Performance of ginger constituents against SARS-CoV-2 virus: A therapeutic and theoretical approach.

In the present research, ginger extracted compounds, namely; Gingerol {(1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone} (1), Zingerone {(4-(4-Hydroxy-3-methoxyphenyl)-2-butanone)} (2), and Shogoals {(E)-1-(4-Hydroxy-3- methoxyphenyl) dec-4-en-3-one)} (3) have been investigated as SARS-Cov-2 inhibitors. The interaction of extracted compounds with the virus's spikes may restrict the virus's reproduction or give time to the body's immune system to detect viruses, consequently producing appropriate antibodies. Gaussian 09 with a 6-311G (d, p) basis set, UCA FUKUI, MGL implement, DSV, and LigPlus software were utilized. The active sites for adsorption were identified using the total electron density (TED), FUKUI function, and Millikan charges. Furthermore, docking analysis clearly showed that the inhibition of viral replication depends on binding energy (Eb) and ligand efficiency (LE). A docking study revealed that the inhibition ability of the studied compounds on SARS-CoV-2 was in the order of 2 > 3 > 1.

A density functional theory study on the potential application of Ni and Co doped ZnO nanosheets as a carrier for ciclopirox anticancer drug.

The Ni and Co doping effect on the ciclopirox (CPX) drug delivery performance of a ZnO nanosheet (ZnO-NS) was investigated theoretically. Doping Ni and Co metals into the ZnO-NS increased the adsorption energy of CPX from -7.9 to -27.4 and -31.7 kcal/mol, respectively. The CPX adsorption reduced the ZnO-NS gap (Eg) from 3.81 to 3.46 eV, while the CPX adsorption reduced the Eg of the Ni- and Co-doped ZnO-NS from 2.74 and 2.68 eV to 1.87 and 1.71 eV, respectively. The CPX adsorption performance increased after doping process. A drug release mechanism was introduced in cancerous tissues based on the PH. .

Screening for chitin degrading bacteria in the environment of Saudi Arabia and characterization of the most potent chitinase from Streptomyces variabilis Am1.

Forty-six promising chitinolytic isolates were recovered during a screening for chitinolytic bacteria in the environment of Saudi Arabia. The top three isolates belonged to the genus Streptomyces. Streptomyces variabilis Am1 was able to excrete the highest amount of chitinases, reaching the maximum at 84 h with 0.5% yeast extract and nitrogen source and 2% galactose as a carbon source. Purification of chitinase by DEAE-Cellulose and Sephadex G75 improved the specific activity to 18.6-fold and the recovery to 23.8% and showed a mass at 56 kDa. The optimal catalysis of the purified chitinase was at 40 °C and pH 8 with high thermostability and pH stability as reflected by a midpoint temperature value of 66.6 °C and stability at pH 4-9. The protein reagents SDS, EDTA, and EGTA significantly inhibited the enzyme and the EDTA-chelated chitinase restored its activity after the addition of Fe2+ ions suggesting a metallo-chitinase type with ferric ions as cofactors. Chitinase exerted high antifungal activity against some phytopathogenic fungi. Interestingly, the tested Streptomyces were able to produce chitosan nanocubes along with chitosan from chitin degradation which may be an additional power in their antifungal activity in nature. This work also reveals the importance of unexplored environments as a pool of promising microorganisms with biotechnological applications.

Molecular modeling for sensing of cisplatin drug by graphdiyne: electronic study via DFT.

CONTEXT: By utilizing first-principles calculations, we studied the electronic properties of graphdiyne nanosheet (GDY) and its Si-doped counterpart, Si-GDY. Both GDY and Si-GDY sheet surfaces were examined for the drug cisplatin (CP) adsorption using adsorption energy, charge transfer, and changes in electrical conductivity as indicators. Pure GDY has little affinity for CP, according to this study. Only 7.83% of the GDY surface's bandwidth energy changed after CP adsorption. CP on Si-GDY has a gaseous energy value of -18.75 kcal/mol and an aqueous energy value of - 49.39 kcal/mol. METHODS: The prescribed medications' water-phase solubility is determined by their solvation energy value. These charges are transferred between CP and the Si-GDY sheet, which is extremely positively charged, and this gives CP the necessary binding energy. After CP adsorption, electrical conductivity of Si-GDY increased by approximately 19.01%.

