The lowest energy structures of SeBn0/− (n = 3–16) clusters tend to form quasi-planar or planar structures. Some selenium-doped boron clusters keep a skeleton of the corresponding pure boron clusters; however, the addition of a Se atom modified and improved some of the pure boron cluster structures. In particular, the Se atoms of SeB7−, SeB8−, SeB10−, and SeB12− are connected to the pure quasi-planar B7−, B8−, B10−, and B12− clusters, which leads to planar SeB7−, SeB8−, SeB10−, and SeB12−, respectively. Interestingly, the lowest energy structure of SeB9− is a three-dimensional mushroom-shaped structure, and the SeB9− cluster displays the largest HOMO–LUMO gap of 5.08 eV, which shows the superior chemical stability. Adaptive natural density partitioning (AdNDP) bonding analysis reveals that SeB8 is doubly aromatic, with 6 delocalized π electrons and 6 delocalized σ electrons, whereas SeB9− is doubly antiaromatic, with 4 delocalized π electrons and 12 delocalized σ electrons. Similarly, quasi-planar SeB12 is doubly aromatic, with 6 delocalized π electrons and 14 delocalized σ electrons. The electron localization function (ELF) analysis shows that SeBn0/− (n = 3–16) clusters have different local electron delocalization and whole electron delocalization effects. The simulated photoelectron spectra of SeBn− (n = 3–16) have different characteristic bands that can identify and confirm SeBn− (n = 3–16) combined with future experimental photoelectron spectra. Our research enriches the geometrical structures of small doped boron clusters and can offer insight for boron-based nanomaterials.A theoretical research of structural evolution, electronic properties, and photoelectron spectra of selenium-doped boron clusters SeB In 2021, researchers synthesized borophene crystal, which was hydrogenated with hydrogen atoms [7 cluster also. The experimental findings of B40− and borophene offer insight for the development of new boron nanomaterials and nanodevices. Research on small boron clusters is promising as a way to provide new ideas for new nanomaterials and nanodevices.Clusters are composed of several to thousands of atoms or molecules whose properties depend on their size and shape. Clusters are ideal model systems for correlating microscopic structure and macroscopic properties of substances, and cluster research is of great significance to deeply understand the laws of matter transformation. Research of structure and properties of clusters can offer insight for the design and manufacture of new materials and new devices at the atomic level. Boron clusters can induce polycentric chemical bonds and adopt several interesting structures with meaningful properties ,3,4,5,6.ructures ,8, and nructures ,10,11. Iructures  B40− hasclusters ,19,20,21clusters , and theen atoms , and it 20− and B22− have a quasi-planar structure [20−, NaB22−, and KB22− display a double-ring configuration [12 (M = Co and Rh) can lead to the quasi-planar B12 form, a semi-sandwich configuration [24 has a double-ring configuration [24 and ScB24 have a cage configuration and three-ring tubular structure, respectively, after adding one Sc or Ti atom [24− has a quasi-planar structure [24− and VB24− have a cage configuration after adding one Ti or V atom [40, NaB40, or KB40 cage is promising for application to the field of nonlinear optics [40 or TiB40 cage is promising for application to the field of hydrogen storage [12− and Rh atom-doped RhB12− can improve chemical activity [40 is promising for application to molecular devices [n−, MnBn−, BiBn−(n = 6–8), CoB16−, La2Bn−(n = 10–11), La3B18−, MB8, and M2B6 have various unique structures [7, small boron clusters with doping are promising as the structural units of borophene and other boron nanomaterials. Selenium is one of the essential microelements in the human body and has obvious inhibitory effects on tumors. The Se atom can combine with metal atoms, such as Cd and Zn, to form semiconductor clusters or quantum dots [n0/− (n = 3–16), extensive geometric configurations were generated and predicted, using the particle swarm optimization (CALYPSO) approach [In recent years, researchers have studied abundant doped boron clusters, and they mainly focused on doping a single metal atom in boron clusters of different sizes. Metal-atom-doped boron clusters can induce new geometrical configuration and properties ,36,37,38tructure . Howeverguration ,40. Co aguration . Neutralguration , while T Ti atom ,42. Anior V atom . In addir optics ; the ScB storage ,17,19; Cactivity ; Co atom devices ; and theructures ,45. Howetum dots ,47. Thestum dots ,49. Se dapproach  in combiapproach .n0/− (n = 3–16) are shown in n0/− (n = 3–16) are displayed in n0/− (n = 3–16) trend to form planar or quasi-planar structures. Interestingly, the ground state configuration of SeB9− is three-dimensional mushroom shaped. Early theoretical and experimental research found that most of the small neutral boron clusters are quasi-planar or planar, and all small anionic boron clusters are quasi-planar or planar structures. n0/− (n = 3–16) have a skeleton of pure boron clusters, such as SeBn  and SeBn−  [n− , each Se atom is connected to two boron atoms to form a three-ring. The lowest energy configurations of SeBn0/− (n = 3–5) have planar structure, and the Se atom is attached to the boron atoms of the pure boron cluster Bn0/− (n = 3–5) [60/− and SeB130/− have quasi-planar structure, and they are different from the ground-state structures of corresponding pure boron clusters B60/− and B130/− [70/−, SeB100/−, and SeB120/−, the lowest energy configurations of neutral clusters and corresponding anionic clusters have similar structure, and the Se atoms of SeB7, SeB10, and SeB12 are connected to the pure quasi-planar B7, B10, and B12 clusters, respectively. However, the Se atoms of SeB7−, SeB10−, and SeB12− are connected to the pure quasi-planar B7−, B10−, and B12− clusters, which leads to the planar SeB7−, SeB10−, and SeB12−, respectively [80/− have same planar structure, and the Se atom of SeB8 is connected to the pure planar B8 cluster. However, the Se atom of SeB8− is connected to the pure quasi-planar B8− cluster, which leads to the planar SeB8−. The pure B90/− have same planar wheel-shape structure [9− to become a three-dimensional mushroom-shaped structure (with C6V symmetry) and causes the neutral SeB9 to become a boat-shaped structure. The lowest energy structures of SeB110/− have similar structure, and the Se atom of SeB11 is connected to the pure planar B11 cluster, which leads to the slight structural change. However, the Se atom of SeB11− is connected to the pure planar B11− cluster [140/− have same planar structure, the lowest energy configurations of SeB160/− have same quasi-planar structure, and the Se atoms are connected to the pure planar B140/− clusters and pure quasi-planar B160/− clusters, respectively [150/− have quasi-planar structure and exhibit two axially chiral isomers, and they are different from the ground-state configurations of pure boron cluster B150/−. Similar to pure B7 clusters, planar and quasi-planar Se-doped boron clusters are promising as the structural units of boron nanomaterials, which can be synthesized further into borophene.Five low-energy structures of SeB–14, 16) ,53. As cn = 3–5) . The lowd B130/− . For SeBectively . The lowtructure . However cluster . The lowectively ,53. The 3−, SeB4, SeB5−, SeB6, SeB7−, SeB8, SeB9−, SeB10, SeB11−, SeB12, SeB13−, SeB14, SeB15−, and SeB16 are 2.48, 3.52, 2.14, 3.92, 2.90, 2.33, 5.08, 2.93, 2.89, 3.29, 2.28, 2.00, 2.91, and 2.27 eV, respectively. For open-shell clusters, α-HOMO–LUMO and β-HOMO–LUMO, energy gaps vary within the range of 1.92-4.39 eV. Meanwhile, the calculated HOMO–LUMO energy gaps of SeBn0/− (n = 3–16) clusters reveal that the SeB9− cluster possesses the largest HOMO-LUMO gap of 5.08 eV, which shows the superior chemical stability.The harmonic frequency analysis confirmed that these lowest-energy structures are actually stable (no imaginary frequency). For closed-shell clusters, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy gaps (HOMO–LUMO energy gaps) of SeBn0/− (n = 3–16), we analyzed the chemical bonding of closed-shell planar SeB8, three-dimensional SeB9−, and quasi-planar SeB12 using the adaptive natural density partitioning (AdNDP) approach. 8. For SeB8, AdNDP analyses reveal that one lone pair   of the ved in 12  cover ein− (n = 3–16), vertical detachment energies (VDEs) were calculated and photoelectron spectra of SeBn− (n = 3–16) were simulated with the time-dependent DFT (TD-DFT) method [Photoelectron spectroscopy in combination with theoretical calculations was used to identify the structures of size-selected boron clusters ,12,55. T) method .n− (n = 3–16). The results indicate that SeB3− has the lowest first VDE, and SeB12− has the largest energy gap (about 1.48 eV) between the first and second peaks. The first several peaks were used to identify boron clusters [9−) come from the calculated ground-state VDEs of SeB3−, SeB4−, SeB5−, SeB6−, SeB7−, SeB8−, SeB10−, SeB11−, SeB12−, SeB13−, SeB14−, SeB15−, and SeB16− at 2.52, 2.62, 2.89, 2.89, 2.89, 2.76, 3.14, 3.51, 2.66, 3.43, 3.15, 3.59, and 3.36 eV, respectively. The calculated ground-state VDEs of these closed-shell clusters originate from the detachment of the electron from the molecular orbital HOMO. However, for open-shell clusters, the calculated ground-state VDE of each cluster originates from the detachment of the electron from the singly occupied molecular orbital α-SOMO. The first peak of SeB9− comes from the second VDE at 4.01 eV, which is smaller than the ground-state VDE of 4.25 eV (second peak). The second peaks of SeB3−, SeB5−, SeB7−, SeB11−, SeB13−, and SeB15− come from the second calculated VDEs at 3.23, 4.07, 4.16, 3.81, 4.30, and 4.12 eV, respectively, which originate from detaching the electrons from HOMO-1. The