV self-sampling of the participants includes 'specimens' spoilage', 'incorrect sampling', 'can't get results in time', and so on. HPV self-sampling can be used to improve cervical cancer screening coverage and participation rates in China.

Cancer screening and can be an alternative primary screening for cervical cancer.•What the results of this study add? This study adds new findings about Chinese women's experience and acceptability of HPV self-sampling. We found that most women had high acceptability for HPV self-sampling in Jiangsu province, China, and high knowledge about HPV as well as good•What is already known on this subject? HPV self-sampling testing was proven to be useful for improving the uptake rate of cervical experience of self-sampling can improve the acceptability for self-sampling in women.•What the implications are of these findings for clinical practice and/or further research? Further research should assess the acceptability of women with less education or who never screened.

The optimal dose of oral tranexamic acid in melasma: A network meta-analysis.

BACKGROUND: Melasma is a chronic skin condition that adversely impacts quality of life. Although many therapeutic modalities are available there is no single best treatment for melasma. Oral tranexamic acid has been used for the treatment of this condition but its optimal dose is yet to be established. OBJECTIVES: We used network meta-analysis to determine the optimal dose of oral tranexamic acid for the treatment of melasma. METHODS: We conducted a comprehensive search of all studies of oral tranexamic acid for the treatment of melasma up to September 2020 using PubMed, EMBASE and the Cochrane Library database. The quality of the studies was evaluated using the Jadad score and the Cochrane's risk of bias assessment tool. Only high quality randomised controlled trials were selected. Some studies lacked standard deviation of changes from baseline and these were estimated using the correlation coefficient obtained from another similar study. RESULTS: A total of 92 studies were identified of which 6 randomized controlled trials comprising 599 patients were included to form 3 pair-wise network comparisons. The mean age of the patients in these studies ranged from 30.3 to 46.5 years and the treatment duration ranged from 8 to 12 weeks. The Jadad scores ranged from 5 to 8. The optimal dose and duration of oral tranexamic acid was estimated to be 750 mg per day for 12 consecutive weeks. LIMITATIONS: Some confounding factors might not have been described in the original studies. Although clear rules were followed, the Melasma Area and Severity Index and the modified Melasma Area and Severity Index were scored by independent physicians and hence inter-observer bias could not be excluded. CONCLUSION: Oral tranexamic acid is a promising drug for the treatment of melasma. This is the first network meta-analysis to determine the optimal dose of this drug and to report the effects of different dosages. The optimal dose is 250 mg three times per day for 12 weeks, but 250 mg twice daily may be an acceptable option in poorly adherent patients. Our findings will allow physicians to balance drug effects and medication adherence. Personalized treatment plans are warranted.

Influences of Dixon anus preserving surgery on curative effect, anorectal function and stress response in patients with low rectal cancer / 中国医师进修杂志

Objective:To investigate the influences of Dixon anus preserving surgery on the curative effect, anorectal function and stress response of patients with low rectal cancer.Methods:A total of 150 patients with low rectal cancer admitted to Zhangjiakou Fifth Hospital from May 2016 to March 2019 were retrospective analysis as the study subjects. According to the different surgical methods, the patients were divided into the study group (75 cases) and the control group (75 cases). The patients in the study group received Dixon operation, and the patients in the control group received Miles operation. The general condition of operation, postoperative urination function, stress response [C reactive protein (CRP), interleukin-6 (IL-6), cortisol (Cor) and adrenocorticotropic hormone (ACTH)], anorectal function [anal resting pressure (ARP), maximal squeeze pressure (MSP), anal longest contraction time (ALCT)], quality of life (PAC-QOL score), postoperative complications, local recurrence rate, distant metastasis rate, 3-year tumor free survival rate and 3-year overall survival rate were compared between the two groups.Results:The operation time, intraoperative blood loss and postoperative drainage volume in the study group were lower than those in the control group: (172.28 ± 23.45) min vs. (189.57 ± 23.68) min, (177.39 ± 21.23) ml vs. (191.35 ± 22.67) ml, (342.36 ± 52.89) ml vs. (489.42 ± 63.33) ml, there were statistical differences ( P<0.05). The levels of CRP, IL-6, Cor and ACTH in the study group were lower than those in the control group: (22.35 ± 4.75) mg/L vs. (35.65 ± 5.28) mg/L, (58.74 ± 15.11) μg/L vs. (79.63 ± 20.23) μg/L, (145.36 ± 27.48) ng/L vs. (156.48 ± 32.55) ng/L, (44.58 ± 5.27) ng/L vs. (49.62 ± 5.68) ng/L, there were statistical differences ( P<0.05). The urination function and anorectal function of the patients in the study group were obviously better than those in the control group 6 months after operation, and the PAC-QOL score was obviously lower than that in the control group: (22.53 ± 2.86) scores vs. (27.54 ± 3.21) scores ( P<0.05); there was no obvious difference between the study group and the control group in the incidence of postoperative complications, local recurrence rate, distant metastasis rate, 3-year tumor free survival rate and 3-year total survival rate ( P>0.05). Conclusions:Dixon operation is effective in the treatment of low rectal cancer. It can preserve the anorectal function of patients to the greatest extent, protect urination function, reduce stress reaction of patients, and it is more conducive to rapid rehabilitation of patients.