Inhibitory behavior and adsorption of asparagine dipeptide amino acid on the Fe(111) surface.

CONTEXT: The inhibitory effect of asparagine (Asn) and its derivatives on iron (Fe) corrosion was studied by performing density functional theory (DFT) calculations. In this paper, the global and local reactivity descriptors of Asn in the protonated and neutral forms were evaluated. Also, the changes in reactivity were investigated when dipeptides were combined with Asn. Due to the increase in the reaction centers within their molecular structure, there was an enhancement in the inhibitory effect of these dipeptides. Moreover, the adsorption energies (Eads) and the adsorption configurations of Asn and small peptides (SPs) with most stability were determined on the surface of Fe(111). It was found that dipeptides had a chemical adsorption on these substrates. In the protonated forms, there was an enhancement in the absolute values of Eads between the inhibitors and the Fe(111) surfaces. Peptides were more likely to be adsorbed on the Fe surfaces, showing the great inhibitory effect of these moieties. The results of the current research demonstrate the possibility of utilizing SPs as efficient "green" corrosion inhibitors. METHODS: DFT computations were undertaken by employing the BIOVIA Material Studio with B3LYP-D3 functional and 6-31 + G* basis set. The theoretical evaluation of the inhibitory effect of asparagine (Asn) dipeptides, and the potential analysis of small peptides to protect against the corrosion of Fe, was done.

Exploring the role of 2D-C2N monolayers in potassium ion batteries.

CONTEXT: In recent years, undivided attention has been given to the unique properties of layered nitrogenated holey graphene (C2N) monolayers (C2NMLs), which have widespread applications (e.g., in catalysis and metal-ion batteries). Nevertheless, the scarcity and impurity of C2NMLs in experiments and the ineffective technique of adsorbing a single atom on the surface of C2NMLs have significantly limited their investigation and thus their development. Within this research study, we proposed a novel model, i.e., atom pair adsorption, to inspect the potential use of a C2NML anode material for KIBs through first-principles (DFT) computations. The maximum theoretical capacity of K ions reached 2397 mA h g-1, which was greater in contrast with that of graphite. The results of Bader charge analysis and charge density difference revealed the creation of channels between K atoms and the C2NML for electron transport, which increased the interactions between them. The fast process of charge and discharge in the battery was due to the metallicity of the complex of C2NML/K ions and because the diffusion barrier of K ions on the C2NML was low. Moreover, the C2NML has the advantages of great cycling stability and low open-circuit voltage (approximately 0.423 V). The current work can provide useful insights into the design of energy storage materials with high efficiency. METHODS: In this research, we used B3LYP-D3 functional and 6-31 + G* basis with GAMESS program to calculate adsorption energy, open-circuit voltage, and maximum theoretical capacity of K ions on the C2NML.

Evaluation of a biosensor-based graphene oxide-DNA nanohybrid for lung cancer.

Lung cancer is nowadays among the most prevalent diseases worldwide and features the highest mortality rate among various cancers, indicating that early diagnosis of the disease is of paramount importance. Given that the conventional methods of cancer detection are expensive and time-consuming, special attention has been paid to the provision of less expensive and faster techniques. In recent years, the dramatic advances in nanotechnology and the development of various nanomaterials have led to activities in this context. Recent studies indicate that the graphene oxide (GO) nanomaterial has high potential in the design of nano biosensors for lung cancer detection owing to its unique properties. In the current article, a nano biosensor based on a DNA-GO nanohybrid is introduced to detect deletion mutations causing lung cancer. In this method, mutations were detected using a FAM-labeled DNA probe with fluorescence spectrometry. GO was synthesized according to Hummers' method and examined and confirmed using Fourier Transform Infrared (FT-IR) Spectrometry and UV-vis spectrometry methods and Transmission Electron Microscopy (TEM) images.