second peaks of SeB4−, SeB6−, SeB8−, SeB10−, SeB12−, SeB14−, and SeB16− come from the second VDEs at 3.36, 4.02, 3.07, 3.48, 4.14, 3.64, and 3.63 eV, respectively, which originate from detaching the electrons from the singly occupied molecular orbital β -HOMO-1. In addition, the peaks with higher binding energy originate from detaching the electrons from lower molecular orbitals. It is noted that some of the doped anionic boron clusters have a similar skeleton as the corresponding anionic pure boron clusters. Comparing their photoelectron spectra, the addition of the Se atom results in a great change in the photoelectron spectra [3- to move 0.30 eV towards the low binding energy side and causes the first two peaks of B5- to move 0.46 eV towards the high binding energy side [4−, SeB8−, and SeB13-, compared with the photoelectron spectra of pure boron clusters, the addition of Se atoms causes different band characteristics [7− and quasi-planar B7- have almost the same first VDE  [10- and quasi-planar B10- have almost the same first VDE  [14− and quasi-planar B14- have almost the same first VDE  [11-, compared to the photoelectron spectra of pure boron clusters B11−, the addition of the Se atom causes the first peak to move 0.08 eV towards the high binding energy side and causes the second peak to move 0.25 eV towards the low binding energy side. For SeB12− and SeB16−, planar SeB12- and quasi-planar B12− have similar band characteristics [16− and quasi-planar B16- have similar band characteristics [n− (n = 3–16) has different spectral features; especially the peaks at the low binding energy side can identify the SeBn− (n = 3–16). As with the discovery of other anionic boron clusters, if the photoelectron spectra of SeBn− (n = 3–16) are obtained in experiments, these simulated values can be used for the identification of SeBn− (n = 3–16).n− n = –16. The  spectra . However spectra . For exaeristics . Figure n0/− (n = 3–16) were performed with CALYPSO 5.0 software in combination with Gaussian 16 software. CALYPSO is a reliable cluster configuration prediction software, and it has successfully predicted boron or doped boron clusters [Configuration searches of Se-doped boron clusters SeBclusters ,59,60,61After the preliminary structural search, low-energy structures were then fully optimized at the PBE0/6-311+G(d) level ,62. Aften0/− (n = 3–16) clusters. The conclusions are summarized as follows. (1) The global minima of SeBn0/− (n = 3–16) clusters tend to form quasi-planar or planar structures. (2) The ground-state structure of SeB9− is a three-dimensional, mushroom-shaped, ground-state structure, and it possesses the largest HOMO–LUMO gap of 5.08 eV, which shows the superior chemical stability. (3) AdNDP bonding analyses reveal that SeB8 is doubly aromatic, with six delocalized σ and six delocalized π electrons, whereas SeB9− is doubly antiaromatic, with twelve delocalized σ and four delocalized π electrons. Similarly, SeB12 is doubly aromatic, with fourteen delocalized σ and six delocalized π electrons. (4) ELF analysis shows that SeBn0/− (n = 3–16) clusters have different local electron delocalization and whole-electron delocalization effects. (5) SeBn− (n = 3–16) have different photoelectron spectra, and especially the first several peaks can be used for the identification of SeBn− (n = 3–16). This research has enriched the structures of doped boron clusters.DFT combined with CALYPSO software is employed to demonstrate the structural evolution of SeB


Chrysopogon zizanioides root essential oil (CZ-EO) In addition, CZ-EO cytotoxicity to LLCMK2 adherent epithelial cells was assessed. The major compounds identified in CZ-EO were khusimol (30.0 ± 0.3%), β-eudesmol (10.8 ± 0.3%), α-muurolene (6.0 ± 0.1%), and patchouli alcohol (5.6 ± 0.2%). CZ-EO displayed optimal antibacterial activity against Prevotella nigrescens, Fusobacterium nucleatum, Prevotella melaninogenica, and Aggregatibacter actinomycetemcomitans, with Minimum Inhibitory Concentration (MIC) values between 22 and 62.5 µg/mL and Minimum Bactericidal Concentration (MBC) values between 22 and 400 µg/mL. CZ-EO was highly active against the L. amazonensis promastigote and amastigote forms  and the T. cruzi trypomastigote form (IC50 = 11.2 µg/mL). Moreover, CZ-EO showed moderate cytotoxicity to LLCMK2 cells, with CC50 = 565.4 µg/mL. These results revealed an interesting in vitro selectivity of CZ-EO toward the L. amazonensis promastigote and amastigote forms  and the T. cruzi trypomastigote form (SI = 50.5) compared to LLCMK2 cells. These results showed the promising potential of CZ-EO for developing new antimicrobial, antileishmanial, and antitrypanosomal drugs. This study aimed to investigate the chemical composition as well as the antibacterial, antiparasitic, and cytotoxic potentialities of the Brazilian  Aggregatibacter actinomycetemcomitans, Prevotella melaninogenica, Prevotella nigrescens, and Fusobacterium nucleatum can be highlighted [Gingivitis and periodontitis, both periodontal diseases characterized by a chronic infection associated with bacteria living in the oral cavity, cause irreversible destruction of tissues supporting the teeth . Gingivihlighted .Trypanosoma cruzi. This protozoan is transmitted to humans when feces of insects contaminated with this microorganism penetrate a skin wound or mucosa. Infection can also occur by vertical, blood, or oral transmission. In the latter case, transmission is possible when the feces of infected vectors or macerated insects contaminate fresh fruit juices or other foods [Trypanosomiasis, or Chagas disease, is a serious public health problem that affects between 6 and 7 million people. This Neglected Tropical Disease (NTD) predominates in Latin American and Caribbean countries, where prevention and treatment have progressed very slowly. However, globalization and migration have caused the number of cases to increase worldwide, including in developing countries . Chagas’er foods . Since tLeishmania, leading to tegumentary (TL) or visceral (VL) manifestations depending mainly on the infecting Leishmania species and the mammalian host’s immunological and nutritional status. Tegumentary leishmaniasis (TL) can be subclinical or cause self-healing skin lesions that can culminate in disfiguring scars, accompanied by extensive tissue destruction in nasopharyngeal mucosal tissues. [Leishmania amazonensis is one of the main species underlying TL, mainly cutaneous leishmaniasis (CL) and diffuse cutaneous leishmaniasis (DCL). TL evolves slowly, covering large skin areas and making an initial diagnosis and treatment difficult [Leishmaniasis is another NTD that impacts global public health, with an estimated 30,000 annual deaths. This complex disease is caused by different species of protozoa belonging to the genus tissues. . In the ifficult . The staifficult .Essential oils (EOs) are mixtures of volatile secondary metabolites that can be obtained from different parts  of medicinal and aromatic plants. EOs display diverse biological activities, like antimicrobial , antileiChrysopogon zizanioides (L.) Roberty (Poaceae), a synonym of Vetiveria zizanioides (L.) Nash, is native to the tropical regions of northern India, but it is distributed worldwide wherever the environmental conditions are appropriate for its development [C. zizanoides roots are strong, spongy, and aromatic, and their distribution is massive and compact. They are useful for treating hyperdipsia, burning sensations, skin diseases, nausea, vomiting, dyspepsia, flatulence, bilious fever, gout, lumbago, sprains, halitosis, cephalalgia, amentia, amenorrhea, helminthiasis, and general debility [C. zizanioides is commonly known as “tranquility oil” or “vetiver oil” and has significant commercial importance due to its unique fragrance, which is not provided by any other synthetic compound. Because of its fragrance, the EO of C. zizanioides is commonly used in foods. This species is also used for treating ulcers, fever, headache, inflammation, and gastritis [elopment . The culdebility . The EO astritis .C. zizanoides roots collected in Southeastern Brazil (CZ-EO), as well as its antimicrobial action against periodontopathogenic bacteria. We also investigate the chemical composition and the cytotoxicity of this EO to LLCMK2 adherent epithelial cells.As part of our ongoing research into the antimicrobial ,17,18 anC. zizanioides roots (CZ-EO), as determined by gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC–MS). A total of 22 compounds were identified, most of which (70.4 ± 0.1 %) are oxygenated sesquiterpenes. The major compounds in CZ-EO are khusimol (30.0 ± 0.3%), β-eudesmol (10.8 ± 0.3%), α-muurolene (6.0 ± 0.1%), and patchouli alcohol (5.6 ± 0.2%). We did not detect monoterpenes or phenylpropanoids in CZ-EO.P. intermedia ATCC 49046 (MIC = 22.0 ± 6.25 µg/mL), A. actinomycetemcomitans ATCC 43717 (MIC = 22.0 ± 6.25 µg/mL), P. melaninogenica ATCC 700524 (MIC = 50.0 ± 0.0 µg/mL), F.nucleatum (MIC = 50.0 ± 0.0 µg/mL), P. gingivalis ATCC 33277 (MIC = 62.5 ± 25.0 µg/mL), and P. nigrescens ATCC 33563 (MIC = 62.5 ± 25.0 µg/mL). CZ-EO also exhibited strong activity against P. gingivalis Clinical Isolate (MIC = 100.0 ± 0.0 µg/mL), P. intermedia Clinical Isolate (MIC = 150.0 ± 58.0 µg/mL), and F. nucleatum Clinical Isolate (MIC = 250.0 ± 100.0 µg/mL) (The Minimum Inhibitory Concentration (MIC) values obtained for CZ-EO ranged from 22.0 ± 6.25 to 250.0 ± 100.0 µg/mL against all the studied bacteria . This EO0 µg/mL) .A. actinomycetemcomitans (22.0 ± 6.25 µg/mL), F. nucleatum (50.0 ± 0.0 µg/mL), P. melaninogenica (50.0 ± 0.0 µg/mL), and P. nigrescens (62.5 ± 25.0 µg/mL) were the same as the respective MIC values , P. intermedia , and F. nucleatum , the MBC values were higher compared to the MIC values.Regarding the Minimum Bactericidal Concentration (MBC), the MBC values obtained for CZ-EO against C values . In the L. amazonensis promastigote and amastigote forms. The half-maximum inhibitory concentration (IC50) values of 7.20 and 16.21 μg/mL against the L. amazonensis promastigote and amastigote forms, respectively, are promising compared to amphotericin B (IC50 = 0.25 μg/mL).50 value obtained for CZ-EO against the T. cruzi trypomastigote form was 11.2 μg/mL. (50 = 9.9 µg/mL. CZ-EO exhibited moderate toxicity to the LLCMK2 cells (CC50 = 565.4 μg/mL) and Selectivity Index (SI = CC50/IC50) values of 78.5, 34.8, and 50.4 against the L. amazonensis promastigote and amastigote forms and T. cruzi trypomastigote forms, respectively   and ketones (9–14%) , predominate in these EOs [C. zizanoides collected in nine different countries [C. zizanioides roots. Although they reported that about 300 compounds have been detected in this EO, khusimol, β-vetispirene, vetiselinenol, and α-vetivone have always been the major compounds in the EO from C. zizanioides roots throughout the world [Sesquiterpenes have been reported as the major compounds in hese EOs . Here, wountries ,22,23. Ahe world .C. zizanioides roots has been investigated against a broad range of bacteria including  [The antimicrobial activity of the EO from s aureus , Staphylphyticus , Staphylpyogenes , Enterocfaecalis , Escherihia coli , Mycobacrculosis ) and funlbicans) . HoweverAccording to the literature, EOs with Minimum Inhibitory Concentration (MIC) values less than or equal to 100 µg/mL, between 101 and 500 µg/mL, between 501 and 1500 µg/mL, and between 1500 and 2000 µg/mL denote very strong, strong, moderate, and weak activity, respectively ,27,28. O50 lower than 10 μg/mL, between 10 and 50 μg/mL, between 50 and 100 μg/mL, and higher than 100 μg/mL are very active, active, moderately active, and inactive [L. amazonensis promastigote forms (IC50 = 7.20 ± 1.14 µg/mL) and active against the L. amazonensis amastigote forms (IC50 = 16.21 ± 1.02 µg/mL). In addition, CZ-EO is active against the T. cruzi trypomastigote form (IC50 = 11.2 µg/mL), with its IC50 resembling the IC50 obtained for benznidazole (IC50 = 9.9 µg/mL), used as a positive control. Concerning cytotoxicity, values of 50% cytotoxic concentration (CC50) lower than 10 μg/mL, between 10 and 100 μg/mL, between 100 and 1000 μg/mL, and higher than 1000 μg/mL are typical of highly toxic, toxic, moderately toxic, and non-toxic EOs [50 = 565.4 ± 1.0 μg/mL)  .C. zizanoides against the L. amazonensis promastigote form. Although the authors did not specify from which part of the plant the EO was obtained, they reported an IC50 of 19.0 ± 3.3 µg/mL and a CC50 = 31.7 ± 2.8 µg/mL, which denotes unspecific activity (SI = 2) [50, CC50, and SI values of 7.20 ± 1.14 µg/mL, 565.4 ± 1.0 µg/mL, and 78.5 for CZ-EO, respectively. These results indicate that CZ-EO is more active compared to the literature—not only does it display a considerably lower IC50 value, but it also has an almost 40 times higher selectivity index (SI). Even though these differences can be due to the distinct susceptibilities of the L. amazonensis strains and the cell lineages used in the antileishmanial and cytotoxic assays conducted herein, they might also be due to differences in the chemical composition of CZ-EO and the commercial EO investigated by Monzote and co-workers [C. zizanoides were not reported by the authors [C. zizanioides against the L. amazonensis promastigote form. The activity displayed by CZ-EO against the L. amazonensis amastigote form is also a noteworthy result because amastigotes are the parasite forms that persist in the infected host [Recently, Monzote and co-workers investigated the in vitro leishmanicidal activity of a commercially available sample of the EO from (SI = 2) . Here, w-workers . However authors . On the ted host .50 = 5.45 µg/mL) against Trypanosoma brucei brucei GUTat 3.1 and displays low cytotoxicity to MRC-5 cells  [Leishmania infantum (IC50 = 52.5 μg/mL) and Trypanosoma cruzi (IC50 > 100 μg/mL) [Guatteria friesiana and Guatteria pogonopus leaves, which are rich in β-eudesmol, display trypanocidal action against the T. cruzi bloodstream trypomastigote and epimastigote forms, with IC50 values of 11.9 and 28 μg/mL against epimastigotes and 10.7 μg/mL and 41.3 μg/mL against trypomastigotes, respectively [The biological activities of EOs can be due to the bioactivities of their components. β-eudesmol, one of the main compounds in CZ-EO, displays antitrypanosomal activity (ECSI > 18) . Bailen 0 μg/mL) . The EOsChrysopogon zizanioides (L.) Roberty (Poaceae) roots were collected in June 2021, in the municipality of Machado, State of Minas Gerais, Southeastern Brazil (21°41′56″ S and 45°52′59″ W). The plant material was identified by the botanist Walnir G. F. Júnior. A voucher specimen (GERAES-CZ01) was deposited at Herbário de Machado of the Departamento de Biologia, Instituto Federal de Educação, Ciência e Tecnologia do Sul de Minas Gerais, Brazil.C. zizanoioides roots (1500 g) were divided into three samples (500 g each) and accommodated into three 500 mL round bottom flasks. Then, 500 mL distilled water was added, and the flask was coupled to a Cleavenger-type apparatus. After hydrodistillation for 4 h, CZ-EO was obtained as a light-yellow oil in a 0.52 ± 0.04% yield (w/w). Next, the EO was dried over MgSO4 and stored at −4 °C until GC analyses and biological assays were performed.2 was used as the carrier gas at a flow rate of 1.0 mL/min. The equipment was set to operate in the injection mode; the injection volume was 0.1 µL (split ratio of 1:10); and the injector and detector temperatures were 240 and 280 °C, respectively. Relative concentrations of the CZ-EO compounds were estimated from the relative peak areas (%) of the GC-FID chromatograms, and expressed as the average of triplicate analyses and the corresponding standard deviations. GC-MS conditions and the identification were based on a previously reported methodology [8–C20). The structures were computer matched with Wiley 7, NIST 08, and FFNSC 1.2, and their fragmentation patterns were compared with literature data [CZ-EO was dissolved in ethyl ether and analyzed by gas chromatography flame- ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC–MS) on the Shimadzu QP5000 Plus and GCMS2010 Plus  systems, respectively. During GC-FID, the column temperature was programmed to rise from 60 to 240 °C at 3 °C/min and held at 240 °C for 5 min. Hhodology . The volure data .Porphyromonas gingivalis ATCC 33277; Aggregatibacter actinomycetemcomitans ATCC 43717, Prevotella intermedia ATCC 49046, Prevotella nigrescens ATCC 33563, Fusobacterium nucleatum ATCC 25586, and Prevotella melaninogenica ATCC 700524) and three clinical isolates  were used. The evaluated clinical isolates were obtained from clinical trials and kept in the Laboratory of Antimicrobial Testing library under cryopreservation at −20 °C. The Minimum Inhibitory Concentration (MIC) values were determined using the microdilution broth method in 96-well microplates  [2, 10% CO2, 85 or 80% N2 atmosphere. After that, resazurin  (30 µL) in an aqueous solution (0.02%) was added to the microplates to indicate microorganism viability [To test CZ-EO against periodontal pathogens, six standard strains from the American Type Culture Collection  (zerland) . To thisL. amazonensis (MHOM/BR/PH8) promastigote forms was evaluated according to the previously described methodology [6 parasites per well. A stock solution of CZ-EO dissolved in DMSO  (1 mg/mL) was added to the cultures to achieve final concentrations from 3.12 to 50 µg/mL. Amphotericin B  at concentrations of 0.19 to 0.011 µg/mL was used as the positive control. RPMI 1640 medium , and RPMI 1640 culture plus 0.1% DMSO (the highest non-toxic concentration) was used as the negative control. Cultures were incubated at 25 °C in BOD ovens (Quimis) for 24 h. The leishmanicidal activity was determined based on the growth inhibition of promastigote forms by counting the total number of alive promastigotes in the Neubauer chamber  considering their flagellar motility. Results were expressed as the mean of the lysis percentage relative to the negative control [The antileishmanial activity of CZ-EO against hodology . The ass control ,46. The L. (L.) amazonensis was assessed as described by Casa and co-workers, with modifications [5 cells/well and seeded onto a 96-well round-coverslip culture plate. They were incubated for 96 h at 37 °C in the presence of a 5% CO2 atmosphere. Adherent macrophages were infected with metacyclic promastigotes  at a concentration of 1 × 106 cells/well (10:1 ratio) for 4 h at 37 °C. Parasites that were not internalized in the macrophages were removed, and the infected cultures were incubated with different concentrations of CZ-EO (3.12 to 50 µg/mL) and Amphotericin B (0.011 to 0.19 μg/mL) for 48 h at 37 °C and 5% CO2. After incubation, the coverslips were washed, fixed with methanol, and stained with Giemsa solution (Synth). The slides were analyzed in an optical microscope  by counting 200 macrophages and which determined the number of amastigote forms within each infected cell. The EC50 was calculated in comparison to the negative control (RPMI 1640 medium containing 0.1% DMSO).The antileishmanial activity of CZ-EO against amastigote forms of ications . BrieflyTrypanosoma cruzi (Y strain) trypomastigote forms. This strain has been maintained at the University of Franca Vivarium through successive tests in Swiss mice by cardiac puncture on the day of the parasitemia peak (seventh day of infection). This procedure was approved by the National Council for Control of Animal Experimentation of the Ethics Committee at the University of Franca, under protocol number 010/14. The assay was performed with blood from infected albino mice by cardiac puncture at the parasitemia peak (seventh day of infection). The infected blood was diluted with a physiological solution to achieve a final