Phenotypic and genetic analysis of a Chinese pedigree affected with Oral-facial-digital syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical phenotype and genetic basis for a Chinese pedigree affected with Oral-facial-digital syndrome type I (OFD1).@*METHODS@#A pedigree with OFD1 who presented at Hebei General Hospital on March 17, 2021 was selected as the subject. Clinical data of the child was collected. Trio-whole exome sequencing (trio-WES) was carried out for the proband and members of her pedigree, and candidate variant was verified by Sanger sequencing.@*RESULTS@#The proband has featured hypotelorism, broad nasal root, flat nasal tip, lobulated tongue, tongue neoplasia, camptodactyly of left fifth finger, syndactyly of right fourth and fifth fingers, and delayed intellectual and language development. Trio-WES revealed that the proband and her daughter, sister and mother have harbored a heterozygous c.224A>G (p.Asn75Ser) variant of the OFD1 gene. The same variant was not found among healthy members from her pedigree.@*CONCLUSION@#The c.224A>G (p.Asn75Ser) variant probably underlay the OFD1 in this pedigree. Above discovery has enriched the spectrum of OFD1 gene variants.

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Intramolecular redox cyclization reaction access to cinnolines from 2-nitrobenzyl alcohol and benzylamine via intermediate 2-nitrosobenzaldehyde.

A transition-metal-free intramolecular redox cyclization reaction for the synthesis of cinnolines has been developed from 2-nitrobenzyl alcohol and benzylamine. Mechanistic investigations disclosed the involvement of a key intramolecular redox reaction, followed by condensation, azo isomerization to hydrazone, cyclization, and aromatization to form the desired products. Notably, the formation of intermediate 2-nitrosobenzaldehyde and (E)-2-(2-benzylidenehydrazineyl) benzaldehyde plays an important role in this transformation.

Copper/TEMPO-Promoted Denitrogenation/Oxidation Reaction for Synthesis of Primary α-Ketoamides Using α-Azido Ketones.

A copper(II)-promoted denitrogenation/oxidation reaction for the preparation of primary α-ketoamides was developed using α-azido ketones as a substrate and TEMPO as an oxidant. α-Azido ketones were denitrogenated in situ to form an imino ketone intermediate, which underwent a radical addition process and radical migration to form α-ketoamides. It is worth noting that the imino ketone intermediate is the key to this reaction.

Higher Risk of Tumor Recurrence in NASH-Related Hepatocellular Carcinoma Following Curative Resection.

The outcomes for patients with NASH-related HCC after curative resection have not been clarified. This study compared the overall survival (OS), time-to-tumor recurrence (TTR), and recurrence-free survival (RFS) associated with NASH-related HCC and virus-related HCC after resection. Methods: Patients with HCC who underwent curative resection were retrospectively enrolled. Baseline characteristics, including disease etiologies and clinical and tumor features, were reviewed. The primary outcomes were OS, TTR, and RFS. Results: Two hundred and six patients were enrolled (HBV: n = 121, HCV: n = 54, NASH: n = 31). Of those with virus-related HCC, 84.0% achieved viral suppression. In both the overall and propensity-score-matched cohorts, those with NASH-related HCC experienced recurrence significantly earlier than those with virus-related HCC (median TTR: 1108 days vs. non-reached; p = 0.03). Through multivariate analysis, NASH-related HCC (hazard ratio (HR), 2.27; 95% confidence interval (CI), 1.25-4.12) was independently associated with early recurrence. The unadjusted RFS rate of the NASH-related HCC group was lower than the virus-related HCC group. There was no difference in the OS between the two groups. Conclusions: NASH-related HCC was associated with earlier tumor recurrence following curative resection compared to virus-related HCC. Post-surgical surveillance is crucial for detecting early recurrence in patients with NASH-related HCC.

The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.