Application of zinc oxide nano-tube as drug-delivery vehicles of anticancer drug.

CONTEXT: Zinc oxide nano-tube (ZnONT) nano-structures, which possess chemical stability and non-toxicity in the human body, are considered promising for delivering different drugs. Within this work, we scrutinized the drug delivery capability of the ZnONT and its adsorptional properties as a drug delivery vehicle (DDV) for hydroxyurea (HU) as an anti-cancer drug through density functional theory along with the solvent impacts. Based on the optimized structures, it can be suggested that Zn atoms of ZnONT are the ideal sites on this nano-tube for the adsorption of HU. HU had a strong physical adsorption through the O atom of carbonyl groups onto the local pyramidal site of the ZnONT. At 1.96 Å and Ead of -39.28 kcal/mol, in the configuration which was favorable in terms of energy, there was an interaction between the O atoms of -C=O group of the drug and a Zn atom of the ZnONT. In order to scrutinize the excited state properties of the HU-ZnONT complex, we also examined the UV/Vis data of the HU/ZnONT interaction system. Following the adsorption of HU onto the surface of the ZnONT, there was a significant red-shift based on the maximum absorption wavelength, showing that the ZnONT is an ideal candidate for optic sensors in order to detect and monitor the drug molecule. HU could be released in the cancer tissues where pH was low based on the drug release mechanism. The current work thoroughly investigated the mechanism of interaction between the ZnONT and HU, showing that ZnONT can be used for the smart drug delivery of HU. Overall, the findings suggest that ZnONT could be used as an efficient drug-delivery system for the HU drug to treat various types of cancer. METHODS: In this work we used B3LYP-gCP-D3 functional and the basis set LANL2DZ on the transition metal (Zn) and the basis set cc-pVDZ on the others. GAMESS software program was employed for performing the calculations. we performed analyses, including charge transport, molecular electrostatic potential surface (MEP), energetic, electronic, natural bond orbitals (NBOs), and structural optimizations.

Theoretical Sensing Performance for Detection of Cyclophosphamide Drug by Using Aluminum Carbide (C3Al) Monolayer: a DFT Study.

Because nanomaterials are highly reactive and electronically sensitive towards a variety of drug molecules, they are thought of as efficient drug sensors. In the present research study, an aluminum carbide (C3Al) monolayer is employed and its interaction is examined with cyclophosphamide (CP) by performing DFT computations. The C3Al monolayer is highly reactive and sensitive towards CP according to the computations. CP interacts with the C3Al monolayer with the adsorption energy of -31.39 kcal/mol. A considerable charge transfer (CT) indicates an enhancement in the conductivity. Also, the charge density is explained based on the electron density differences (EDD). The decrease in CP/C3Al energy gap (Eg) by approximately 52.91% is due to the remarkable effect of adsorption on the LUMO and the HOMO levels. Therefore, due to the decrease in Eg which can generate an electrical signal, the electrical conductivity is considerably increased. These results suggest that the C3Al monolayer can be employed as a proper electronic drug sensor for CP. Also, the recovery time for the desorption process of CP form the surface of C3Al is 351 s at 598 K.

BeO nanotube as a promising material for anticancer drugs delivery system.

In this research, the application of BeO nanotube (BeONT) as a nanocarrier for Fluorouracil (5-FU) anticancer drug has been studied by density functional theory (DFT) approach. The method ωB97XD with 6-31 G** basis set were employed. A precise surface study, shows that there are two directions for 5-FU adsorption that did not deliver any of the imaginary frequency vibrational spectra, identifying that all relaxation structures are at the lowest energy level. Based on our calculations, the energy of adsorption for 5FU@BeONT structures are range -120 to -168 kJ/mol, in the gas phase and -395 to 4-00 kJ/mol in the aqueous phase. The highest and the lowest values of adsorption energy are both in strong physical adsorption. Due to receiving an electronic charge from 5-FU, BeONT exhibited a p-type semiconducting feature for all positions. In addition, based on natural bond orbital (NBO) analysis, the direction of charge transfer was from fluorine's σ orbitals of the drug to n* orbitals (O and Be atoms) of BeONT with a considerable amount of transferred energy. BeONT can be employed as a potential strong carrier for 5-FU drugs for practical purposes based on our findings.