blood concentration of 1 × 106 trypomastigote forms/mL. Aliquots of CZ-EO diluted in DMSO (1 mg/mL) were added to the infected blood on the microtiter plate (96 wells) to achieve a final volume of 200 μL containing CZ-EO at final concentrations from 12.5 to 200 µg/mL. Benznidazole  and 0.5% DMSO (the highest non-toxic concentration for this cell type) were used as the positive and negative controls, respectively. The microplate was incubated at 37 °C for 24 h. The activity was quantitatively verified by counting the trypomastigote forms as previously described in the literature [The in vitro trypanocidal assays were performed using terature . The par2 cells were counted in a Neubauer chamber and adjusted on a 96-well microplate to the concentration of 2 × 105 cells/mL. The cells were cultivated in a supplemented RPMI 1640 medium at 37 °C with 5% CO2 atmosphere for 24 h. Next, CZ-EO that had been previously dissolved in DMSO was added to the wells at concentrations ranging from 6.25 to 400 µg/mL, and the cells were cultivated in the same previously described conditions for 48 h. Afterward, the cells were washed, and 20 µL of a PBS  solution containing 7 mg of 3--2,5-diphenyltetrazolium bromide) (MTT)  was added to each well. Cell viability was determined by the colorimetric metabolic activity assay, which assesses the ability that metabolically active cells have to reduce MTT by converting their yellow salts into purple formazan crystals. The plates were incubated at 37 °C and in a 5% CO2 atmosphere for 4 h, and the formazan precipitate was solubilized with 100 μL of isopropyl alcohol . The absorbance was read at 550 nm with a spectrophotometer . The negative, solvent, and positive control groups consisted of LLCMK2 cells incubated with a RPMI 1640 medium containing 0.1% DMSO, and 10% DMSO, respectively. Experiments were performed in triplicate.The LLCMK50 (the inhibitory concentration that inhibited trypomastigote and promastigote viability or intracellular amastigote growth by 50%) and CC50 (the CZ-EO that was cytotoxic to 50% of the cells) values were calculated by a non-linear regression dose-response inhibition curve. The selective index (SI), which indicates the parasite toxicity as compared to the host, was calculated as the ratio between CC50 and IC50 [All the experiments were performed in triplicate and repeated at least two times. The ICand IC50 . Data weC. zizanioides roots (CZ-EO) is effective against Trypanosoma cruzi, Leishmania amazonensis, and periodontopathogenic bacteria in vitro. Moreover, it displays moderate cytotoxicity to LLCMK2 cells and a very interesting selective index (SI) with regard to the L. amazonensis promastigote and amastigote forms and the T. cruzi trypomastigote form. These results suggest that CZ-EO can be potentially used as a component of new oral care products, and can be further exploited for the development of new antileishmanial and antitrypanosomal drugs. To this end, further in vitro studies aiming to elucidate the mode of action of CZ-EO and to identify the compounds underlying its antileishmanial and antitrypanosomal activities are needed, and the possible synergistic, additive or even antagonistic effects among them must be investigated.The essential oil from


Dandelion is an herb with high nutritional and medicinal values, which has been listed in Chinese Pharmacopeia, European Pharmacopoeia and British Pharmacopoeia, gaining increasing acceptance around the world. However, the current quality control of dandelion is lagging behind. Only in Chinese Pharmacopeia, cichoric acid is used as a marker compound for its quality evaluation, whereas, it can not comprehensively reflect the bioactivity of dandelion.This study developed a strategy by integrating chemometrics with in silico pharmacology to reveal the bioactive markers of dandelion for its quality control. Firstly, the major chemicals in dandelion were characterized using HPLC-DAD-MS/MS, and the corresponding antioxidant and anti-inflammatory activities were evaluated in vitro. Subsequently, the active components were screened by relating the chemicals and bioactivity of dandelion via grey relational assay and partial least squares regression analysis. The potential active components were then subjected to a validation for their activities. Moreover, in silico pharmacology was utilized to evaluate the contribution of active components to efficacy.Taraxacum mongolicum Hand.-Mazz. (TAM) and T. officinale F. H. Wigg. (TAO). TAM, with acceptably higher content of cichoric acid and caffeic acid, showed better antioxidant activity than TAO. While TAO included higher content of luteolin, presenting slightly more effective in anti-inflammation.A total of 22 phenolic compounds were characterized. Among them, cichoric acid, caffeic acid and luteolin were identified as quality markers owing to their good correlations with the bioactivities of dandelion. These three markers were quantified in frequently-used dandelion species, viz. An useful strategy for the quality marker discovery was successfully designed. And the results provided more knowledge for the quality evaluation of dandelion.The online version contains supplementary material available at 10.1186/s13020-022-00679-4. Taraxacum mongolicum Hand.-Mazz. (TAM), T. officinale F. H. Wigg. (TAO) or other sibling plants of Compositae family − ion at m/z 609.1461 and generated a base peak at m/z 285.0374  corresponded to a substitute of disaccharide, thus, it was putatively identified as luteolin-7-O-β-d-gentiobioside  or electron transfer rate (FRAP). Gallic acid with good antioxidant activity was used as positive control drug. As shown in Fig. At first, the cytotoxicity of dandelion sample on RAW 264.7 cells was measured by CCK-8 assay. RAW 264.7 cells treated with dexamethasone were utilized as positive control. As shown in Fig. GRA was primarily used to assess the correlation of each phenolic component annotated in dandelion with antioxidant and anti-inflammatory activities. The GRD values of 22 phenolic compounds were over 0.9 (Table PLSR was utilized to construct the relationship between 22 phenolic compounds and corresponding activities for the screening of efficacy-related markers.2Y (cum) & Q2 (cum) in the PLSR models about DPPH and FRAP were 0.904 & 0.753 and 0.861 & 0.656, respectively, indicating a satisfactory explanation and prediction ability. Permutation tests verified the models not overfitted , 0.619 & Q2 (cum), 0.507). The same standards were applied to screen out the potential anti-inflammatory components. As shown in Table 8, 9, 13, 17, 18, 19, 20 and 21 were considered to be responsible for the anti-inflammatory activity of dandelion.Similarly, the PLSR model about anti-inflammatory activity was also constructed in Additional file O-caffeoylquinic acid, caffeic acid and caftaric acid presented good antioxidant activity and their IC50 values were 13.15 ± 0.52 µM, 14.17 ± 2.50 µM, 36.76 ± 0.85 µM, 19.63 ± 0.60 µM and 26.53 ± 0.40 µM, respectively. Take Trolox (or FeSO4) as a reference, the antioxidant capacities of the four compounds were signed as 6.46 ± 1.49 mmol Trolox/g (11.01 ± 1.49 mmol FeSO4/g), 3.93 ± 0.49 mmol Trolox/g (9.39 ± 0.87 mmol FeSO4/g), 2.54 ± 0.64 mmol Trolox/g (4.66 ± 1.20 mmol FeSO4/g), 8.25 ± 0.78 mmol Trolox/g (18.70 ± 3.96 mmol FeSO4/g)) and 3.94 ± 0.30 mmol Trolox/g (10.46 ± 1.27 mmol FeSO4/g).As shown in Fig. 50, 17.70 µM), which was comparable to that of apigenin  but far superior to kaempferol  and luteolin-7-O-β-d-glucoside . Comparing their structural characteristics, it could be deduced that the chromonyl group was the anti-inflammatory pharmacophore.As shown in Table O-β-d-glucoside presented a mild dose-dependent toxicity.Additionally, the cytotoxicity of the anti-inflammatory compounds was investigated , was applied to predict the pharmacokinetics parameters (GA and drug-likeness) of these candidate markers. Drug-likeness was evaluated by Lipinski, Ghose, Veber, Ega, and Muegge rules, which were developed by major pharmaceutical companies based on the structural or physicochemical inspections to exclude molecules with properties most probable incompatible with an acceptable pharmacokinetics profile [18, 20, 19, 5, 8 and 21 conformed to the above conditions. Thus, they were theoretically reasonable to be markers for the quality control of dandelion. Moreover, although the GA of peak 7 was low, it could be detected in vivo [7 was exceptionally involved in the quality markers.To be effective markers, the molecules should reach their targets in the body in sufficient concentration. SwissADME  were predicted based on their structures using SwissTargetPrediction web tool (http://www.swisstargetprediction.ch/). The obtained targets were annotated via DAVID and then a total of 49 targets related to oxidation and inflammation were discovered. The results were visualized in Fig. 5) and cichoric acid (peak 7) targeted ALOX5, CA3, FYN, MAPK1, APP, PTGS1 and AKR1B1, while the 5 anti-inflammatory markers targeted up to 47 unique genes. As shown in Fig. The targets of the above 7 markers  higher than 0.9994 in a in a relatively wide concentration range. The contents of three markers in the frequently used dandelion species of TAM and TAO were determined. As shown in Fig. A simultaneous quantitation method was developed based on HPLC-UV. Methodology investigation was performed , and base peak chromatogram (b). Figure S2. PLSRmodels. The summary of fit (a-c) and permutation tests (d-f) forantioxidative activity by DPPH and FRAP, and anti-inflammatory activity,respectively. Figure S3. The toxicity of luteolin (a), apigenin (b),kaempferol (c) and luteolin-7-O-β-d-glucoside (d) on RAW264.7 cells. Figure S4. Chromatogramsof Taraxacum extracts with different extraction solvents. 1caffeic acid, 2 cichoric acid, 3 p-coumaric acid, 4luteolin, 5 kaemferol, 6 apigenin, 7 eupalitin.


KRAS, NRAS and BRAF mutations in patients with colorectal cancer (CRC) in North Africa and to compare its prevalence with that shown in other populations and (ii) the possible role of dietary and lifestyle factors with CRC risk. Our review discuss (i) the findings from analyzed data that have examined KRAS, NRAS, and BRAF mutations in CRC patients from Morocco, Tunisia, Algeria and Lybia. Using electronic databases, a systematic literature search was performed for the KRAS, NRAS and BRAF were 46.4%, 3.2% and 3.5% of all patients, respectively. A broad range of reported KRAS mutation frequencies have been reported in North Africa countries. The KRAS mutation frequency was 23.9% to 51% in Morocco, 23.1% to 68.2% in Tunisia, 31.4% to 50% in Algeria, and 38.2% in Libya. The G12D was the most frequently identified KRAS exon 2 mutations (31.6%), followed by G12V (25.4%), G13D (15.5%), G12C (10.2%), G12A (6.9%), and G12S (6.4%). G12R, G13V, G13C and G13R are less than 5%. There are important differences among North Africa countries. In Morocco and Tunisia, there is a higher prevalence of G12D mutation in KRAS exon 2 (≈50%). The most frequently mutation type in KRAS exon 3 was Q61L (40%). A59T and Q61E mutations were also found. In KRAS exon 4, the most common mutation was A146T (50%), followed by K117N (33.3%), A146P (8.3%) and A146V (8.3%).Seventeen studies were identified through electronic searches with six studies conducted in Morocco, eight in Tunisia, two in Algeria, and one in Libya. A total of 1843 CRC patients were included 576 (31.3%) in Morocco, 641 (34.8%) in Tunisia, 592 (32.1%) in Algeria, and 34 (1.8%) in Libya. Overall, the average age of patients was 52.7 years old. Patients were predominantly male (56.6%). The mutation rates of KRAS mutated CRC patients in North Africa have been identified with incidence closer to the European figures. Beside established anti-CRC treatment, better understanding of the causality of CRC can be established by combining epidemiology and genetic/epigenetic on CRC etiology. This approach may be able to significantly reduce the burden of CRC in North Africa. Colorectal cancer (CRC) is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women. According to GLOBOCAN 2020 data, there are 1.15 million new cases of colon cancer, 0.7 million new cases of rectal cancers, and 50,000 new cases of anal cancer in 2020 globally . Over thStudies of CRC in the North of Africa  have shown rising trend in incidence rate. It was 7.1/100,000 . When coRAS/RAF/MEK/ERK/MAPK is the most well-known pathway in the pathogenesis of CRC. Mutations in genes of this pathway have been reported in about 50% of patients with CRC [RAS and BRAF are members of the RAS/RAF/MEK/ERK/MAPK pathway which mediates cellular response to growth signals. Both RAS and RAF are members of multi-gene families and there are three RAS members  and three RAF members  and ARAF). The KRAS proto-oncogene (locus 1p13.2) is the most commonly mutated RAS family member (75% of RAS mutations). The KRAS proto-oncogene encodes a protein (p21-ras) belonging to the family of GTP/GDP-binding proteins with GTPase activity and is involved in the transduction of mutagenic signals. KRAS gene mutations occur commonly in colon [NRAS proto-oncogene (locus 1p13.2) is also a member of the RAS gene family which encodes proteins involved in signal transmission in cells and participates in the regulation of cell growth. NRAS gene mutations is associated with of CRC tumors [BRAF gene on chromosome 7 (7q34) encodes for a serine/threonine protein kinase) that sends signals from outside of the cell to the nucleus that in turn drives the growth of a cell. BRAF is a downstream target of RAS, playing a pivotal role in the MAPK/ERK signaling pathway. Mutations of BRAF are found in CRCs [CRC is a complex and genetically heterogeneous disease with involving in oncogene and tumor suppressor genes, as well as genes involved in DNA damage recognition and repair. CRC includes also different categories of tumors based on their specific spectrum of mutations and molecular phenotype, driving various oncogenic signaling pathways . The RASwith CRC . RAS andin colon , 15. TheC tumors , 15. The in CRCs  and are  in CRCs .KRAS, NRAS and BRAF mutations in patients with CRC in North Africa and to compare its prevalence with that shown in other populations and (ii) the possible role of dietary and lifestyle factors with CRC risk.This systematic review aims to discuss (i) the findings from analyzed data that have examined KRAS, NRAS, or BRAF gene mutations in CRC patients in North Africa. Pub Med, Science Direct and Google Scholar were searched up to December 2021 for eligible studies using the following keywords: “colorectal cancer”, or “colon cancer”, or “rectum cancer”, or “RAS”, or “KRAS”, or “NRAS”, or “BRAF”, or “mutation”, or “oncogenic mutation”, or “oncogenic driver mutation”, or “activating mutation”, or “prevalence”, or “rate”, or “incidence”, or “frequency”.We included articles that described the prevalence of An additional literature search was also conducted using North Africa and specific country names belonging to the considered region and any other variant names for any of North Africa countries . We manually checked reference lists of the included studies and relevant reviews to identify additional studies. We also searched relevant abstracts reported in the most important multi-disciplinary societies of medical oncology such as the American Society of Clinical Oncology (ASCO) meetings to identify unpublished studies.KRAS, NRAS, and BRAF mutations in a specific or selected coding region of KRAS, NRAS genes  and BRAF (exon 15). Studies published as original articles on human subjects, evaluating KRAS, NRAS, and BRAF mutation status and recording clinico-pathological features in CRC patients, and written in English, were included for analysis.Eligible analytical study designs were prospective or retrospective cohort studies, cross-sectional, and case–control studies and were conducted in CRC patients. The study must give information on the prevalence of The following information data were abstracted from each of the eligible studies: 1) characteristics of the study ; 2) characteristics of study participants ; 3) characteristics of study outcomes ; 4) clinicopathological characteristics .Letters, comments, and review articles or meta-analysis without original data, were excluded from analysis.n = 6) [n = 8) [n = 2) [n = 1) [The search of the databases yielded 17 relevant references which are closely related to defining the inclusion criteria and was included in this review. The retrieved articles describe studies conducted in Morocco (n = 6) –23, Tuni [n = 8) –31, Alge [n = 2) , 33, and [n = 1) . A total [n = 1) –23, 641  [n = 1) –31, 592  [n = 1) , 33, and [n = 1) . The ave [n = 1) , 31–33,  [n = 1) , 29, 34 From the specimens where tumor site information was available, 31.4% 344/1095) of them were in the rectum , 31–34, 4/1095 ofKRAS, NRAS and BRAF mutation analysis. Both exon 2, 3, and 4 mutations of KRAS and NRAS genes were analyzed in 23.5% (4/17) [KRAS gene were evaluated in one study  [KRAS exons (exon 2 and/or exon3) were assessed in 58.8% (10/17) [NRAS exons (exon 2 and 3) in 11.8% (2/17) investigations [KRAS mutations in exon 2 codon 12 and 13 have been assessed in the most of the studies  [BRAF gene mutation was analyzed in 41.2% (7/17) studies [Among the 17 studies, 13 (76.5%) , 30–33 u% (4/17) , 25, 32  (10/17) , 26–30 aigations , 24. KRA, 16/17) . BRAF ge studies , 33.KRAS, NRAS and BRAF mutations screening. Sequencing assay was broadly used, as it was used in 64.70% (11/17) studies [KRAS, NRAS and BRAF genes in North Africa are presented in Table Different molecular methods were used for  studies , 26–34.  studies , 20, 25, studies , 22, amp studies , Allele- studies , End-poi studies  and Dena studies . The detKRAS, NRAS and BRAF mutations has been reported in 16 [KRAS mutations were most frequently detected among North Africa patients with CRC, accounting for 41.7% (749/1795) [KRAS mutations rates, ranging from 23.1% (12/52) [KRAS Mutations were distributed among the different exons as follows: 95.9% (718/749) exon 2 [The prevalence of ed in 16 , 32–34, ed in 16 , 25, 32,ed in 16 , 31, 33 49/1795) , 32–34.  (12/52)  to 68.2%: 95.9% 7/749 exon) exon 2 , 32–34, : 95.9% 7/749 exon) exon 2 , 25, 32,: 95.9% 7/749 exon) exon 2 , 25 (Tab: 95.9% 7/749 exon) exon 2 , 27, 34 : 95.9% 7/749 exon) exon 2 , 27, 34 As seen in Table NRAS total mutations were identified in 3.2% (35/1090) [NRAS mutations rates, ranging from 2% (1/51) to 5.3% (6/114) [NRAS mutations has been reported in Tunisia  [NRAS gene was located in exons 2 and 3, with 28.6% (10/35) [NRAS mutations were more common in codon 61, accounting for 48.6% (17/35)[35/1090) , 25, 32  6/114) , 20. A h%, 7/96) . Overall (10/35) , 24, 25  , 20. A % (17/35), 25 and % (17/35), 20, 25 BRAF gene was 2.8% (17/602) [BRAF mutations was found in Tunisia and reported in 8.3% (4/48) of patients with CRC [BRAF V600E mutation was 2.8% (17/602) [The total mutation frequency in the (17/602) , 31, 33.(17/602) , 31, 33 RAS/RAF/MEK/ERK/MAPK pathway has been used to predict the outcomes of EGFR-targeted therapy for CRC. KRAS mutations were found in ≈33% of in the COSMIC dataset comprising 75,000 tested specimens samples. In other datasets based on small sample sizes (< 500), KRAS was found mutated in ≈40%-45% CRCs. Notably however, the private Foundation Medicine dataset comprising 13,336 colorectal samples reports a KRAS mutation frequency of ≈50% [http://cancer.sanger.ac.uk/cosmic), mutations at codon 12  are the most prominent (> 90%), followed by those affecting codon 13 , codon 61 , codon 146 , and codon 117 (K117N). NRAS mutations are found in 5%-10% of CRC. NRAS is mutated in the same codon of KRAS particularly in exon 2 (3%-5%) and exon 3 (2%-6%). BRAF mutations are identified in about 8%-12% of CRC patients and 90% of all identified mutations are a T1799A transversion in exon 15, which results in a valine amino acid substitution (V600E) [CRC is a pathologically and clinically heterogeneous malignancy. Phenotypic and molecular characteristics of CRC represents a significant key step in defining diagnosis, prognosis and treatment predictive value both in localized and in the setting of CRC . Mutatio (V600E) .KRAS mutation was detected in the CRC tumor of 41.7% of the patients among whom 95.9% had a single mutation at exon 2, 2.7% at exon 3, and 2.9% at exon 4. Among mutations in KRAS exon 2, 79.7% of cases had single mutations in codon 12 and 20.3% of cases in codon 13. The G12D was the most frequently identified exon 2 mutations (31.6%), followed by G12V, G13D, G12C, G12A, and G12S. The most frequently mutation type in exon 3 was Q61L (40%). In exon 4, the most common mutation was A146T, K117N, A146P and A146V. NRAS total mutations were identified in 3.2% tumor samples. The common mutation site of NRAS gene was located in exons 2 and 3, with 28.6% and 48.6% of CRC patient. NRAS mutations were more common in codon 61, accounting for 48.6%. The most common mutations were G12D in exon 2 and Q61K in exon 3 and accounted for 40% and 35.3% of CRC patients, respectively. In BRAF gene, the overall frequency of the BRAF V600E mutation was 2.8%.In our review, RAS (KRAS and NRAS) and BRAF mutations. Interestingly, our finding showed that the KRAS mutation rate was different with more investigation that therefore the geographic location and ethnicity/race can impact on the prevalence of KRAS mutation in CRC patients. The KRAS mutation frequency was 23.9% to 51% in a Moroccan population [KRAS mutation frequencies may be related with diverse ethnic background. Arabs and Berbers make up the overwhelming majority of the population throughout the North Africa today. However, throughout its history, North Africa has been the site of invasions and migratory waves of ethnic groups such as Phoenicians, Romans, Vandals, Arabs, Ottomans and Europeans.Our review of mutation prevalence among CRC patients in North Africa identified a significant difference in prevalence of both pulation –23, 23.1pulation –30, 31.4pulation , 33, andpulation . This brKRAS mutation frequency between race/ethnic groups. In Nigerian, the frequency of CRC with KRAS mutations is 21% [KRAS mutations vary from 37.9% (Japan) to 52.7% (China) [KRAS mutation rate slightly higher among African American compared to Caucasians (47% versus 43%) [KRAS mutation