Facile synthesis and comparative study of the enhanced photocatalytic degradation of two selected dyes by TiO2-g-C3N4 composite.

Photocatalysis is considered a useful technique employed for the dye degradation through solar light, visible or UV light irradiation. In this study, TiO2, g-C3N4, and TiO2-g-C3N4 nanocomposites were successfully synthesized and studied for their ability to degrade Rhodamine B (RhB) and Reactive Orange 16 (RO-16), when exposed to visible light. The analytical techniques including XRD, TEM, SEM, DRS, BET, XPS, and fluorescence spectroscopy were used to explore the characteristics of all the prepared semiconductors. The photocatalytic performance of synthesized materials has been tested against both the selected dyes, and various experimental parameters were studied. The experimental results demonstrate that, in comparison to other fabricated composites, the TiO2-g-C3N4 composite with the optimal weight ratio of g-C3N4 (15 wt%) to TiO2 has shown outstanding degrading efficiency against RhB (89.62%) and RO-16 (97.20%). The degradation experiments were carried out at optimal conditions such as a catalyst load of 0.07 g, a dye concentration of 50 ppm, and a temperature of 50 ℃ at neutral pH in 90 min. In comparison to pure TiO2 and g-C3N4, the TiO2-g-C3N4, a semiconductor, has shown higher degradation efficiency due to its large surface area and decreased electron-hole recombination. The scavenger study gave an idea about the primary active species (-OH radicals), responsible for dye degradation. The reusability of TiO2-g-C3N4 was also examined in order to assess the composite sustainability.

Analysis of the protection of copper corrosion by using amino acid inhibitors.

Since the human body is one of the highly aggressive environments, the materials utilized for an implant should have high resistance to degradation and corrosion. One of the commonly used biomaterials in medicine is copper (Cu). The Cu corrosion can result in the release of ions in the body with high toxicity, thereby causing inflammatory diseases. Based on the literature, as biomolecules, amino acids act as a corrosion inhibitor in aggressive solutions. The current work aims at scrutinizing the inhibition impact of L-arginine (L-Arg) and L-Valine (L-Val), which have been rarely investigated, upon the corrosion process of Cu. We undertook density functional theory computations to scrutinize the inhibitory impact of L-Arg and L-Val as well as their conformers upon Cu corrosion. Also, we scrutinized the computed parameters according to the back donation of electrons between Cu and the inhibitors, transported electron fraction, energy gap, softness, hardness, EHOMO, and ELUMO. According to the theoretical indices of L-Arg, it prefers adsorption. We examined the inhibitory efficiency of L-Arg against corrosion and found that it is a promising inhibitor.

A molecular modeling on the potential application of beryllium oxide nanotube for delivery of hydroxyurea anticancer drug.

Within this work, we scrutinized the use of BeO nanotube (BeONT) as a nanocarrier for the anticancer drug hydroxyurea (HU) through density functional theory (DFT) calculations. We utilized the functional ꞷB97XD and the basis set 6-31G**. Based on a detailed surface analysis, HU was adsorbed on the surface of the nanotube through 4 different orientations. Also, no vibrational spectra exhibited imaginary frequencies, showing the minimum energy of the relaxed structures. The maximum adsorption energy and the minimum adsorption energy are in strong physical adsorption. The BeONT exhibited p-type semiconducting characteristics in all orientations since it received electronic charge from HU. The results demonstrate the possibility of using the BeONT as a promising carrier for HU drugs.

Coronavirus disease 2019 (COVID-19) vaccine hesitancy among physicians, physician assistants, nurse practitioners, and nurses in two academic hospitals in Philadelphia.