prevalence ranging from 13% (Colombia and Venezuela) to 40% (Chile) [KRAS mutation gene has been reported to be in the range of 11% to 49.1% [KRAS mutation prevalence may also vary in the same population. Studies from Japan with homogeneous ethnic groups found a wide range of KRAS mutation frequencies (9–71%) [KRAS mutation (14–83%) [KRAS and BRAF gene mutations in cancerous tissue obtained from Saudi CRC patients were typical of those observed elsewhere, and may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences in Saudi Arabia [Worldwide, a persistent finding in the literature is the substantial variation in s is 21% . In Asia (China) , 40, whi (China) , 42. Dat (Chile) . The Eur (Chile) , 46. In to 49.1% . Turkey to 49.1% . The KRA (9–71%) . Studies(14–83%) , 55. Thei Arabia , 57.KRAS mutation in North Africa. However, there are important differences among North Africa populations. In Morocco and Tunisia there is a higher prevalence of G12D mutation (≈50%) [KRAS mutations are predominantly found in codon G12 and mutations in codon 12 diminish both inherent and GAP-mediated hydrolysis without affecting the rate of nucleotide exchange, except for G12C, which exhibits GTPase activity similar to that of wild type [KRAS-mutant CRC [KRAS testing. A series of novel inhibitors that act against G12C in its GDP-bound state have been developed (such as Sotorasib and Adagrasib) and provides new therapeutic strategies to improve patient outcomes.In agreement with COSMIC, G12D and G12V mutation frequencies were the most frequent mutations among all CRC patients with n (≈50%) , 27. G12n (≈50%) , 59. Genild type . G12C hatant CRC , adding KRAS gene mutations in CRC in North Africa populations are the change in dietary patterns and nutrient intakes. In Morocco, The major pattern of nutritional change includes a large increase in the consumption of high calorie diets and fatty foods. Consumption of high amounts of these foods was associated with increased body weight, BMI, and risk of overweight and obesity. Between 2001 and 2014, the consumption of high-fat diets increased following the intake of oils which increased on average by 5.4 L (22.4 L against 17.02 L per person per year) [KRAS mutations [KRAS oncogene in Moroccan population. El Asri et al., showed that an increase in the consumption of PUFA above 16.9 g/day was associated with an increase in the presence of KRAS mutations  as compared to the reference group whose consumption was less than 16.9 g [KRAS mutated colon tumors, possibly by generating G > A transitions or G > T or G > C transversions in the KRAS oncogene [al., showed that consumption of red meat was positively associated with colon cancer  and CRC risk in Moroccan patients  [The aspects that seem to play an important role in the incidence of er year) . Signifier year) . High leutations . Polyunsn 16.9 g . A high oncogene . S Deoul02–1.27) . In Tuni02–1.27)  and 80% 02–1.27) . A high 02–1.27) . The gro02–1.27) . The Wor02–1.27) . A recen02–1.27) . The Wor02–1.27) .KRAS mutations [KRAS G12D/G13D [Red meat consumption may increase colon cancer risk by inducing the endogenous production of N-nitroso compounds and their precursors, which may induce utations , 71. Ana12D/G13D . Heteroc12D/G13D , 74. HCA12D/G13D , 76.KRAS-specific context, the role of KRAS signaling on the tumor microbiome is still to be determined, even though, it is accepted that the exposure to bacterial can shape the development of CRC of which KRAS can be one major player [KRAS-driven CRCs, it has been shown that genotoxic stress and some other factors, including metabolites produced by the microbiota, can facilitate genetic and epigenetic changes leading to carcinogenesis [Interestingly, the use of antibiotics such as Fluoroquinolone and the third-generation cephalosporins was increasing in North Africa . An incrr player , 87. Nevogenesis . A priorogenesis . MicrobiModifiable dietary and lifestyle risk factors could prevent most cases of CRC and. Up to 47% of CRC cases could be prevented by staying physically active, maintaining a healthy body weight and eating a healthy diet . In NortBeside established anti-CRC treatment, better understanding of the causality of CRC can be established by combining epidemiology and genetic/epigenetic on CRC etiology. This approach may be able to significantly reduce the burden of disease in North Africa population. Furthermore, the government should develop policy on CRC prevention and public health programs which may serve as a feasible setting to increase public awareness on lifestyle risk factors.


Consequently, enzymes combination can significantly improve the quality of oat bran CSB whereas reduce the nutritional value of oat bran CSB.The present study investigates the individual and interactional effects of α-amylase (6 and 10 ppm), xylanase (70 and 120 ppm) and cellulase (35 and 60 ppm) on the physicochemical characteristics and nutritional quality of Chinese steamed bread (CSB) incorporated with 15% oat bran. As a result, the single enzyme can significantly improve the specific volume and texture of CSB. Compared to the single enzyme, the combined enzymes improved the specific volume of CSB up to the highest value (2.51 mL/g) and decreased the hardness to the minimum value (233.61 g) when the concentration was 6, 70 and 35 ppm. With respect to chemical and nutritional properties, the addition of single enzyme had no great changes, while the combined enzymes  significantly ( In recent years, consumers have developed a growing awareness surronding the link between diet and nutrition, and thus there has been an increased demand for healthier products with a consequent rise in interest in functional and nutritional items by the food industry . As a fuTo improve the quality of CSB incorporated with 15% oat bran, enzymes were used as individual and combination. A previous study by Liu et al.  reportedThus, the aim of this study was to investigate the effect of α-amylase, xylanase and cellulase on the physicochemical and nutritional properties of CSB incorporated with 15% oat bran.Wheat flour , oat bran , yeast powder and salt  were purchased at a local supermarket. Megazyme Dietary Fibre and Total Starch analysis kits were purchased from Novozymes Australia Pty Ltd. .Three commercial enzymes were used: Fungamyl 2500 SG (2–10 ppm), Pentopan Mono BG (20–120 ppm) and Cellulast BG (10–60 ppm) .Firstly, the effect of single enzyme use on the quality of CSB incorporated with 15% of oat bran was analysed by analysis of variance (ANOVA). According to the manufacture recommendations and previous research , the dos3 design of experiments was used to investigate the effect of enzymes combinations on the quality of CSB incorporated with 15% of oat bran. Generally, there are three factors  at two levels  resulting in 8 different combinations of experiments and the coded values per each level of each factor are presented in i and βij), the theoretical response function (W) was calculated as following linear regression model:Secondly, a full factorial 2Factors: A—α-amylase; B—xylanase; C—cellulase; AB—α-amylase*xylanase; AC—α-amylase*cellulase; BC—xylanase*cellulase; ABC—α-amylase*xylanase*cellulase.0—The global mean; βi—The regression coefficient corresponding to main factor; βij and β—ijkThe regression coefficient corresponding to the interactions.W—The theoretical response variable; βThis multiple linear regression model with three independent variables describes the bread quality is related to the α-amylase, xylanase and cellulase.w/w based on wheat flour dry weight). The dough was formed by using stand mixer  for 5 min and kneading by hand for 5 min, then it was rested at 28 °C for 5 min. After that, the dough was kneaded for a further 8 min before fermentation in an incubator at 30 °C for 30 min. After fermentation, the dough was rolled out, and allowed to rise at 30 °C for 25 min. Finally, the dough pieces were placed in a Convotherm mini easyTouch oven  and steamed for 20 min. Steamed bread loaves were cooled to room temperature and then analysed.Chinese steamed bread loaves were produced using the formulation of Liu et al. . The recMoisture content of steamed bread was determined by an oven drying method (105 ± 2 °C overnight) described by the AACC International Approved Method 44-16.01 . AnalysiThe volume of steamed bread loaves was measured using the rapeseed displacement method, following the AACC International Approved Method 10-05. 01 . The meaSpecific volume of steamed bread was calculated by dividing loaf volume by loaf weight, according to the AACC International Approved Method 10-05. 01 .The texture properties of steamed bread were determined using TA-XT2 Texture Analyser  equipped with a 25 mm diameter cylinder probe . Steamed2), the overall mean cell area (mm2), the size of cell (mm).Image analysis was carried out following the method described by Dewaest et al.  with somThe determination of total starch in the steamed bread was carried out by the Megazyme Total Starch analysis kit following the AACC standard method.The determination of total (TDF), soluble (SDF) and insoluble (IDF) dietary fibre in the steamed bread incorporated with 15% oat bran was performed by the Megazyme Dietary Fibre analysis kit following the AACC standard method.In vitro Method Analysis: An in vitro glycaemic measurement as described by Brennan et al. . Milled p < 0.05.All data were treated by ANOVA and multiple regression analysis using Minitab 17 statistical software, version 17. 2. 1  at a significance level of p < 0.05) improved the physical properties of CSB. For example, the volume, height, moisture, cohesiveness, springiness and cell size of oat bran CSB increased when adding 10 ppm α-amylase. Additionally, the addition of 10 ppm α-amylase resulted in a reduction of hardness, gumminess, chewiness and cell density. Similar results were observed by Barrera et al. [Individual effect of α-amylase, xylanase and cellulase on physicochemical properties of CSB enriched with 15% oat bran are shown in a et al. , who repa et al.  also indp < 0.05) decreased hardness, gumminess, chewiness, springiness and cell density of oat bran CSB. Xue et al. [In terms of xylanase, oat bran supplemented with 70 ppm xylanase had higher value of specific volume, loaf height, moisture cohesiveness, springiness and cell size compared to the control. Moreover, xylanase addition significantly W (Specific volume) = 2.32 − 0.09A − 0.04B − 0.04C + 0.01AB + 0.01AC + 0.01BC + 0.02ABC (R2 = 0.90)W (Loaf height) = 58.22 − 2.02A − 0.33B − 1.01C + 0.16AC (R2 = 0.99)W (Moisture) = 46.85 + 0.53A + 0.24B + 0.66C − 1.89AB + 0.43AC + 0.36BC − 0.46ABC (R2 = 0.98)W (Hardness) = 313.81 + 37.20A + 21.36B − 3.45AB − 9.99AC − 24.67BC − 17.25ABC (R2 = 0.98)W (Chewiness) = 311.19 + 29.63A + 7.92B + 17.61C − 7.44AB − 11.53BC − 4.09BC − 24.21ABC (R2 = 0.92)W (Cells) = 48.92 + 3.83A + 1.46B − 1.96C + 4.29AB − 3.66BC (R2 = 0.97)W (Cell size) = 0.72 − 0.08A − 0.04B − 0.05AB + 0.03AC + 0.08BC − 0.03ABC (R2 = 0.85)W (Cell area) = 21.72 − 0.58A − 0.47B − 0.33C + 0.37AC + 0.22BC (RFactors:A—α-amylase; B—xylanase; C—cellulase; AB—α-amylase*xylanase; AC—α-amylase*cellulase; BC—xylanase*cellulase; ABC—α-amylase*xylanase*cellulase.According to In terms of texture of oat bran CSB, the combination of α-amylase, xylanase and cellulase significantly improved the texture of CSB. As a result, there was a significant decrease in hardness and increase in springiness, cohesiveness and chewiness. However, the interaction of α-amylase, xylanase and cellulase showed a negative synergistic effect on hardness of CSB. Altuna et al.  illustra2. However, there was an increase in cell size and cell area. The interaction of xylanase and cellulase shows a negative synergistic effect on cell density and a positive synergistic effect on cell size and cell density. It can be seen from The crumb structure of oat bran CSB was significantly influenced when enzymes combinations were added. With respect to cell density, the addition of α-amylase, xylanase and cellulase combination decreased cell density from 80.50 to 45.33 cells/cmIn this study, we investigated the individual and combined effects of α-amylase, xylanase and cellulase on the physicochemical properties of CSB substituted with 15% oat bran. Compared to the single enzyme, the combined enzymes increased the specific volume and cell size to higher value and decreased hardness to lower value due to the synergistic effect of enzymes.


SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel that drives the electrical excitability of cardiac myocytes and contributes to slow waves of the human gastrointestinal smooth muscle cells. NaV1.5 is mechanosensitive: mechanical force modulates several facets of NaV1.5’s voltage-gated function, and some NaV1.5 channelopathies are associated with abnormal NaV1.5 mechanosensitivity (MS). A class of membrane-active drugs, known as amphiphiles, therapeutically target NaV1.5’s voltage-gated function and produce off-target effects including alteration of MS. Amphiphiles may provide a novel option for therapeutic modulation of NaV1.5’s mechanosensitive operation. To more selectively target NaV1.5 MS, we searched for a membrane-partitioning amphipathic agent that would inhibit MS with minimal closed-state inhibition of voltage-gated currents. Among the amphiphiles tested, we selected capsaicin for further study. We used two methods to assess the effects of capsaicin on NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macro-patch and whole-cell Na+ current parameters with and without force. Capsaicin abolished the pressure- and shear-mediated peak current increase and acceleration; and the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of NaV1.5 MS makes capsaicin a promising candidate for therapeutic interventions targeting MS. SCN5A-encoded voltage-gated sodium channel, NaV1.5, is an ion channel gated by the electrical transmembrane potential present in excitable cells [V1.5 is also mechanosensitive: mechanical force modulates NaV1.5's voltage-dependent operation [V1.5 is responsible for the action potential upstroke in cardiac myocytes [V1.5 channelopathies with abnormal MS are found in human cardiac and GI diseases [SCN5A mutations responsible for cardiac arrhythmias result in impaired stretch modulation [V1.5 are found in patients with irritable bowel syndrome (IBS) [SCN5A mutations have relatively unchanged voltage-dependent gating but a loss of MS [V1.5 MS dysfunction are poorly studied, and pharmacological treatments targeting MS remain unexplored [The le cells . In addiperation . This memyocytes ; and elemyocytes . NaV1.5 diseases . Some SCdulation ,17; otheme (IBS) ,18. Someexplored ,18,19.V1.5's voltage-dependent function by increasing whole-cell conductance, shifting the voltage-dependence to hyperpolarized potentials, and accelerating kinetics [V1.5 and other mechanosensitive channels detect mechanical stimuli through lipid-bilayer (membrane) tension and cytoskeletal deformation [Ion channels are prime pharmacological targets because they are involved in many diseases and being embedded in the cells’ plasma membrane—are highly accessible ,21. In pkinetics . NaV1.5 ormation . A classormation ,27. The ormation .V1.5's voltage-dependent function and MS [V1.5 late current and MS in primary human GI SMCs, in addition to stretch-dependent function in GI smooth muscle [V1.5 and other channels.Interestingly, some amphipathic drugs modulate both Nan and MS ,26,29. Rn and MS . Ranolazh muscle ,26. Thesh muscle  and helph muscle ,30. Otheh muscle ; and theh muscle ; also alV1.5's voltage-dependent opening is usually undesired. Yet, selectively targeting NaV1.5 MS while sparing voltage-gated activation could have novel therapeutic applications: this would allow mechano-electrical feedback modulation without direct inhibition of electrical activity. Amphiphilic drugs, including some antiarrhythmics, alter the membrane bilayer within the therapeutic range, and their efficacy has been correlated with membrane-modifying capacity [V1.5's voltage activation. Among the candidates, capsaicin shows promise; accordingly, we characterized its ability to modulate NaV1.5 MS.Inhibiting Nacapacity ,33. Nonscapacity . TherefoSCN5A variant, Q1077del NaV1.5 [V1.5 in the heart [SCN5A was co-transfected with pEGFP-C1 into HEK-293 cells using Lipofectamine 3000 .We used the wild-type ow): amiodarone [V’s [The amphiphiles chosen were readily partitionable based on the octanol-water partition coefficients (logPiodarone  (7.2 µM)iodarone , Triton iodarone  (4.6 µM)iodarone   from KG12 glass to a resistance of 2–5 MΩ. For cell-attached patch experiments, electrodes were pulled from 8250 glass  then fire-polished to wide-bore, bullet-shaped tips with a final resistance of 1–2 MΩ. Electrodes were coated with R6101 elastomer  and then cured with a heat gun to reduce capacitive transients.Whole-cell and cell-attached patch data from HEK-293 cells were recorded at 20 kHz with an Axopatch 200B patch-clamp amplifier, Digidata 1550, and pClamp11 software .2, 10 HEPES, and 5.5 glucose; with an osmolality of 290 mmol/kg. GdCl3 (10 µM) was included in the pipette solution to inhibit endogenous stretch-activated channels [2, 10 HEPES, and 5.5 glucose; with an osmolality of 305 mmol/kg. Both solutions were made to a pH of 7.35. Where applicable, capsaicin was diluted 1000-fold in bath solution from a 20 mM ethanol stock then added to the recording chamber. Seal pressures were digitally controlled and monitored by High-Speed Pressure Clamp . Suction ≤10 mmHg was applied to establish giga-seals.The pipette solution contained (in mM): 149 NaCl, 4.7 KCl, 2.5 CaClchannels . The bat+ currents in macroscopic patches were elicited by an identical pair of voltage ladders with 31-ms pressure steps up to −50 mmHg encompassing the second voltage ladder. Patches were held at +100 mV, stepped briefly for 10 ms to +190 mV to close NaV channels, then stepped through a 10-step voltage ladder from +100 to 0 mV in 21-ms long, 10-mV increments with a total duration of 280 ms per sweep. Recordings were an average of 5 runs. Capsaicin (20 µM) was added to the chamber 5 min before testing the effects of the drug.NaV1.5 from inactivation, cells were held at 120 mV and stepped to (1) 20 mV for 30 ms, next to (2) 120 mV for a variable duration to recover, then to (2) 20 mV for 30 ms. The time between the beginning of each sweep was 5 s. The duration of the recovery time in stage (2) was varied between 1 and 300 ms in half-log unit increments. The pressure step per sweep was 400 ms regardless of recovery time.To test the effect of pressure on the recovery of NaV1.5 inactivation, cells were held at 120 mV and stepped 20 times to 20 mV, with the frequency between steps varying between 33.33 and 3.33 Hz. The duration of the pressure step per sweep was 30 ms.To test the effect of pressure on the onset (use dependence) of Na3SO3, 20 CsCl, 5 NaCl, 5 MgCl2, 10 HEPES, and 2 EGTA; with an osmolality of 290 mmol/kg; and pH of 7.0. The extracellular solution contained (in mM): 140 CsCl, 15 NaCl, 5 KCl, 2.5 CaCl2, 10 HEPES and 5.5 glucose; with an osmolality of 300 mmol/kg; and pH of 7.35.The intracellular solution contained (in mM): 125 CsCH+ current density, cells transfected with NaV1.5 were held at −120 mV then stepped through a 2-stage, 19-step voltage ladder (1) from −110 to −30 mV in 5 mV intervals for 2.9 s each and (2) to −30 mV for 100 ms. The time from the start of each sweep to the next was 5 s. Peak currents at each voltage step were normalized to the cell capacitance (pF) to quantify current densities  −30 mV for 100 ms, next to (2) −130 mV for a variable duration to recover, then to (2) −30 mV for 100 ms. The time between each sweep start was 2.5 s. The length of the recovery time in stage (2) of sweep V1.5 inactivation, cells were held at −130 mV and depolarized 10 times to −40 mV, in which the frequency of steps recorded ranged between 0.3 and 50 Hz. Mechanical stimulation by shear stress. When testing the effect of shear stress, the extracellular (bath) solution was perfused by gravity drip (at 10 mL/min) for the duration of the voltage protocol.To measure the onset of Na+ current and voltage dependence of activation were determined by fitting the NaV1.5 current-voltage (I–V) plots with MAX*(V-EREV)/(1 + e(V–V1/2A)/δV)I = G, where MAXG is the maximum Na+ conductance in whole cells (MAXI substitutes MAXG for the maximum Na+ current in patches), V is the voltage, REVE is the reversal potential, 1/2AV is the voltage of half-maximal activation, and δV is the slope. Conductance measurements were performed for the whole-cell configuration. Activation kinetics were determined by fitting currents with a two-term weighted exponential function: 1e(-t/τA)+A2e(-t/τI)+CI(t) = A, where Aτ and Iτ are the time constants of activation and inactivation, respectively, and 1,A2A, and C are constants. Steady-state inactivation was obtained by fitting remaining peak Na+ currents with a 3-parameter sigmoid curve: ((V–V1/2I)/δVI))I = 1/, and IδV the slope. For graphing, each IV curve was normalized to the peak effect without mechanical stimulation to demonstrate the increases in peak current with mechanical stimulation. To calculate recovery from inactivation, peak Na+ currents were fit with the equation: 0 = 1/(1 + t/t1/2)bI/I, in which 0I/I is the ratio of Na+ current recovered following inactivation from the control current, b is the rate of inactivation recovery, t is time, and 1/2t is the midpoint in which half of the Na+ current has recovered from inactivation. To calculate the plateau for use-dependent inactivation, peak Na+ currents of successive pulses were fit with the 3-parameter exponential decay equation: 10/I1 = Iƒeb/(t+c)I, in which 10/I1I is the peak Na+ current of step 10 normalized to the peak of step 1, and ƒI is the maximally inactivated peak Na+ current at frequency ƒ, t is time, and b or c is the rate or constant of use-dependent inhibition, respectively. To determine the voltage-step frequency at which peak Na+ currents were inhibited by 50% , ƒI was plotted vs. step frequency ƒ and fit with ƒ = (1-a)/(1 + e(ƒ1/2-ƒ)/δV)I, where a is the limit of use-dependent inhibition, 1/2ƒ the half-frequency of use-dependent inhibition, ƒ the frequency and δV the slope. Data are expressed as the mean ± standard error of the mean (SEM). Significance was assigned when P < 0.05 by a 2-way ANOVA and Tukey posttest when comparing force to rest or capsaicin to drug-free.The maximum peak NaV1.5 MS [−9 to 10−4 M) for its ability to inhibit peak voltage-gated Na+ currents  similar to capsaicin, was an 8-fold more potent NaV1.5 inhibitor than the latter, indicating that logPOW is not a good predictor of the drug’s effect on NaV1.5 voltage-gated function, consistent with the previous literature [50 8.4 µM) was comparable to the two most potent current inhibitors: Triton X-100 and propranolol . Overall, capsaicin, compared against the other amphiphiles tested, was an order of magnitude less potent for current inhibition  for further investigation, as this dose inhibited voltage-dependent Na+ current by ≤25%  by 16.6 ± 2.4%  by 16.0 ± 3.1% in whole cells , and both pressure (+4.8 ± 3.0%) and shear sensitivity  were lost .To test the effect of capsaicin on Namulation : (1) themulation ,44–47. Tmulation ,26,48, w+ current activation, decreasing the activation constant (τACT) by 20.0 ± 5.3% or 20.4 ± 3.3%, respectively  in whole cells but not in patches, and it inhibited the acceleration of activation induced by pressure and shear, as τACT did not accelerate with pressure or shear , and shear stress induced a smaller but significant shift of −1.5 ± 0.6 mV in the V1/2A  in whole cells. With force, capsaicin inhibited the shear-induced shift in the V1/2A  but not the pressure-induced shift . Similar to shear-induced shifts in the V1/2A, pressure or shear shifted the voltage dependence of inactivation or availability (V1/2I) in the absence of capsaicin , though the technical limitations of holding the voltage to more negative potentials prevented us from reaching the plateau in whole-cell experiments , suggesting loss of the MS of NaV1.5 inactivation. Overall, our results show that capsaicin inhibited the mechanosensitive shifts in NaV1.5 voltage-gating.Pressure ,21,26,48eriments . Withoutobserved . In the V1.5 from fast inactivation [+ currents recovered within ~100 ms in either configuration (V1.5 inactivation recovery (t1/2R) at rest was 13.2 ± 2.5 ms in the patch and 18.8 ± 1.7 ms in whole-cell , whereas pressure delayed the t1/2R  . With capsaicin present, pressure increased the t1/2R by 19.2 ± 8.2 ms (P < 0.05 to drug with no pressure), whereas shear reduced the t1/2R in whole cells by 9.5 ± 0.9 ms (P < 0.05 to drug with no shear). In all, the recovery from inactivation was delayed by capsaicin across both approaches. In the presence of capsaicin, pressure further delayed recovery in pressurized patches, but shear accelerated recovery in whole cells.Both capsaicin and pressure delay the recovery of Nativation ,48. Therguration ; the halole-cell . In addi30 mmHg) . In wholV1.5 through use-dependent inhibition [V1.5 in the absence of capsaicin and reexamined use-dependent inactivation in the presence of capsaicin  of 22.3 ± 2.6 Hz in patches  in the whole-cell configuration. In the presence of capsaicin, shear produced a modest decrease in the maximum use-dependent inhibition , and ƒ1/2 was unaffected, suggesting that shear partially reverses the use-dependent inhibition of NaV1.5 promoted by capsaicin. In patches, capsaicin affected neither the use-dependent inhibition nor ƒ1/2 at rest  but increased the pressure-sensitivity . Together, our results suggest that, though capsaicin enhances use-dependent inhibition, its effect on force-dependent changes to NaV1.5 use dependence may be specific to the type of force applied.Capsaicin can stabilize the inactivated state of Nahibition . Therefoapsaicin . To meas patches  and 64.6le cells . In the V1.5 serves as a key electrically excitable component in both the cardiac action potential and GI smooth muscle contraction [V1.5 is mechanosensitive [V1.5 MS [The MS of voltage-gated ion channels contributes to a mechano-electrical feedback system that has important implications in organs with primarily mechanical functions ,48,49. Etraction ,50. Besiensitive ; this pr[V1.5 MS ,21,51 anV1.5, and whether they do so by a mechanism separate from voltage-dependent current inhibition remain critical points for understanding amphiphile-mediated effects [V1.5's peak current, and at lower concentrations, inhibits MS [How membrane-permeable amphipathic drugs alter the MS of voltage-gated channels, like Na effects ,26,29. Fibits MS ,26. Suppibits MS ; while tV1.5 MS without significant inhibition of the voltage-gated channel opening. In principle, this would allow selective targeting NaV1.5 MS in conditions where it is abnormal. We selected amphiphiles with high partition coefficients and tested each candidate’s inhibition of peak voltage-gated currents in NaV1.5. Our choices were motivated by these molecules’ therapeutic use and ability to alter membrane stiffness, as demonstrated with the gA channel assay [V1.5's voltage-gated function. Our results, interpreted together with earlier studies with membrane-impermeable quaternary ammonium amphiphilic local anesthetics [ow does not determine the level of NaV1.5 current inhibition.Amphiphiles often have significant effects on both voltage-gating and MS . Thus, wel assay ,38,40–42sthetics ,53, sugg+ channel. Using the KcsA channel as a model for the voltage-gated ion channel [The class of therapeutic amphiphiles, local anesthetics  have bee channel , amphiphV1.5 MS in a manner comparable to other amphipaths—such as lidocaine [V1.5 in membrane patches and whole cells, respectively, by inhibiting the: (1) mechanosensitive increases in Na+ current, (2) shifts in steady-state voltage-dependence, and (3) acceleration of NaV1.5 gating kinetics.Both amiodarone and propranolol are antiarrhythmics, yet amiodarone exerts membrane-perturbing effects within the therapeutic range of use while propranolol-induced perturbations occur above therapeutic concentrations . This maidocaine ,26, and idocaine —yet withV1.5 MS using both whole-cell and macroscopic patch modes in parallel [V1.5 MS in the absence or presence of a drug in detail. As with other amphiphiles studied [1/2A and V1/2I to hyperpolarized potentials [V1.5's mechanosensitive responses and capsaicin’s effects on NaV1.5 MS were similar across techniques. Both produced an increase in peak Na+ current, shifts in the V1/2A and V1/2I, and an acceleration in τA. Capsaicin in both approaches inhibited most of these changes. These effects are important in the context of MS channel function—mechanical strain leads to faster and greater Na+ influx, which increases NaV channel availability and further depolarizes the membrane. When NaV1.5 is mechanically stimulated in the presence of capsaicin, there would be a reduction in Na+ influx and consequently a slower membrane depolarization, which would reduce the effect of mechanical force on NaV channel availability.Quantifying ion channel MS is challenging , and difparallel ,21,48. T studied , capsaictentials . ComparaV channels [V1.5 inactivation recovery and use-dependent inactivation. Pressure applied to patches increased the time to recover from inactivation, whereas shear stress applied in the whole-cell configuration decreased it. As previously reported [V1.5's recovery from inactivation in patches and whole cells. Intriguingly, the opposing effects on recovery with mechanical stimuli were further amplified with capsaicin. Capsaicin and pressure cooperatively delayed recovery further, in contrast to whole cells, where shear stress accelerated inactivation recovery, and thus reduced capsaicin mediated recovery delays.Many membrane modulating amphiphilic drugs alter recovery from inactivation and exert use-dependent block on Nachannels : these rchannels . Changeschannels . Thereforeported , the addV1.5 inactivation recovery time in patches [V1.5 was previously unknown.At the concentration we tested, capsaicin alone did not significantly alter the half-frequency for use-dependent inactivation in patches, but it did in whole cells. When capsaicin and pressure were applied together, they decreased the frequency of use-dependent inactivation in patches; while in whole cells, capsaicin alone lowered use-dependence frequency, a process unaffected by shear stress. Pressure has previously been shown to prolong Na patches , but theV1.5 MS. Negative pressures in the patch may alter the properties of voltage-gating through a lipid-stretch mechanism, whereas shear stress across the membrane may alter gating though a mechanism involving an asymmetric tension across on the entire cell that is attached to the electrode [The opposing responses in use-dependence and recovery using the two approaches are independent of capsaicin, suggesting that these mechanical stimuli may act through different mechanisms ,62. Conclectrode ,62,64.V channel gating mechanism [Lipid compositions surrounding channels play critical roles in force transduction and gating mechanisms. Changing the lipid composition of synthetic bilayers alters mechano-gated MscL channel gating parameters—adding lysophosphatidylcholine to vesicles composed of phosphatidylcholine was sufficient to measurably open MscL channels . In anotechanism . Two gA echanism . Amphiphechanism ,42. ThesMechanical stimuli modulate components of cardiac and intestinal contractility . In the V1.5 voltage-gating and MS with therapeutic potential. The NaV1.5 targeting amphiphilic drug, ranolazine is a common anti-ischemic and anginal medication. Ranolazine inhibits the increase in peak Na+ current and the hyperpolarization of voltage-dependence of activation induced by pressure or shear stress in a manner comparable to capsaicin [V1.5 peak current and MS [V1.5 MS, since TRPV1 is only expressed in extrinsic sensory fibers, which are not primary regulators of motility [SCN5A/NaV1.5) processes by different mechanisms in the GI tract. Amphipathic drugs are widely used in clinical practice to target ion channels but are rarely used for mechano-modulation [V1.5 MS demonstrates that membrane-partitioning amphipathic agents can effectively alter MS and may have pharmacologic potential. Such amphiphiles may be viable candidates for therapeutic modulation of NaV1.5 MS and for targeting dysfunction in channelopathies with disordered MS. Channelopathies involving mechanosensitive dysfunction are an emerging area of study [V1.5 MS channelopathies currently lack targeted treatment options. While continued progress is required, this study suggests that therapeutically targeting the voltage-gated and mechanosensitive functions of NaV1.5 separately in human diseases may hold promise for MS-associated disorders.Capsaicin joins a growing group of amphipaths that modulate Naapsaicin . Abnormaapsaicin . This cot and MS . Capsaict and MS ; howevert and MS  and is mt and MS ,71. Capst and MS . Fascinat and MS , possiblmotility . Buildindulation ,13. The dulation , and randulation  to inhibof study ,19,76,77of study  and NaV1