OBJECTIVE: To evaluate coronavirus disease 2019 (COVID-19) vaccine hesitancy among healthcare personnel (HCP) with significant clinical exposure to COVID-19 at 2 large, academic hospitals in Philadelphia, Pennsylvania. DESIGN, SETTING, AND PARTICIPANTS: HCP were surveyed in November-December 2020 about their intention to receive the COVID-19 vaccine. METHODS: The survey measured the intent among HCP to receive a COVID-19 vaccine, timing of vaccination, and reasons for or against vaccination. Among patient-facing HCP, multivariate regression evaluated the associations between healthcare positions (medical doctor, nurse practitioner or physician assistant, and registered nurse) and vaccine hesitancy (intending to decline, delay, or were unsure about vaccination), adjusting for demographic characteristics, reasons why or why not to receive the vaccine, and prior receipt of routine vaccines. RESULTS: Among 5,929 HCP (2,253 medical doctors [MDs] and doctors of osteopathy [DOs], 582 nurse practitioners [NPs], 158 physician assistants [PAs], and 2,936 nurses), a higher proportion of nurses (47.3%) were COVID-vaccine hesitant compared with 30.0% of PAs and NPs and 13.1% of MDs and DOs. The most common reasons for vaccine hesitancy included concerns about side effects, the newness of the vaccines, and lack of vaccine knowledge. Regardless of position, Black HCP were more hesitant than White HCP (odds ratio [OR], ∼5) and females were more hesitant than males (OR, ∼2). CONCLUSIONS: Although most clinical HCP intended to receive a COVID-19 vaccine, intention varied by healthcare position. Consistent with other studies, hesitancy was also significantly associated with race or ethnicity across all positions. These results highlight the importance of understanding and effectively addressing reasons for hesitancy, especially among frontline HCP who are at increased risk of COVID exposure and play a critical role in recommending vaccines to patients.

Human splenic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are strategically located immune regulatory cells in cancer.

In contrast to the mouse, functional assets of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in the human spleen remain to be better elucidated. Here, we report that the spleen in gastric and pancreatic cancer adopts an immune regulatory character, harbors excessive amount of PMN-MDSC, and anatomically enables their interaction with T cells. Compared to the peripheral blood, the spleen from cancer patients contained significantly higher levels of low-density PMN-MDSC, but not early-stage MDSC (e-MDSC) and monocytic-MDSC (M-MDSC). Low-density fraction of polymorphonuclear (PMN) cells was enriched in immature myeloid cells and displayed higher levels of CD10, CD16, and ROS than their blood-derived counterparts. They were also positive for PD-L1, LOX-1, and pSTAT3. The white pulp and periarteriolar lymphoid sheath (PALS) were strategically surrounded by PMN cells that were in contact with T cells. Unlike those from the blood, both low-density and normal-density PMN cells from the human spleen suppressed T cell proliferation and IFN-γ production. Independent of clinical grade, high PMN-MDSC percentages were associated with decreased survival in gastric cancer. In summary, our results outline the immune regulatory role of the spleen in cancer where neutrophils acquire MDSC functions and feasibly interact with T cells.

Association Between HIV-Related Tweets and HIV Incidence in the United States: Infodemiology Study.

BACKGROUND: Adolescents and young adults in the age range of 13-24 years are at the highest risk of developing HIV infections. As social media platforms are extremely popular among youths, researchers can utilize these platforms to curb the HIV epidemic by investigating the associations between the discourses on HIV infections and the epidemiological data of HIV infections. OBJECTIVE: The goal of this study was to examine how Twitter activity among young men is related to the incidence of HIV infection in the population. METHODS: We used integrated human-computer techniques to characterize the HIV-related tweets by male adolescents and young male adults (age range: 13-24 years). We identified tweets related to HIV risk and prevention by using natural language processing (NLP). Our NLP algorithm identified 89.1% (2243/2517) relevant tweets, which were manually coded by expert coders. We coded 1577 HIV-prevention tweets and 17.5% (940/5372) of general sex-related tweets (including emojis, gifs, and images), and we achieved reliability with intraclass correlation at 0.80 or higher on key constructs. Bivariate and multivariate analyses were performed to identify the spatial patterns in posting HIV-related tweets as well as the relationships between the tweets and local HIV infection rates. RESULTS: We analyzed 2517 tweets that were identified as relevant to HIV risk and prevention tags; these tweets were geolocated in 109 counties throughout the United States. After adjusting for region, HIV prevalence, and social disadvantage index, our findings indicated that every 100-tweet increase in HIV-specific tweets per capita from noninstitutional accounts was associated with a multiplicative effect of 0.97 (95% CI [0.94-1.00]; P=.04) on the incidence of HIV infections in the following year in a given county. CONCLUSIONS: Twitter may serve as a proxy of public behavior related to HIV infections, and the association between the number of HIV-related tweets and HIV infection rates further supports the use of social media for HIV disease prevention.

Serine protease inhibitor-6 differentially affects the survival of effector and memory alloreactive CD8-T cells.

The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6(-/-) mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6(-/-) CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells.

Etiological profile of Omani women with recurrent pregnancy loss.

OBJECTIVE: To define the different causes of recurrent pregnancy loss (RPL) among Omani women. METHODS: This retrospective study included all women with RPL seen between June 2006 and March 2012 in the RPL outpatient clinic in Sultan Qaboos University hospital, Muscat, Oman. The data were collected from the Hospital Information System by screening the electronic records of these patients. The sample size gathered during the study period was 290 women. RESULTS: One hundred and forty (48%) of the examined patients had an identifiable cause for RPL, while in 150 (52%), no cause was identified. The most common causes were immunological factors (35.4%) and the least common were environmental factors (1.7%). Other causes implicated included: chromosomal abnormalities (8%), anatomical factors (9.4%), endocrine disorders (29.8%), infectious causes (3%), and thrombotic causes (12.7%). CONCLUSION: Recurrent pregnancy loss is prevalent among Omani women. The etiological profile of RPL in Omani women is consistent with that reported elsewhere according to previously published studies, with minor variations.

Periodontal status of rheumatoid arthritis patients in khartoum state.

BACKGROUND: Few studies have investigated the periodontal condition among Rheumatoid arthritis in Sudan. The present study described the periodontal condition among Sudanese patients suffering from rheumatoid arthritis and to compare them with those of non-rheumatic subjects. METHODS: A group of eighty rheumatoid arthritis patients was selected from Patient's Rheumatoid Clinics in Khartoum State during the period of January to May 2010. A control group of eighty patients with the same age and gender was selected for the study. Both Rheumatoid arthritis patients and the control group were examined for their plaque index, gingival index, and clinical attachment loss. RESULTS: The results revealed that there were no significant differences in plaque and gingival index among study and control groups, with mean plaque index of (1.25 ± 0.4) for patients and (1.17 ± 0.28) for the control group (p-value is 0.3597). The mean gingival index was (1.2 ± 0.24) for the patients and (1.2 ± 0.33) for the control (p = is 0.3049). The results showed statistically significant differences in clinical attachment loss between study and control groups, with mean clinical attachment loss of (1.03 ± 0.95) for the study group and (0.56 ± 0.63) for the control group (p = 0.0002). The study revealed that no association exists between the type of drug used to treat rheumatoid arthritis (NSAIDs & DMARDs) and the periodontal parameters (plaque index, gingival index, and clinical attachment loss). CONCLUSION: A significant relationship between periodontal disease and Rheumatoid Arthritis does exist, but no difference between plaque and gingival index has been detected among study and control groups.

Reading Saedi's Ahl-e Hava: pattern and significance in spirit possession beliefs on the southern coasts of Iran.

Based on Saedi's description of spirit possession beliefs and practices on the southern coasts of Iran, this paper attempts to isolate the culturally and psychologically significant categories through which the possessed define and differentiate their possessing spirits or "Winds." It examines the play of these categories in the complex of relationships between the possessing spirits, the possessed, ritual specialists, "symptoms" of possession, and their "negotiations" in rituals of possession or exorcism. It examines from a semiotic perspective the double transformations of spirit and host which in the beliefs and practices of the People of the Air constitute "therapy." It points out areas for further research and analysis.